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TomTBT

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[ "<title>Background</title>", "<p>In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe, including the Netherlands, Belgium, Germany, Luxembourg and the North of France [##REF##18620767##1##,##REF##18620768##2##]. In order to improve the understanding of the epidemiological situation of this disease, it was necessary to execute a cross-sectional serological study at the end of the vector season of 2006. The Community legal framework on bluetongue monitoring and surveillance was laid down in Council Directive 2000/75/EC and Commission Decision 2005/393/EC and these are in line with the Terrestrial Animal Health Code of the OIE. Cattle were the target species for the cross-sectional serological study at the end of 2006 [##UREF##0##3##,##UREF##1##4##].</p>", "<p>Many hoped that the winter season of 2006/2007 would halt the BTV epidemic, assuming that the chain of transmission would be broken by the dying off of infected adult vectors and a halt in the life cycle of the vector because of low temperatures. However, in the course of 2007 it became evident that BTV8 somehow had survived the winter in North-West Europe and a re-emerging epidemic spread exponentially within the original affected countries. Moreover, BTV8 was introduced into the United Kingdom, Denmark, Czech Republic and Switzerland [##UREF##2##5##].</p>", "<p>The scale of the epidemic in 2007 was so huge that the European Union decided to start vaccination against BTV8 in 2008. The vaccination campaign aims to achieve a 80% or more coverage of animals protected (either by vaccination or by immunity acquired through natural infection) [##UREF##3##6##].</p>", "<p>The sentinel monitoring system, set up at the beginning of 2007 to detect re-emergence of BTV8, already provided some insights into the extend of the BTV8 spread in the cattle population in 2007. However, with respect to goats and sheep there was no information on the BTV8-seroprevalence in North-West Europe. This paper presents the seroprevalence and geographical spread of BTV8 on animal and herd levels in goats and sheep in the Netherlands in 2006 and 2007.</p>" ]

[ "<title>Methods</title>", "<p>Blood samples from Dutch sheep and goats were serologically tested at the Central Veterinary Institute (CVI) in Lelystad for antibodies against BTV8 using a competitive ELISA (Institute Pourquier, Montpellier, France). This ELISA has a high sensitivity (~100%) and specificity (&gt;99.8%) [##REF##17553280##7##]. The blood samples were collected in the framework of obligatory and voluntary health programmes (e.g. certified disease-free programmes within the European Union) executed by the Dutch Animal Health Service. For the 2006 seroprevalence estimates, we used blood samples collected in the first months of 2007. For the 2007 seroprevalence estimates, we used blood samples collected in the last months of 2007. Locations with animal sampled were selected proportional to the population distribution of locations with goats and sheep in the Netherlands in the different provinces. Sample size within locations for the health programmes was set at detecting at least a prevalence of disease of 5%. In smaller flocks this often meant that almost all animals within the flock were sampled.</p>", "<p>There are approximately 41,000 locations with sheep and 23,000 locations with goats in the Netherlands registered at the Animal Health Service. Based on these data and an a priori estimated seroprevalence of BTV8-infected locations with goats of 5% and a maximum acceptable error in the estimated prevalence of 5% and a 95% confidence level required for the estimated prevalence, a sample size of approximately 70 locations with goats was calculated using WinEpiscope version 2.0 [##REF##17553280##7##] for our cross-sectional study in 2006. For 2007, we adjusted the a priori estimated seroprevalence of BTV-8 infected locations with goats to 30% with a maximum acceptable error in the estimated prevalence of 10% and a 95% confidence level required for the estimated prevalence, resulting in a sample size of approximately 81 locations with goats for 2007. For the locations with sheep, we used an a priori estimated prevalence of BTV8-infected locations with sheep of 10% and a maximum acceptable error in the estimated prevalence of 5%, resulting in a calculated sample size of approximately 140 locations with sheep for 2006. For 2007, we adjusted the a priori estimated seroprevalence to 50% with a maximum acceptable error in the estimated prevalence of 7.5% and a 95% confidence level required for the estimated prevalence, resulting in a sample size of approximately 170 locations. Since there is also an unknown number (not registered) of small-holder locations in the Netherlands with goats and sheep with a small numbers of animals (1 to 5 animals per location), we arbitrarily increased the actual sample size with approximately 15 to 20%. Exact confidence intervals for the estimated seroprevalence were calculated according to Fleiss [##UREF##4##9##].</p>" ]

[ "<title>Results</title>", "<title>Seroprevalence of BTV8-infected locations with goats and sheep in 2006</title>", "<p>A total of 1,975 goat sera from 83 locations with goats and 2,555 sheep sera from 143 locations with sheep were collected. All goat samples were seronegative, a total of 17 sheep samples (0.7%) from 10 locations with sheep (7.0%) were seropositive. The BTV8-seropositve locations with sheep were located in the provinces of Limburg, North Brabant, Zeeland, and Gelderland. Within-location seroprevalence ranged from 3 to 50% (almost all sheep present in the flocks were sampled). The location of the provinces in the Netherlands is shown in Figure ##FIG##0##1##. Based on our sampled population we estimated the proportion of BTV8-seropositive locations in the Netherlands with sheep and goats. On a national level the estimated seroprevalence of BTV8-exposed locations in 2006 was 0% for goats (95% confidence interval: 0 – 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 – 12.9%).</p>", "<title>Seroprevalence of BTV8-infected locations with goats and sheep in 2007</title>", "<p>A total of 1,995 goat sera from 81 locations with goats and 4,252 sheep sera from 214 locations with sheep were collected. A total of 204 goat samples (10.2%) from 38 locations with goats (46.9%) were seropositive. The BTV8-seropositve locations with goats in 2007 were located in the provinces of North Holland, South Holland, Utrecht, Gelderland, North Brabant, and Limburg.</p>", "<p>A total of 1,627 sheep samples (38.3%) from 149 locations with sheep (69.6%) were seropositive. The BTV8-seropositve locations with sheep were located in all provinces of the Netherlands in 2007. On a national level the estimated seroprevalence of BTV8-exposed locations in 2007 was 47% for goats (95% confidence interval: 36 – 58%) and 70% for sheep (95% confidence interval: 63 – 76%). The median within-location seroprevalence on BTV8-infected locations with goats was 21% (min – max: 1 – 100%) and with sheep 67% (min – max: 1 – 100%) (Figure ##FIG##1##2##).</p>" ]

[ "<title>Discussion</title>", "<p>There is very sparse information on seroprevalence of BTV-infected locations with goats and sheep during outbreaks. A cross-sectional study in Kazakhstan showed a within-herd seroprevalence in cattle, sheep and goats varying between 0 and 100% [##REF##12523989##10##]. A cross-sectional study in sheep flocks in Northern Pakistan [##REF##9234437##11##] indicated 90% of the flocks seropositive. In these seropositive sheep flocks, within-flock seroprevalence ranged from 12 to 100% (median: 47%). A cross-sectional serological study in Queensland, Australia, found within-flock seroprevalences in infected sheep flocks ranging from 1 – 42% and in infected goat flocks ranging from 5 – 16% [##UREF##5##12##].</p>", "<p>During the 2006-epidemic in North-West Europe, no clinically affected goats were reported [##UREF##0##3##]. The results of our seroprevalence study is in line with those findings. However, in week 35 of 2007, the first clinical disease in goats caused by BTV8 in North-West Europe was reported from the Netherlands [##REF##17990633##13##]: in a holding containing 600 milking goats, 10 goats demonstrated clinical signs of BT, starting with acute drop in milk yield and pyrexia, followed by edema of lips and face, crusts on lips and muzzle, nasal discharge, conjunctivitis and erythema of the udder. Up to the end of 2007, a total of 25 holdings reported clinical disease (BT indicative) in goats in the Netherlands. The results of our seroprevalence study indicate a seroprevalence of locations with goats of approximately 50% in the Netherlands in 2007. This is much higher than can be inferred from the 25 locations that reported clinical outbreaks in goats in 2007. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep [##REF##17965371##14##].</p>", "<p>In the first year (2006) of the BTV8-epidemic in the Netherlands, a total of 270 sheep flocks and 200 cattle herds reported clinical disease. In 2007, the epidemic really took off: about 3,200 clinical outbreaks were reported by locations with sheep on a total of 6,500 outbreaks reported. The results of the seroprevalence study in sheep in 2006 and 2007 are in line with the number of clinical outbreaks reported.</p>", "<p>In our study we see a wide range in within-location seroprevalence in locations with goats and sheep. This means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between locations. Two options for a vaccination strategy are open: either one vaccinates all ruminants on the locations with animals irrespective of the proportion of animals protected by a natural infection in 2006 or 2007 or one determines the proportion of animals protected by a natural infection within the flock: if a low to moderate proportion of animals is naturally protected one has a good reason to vaccinate all the animals; if a high proportion of animals is naturally protected, this might be a reason not to vaccinate the flock.</p>" ]

[ "<title>Conclusion</title>", "<p>Seroprevalence of BTV8-exposed locations with goats and locations with sheep was much higher in 2007 than in 2006. There was a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. There was a wide range in within-location seroprevalence in locations with goats and sheep. This means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats.</p>", "<title>Results</title>", "<p>On the basis of our cross-sectional study, the estimated seroprevalence of BTV8-exposed locations in the Netherlands in 2006 was 0% for goats (95% confidence interval: 0 – 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 – 12.9%). The estimated seroprevalence of BTV-8 exposed locations in 2007 was 47% for goats (95% confidence interval: 36 – 58%) and 70% for sheep (95% confidence interval: 63 – 76%). There was a wide range in within-location seroprevalence in locations with goats and sheep (1 – 100%). A gradient in seroprevalence was seen, with the highest level of seroprevalence in the southern Netherlands, the area where the epidemic started in 2006, and a decreasing seroprevalence when going in a northern direction.</p>", "<title>Conclusion</title>", "<p>There is a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. The wide range in within-location seroprevalence observed means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.</p>" ]

[ "<title>Authors' contributions</title>", "<p>ARWE, PAvR and EMAvR designed the study. PV facilitated the use of sera collected by the Animal Health Service. JP and SO performed the laboratory analyses. ARWE performed the data analyses and drafted the manuscript. JP, PAvR, PV and EMAvR commented on the draft. All authors read the manuscript and approved the manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>This work was commissioned and financially supported by the Dutch Ministry of Agriculture, Nature and Food Quality.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Geographical location of the twelve provinces in the Netherlands</bold> (DR: Drente; FL: Flevoland; FR: Friesland; GL: Gelderland; GR: Groningen; L: Limburg; NB: North Brabant; NH: North Holland; OV: Overijssel; UT: Utrecht; ZH: South Holland; ZL: Zeeland).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Distribution of within-location seroprevalence on locations with antibodies against bluetongue virus serotype 8 in goats (N = 38) and sheep (N = 149) in the Netherlands in 2007.</p></caption></fig>" ]

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[ "<title>Background</title>", "<p>Inflammatory mechanisms are pivotal in many disease states, including atherosclerosis, autoimmune disorders and ischemia/reperfusion injury [##REF##14654305##1##, ####REF##7522621##2##, ##REF##7504883##3##, ##REF##8975870##4####8975870##4##]. Under inflammatory conditions there is activation of vascular endothelial cells that involves various morphological and metabolic changes [##REF##8204088##5##]. There is induction of specific cell adhesion molecules, such as, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. These interact with their corresponding ligands on leukocytes namely, lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA-4) and carbohydrate moieties respectively [##REF##7522621##2##,##REF##10430040##6##]. The process of infiltration involves sequential capture, rolling, firm adhesion and transmigration across the endothelial barrier [##REF##12003992##7##]. Blockade of CAMs that mediate the accumulation of mononuclear cells under inflammation is now considered as an effective treatment strategy in clinical inflammatory disorders.</p>", "<p>TNFα is one of the major proinflammatory cytokines that is dysregulated in inflammatory diseases mentioned earlier and has been shown to contribute to endothelial dysfunction [##REF##10493176##8##]. TNFα causes endothelial dysfunction by various mechanisms that includes activation of transcription factor NF-κB [##REF##11418010##9##]. Transcriptional regulation of many pro-inflammatory genes, including CAMs, is under the control of different transcriptional factors including NF-κB [##REF##7540353##10##,##REF##7542214##11##]. NF-κB is a redox sensitive transcription factor that most commonly exists as a p50/p65 heterodimer. This heterodimer remains sequestered in the cytoplasm when associated with inhibitor of kappa B (IκB) proteins. Upon stimulation (e.g. by TNFα) IκB proteins get phosphorylated by upstream IκB kinases (IKKs) followed by degradation, releasing the active dimer to translocate into the nucleus to transcribe its target genes [##REF##15371334##12##,##REF##7822333##13##].</p>", "<p>Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone superfamily of ligand-activated transcriptional factors. PPARs heterodimerize with retinoid × receptor (RXR) and bind to peroxisome proliferator-response elements in target genes [##REF##8521507##14##]. The subtype PPARγ is a regulator of adipogenesis [##REF##8001151##15##]. A number of studies have demonstrated that PPARγ may play a role in regulating inflammatory responses [##REF##11375115##16##,##REF##12360213##17##]. 15-deoxy-d 12, 14-prostaglandin J2, the ultimate metabolite of prostaglandin (PG) D₂, is a natural ligand of PPARγ. 15d-PGJ2 has been shown to inhibit expression of iNOS and TNFα in several cell types that are dependent on PPARγ [##REF##8521498##18##,##REF##9422508##19##]. However, there are also anti-inflammatory responses of 15d-PGJ2 that are PPARγ independent [##REF##10200320##20##,##REF##10570310##21##]. There are studies that report protective effects mediated by 15d-PGJ2 via inhibition of infiltration of immune cells in various models of inflammation e.g. endotoxic shock [##REF##15988322##22##], lung injury [##REF##15684285##23##], ischemia/reperfusion injury [##REF##12970094##24##] and experimental autoimmune encephalomyelitis (EAE) [##REF##11960303##25##,##REF##11859145##26##]. Thus, based on these studies, we hypothesized that 15d-PGJ2 inhibits the adhesion of mononuclear cells to the endothelial cells and thereby attenuates their transmigration. We observed that 15d-PGJ2 inhibited the adhesion of monocytes to bEND.3 endothelial cell line, activated by TNFα, by downregulation of endothelial CAMs via inhibition of IKK-NF-κB pathway.</p>" ]

[ "<title>Methods</title>", "<title>Reagents and Antibodies</title>", "<p>DMEM (4.5 g/L glucose), minimum essential medium alpha (MEM alpha) with ribonucleotides and deoxyribonucleotides, RPMI-1640 medium and FBS were purchased from Gibco BRL (Carlsbad, CA, USA). Granulocyte macrophage colony stimulating factor (GMCSF) and recombinant mouse TNFα were from R &amp; D Systems (Minneapolis, MN, USA). Vybrant Cell adhesion kit containing Calcein AM fluorescent dye was from Molecular Probes (Eugene, OR, USA). ECL detection kit was from GE healthcare (Piscataway, NJ, USA). Antibodies for p65, p50, IκBα, VCAM-1 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Texas red conjugated rabbit IgG antibody was from Vector Lab. Inc. (Burlington, CA, USA). Trizol reagent and Lipofectamine Plus were from Invitrogen (Carlsbad, CA, USA). Fluoromount-G was from Electron Microscopy Sciences (Hartfield, PA, USA). Antibodies against VCAM-1 (FITC labeled), ICAM-1 and E-selectin (PE labeled) were from BD Pharmingen (Franklin Lakes, NJ). Luciferase assay system was purchased from Promega (Madison, WI).</p>", "<title>Cell culture</title>", "<p>The bEND.3 mouse brain endothelial cells were from ATCC (American Type Culture Collection, Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% Fetal Bovine serum (FBS) and antibiotics. Cells were grown to confluence, made serum free for further treatments, and stimulation with TNFα (50 ng/ml) for all the experiments. JAWS II, a mouse monocyte cell line (ATCC) was maintained in MEM Alpha medium with 10% heat inactivated FBS, 0.5% gentamycin and granulocyte-macrophage colony-stimulating factor (GMCSF) (1 ng/mL; R &amp; D Systems).</p>", "<title>Plasmids and Transfection</title>", "<p>NF-κB-luciferase was kindly provided by Dr. George Rewadi (Institut Pasteur, Laboratoire des Mycoplasmes, Paris, France), flag-IKKα was a gift from Dr. Zheng-Gang Liu (National Institute of Health, Bathesda, MD) and FLAG-tagged wild-type (wt) PPARγ and FLAG-tagged L468A/E471A PPARγ were provided by Dr. V. Chatterjee (University of Cambridge, Cambridge, U.K.). The peroxisome proliferator-response element (PPRE)-containing reporter plasmid (J6-thymidine kinase (TK)-Luc) was provided by B. Staels (Institut Pasteur de Lille, Lille, France). PTL-luciferase, Gal-p65 and Gal-DBD (DNA binding domain) were purchased from Panomics (Fremont, CA). The endothelial cell line was transfected with the indicated plasmid (0.5 μg/well) using Lipofectamine Plus Reagent under serum free conditions as described before [##REF##15953363##27##]. pcDNA3.1 was used to normalize the total content of DNA in all transfection experiments.</p>", "<title><italic>In vitro </italic>Adhesion assay model</title>", "<p>As described earlier, bEND.3 cells were grown as monolayers in double chamber slides (Nalge Nunc, Naperville, IL, USA) [##REF##15953363##27##]. Cells were pre-treated with 15d-PGJ2 for 30 min followed by TNFα for 6 h. Dye labeled monocytes at the concentration of 2 × 10⁶cells/ml were added per chamber on the bEND.3 cells and allowed to interact for 30 min with gentle shaking at 37°C. Adherent fluorescent cells were observed using a fluorescence microscope (Olympus, BX60) and images were captured in Adobe Photoshop 7.0 at 100×. Adherent fluorescent cells were counted using Image Pro-Plus 4.0 software. Mean and SD were calculated for independent experiments. Results were plotted as fold change compared to the control values for all the experiments.</p>", "<title>Immunocytochemistry</title>", "<p>BEND.3 cells were grown in chamber slides and treated with 15d-PGJ2 and stimulated with TNFα for 20 min. Cells were fixed with paraformaldehyde (4%) followed by blocking in blocking reagent. Cells were then incubated in anti-p65 antibody followed by incubation in secondary antibody and mounting with Flouromount-G. The stained sections were analyzed by immunofluorescence microscopy (Olympus BX-60 from Opelco, Dulles, VA, USA) with images captured using an Olympus digital camera (Optronics, Goleta, CA, USA) at 400× magnification. Captured images were processed using Adobe Photoshop 7.0 and were adjusted using brightness and contrast tools. Three independent experiments were done and 5 fields for each treatment were taken. Representative images are shown.</p>", "<title>Real-time or quantitative (q) PCR</title>", "<p>Cells were harvested in Trizol reagent and RNA was isolated per the manufacturer's protocol. cDNA synthesis was done using iScript CDNA synthesis kit (BIO-RAD Laboratories, Hercules, CA, USA) per the manufacturer's protocol. qPCR was performed using SYBR GREEN PCR master mix (Applied Biosciences, Foster city, CA, USA) and BIO-RAD laboratories iCycler iQ PCR using primers as described before [##REF##15953363##27##]. primers of CAMs and 18S are as follows, ICAM-1 FP 5'-gca gag tgt aca gcc tct tt-3' RP 5'-ctg gta tcc cat cac ttg-3', VCAM-1 FP 5'-gca gag tgt aca gcc tct tt-3', RP 5'-ctg gta tcc cat cac tcg ag-3'; E-selectin FP 5'-act tca gtg tgg tcc aag ag-3' RP 5'-gca cat gag gac ttg tag gt-3'; 18S FP 5'-gaa aac att ctt ggc aaa tgc ttt-3' RP5'-gccgct aga ggt gaa att ctt-3'. The normalized mRNA expression was computed with that of 18s expression. Values are expressed as fold change from the control values and plotted.</p>", "<title>Preparation of cytosolic and nuclear extracts</title>", "<p>Cytosolic and nuclear extracts from bEND.3 cells were prepared using the method of Digman et al [##REF##6828386##28##] with slight modification [##REF##15470065##29##]. Cells were harvested, washed twice with ice-cold PBS, and lysed in 400 μl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl₂, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin) containing 0.1% Nonidet P-40 for 15 min on ice, vortexed vigorously for 15 s, and centrifuged at 14,000 rpm for 30 s. The pelleted nuclei were resuspended in 40 μl of buffer B [20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin]. After 30 min on ice, lysates were centrifuged at 14,000 rpm for 10 min. Supernatants containing the nuclear proteins were diluted with 20 μl of modified buffer C [20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.05 M KCl, 0.2 mM EDTA and 0.5 mM PMSF] and stored at -70°C until use. Cytosolic fraction (50 μg) was used for western blot analysis for the detection of IκBα and IKKα using their specific antibodies as described before [##REF##15470065##29##].</p>", "<title>Western blot</title>", "<p>Cell extracts were prepared as previously described with lysis buffer (50 m<sc>M</sc> Tris-HCl, pH 7.4, containing 50 mM NaCl, 1 m<sc>M</sc> EDTA, 0.5 m<sc>M</sc> EGTA, 1% Triton X-100, 10% glycerol, and protease inhibitor mixture) [##REF##15953363##27##,##REF##15470065##29##]. Protein (50 μg) was loaded with appropriate marker (Bio-Rad Laboratories, Hercules, CA, USA) on 8% sodium dodecyl sulfate-polyacrylamide gel (SDS_.PAGE), followed by transfer to nitrocellulose membrane. The membrane was blocked with 5% milk or 3% BSA in Tris buffered saline-tween (TBST). Primary anti-p65, -IκBα, -pIKKα was added. Blots were washed, followed by incubation in secondary antibody and then detection by ECL-chemiluminescence method.</p>", "<title>Electrophoretic mobility shift assay (EMSA)</title>", "<p>Nuclear extracts from treated and untreated cells were prepared and EMSA was performed as described previously [##REF##15470065##29##,##REF##14724246##30##] using NF-κB consensus sequence that was end-labeled with [γ-³²P] ATP. Nuclear extracts were normalized on the basis of protein concentration and equal amounts of protein (5 μg) were loaded. The gels were dried and then autoradiographed at -70°C using x-ray film.</p>", "<title>Flow cytometry</title>", "<p>15-PGJ2 treated and untreated bEND.3 cells in the presence or absence of TNFα (50 ng/ml) were harvested and processed as described earlier [##REF##14707106##31##]. Cells were blocked with anti-CD16/CD32 and incubated with FITC- or PE-labeled antibodies against ICAM-1, VCAM-1 and E-selectin. The cells were acquired by FACS and analyzed by CellQuest (BD PharMingen, Franklin Lakes, NJ).</p>", "<title>Statistical analysis</title>", "<p>Results shown represent mean ± SD. Statistical analysis was performed by ANOVA by the Student-Neumann-Keuls test using GraphPad InStat software (San Diego, CA, USA).</p>" ]

[ "<title>Results</title>", "<title>15d-PGJ2 inhibits monocyte adhesion to a brain-derived endothelial cell line</title>", "<p>Activated bEND.3 endothelial cells under pro-inflammatory environment allows increased adherence of leukocytes to its surface to facilitate their migration [##REF##10430040##6##]. In our <italic>in vitro </italic>system, bEND.3 cells were activated with TNFα that caused a significant increase in the adhesion of monocytes (~9 fold) compared to untreated cells. However, treatment with15d-PGJ2 (1–10 μM) 30 min prior to the addition of TNFα significantly inhibited the adhesion of monocytes (Fig. ##FIG##0##1a, b##). Prostaglandin production begins with the liberation of arachidonic acid which under cyclooxygenase enzymes 1 and 2 gets converted to PGH2. Specific prostaglandin synthase convert PGH2 into a series of prostaglandins including PGI2, PGF2α, PGD2 and PGE2 [##REF##11864843##32##]. We also treated the bEND.3 cells with different prostaglandins (PGA1, PGB2, PGD2, PGE1, PGE2, PGF1α, 15d-PGJ2, PGJ2), arachidonic acid, leukotriene (LTB4) and thromboxane (TXB4) and observed that PGA1 and PGD2 treatment showed a significant decrease in TNFα induced adhesion of monocytes, as these are precursors of 15d-PGJ2 (Fig. ##FIG##0##1c##). These results suggest the specificity of 15d-PGJ2 in mediating the inhibition of the adhesion process of monocytes on activated bEND.3 cells. 15d-PGJ2 did not cause any cell death (assessed by MTT and LDH release assays) at the concentrations used (data not shown).</p>", "<title>15d-PGJ2 inhibits expression of endothelial CAMs</title>", "<p>Extravasation of mononuclear cells the recruitment cascade are orchestrated by cell adhesion molecules on both endothelial and immune cells [##REF##14654305##1##]. Accordingly, we examined the effect of 15d-PGJ2 on TNFα induced expression of CAMs (VCAM-1, ICAM-1 and E-selectin). For this, bEND.3 cells were pretreated with15d-PGJ2 (5–10 μM) followed by TNFα (50 ng/ml) treatment. After 2 h of incubation, bEND.3 cells were processed for RNA isolation and quantitative analysis of CAMs using real time PCR (qPCR). Treatment with TNFα significantly increased the mRNA expression of VCAM-1, ICAM-1 and E-selectin as compared to control cells. 15d-PGJ2 markedly downregulated their expression with a most pronounced effect observed on expression of VCAM-1 as compared to E-selectin or ICAM-1 (Fig. ##FIG##1##2a, b## and ##FIG##1##2c##). These observations are in agreement with flow cytometry analysis which also showed that 15d-PGJ2 treatment significantly reduced the expression of endothelial CAMs with maximum affect on VCAM-1 expression (Fig. ##FIG##1##2d##).</p>", "<title>15d-PGJ2 inhibits VCAM-1 expression in a PPARγ independent manner</title>", "<p>To determine whether 15d-PGJ₂mediates its inhibitory effect through PPARγ, we employed GW9662, an irreversible PPARγ antagonist. GW9662 (10 μM) did not reverse 15d-PGJ2 mediated inhibition of TNFα induced expression of VCAM-1 in the endothelial cell line (Fig. ##FIG##2##3A##). Another activator of PPARγ, troglitazone, was used to examine if PPARγ plays any role in expression of VCAM-1 in bEND.3 cells. Troglitazone treatment, similar to 15d-PGJ2 treatment, inhibited the expression of VCAM-1, which could not be reversed by GW9662 (Fig. ##FIG##2##3a##). To examine the ability of GW9662 on 15d-PGJ2 and troglitazone mediated induction of PPARγ transcription, we used a chimeric receptor system in which the putative ligand-binding domain of the PPARγ is fused to the DNA binding domain of the yeast transcription factor galactose-responsive gene 4 (GAL4). The 15d-PGJ₂and troglitazone potently activated the PPARγ-dependent chloramphenicol acetyltransferase (CAT) reporter activity, which was completely blocked by GW9662 treatment (Fig. ##FIG##2##3b##). To confirm this observation, bEND.3 cells were transfected with PPARγ wild type (Wt) and dominant-negative (DN) expression vectors and determined the effects on VCAM-1mRNA expression. 15d-PGJ2 was able to inhibit the TNFα induced expression of VCAM-1 in both control and PPARγ Wt transfected cells. However, transfection with PPARγ DN was not able to attenuate the 15-dPGJ2 mediated inhibition of VCAM-1 mRNA expression indicating that the inhibitory effect of 15d-PGJ2 is independent of PPARγ (Fig. ##FIG##2##3c##). Treatment with 15d-PGJ2 induced the PPRE-luciferase activity in transiently transfected PPARγ Wt expression vector, whereas, it had no effect in PPARγ DN transfected cells (Fig. ##FIG##2##3d##) suggesting that 15d-PGJ2 has the ability to activate PPARγ but its effect on VCAM-1 expression in the bEND.3 endothelial cell line is independent of PPARγ.</p>", "<title>15d-PGJ2 inhibits NF-κB function in brain-derived endothelial cell line</title>", "<p>To further understand the mechanism of inhibitory action of 15d-PGJ2 on endothelial CAMs and the process of adhesion we examined the effect of 15d-PGJ2 on NF-κB pathway, which is a pleiotropic regulator of many genes involved in inflammation including CAMs [##REF##7542214##11##]. Using EMSA, we observed that 15d-PGJ2 inhibited the TNFα induced binding of the NF-κB complex, in a time and dose-dependent manner (Fig. ##FIG##3##4a##). To further define the inhibitory effect of 15d-PGJ2 on TNFα mediated activation of the NF-κB pathway, the bEND.3 cells were transfected with the p65/p50 complex along with the NF-κB luciferase reporter construct. Cells transfected with p65/p50 exhibited increased reporter activity, which was markedly reduced in a dose-dependent manner with 15d-PGJ2 treatment (Fig. ##FIG##3##4b##). These observations obtained from EMSA and transfection studies were further confirmed by immunostaining for p65 nuclear translocation. Under TNFα stimulation, p65 translocated to the nucleus and was markedly attenuated by 15d-PGJ2 treatment (Fig. ##FIG##3##4c##). Correspondingly, we also observed that 15d-PGJ2 inhibited the TNFα induced nuclear translocation of p65 and degradation of IκBα protein in a time and dose-depended manner (Fig. ##FIG##3##4d##).</p>", "<p>To support the 15d-PGJ2 mediated inhibition on NF-κB pathway, we examined the effect of 15d-PGJ2 on p65-DNA binding domain-gal4 transcriptional activity. The p65-DNA binding domain-gal4 (p65-DNA-gal4) is a chimeric-transactivator, which consists of transcriptional activation domain of NF-κB p65 protein fused to the DNA-binding domain of GAL4 protein from yeast. As evident from figure ##FIG##4##5##, treatment with TNFα induced the transcriptional activity of p65-DBD-gal4 which was completely blocked by 15d-PGJ2 treatment.</p>", "<title>Inhibition of IKK activity by 15d-PGJ2</title>", "<p>Based on preceeding results, we examined the effect of 15d-PGJ2 on the activity of IKK, the upstream kinase of the NF-κB pathway. Cells were treated with 15d-PGJ2 followed by TNFα for 15 min and phosphorylation of IKKα was detected using a specific antibody. As shown in figure ##FIG##5##6a##, TNFα treatment induced phosphorylation of IKKα in bEND.3 cells which was completely blocked by 15d-PGJ2 treatment. bEND.3 cells were further cotransiently transfected with IKKα and NF-κB luciferase reporter constructs and after 24 h, cells were treated with TNFα with or without 15d-PGJ2. TNFα induced the IKKα mediated NF-κB-reporter activity, which was a significantly downregulated by 15d-PGJ2 treatment (Fig. ##FIG##5##6b##). This observation was further supported when bEND.3 cells were transiently cotransfected with p65-DBD-gal4 and IKKα expression vectors. Transient transfection with IKKα significantly induced p65 transcriptional activity which was completely blocked by 15d-PGJ2 treatment (Fig. ##FIG##5##6c##) suggesting that 15d-PGJ2 inhibits NF-κB function by inhibiting IKKα activity in bEND.3 cells.</p>", "<title>Post treatment of 15d-PGJ2 inhibits adhesion of monocytes on activated brain-derived endothelial cell line</title>", "<p>Our results suggested that 15d-PGJ2 inhibits the adhesion of mononuclear cells on activated endothelial cells by inhibiting the CAMs expression via downregulation of NF-κB pathway when pretreated before stimulation with TNFα. We wanted to examine if post treatment with 15d-PGJ2 could inhibit the adhesion of mononuclear cells on TNFα-stimulated cells. For this, bEND.3 cells were stimulated with TNFα for 6 h followed by addition of various concentrations (5–20 μM) of15d-PGJ2. After 30 min of treatment with 15d-PGJ2, cells were washed and labeled monocytes were added for adhesion assay. Interestingly, post treatment with 15d-PGJ2 inhibited adhesion of mononuclear cells on activated bEND.3 cells (Fig. ##FIG##6##7##) suggesting that 15d-PGJ2 probably inhibits multiple pathways including NF-κB-CAMs expression and other signaling pathway required for monocyte-endothelial cell adhesion,.</p>" ]

[ "<title>Discussion</title>", "<p>PGs are small lipid molecules that regulate numerous processes in the body and their biological effects is an area of concentrated research [##REF##15639643##33##]. The J series of PGs have been demonstrated to regulate processes like adipogenesis, inflammation and tumorigenesis [##REF##11864843##32##]. 15d-PGJ2 is a metabolite of PGD2 and is produced by mast cells, T cells, platelets and alveolar macrophages [##REF##7649404##34##]. 15d-PGJ2 is emerging as a key anti-inflammatory mediator. Consistent with this we have previously shown that 15d-PGJ2 has an anti-inflammatory role in primary astrocytes [##REF##15470065##29##]. This study reports for the first time that 15d-PGJ2 inhibits adhesion of monocytes to TNFα activated bEND.3 endothelial cells by downregulating endothelial CAMs via inhibition of IKKα-NF-κB pathway but in a PPARγ independent manner.</p>", "<p>Infiltration of leukocytes is a crucial response in inflammatory reactions in numerous disorders where these leukocytes are intended to induce inflammation in CNS when BBB is compromised. However, when misdirected, they destroy healthy cells and matrix components causing tissue damage [##REF##14654305##1##]. Therefore, in recent years efforts have been directed to limit the infiltration of mononuclear cells so as to minimize the tissue injury during the disease process. In earlier studies in different disease models, it was reported that 15d-PGJ2 inhibits infiltration of leuckocytes to site of inflammation [##REF##15470065##29##,##REF##10638762##35##]. Since adhesion of infiltrating cells to endothelium, is a prerequisite for infiltration, we investigated the effect of PPAR activator 15d-PGJ2 on the adhesion process. 15d-PGJ2 was observed to inhibit the adhesion of monocytes to activated bEND.3 endothelial cells in a dose-dependent manner. These Results were consistent with previous studies where 15d-PGJ2 inhibited the adhesion of mononuclear cells to PMA, IFNγ or IL-1β activated endothelial cells [##REF##12406386##36##,##REF##10479650##37##]. The inhibition of the adhesion process by15d-PGJ2 was mediated by down regulation of TNFα induced endothelial CAMs expression, namely, VCAM-1, E-selectin and ICAM-1. Further, this effect was found to be PPARγ independent. Our Results were consistent with other reports in which 15d-PGJ2 and other PPAR activators negatively modulate endothelial CAMs <italic>in vitro </italic>[##REF##10479650##37##, ####REF##10645917##38##, ##REF##15662020##39####15662020##39##]. Treatment of bEND.3 cells with 15d-PGJ2 showed effects by attenuating signaling taking place during adhesion process as well as downregulating endothelial CAMs expression, thereby giving a significant additive effect on inhibition on adhesion of monocytes. To further understand the mechanism of inhibition mediated by15d-PGJ2, we determined its effect on the NF-κB transcription factor which is known to be activated by TNFα [##REF##11418010##9##]. 15d-PGJ2 was observed to inhibit DNA binding of the NF-κB complex in a gel shift assay. Interestingly, this inhibition was through modulation of upstream targets of the NF-κB pathway. There was inhibition of TNFα induced degradation of IkBα protein thereby preventing p65 nuclear translocation. Our study is supported by other reports of inhibition of NF-κB by 15d-PGJ2, though in different cell types [##REF##15470065##29##,##REF##10638762##35##,##REF##12474221##40##,##REF##12353085##41##]. Thus, our data showed that 15d-PGJ2 inhibits TNFα induced NF-κB activity and consequently the expression of endothelial CAMs under our experimental model. Moreover, we have previously suggested IKK as a target of 15d-PGJ2 in modulating NF-κB pathway in brain glial cells [##REF##15470065##29##,##REF##10638762##35##], which is consistent in endothelial cells too. We can conclude from our <italic>in vitro </italic>data that 15d-PGJ2 inhibits endothelial-monocyte interactions via IKK-NF-κB-CAMs pathway in endothelial cells. PI3 kinase and Akt are also known to play an important role in the adhesion process [##REF##11278864##42##]. The activation of IKK is also regulated via phosphorylation by Akt [##REF##10485710##43##]. 15d-PGJ2 has been demonstrated to inhibit the PI3 kinase/Akt pathway in brain glial cells [##REF##15470065##29##]. PI3 kinase and Akt pathway play important role in adhesion as we have documented before that inhibition of PI3Kinase and Akt is able to inhibit the adhesion of monocytes [##REF##15953363##27##].</p>", "<p>Thus, 15d-PGJ2 might be modulating PI3 kinase-Akt-IKK-NF-κB-CAMs pathway. Interestingly, post treatment with 15d-PGJ2 was also able to inhibit monocyte adhesion on activated bEND.3 cells, suggesting the possibility that15d-PGJ2 may also inhibit other signaling pathway/s important for firm and sustained adhesion of monocyte on endothelial cells.</p>", "<p>15d-PGJ2 is a natural ligand of PPARγ and has numerous effects which are PPARγ dependent. Moreover, it has been shown to has therapeutic potential in various human autoimmune diseases as well as animal models of autoimmunity, including arthritis [##REF##11263774##44##, ####REF##10903334##45##, ##REF##15213234##46####15213234##46##], ischemia-reperfusion injury [##REF##11159886##47##,##REF##17589386##48##], Alzheimer's disease [##REF##10632585##49##, ####REF##10995830##50##, ##REF##16061222##51####16061222##51##], lupus nephritis [##REF##10640767##52##,##REF##11237557##53##] and EAE [##REF##11859145##26##,##REF##16844232##54##,##REF##15723383##55##]. More recent evidences have shown that there are effects of 15d-PGJ2 that are independent of PPARγ activation [##REF##11864843##32##], while, the exact mechanism of action of 15d-PGJ2 in different systems is unknown. There are various propositions such as presence of another cytoplasmic PG receptor [##REF##11208866##56##], recruitment of p300 by NF-κB [##REF##15470065##29##], inhibition of NF-κB DNA binding by alkylation of cysteine residue of p65 [##REF##10781090##57##], or ROR dependent mechanism [##REF##15662020##39##].</p>" ]

[ "<title>Conclusion</title>", "<p>All together, the present data shows that 15d-PGJ2 regulates inflammatory responses by inhibiting the infiltration of leukocytes across the endothelial barrier, which it does so by inhibiting monocyte adhesion to activated endothelial cells via downregulation of IKK-NF-κB-CAMs pathway in endothelial cells, independent of PPARγ.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The Infiltration of leukocytes across the brain endothelium is a hallmark of various neuroinflammatory disorders. Under inflammatory conditions, there is increased expression of specific cell adhesion molecules (CAMs) on activated vascular endothelial cells which increases the adhesion and infiltration of leukocytes. TNFα is one of the major proinflammatory cytokines that causes endothelial dysfunction by various mechanisms including activation of transcription factor NF-κB, a key transcription factor that regulates expression of CAMs. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone superfamily of ligand-activated transcriptional factors. 15-deoxy-δ 12, 14-prostaglandin J2 (15d-PGJ2) is a well recognized natural ligand of PPARγ and possesses anti-inflammatory properties both <italic>in vitro </italic>and <italic>in vivo</italic>. This study aims to elucidate the mechanism of 15-PGJ2 on the adhesion of mononuclear cells to activated endothelial cells.</p>", "<title>Methods</title>", "<p>To delineate the signaling pathway of 15d-PGJ2 mediated effects, we employed an <italic>in vitro </italic>adhesion assay model of endothelial-monocyte interaction. Expression of CAMs was examined using flow cytometry and real time PCR techniques. To define the mechanism of 15d-PGJ2, we explored the role of NF-κB by EMSA (<underline>E</underline>lectrophoretic <underline>M</underline>obility <underline>S</underline>hift <underline>A</underline>ssay) gels, NF-κB reporter and p65-transcriptional activities by transient transfection in the brain-derived endothelial cell line (bEND.3).</p>", "<title>Results</title>", "<p>Using an <italic>in vitro </italic>adhesion assay model, we demonstrate that 15d-PGJ2 inhibits TNFα induced monocyte adhesion to endothelial cells, which is mediated by downregulation of endothelial cell adhesion molecules in a PPARγ independent manner. 15d-PGJ2 modulated the adhesion process by inhibiting the TNFα induced IKK-NF-κB pathway as evident from EMSA, NF-κB reporter and p65 mediated transcriptional activity results in bEND.3 cells.</p>", "<title>Conclusion</title>", "<p>These findings suggest that 15d-PGJ2 inhibits inflammation at multiple steps and thus is a potential therapeutic target for various inflammatory diseases.</p>" ]

[ "<title>Abbreviations</title>", "<p>15d-PGJ 2: 15-deoxy-Delta (12, 14)-prostaglandin J; CAM: cell adhesion molecule; ICAM: Intercellular cell adhesion molecule-1; VCAM-1: Vascular cell adhesion molecule-1; NF-κB: Nuclear factor kappa B; IκB: Inhibitory kappa B; IKK: Inhibitory kappa B kinase.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>This study is based on an original idea of SG and IS. RP and SG wrote the manuscript. SG directed and RP performed the <italic>in vitro </italic>experiments. AKS helped in finalizing manuscript. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>RP and SG are equal contributors for this work. We would like to thank Drs. Anne G. Gilg and Ramandeep Rattan for editing manuscript and Ms Joyce Bryan for procurement of chemicals used in this study. These studies were supported by grants (NS-40144, NS-22576, NS-34741, NS-37766, and NS-40810) from the NIH and (SCIRF 0406 and SCIRF 0506) from State of South Carolina Spinal Cord Injury Research Fund Board. This work was supported by the NIH (NS-22576, NS-34741, NS-37766 and NS-40810) and from the Extramural Research Facilities Program of the National Center for Research Resources (Grants C06 RR018823 and No C06 RR015455).</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>5d-PGJ2 inhibits monocyte adhesion to endothelial cells</bold>. bEND.3 cells were incubated with different concentrations of 15d-PGJ2 (1–20 μM) (A) or mentioned prostaglandins (5 μM), arachidonic acid (5 μM), Leukotriene 4 (LTB 4, 5 μM) and Thromboxanes 4 (TXB 4, 5 μM) (C) for 30 min followed by TNFα (50 ng/ml) stimulation for 6 h. Fluorescently labeled monocytes were allowed to interact with activated bEND.3 cells. Adhered monocytes were counted as mentioned in 'Material and Methods'. Data calculated as mean ± SD of 21 fields from 3 different experiments. *** p &lt; 0.001 compared to untreated control cells and !!! p &lt; 0.001 compared to TNFα treated cells. (B) is the pictorial representation of adhesion under TNFα (50 ng/ml) and 15d-PGJ2 (10 μM) treatment.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>15d-PGJ2 inhibits mRNA and protein expression of endothelial CAMs</bold>. bEND.3 cells were pretreated with 15d-PGJ2 (5–20 μM) for 30 min followed by stimulation with TNFα (50 ng/ml) for 2 h. Cells were harvested in Trizol reagent for RNA isolation and cDNA synthesis. RT-PCR analysis was done for ICAM-1 (A), VCAM-1 (B) and E-selectin (C). Results were calculated as mean ± SD for 3 independent experiments. Samples were examined in triplicates. &amp;&amp;&amp; p &lt; 0.001 compared with control (untreated and unstimulated cells) and !!! p &lt; 0.001 as compared to TNFα treatment. For the quantitation of expression of surface CAMs, bEND.3 cells were treated with TNFα (50 ng/ml) in the presence or absence of 15d-PGJ2 (5–20 μM) for 6 h followed by flow cytometry analysis (D) (n = 2).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>15d-PGJ2 inhibits VCAM-1 in PPARγ independent manner</bold>. bEND.3 cells were treated with GW9662 (10 μM) 30 min prior to treatment with 15d-PGJ2 (10 μM) or troglitazone (10 μM) followed by TNFα treatment (50 ng/ml). bEND.3 cells were lysed and processed for immunoblot analysis for VCAM-1 and β actin expression (A). Endothelial cell line was cotransfected with PPARγ-GAL4 chimeras and the reporter plasmid (upstream activating sequences)₅-TK-CAT. After 48 h, cells were treated with 15d-PGJ₂or trogliatzone in the presence or absence of GW9662 (10 μM) for 24 h. Cell extracts were subsequently assayed for CAT activity by ELISA (Roche) (B). pCMV-GAL4-binding domain (without insert) and (upstream activating sequences)₅-TK-CAT were transfected as a control to detect the basal levels of CAT activity (first lane). Data are mean of three values ± SD. *** <italic>p </italic>&lt; 0.001 as compared with untreated cells; !!! <italic>p </italic>&lt; 0.001 as compared with 15d-PGJ2 treated cells. (C) Cells were transfected with PPARγ wild type (Wt) and dominant negative (DN) constructs followed by treatment with 15-dPGJ2 (5 and 10 μM; 30 min) and TNFα (50 ng/ml, 2 h) and processed for qPCR for detection of VCAM1 mRNA expression as described in 'Material and Methods' (C). Results were calculated as mean ± SD for 3 independent experiments. Samples were run in triplicates. &amp;&amp;&amp; p &lt; 0.001 compared with control (untreated and unstimulated cells) and !!! p &lt; 0.001 as compared to TNFα treatment. Cells were co-transfected with PPARγ wild type (Wt) and dominant negative (DN) (0.5 μg/well) constructs along with PPRE-luc reporter (0.5 μg/well) and pRL-TK (0.5 μg/well) followed by treatment with 15-dPGJ2 (10 μM) after 24 h. After 24 h incubation, luciferase activity was performed, as described before pcDNA3.1 was added to normalize the total content of DNA for transfection. Data are mean ± SD of three different values. ***, <italic>p </italic>&lt; 0.001 as compared with untreated cells; !!!, <italic>p </italic>&lt; 0.001 as compared with 15d-PGJ₂-treated PPAR wt transfected cells.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>15d-PGJ2 inhibits TNFα induced NF-κB function in endothelial cells</bold>. bEND.3 cells were treated with 15d-PGJ2 (1–10 μM) and TNFα (50 ng/ml) for various time periods (5–40 min) and processed for EMSA as described in 'Material and Methods' (A). bEND.3 cells were transiently transfected with p65, p50 expression vectors along with NF-κB luciferase reporter construct (0.5 μg/well) and pCMV-β-galactosidase (0.5 μg/well) followed by treatment with 15d-PGJ2 (5–20 μM) for 4 h and processed for luciferase and β-galactosidase activities. Luciferase activity was normalized with respect to β-gal activity (B). Results were calculated as mean ± SD for 3 independent experiments. Samples were run in triplicates. &amp;&amp;&amp; p &lt; 0.001 compared with control and !!! p &lt; 0.001 compared with TNFα treatment (50 ng/ml). Cells were treated with 15d-PGJ2(10 μM) for 30 min followed by TNFα for 20 min and stained with anti-p65 antibody as described in 'Material and Methods' (C). Images taken at 200× magnification are representative of 6 fields from each treatment and 3 independent experiments. Treated and untreated cells were processed for immunoblot analysis for p65 and IκBα levels (D). Representative blot from two independent experiments are shown.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>15d-PGJ2 inhibits p65 transcriptional activity in endothelial cells</bold>. bEND.3 cells were transfected with Gal-p65 or Gal-DBD along with PTL-luciferase and PRL-TK reporter constructs as described in Material and Method. bEND.3 cells were pretreated with 15d-PGJ2 (10 μM) for 30 min followed by TNFα treatment (50 ng/ml). After 6 h of TNFα treatment, cells were processed for luciferase assay and results were normalized with PRL-TK luciferase activity in each sample. Results were calculated as mean ± SD for 3 independent experiments. *** and !!! p &lt; 0.001 compared with control, @@@ p &lt; 0.001 compared with TNFα treatment.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>15d-PGJ2 inhibits TNFα induced IKKα mediated NF-κB reporter activity</bold>. bEND.3 cells were treated with TNFα (50 ng/ml) in the presence or absence of 15d-PGJ2 (10 μM) followed by detection of pIKKα using its specific antibody (Cell Signaling) (A). β actin was used as a control for equal content of protein loaded. bEND.3 cells were transfected with IKKα, NF-κB luciferase and pCMV-β-galactosidase constructs and treated with 15d-PGJ2 (5–20 μM) and TNFα (50 ng/ml). After 4 h of TNFα treatment, cells were processed for luciferase assay as described in 'Material and Methods' (B). Results were calculated as mean ± SD for 3 independent experiments. &amp;&amp;&amp; p &lt; 0.001 compared with control, !!! p &lt; 0.001 compared with TNFα treatment and ### p &gt; 0.001 compared with IKKα. bEND.3 cells were transfected with Gal-p65 or Gal-DBD in the presence or absence of flag-IKKα along with PTL-luciferase and PRL-TK reporter constructs as described in Material and Method. bEND.3 cells were pretreated with 15d-PGJ2 (10 μM) for 30 min followed by TNFα treatment. After 6 h of TNFα treatment (50 ng/ml), cells were processed for luciferase assay and results were normalized with PRL-TK luciferase activity in each sample (C). Total DNA content was normalized with pcDNA3. Results were calculated as mean ± SD for 3 independent experiments.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p><bold>Post treatment of 15d-PGJ2 inhibits monocyte adhesion to activated endothelial cells</bold>. bEND.3 cells were treated with TNFα (50 ng/ml) for 6 h, followed by addition of different concentrations of 15d-PGJ2 (5–20 μM). After 30 min of incubation with 15-PGJ2, fluorescently labeled monocytes were allowed to interact with activated bEND.3 cells. Adhered monocytes were counted as mentioned in 'Material and Methods'. Data calculated as mean ± SD of 21 fields from 3 different experiments. *** p &lt; 0.001 compared to untreated control cells and @ p &lt; 0.001 compared to TNFα treated cells.</p></caption></fig>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Non-Hodgkin's Lymphoma (NHL) is a cancer of the lymphatic system [##REF##12867117##1##,##UREF##0##2##]. Even though NHL is a relatively rare disease, its incidence rates have been increasing worldwide for both men and women. The incidence rates in Canada, for both males and females were increased by about 50% between 1978 and the late 1990s. After the latter time, incidence rates have stabilized. Mortality rates of NHL have followed a similar pattern [##UREF##1##3##]. Age-standardized rates have increased faster among males than among females [##REF##12867117##1##, ####UREF##0##2##, ##UREF##1##3##, ##UREF##2##4##, ##UREF##3##5####3##5##]. A number of factors, including inherited and acquired immunodeficiency states [##REF##6611504##6##] as well as infectious, physical, and chemical agents have been associated with an increased risk for NHL [##REF##6611504##6##,##REF##1317221##7##].</p>", "<p>Epidemiological studies have reported positive associations between NHL and certain occupations including those of farmers [##REF##8427258##8##, ####REF##2789947##9##, ##UREF##4##10##, ##REF##7627313##11##, ##REF##3471999##12##, ##REF##12024692##13##, ##UREF##5##14##, ##REF##1568215##15##, ##REF##1394162##16####1394162##16##], pesticide applicators [##REF##3471999##12##,##REF##1460670##17##, ####REF##2930251##18##, ##REF##8732923##19##, ##REF##8329071##20####8329071##20##], drivers [##REF##11333408##21##,##REF##6361201##22##], and managers [##REF##11138825##23##,##REF##7863296##24##]. Several studies have reported no association between development of NHL and the agricultural occupations (farmers, agricultural and forestry workers and pesticide applicators [##REF##8162585##25##, ####REF##3342183##26##, ##UREF##6##27####6##27##]). Occupational exposures of a priori interest include pesticides [##REF##9681531##28##, ####UREF##7##29##, ##REF##9065230##30##, ##REF##2029048##31##, ##UREF##8##32##, ##REF##2078610##33####2078610##33##], dusts (metal, wood, paper [##REF##8427258##8##], etc), paints [##REF##8427258##8##,##REF##1394163##35##], diesel exhaust fumes [##REF##11333408##21##,##REF##6361201##22##,##REF##3354585##34##,##REF##1394163##35##], cleaning fluids [##REF##8427258##8##], cutting oils [##REF##10707783##36##], and solvents [##UREF##9##37##,##REF##9270958##38##]. In this paper, we examined the association between NHL and (1) selected long term occupations, and (2) occupational exposures based on an individual's occupational history, and (3) duration of employment.</p>" ]

[ "<title>Methods</title>", "<p>Details of the study design and methodology have been previously published [##REF##11700263##39##, ####REF##16531830##40##, ##REF##16093930##41####16093930##41##]. Briefly, we conducted a six province Canadian population based case-control study of men with an incident first diagnosis of NHL between 1991 to 1994; control subjects were frequency matched by age ± 2 years to be comparable with the age distribution of the entire case group (Soft Tissue Sarcoma (STS), Hodgkin's Disease (HD), NHL, and Multiple Myeloma (MM)) within each province of residence. The study had approximately three matched controls for each NHL case. Deceased subjects were ineligible as either cases or controls. All participating control subjects were used in the statistical analysis of each cancer site. Cases were identified from provincial cancer registries – except in Quebec where hospital records were used – and were coded using ICD-O 2^ndedition except Quebec which used ICD-O 1^stedition [##UREF##10##42##]. Malignant morphology codes 9591, 9642, 9670–9764, and 9823 were included. A reference pathologist reviewed the tumour tissue slides for 60% of the NHL cases, and confirmed NHL in all but 2% of cases. Cases not confirmed as NHL were eliminated. Control subjects were identified through provincial health insurance programs except in Ontario (telephone listing) and British Columbia (voter's lists), as generally described [##REF##11700263##39##, ####REF##16531830##40##, ##REF##16093930##41####16093930##41##].</p>", "<p>The study design consisted of two stages: Stage 1 was a self-administered postal questionnaire; and Stage 2 was a detailed pesticide exposure information collected via telephone interview. With permission, we modified a pesticide exposure questionnaire developed by Hoar et al. [##UREF##11##43##] to create the study questionnaire. The results in this manuscript are based on the Stage 1 postal questionnaire only.</p>", "<p>The postal questionnaire captured demographic details, personal medical history, lifetime occupational history and specific occupational exposures of interest. Occupational information included a list of all full time jobs held by the respondent for at least one year. For each job held, we collected information on job titles, business organization – whether service or industry – and duration of employment. A list of occupational exposures that have been epidemiologically linked to NHL or to one of the other three types of cancers which we studied simultaneously was grouped into dusts, coal products, printing products, paints, metals, pesticides, radiation and miscellaneous. Additional details of exposure to agricultural chemicals in broad classes i.e. herbicides, fertilizers etc, were obtained. Job titles and each industry's coding were provided by Statistics Canada [##UREF##12##44##].</p>", "<title>Statistical analysis</title>", "<p>Data were entered into a custom designed SPSS-data entry program. Results were presented as frequencies for categorical variables; mean, standard deviation (SD) for continuous variables for cases and controls were presented separately. We obtained information about the duration of employment (measured in years) for each individual. The occupations were selected for analysis if the occupant worked in a particular occupation at least for one year and at least 2% of cases for that occupational category. Based on that information, we derived two new variables called ever held occupations and long held occupations. Occupations were defined as ever held occupation if respondents worked at least for one year in that occupation. Occupations were defined as long held occupation if respondents worked for 10 years or more in that occupation. Duration of employment is the total of number of years in each long held occupation. A bivariate analysis was conducted to determine the association between each explanatory variable and the NHL outcome. Based on this model, building procedure explanatory variables with p &lt; 0.20 were selected for the multivariate model. Statistically significant (p = 0.05) variables and important explanatory variables were considered for the final multivariate model adjusting for age and province of residence. Conditional logistic regression was used to compute adjusted odds ratios (OR) and 95% confidence intervals (95% CI).</p>", "<title>Ethics</title>", "<p>The letters of informed consent, questionnaires, and all other correspondence with study participants were approved by the relevant ethics agencies in each province. All of the information that could be used to identify study participants remained within each province of origin under the supervision of the provincial principal investigators.</p>" ]

[ "<title>Results</title>", "<p>This study includes responses from 513 cases with NHL and 1506 control subjects. The mean age ± standard deviation (SD) of cases was 57.7 ± 14.0 years and, of the controls, 54.1 ± 16.0 years. More cases (n = 74, 14.4%) than controls (n = 87, 5.8%) had a personal history of cancer other than NHL (OR_adj(95 % CI): 2.56 (1.81, 3.62)). There were no significant differences between NHL cases and controls with respect to their education level and to whether they ever lived or worked on a farm. Results are shown in Table ##TAB##0##1##.</p>", "<p>Table ##TAB##1##2## shows the distribution of ever held occupations and long held occupations during a lifetime stratified by case-control status. None of the ever held occupations were statistically significant. The long held occupations (10 years or more) as farmer and machinist showed a significant risk increase for NHL. The adjusted odds ratios (OR_adj) and 95% confidence intervals (95% CI) for a long held occupation during the lifetime as farmer and machinist were 1.54 (1.05, 2.27) and 2.21 (1.02, 4.79) respectively. Using four categories (no exposure, &lt; 10 years, 10–20 years, and &gt; 20 years), further models with years in these industries were used to investigate whether or not there is a dose-response relationship between the long held occupation as a farmer and a machinist and NHL (Table ##TAB##2##3##). A dose-response relationship between duration of exposure as farmer and incidence of NHL was observed. Those who worked as a farmer for more than 20 years were 1.5 times more likely to be diagnosed with NHL than non-exposed subjects. Similarly, we observed a dose-response relationship between duration of exposure as a machinist and incidence of NHL. Those who worked as a machinist for more than 20 years were 2.3 times more likely to be diagnosed with NHL than non-exposed subjects (Table ##TAB##2##3##).</p>", "<p>Of the 45 specific occupational exposures grouped into six classes (dusts, coal products, printing, paints, metals and miscellaneous), only exposure to diesel exhaust fumes showed an association with NHL (Table ##TAB##3##4##). Ever exposure to solvents and exposure to wood or paper dust were not associated with NHL. Ever exposure to ionizing radiation (radium) showed a significant association with the risk of NHL incidence (OR adj (95% CI): 3.26 (1.38, 7.73)).</p>", "<p>Table ##TAB##4##5## shows the results of multivariate conditional logistic regression models for the long held jobs of farmer and machinist. The variables that remained statistically significantly associated with increased risk of NHL for long held job as a farmer were personal history of another cancer and exposure to ionizing radiation (radium). The variables for the long held job as a machinist associated with increased risk of NHL were personal history of another cancer, exposure to ionizing radiation (radium) and exposure to diesel. Duration of exposure for the long held jobs of farmer and machinist were borderline significant at 5% level (p = 0.08 and p = 0.059), but there was evidence of an increase risk of NHL with longer duration of exposure.</p>" ]

[ "<title>Discussion</title>", "<p>Our study investigated the association between NHL and several occupations and occupational exposures. The findings revealed that two long held occupations (10 years or more), farmer and machinist, were significantly associated with increased risk of developing NHL. One of the possible explanations is that farmers and drivers might be exposed to pesticides and engine exhaust and machinists might be exposed to solvents or engine exhaust at the work place. The increased risk of NHL for farmer and machinist seen in our study is consistent with the findings from other studies [##REF##8427258##8##, ####REF##2789947##9##, ##UREF##4##10##, ##REF##7627313##11##, ##REF##3471999##12##, ##REF##12024692##13##, ##UREF##5##14##, ##REF##1568215##15##, ##REF##1394162##16####1394162##16##].</p>", "<p>Pesticides including herbicides and insecticides have been associated with Non-Hodgkin's Lymphoma in studies of farmers, agricultural related workers, other pesticide applicators, manufacturing workers and other exposed populations [##REF##11700263##39##,##REF##1394159##45##]. Grain handlers exposed to pesticides, grain dusts, and organic solvents were shown a five-fold risk of NHL [##REF##2332902##46##]. Our study confirms that those who held the long held job title as a farmer (farmer, farm labourer and farm managers) had 1.5 times higher risk of being diagnosed with NHL than those who held a job title from the category of non-farmer.</p>", "<p>Our results confirm previously reported associations of NHL and a personal history of cancer [##REF##9048836##47##,##REF##1394165##48##]. Occupational exposure to dust (wood, paper, metal etc.), coal products, paints, metal, and printing are unlikely to increase the risk of NHL, as is evident from our analysis. In contrast, Kawachi et al [##REF##2819671##49##] found a significant association between working with wood and NHL. In addition, Kogevinas et al [##UREF##13##50##] found an increased risk of Lymphomas in pulp and paper workers. Ever exposure to diesel exhaust fumes is likely to increase the risk of NHL, as is evident from our analysis. Our finding is agreement for diesel exhaust fumes with Baris et al [##REF##11333408##21##] and Maizlish et al [##REF##3354585##34##].</p>", "<p>The mechanism of cancer induction by radiation suggested in our study is not clear. The most widely accepted hypothesis is that some of the ionizing events, which occur when radiation is absorbed in tissue, produce a change in the genes or chromosomes of one or more cells [##REF##322845##51##]. A case-referent study conducted to investigate the possible association between occupation and occupational exposures and risk of hematological malignancies showed that exposure to asbestos, hydrocarbons, fertilizer, radiation, pesticides and mineral oils were highly associated with hematological malignancies [##UREF##4##10##]. Another matched case-control study in the nuclear industry [##REF##8499814##52##] found no significant excess of NHL at any radiation exposure level. Archer [##REF##322845##51##] stated that uranium mill workers appeared to have excess Lymphomas. In our study, any form of radiation exposure at work was considered. Exposure to ionizing radiation (radium) is significantly associated with increase risk of NHL, which suggests equivocal evidence of an association with NHL presented by Ron [##REF##11956701##53##].</p>", "<p>There are many potential sources of non-ionizing radiation to workers. One of them is ultraviolet (UV) radiation. There is suggestive evidence that exposure to ultraviolet (UV) light, an established cause of immune suppression, may increase the risk of NHL [##REF##1394156##54##, ####REF##2201784##55##, ##REF##17334774##56##, ##UREF##14##57####14##57##]. The most recent epidemiologic literature suggests that there is no association or protective effect between exposure to sunlight and NHL [##UREF##15##58##, ####REF##10048959##59##, ##UREF##16##60##, ##UREF##17##61##, ##REF##16912575##62##, ##REF##17708556##63####17708556##63##]. Our study did not find any association between exposure to ultraviolet (UV) light with NHL.</p>", "<p>Solvents have been associated with NHL in a number of studies [##REF##2775671##64##, ####REF##87845##65##, ##REF##3175557##66####3175557##66##], including studies of rubber workers [##UREF##18##67##], aircraft maintenance workers [##REF##1878308##68##], and dry cleaners [##REF##2924893##69##]. In particular, benzene exposure is common in above mention occupations and this may be due to its effects on the immune system [##REF##3175557##66##]. Other occupations which might involve exposure to solvents or related chemicals and which are reported as being at increased risk of NHL include those of highway workers [##REF##3354585##34##], petroleum refinery employees [##REF##3947563##70##, ####REF##1878307##71##, ##REF##7057282##72####7057282##72##], styrene workers [##REF##4070999##73##], chemists [##REF##5353243##74##,##REF##62154##75##], and chemical manufacturers [##REF##2556914##76##,##REF##3284337##77##]. We could not find any association between NHL and exposure to solvents, cleaning fluids, or preservatives.</p>", "<p>A major strength of this study is the large number of cases and controls from residents of six Canadian provinces. Questions were designed to obtain a complete occupational history and extensive list of potential occupational exposures. A reference pathologist validated 84% of the NHL tumours.</p>", "<p>There are, however, several limitations in this study. One of the limitations is the potential for recall bias and misclassification of pesticide exposures. Also, occupational exposures in this study were self-reported and this might also bias results. Due to budget constraints, the study was restricted to males. The response rates of 67.1% for cases and 48% for controls represent another potential limitation that could create misleading conclusions if the non-respondents differ significantly from the respondents with respect to the variables under investigation. We compared non-respondents to respondents using postal codes as an indicator of rural residence and did not find a rural bias among respondents. The most common reasons for not participating were death, change of address, and refusal for both cases and controls. Another limitation was the possibility of false-positive findings given the large number of jobs and exposures assessed.</p>" ]

[ "<title>Conclusion</title>", "<p>Our results support previous findings of an association between NHL and specific job titles and occupational exposures. In our analysis, NHL was associated with personal history of cancer, exposure to diesel exhaust fumes, exposure to ionizing radiation (radium) and long held occupations such as farmer and machinist. Also, we have supportive evidence of increased risk of NHL with longer durations of exposure.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The objective was to study the association between Non-Hodgkin's Lymphoma (NHL) and occupational exposures related to long held occupations among males in six provinces of Canada.</p>", "<title>Methods</title>", "<p>A population based case-control study was conducted from 1991 to 1994. Males with newly diagnosed NHL (ICD-10) were stratified by province of residence and age group. A total of 513 incident cases and 1506 population based controls were included in the analysis. Conditional logistic regression was conducted to fit statistical models.</p>", "<title>Results</title>", "<p>Based on conditional logistic regression modeling, the following factors independently increased the risk of NHL: farmer and machinist as long held occupations; constant exposure to diesel exhaust fumes; constant exposure to ionizing radiation (radium); and personal history of another cancer. Men who had worked for 20 years or more as farmer and machinist were the most likely to develop NHL.</p>", "<title>Conclusion</title>", "<p>An increased risk of developing NHL is associated with the following: long held occupations of faer and machinist; exposure to diesel fumes; and exposure to ionizing radiation (radium). The risk of NHL increased with the duration of employment as a farmer or machinist.</p>" ]

[ "<title>Abbreviations</title>", "<p>NHL: Non-Hodgkin's Lymphoma; ICD: International Classification of Diseases; STS: Soft Tissue Sarcoma; HD: Hodgkin's Disease; MM: Multiple Myeloma.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>CPK analyzed data and prepared the manuscript. HHM designed, coordinated the study and collect the data. JAD participated in study design, coordination, data collection and manuscript preparation. JJS participated in the design of the study and data collection. PP designed and coordinated the study as well as collected and prepared the manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>Special thanks go to the collaborators Drs. G. Theriault, J. McLaughlin, D. Robson, S. Fincham, L. Skinnider, D. White, T. To and Late N.W. Choi. Also, the authors are indebted to the following members of the Advisory Committee: Drs. G.B. Hill, A. Blair, L. Burmeister, H. Morrison, R. Gallagher, and D. White. We owe a debt of gratitude to the provincial coordinators across Canada and data managers for their meticulous attention to detail: T. Switzer, M. Gantefor, J. Welyklowa, J. Ediger, I. Fan, M. Ferron, E. Houle, S. de Freitas, K. Baerg, L. Lockinger, E. Hagel, P. Wang, G. Dequiang, J. Hu. We thank Drs. G. Theriault and N. Choi for supervising the collection of data in Quebec and Manitoba respectively; and to Dr. L. Skinnider for reviewing the pathological specimens. The study participants gave freely of their time and shared personal details with us and we sincerely thank each of them. Written consent for publication was obtained from the participants. This work was funded by Health Canada National Health Research Programs Directorate Grant 6608-1258, the British Columbia Health Research Foundation and Institute of Agricultural, Rural and Environmental Health, University of Saskatchewan.</p>" ]

[]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characterization of study participants stratified by NHL case- control status: demographics and selected medical history</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\">NHL (<italic>N = 513</italic>)</td><td align=\"center\">Controls (<italic>N = 1506</italic>)</td><td align=\"center\">OR^b_adj[P1](95% CI)</td></tr></thead><tbody><tr><td align=\"left\"><bold>Demographics</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> Mean age ± SD (years)</td><td align=\"center\">57.7 ± 14.0</td><td align=\"center\">54.1 ± 16.0</td><td/></tr><tr><td align=\"left\"> Education Level^a</td><td/><td/><td/></tr><tr><td align=\"left\">  University and Vocational</td><td align=\"center\">28 (6.6)</td><td align=\"center\">96 (5.5)</td><td align=\"center\">1.23 (0.81, 1.88)</td></tr><tr><td align=\"left\">  University</td><td align=\"center\">94 (18.5)</td><td align=\"center\">310 (20.8)</td><td align=\"center\">1.08 (0.68, 1.70)</td></tr><tr><td align=\"left\">  Vocational</td><td align=\"center\">111 (21.9)</td><td align=\"center\">358 (24.1)</td><td align=\"center\">1.06 (0.67, 1.70)</td></tr><tr><td align=\"left\">  Elementary/High school</td><td align=\"center\">274 (54.0)</td><td align=\"center\">723 (48.6)</td><td align=\"center\">1.00</td></tr><tr><td align=\"left\"> Ever lived/worked on a farm</td><td/><td/><td/></tr><tr><td align=\"left\">  Yes n (%)</td><td align=\"center\">235 (45.8)</td><td align=\"center\">673 (44.7)</td><td align=\"center\">1.02 (0.82, 1.27)</td></tr><tr><td align=\"left\">  No n (%)</td><td align=\"center\">278 (54.2)</td><td align=\"center\">833 (55.3)</td><td align=\"center\">1.00</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Medical History</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> Previous diagnosis of Cancer</td><td/><td/><td/></tr><tr><td align=\"left\">  Yes n (%)</td><td align=\"center\">74 (14.4)</td><td align=\"center\">87 (5.8)</td><td align=\"center\"><bold>2.56 (1.81, 3.62)</bold>^c</td></tr><tr><td align=\"left\">  No n (%)</td><td align=\"center\">439 (85.6)</td><td align=\"center\">1419 (94.2)</td><td align=\"center\">1.00</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Adjusted odds ratio (OR) and 95% confidence interval (95% CI) for different occupations (job titles).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Job Title (code#)</td><td align=\"center\">NHL cases<break/> n (%)</td><td align=\"center\">Controls<break/> n (%)</td><td align=\"center\">OR _adj a (95% CI)</td></tr></thead><tbody><tr><td align=\"left\"><bold>Ever held Occupations</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Accountant (1)</td><td align=\"center\">30 (5.8)</td><td align=\"center\">81 (5.4)</td><td align=\"center\">1.21 (0.77, 1.89)</td></tr><tr><td align=\"left\">Administrator (2)</td><td align=\"center\">11 (2.1)</td><td align=\"center\">52 (3.4)</td><td align=\"center\">0.58 (0.30, 1.15)</td></tr><tr><td align=\"left\">Carpenter (12)</td><td align=\"center\">21 (4.1)</td><td align=\"center\">55 (3.6)</td><td align=\"center\">1.06 (0.63, 1.79)</td></tr><tr><td align=\"left\">Clerk (17)</td><td align=\"center\">14 (2.7)</td><td align=\"center\">92 (6.1)</td><td align=\"center\">0.44 (0.24, 0.79)</td></tr><tr><td align=\"left\">Constructor (19)</td><td align=\"center\">14 (2.7)</td><td align=\"center\">78 (5.2)</td><td align=\"center\">0.51 (0.28, 0.93)</td></tr><tr><td align=\"left\">Driver (25)</td><td align=\"center\">55 (10.7)</td><td align=\"center\">133 (8.8)</td><td align=\"center\">1.29 (0.91, 1.82)</td></tr><tr><td align=\"left\">Electrician (26)</td><td align=\"center\">16 (3.1)</td><td align=\"center\">47 (3.1)</td><td align=\"center\">0.99 (0.54, 1.78)</td></tr><tr><td align=\"left\">Engineer (27)</td><td align=\"center\">13 (2.5)</td><td align=\"center\">68 (4.5)</td><td align=\"center\">0.54 (0.29, 1.02)</td></tr><tr><td align=\"left\">Factory worker (29)</td><td align=\"center\">13 (2.5)</td><td align=\"center\">46 (3.0)</td><td align=\"center\">1.14 (0.59, 2.17)</td></tr><tr><td align=\"left\">Foreman (30)</td><td align=\"center\">11 (2.1)</td><td align=\"center\">39 (2.6)</td><td align=\"center\">0.64 (0.32, 1.28)</td></tr><tr><td align=\"left\">Farmer (31, 33, 89)</td><td align=\"center\">86 (16.7)</td><td align=\"center\">230 (15.3)</td><td align=\"center\">1.14 (0.85, 1.54)</td></tr><tr><td align=\"left\">Armed forces (138)</td><td align=\"center\">28 (5.5)</td><td align=\"center\">92 (6.1)</td><td align=\"center\">0.76 (0.48, 1.18)</td></tr><tr><td align=\"left\">Janitor (41)</td><td align=\"center\">14 (2.7)</td><td align=\"center\">40 (2.7)</td><td align=\"center\">1.07 (0.57, 2.02)</td></tr><tr><td align=\"left\">Labourer (44)</td><td align=\"center\">31 (6.0)</td><td align=\"center\">99 (6.6)</td><td align=\"center\">0.86 (0.56, 1.33)</td></tr><tr><td align=\"left\">Lumberman (46)</td><td align=\"center\">17 (3.3)</td><td align=\"center\">38 (2.5)</td><td align=\"center\">1.12 (0.61, 2.03)</td></tr><tr><td align=\"left\">Machinist (47)</td><td align=\"center\">22 (4.3)</td><td align=\"center\">49 (3.2)</td><td align=\"center\">1.41 (0.83, 2.40)</td></tr><tr><td align=\"left\">Manager (48)</td><td align=\"center\">63 (12.3)</td><td align=\"center\">183 (12.1)</td><td align=\"center\">0.97 (0.70, 1.33)</td></tr><tr><td align=\"left\">Mechanic (49)</td><td align=\"center\">26 (5.1)</td><td align=\"center\">88 (5.8)</td><td align=\"center\">0.83 (0.52, 1.31)</td></tr><tr><td align=\"left\">Salesman (73)</td><td align=\"center\">44 (8.6)</td><td align=\"center\">127 (8.4)</td><td align=\"center\">1.06 (0.73, 1.53)</td></tr><tr><td align=\"left\">School Teacher (74)</td><td align=\"center\">31 (6.0)</td><td align=\"center\">88 (5.8)</td><td align=\"center\">0.96 (0.62, 1.48)</td></tr><tr><td align=\"left\">Welder (86)</td><td align=\"center\">13 (2.5)</td><td align=\"center\">33 (2.2)</td><td align=\"center\">1.25 (0.64, 2.44)</td></tr><tr><td align=\"left\">Office worker (97)</td><td align=\"center\">17 (3.3)</td><td align=\"center\">68 (4.5)</td><td align=\"center\">0.70 (0.40, 1.22)</td></tr><tr><td align=\"left\">Equipment hander (134)</td><td align=\"center\">14 (2.7)</td><td align=\"center\">37 (2.5)</td><td align=\"center\">1.34 (0.70, 2.56)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Long held Occupations</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Accountant (1)</td><td align=\"center\">20 (3.9)</td><td align=\"center\">41 (2.7)</td><td align=\"center\">1.39 (0.79, 2.42)</td></tr><tr><td align=\"left\">Driver (25)</td><td align=\"center\">27 (5.3)</td><td align=\"center\">48 (3.2)</td><td align=\"center\">1.45 (0.88, 2.37)</td></tr><tr><td align=\"left\">Farmer (31, 33, 89)</td><td align=\"center\">50 (9.8)</td><td align=\"center\">106 (7.0)</td><td align=\"center\"><bold>1.54 (1.05, 2.27)</bold>^c</td></tr><tr><td align=\"left\">Machinist (47)</td><td align=\"center\">12 (2.3)</td><td align=\"center\">16 (1.1)</td><td align=\"center\"><bold>2.21 (1.02, 4.79)</bold>^c</td></tr><tr><td align=\"left\">Manager (48)</td><td align=\"center\">31 (6.0)</td><td align=\"center\">96 (6.4)</td><td align=\"center\">0.86 (0.56, 1.32)</td></tr><tr><td align=\"left\">Mechanic (49)</td><td align=\"center\">15 (2.9)</td><td align=\"center\">49 (2.2)</td><td align=\"center\">1.00 (0.99, 1.02)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Duration of exposure as a farmer and machinist and risk of NHL</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"right\">Duration (in years)</td><td align=\"center\">NHL (<italic>N = 513</italic>)</td><td align=\"center\">Control (<italic>N = 1506</italic>)</td><td align=\"center\">OR (95% CI)^a</td></tr><tr><td/><td align=\"center\">n (%)</td><td align=\"center\">n (%)</td><td/></tr></thead><tbody><tr><td align=\"right\"><bold>Job Title: Farmer</bold></td><td/><td/><td/></tr><tr><td align=\"right\">No exposure</td><td align=\"center\">427 (83.2)</td><td align=\"center\">1276 (84.7)</td><td align=\"center\">1.00</td></tr><tr><td align=\"right\">&lt;10 years</td><td align=\"center\">36 (7.0)</td><td align=\"center\">124 (8.2)</td><td align=\"center\">0.84 (0.51, 1.41)</td></tr><tr><td align=\"right\">10–20 years</td><td align=\"center\">7 (1.4)</td><td align=\"center\">23 (1.5)</td><td align=\"center\">1.40 (0.57, 3.43)</td></tr><tr><td align=\"right\">&gt; 20 years</td><td align=\"center\">43 (8.4)</td><td align=\"center\">83 (5.5)</td><td align=\"center\"><bold>1.55 (1.02, 2.36)</bold>^c</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"right\"><bold>Job Title: Machinist</bold></td><td/><td/><td/></tr><tr><td align=\"right\">No exposure</td><td align=\"center\">491 (95.7)</td><td align=\"center\">1457 (96.7)</td><td align=\"center\">1.00</td></tr><tr><td align=\"right\">&lt;10 years</td><td align=\"center\">10 (1.9)</td><td align=\"center\">33 (2.2)</td><td align=\"center\">0.75 (0.30, 1.88)</td></tr><tr><td align=\"right\">10–20 years</td><td align=\"center\">2 (0.4)</td><td align=\"center\">4 (0.3)</td><td align=\"center\">1.77 (0.31, 10.22)</td></tr><tr><td align=\"right\">&gt; 20 years</td><td align=\"center\">10 (1.9)</td><td align=\"center\">12 (0.8)</td><td align=\"center\"><bold>2.33 (1.00, 5.52)</bold>^c</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Adjusted odds ratio (OR) and 95% confidence interval (95% CI) for different occupational exposures.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\">NHL (<italic>N = 513</italic>)</td><td align=\"center\" colspan=\"2\">Control (<italic>N = 1506</italic>)</td><td/></tr><tr><td/><td colspan=\"2\"><hr/></td><td colspan=\"2\"><hr/></td><td/></tr><tr><td align=\"left\">Exposure</td><td align=\"center\">n^b</td><td align=\"center\">%</td><td align=\"center\">n^b</td><td align=\"center\">%</td><td align=\"center\">OR_adj(95% CI)^a</td></tr></thead><tbody><tr><td align=\"left\"><bold>Dusts</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Cement dust</td><td align=\"center\">134</td><td align=\"center\">26.1</td><td align=\"center\">432</td><td align=\"center\">28.7</td><td align=\"center\">0.93 (0.73, 1.18)</td></tr><tr><td align=\"left\"> Fiberglass dust</td><td align=\"center\">102</td><td align=\"center\">19.9</td><td align=\"center\">319</td><td align=\"center\">21.2</td><td align=\"center\">1.02 (0.78, 1.33)</td></tr><tr><td align=\"left\"> Coal dust</td><td align=\"center\">63</td><td align=\"center\">12.3</td><td align=\"center\">149</td><td align=\"center\">9.9</td><td align=\"center\">1.19 (0.86, 1.66)</td></tr><tr><td align=\"left\"> Soil/field dust</td><td align=\"center\">142</td><td align=\"center\">27.7</td><td align=\"center\">375</td><td align=\"center\">24.9</td><td align=\"center\">1.26 (0.99, 1.61)</td></tr><tr><td align=\"left\"> Whey dust</td><td align=\"center\">12</td><td align=\"center\">2.3</td><td align=\"center\">38</td><td align=\"center\">2.5</td><td align=\"center\">0.89 (0.45, 1.77)</td></tr><tr><td align=\"left\"> Paper dust</td><td align=\"center\">68</td><td align=\"center\">13.3</td><td align=\"center\">180</td><td align=\"center\">11.9</td><td align=\"center\">1.22 (0.89, 1.67)</td></tr><tr><td align=\"left\"> Wood dust</td><td align=\"center\">143</td><td align=\"center\">27.9</td><td align=\"center\">445</td><td align=\"center\">29.5</td><td align=\"center\">0.95 (0.75, 1.20)</td></tr><tr><td align=\"left\"> Coke dust</td><td align=\"center\">10</td><td align=\"center\">1.9</td><td align=\"center\">58</td><td align=\"center\">3.8</td><td align=\"center\">0.53 (0.26, 1.06)</td></tr><tr><td align=\"left\"> Stone dust</td><td align=\"center\">55</td><td align=\"center\">10.7</td><td align=\"center\">173</td><td align=\"center\">11.5</td><td align=\"center\">0.99 (0.71, 1.40)</td></tr><tr><td align=\"left\"> Grain Dust</td><td align=\"center\">117</td><td align=\"center\">22.8</td><td align=\"center\">347</td><td align=\"center\">23.0</td><td align=\"center\">0.99 (0.76, 1.29)</td></tr><tr><td align=\"left\"> Sand</td><td align=\"center\">90</td><td align=\"center\">17.5</td><td align=\"center\">303</td><td align=\"center\">20.1</td><td align=\"center\">0.89 (0.67, 1.16)</td></tr><tr><td align=\"left\"> Cardboard dust</td><td align=\"center\">50</td><td align=\"center\">9.7</td><td align=\"center\">170</td><td align=\"center\">11.3</td><td align=\"center\">1.01 (0.71, 1.44)</td></tr><tr><td align=\"left\"> Metal dust</td><td align=\"center\">120</td><td align=\"center\">23.4</td><td align=\"center\">368</td><td align=\"center\">24.4</td><td align=\"center\">1.06 (0.82, 1.36)</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Coal Products</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Pitch</td><td align=\"center\">17</td><td align=\"center\">3.3</td><td align=\"center\">38</td><td align=\"center\">2.5</td><td align=\"center\">1.24 (0.68, 2.25)</td></tr><tr><td align=\"left\"> Asphalt</td><td align=\"center\">46</td><td align=\"center\">8.9</td><td align=\"center\">142</td><td align=\"center\">9.4</td><td align=\"center\">0.96 (0.67, 1.38)</td></tr><tr><td align=\"left\"> Crude petroleum</td><td align=\"center\">30</td><td align=\"center\">5.8</td><td align=\"center\">84</td><td align=\"center\">5.6</td><td align=\"center\">1.00 (0.64, 1.57)</td></tr><tr><td align=\"left\"> Tar/tar products</td><td align=\"center\">53</td><td align=\"center\">10.3</td><td align=\"center\">143</td><td align=\"center\">9.5</td><td align=\"center\">1.20 (0.84, 1.69</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Printing</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Printing inks</td><td align=\"center\">35</td><td align=\"center\">6.8</td><td align=\"center\">134</td><td align=\"center\">8.9</td><td align=\"center\">0.90 (0.60,1.36)</td></tr><tr><td align=\"left\"> Printing fluid</td><td align=\"center\">28</td><td align=\"center\">5.5</td><td align=\"center\">96</td><td align=\"center\">6.4</td><td align=\"center\">0.93 (0.59, 1.47)</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Paints</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Paints, dyes</td><td align=\"center\">148</td><td align=\"center\">28.8</td><td align=\"center\">442</td><td align=\"center\">29.3</td><td align=\"center\">1.06 (0.84, 1.33)</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Metals</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Arsenic</td><td align=\"center\">13</td><td align=\"center\">2.5</td><td align=\"center\">28</td><td align=\"center\">1.9</td><td align=\"center\">1.45 (0.72, 2.93)</td></tr><tr><td align=\"left\"> Nickel</td><td align=\"center\">29</td><td align=\"center\">5.6</td><td align=\"center\">85</td><td align=\"center\">5.6</td><td align=\"center\">1.11 (0.71, 1.74)</td></tr><tr><td align=\"left\"> Cadmium</td><td align=\"center\">20</td><td align=\"center\">3.9</td><td align=\"center\">55</td><td align=\"center\">3.6</td><td align=\"center\">1.07 (0.62, 1.84)</td></tr><tr><td align=\"left\"> Zinc</td><td align=\"center\">38</td><td align=\"center\">7.4</td><td align=\"center\">103</td><td align=\"center\">6.8</td><td align=\"center\">1.12 (0.75,1.67)</td></tr><tr><td align=\"left\"> Mercury</td><td align=\"center\">20</td><td align=\"center\">3.9</td><td align=\"center\">63</td><td align=\"center\">4.2</td><td align=\"center\">0.84 (0.49, 1.43)</td></tr><tr><td align=\"left\"> Chromium</td><td align=\"center\">24</td><td align=\"center\">4.7</td><td align=\"center\">58</td><td align=\"center\">3.8</td><td align=\"center\">1.33 (0.79, 2.22)</td></tr><tr><td align=\"left\"> Iron</td><td align=\"center\">40</td><td align=\"center\">7.8</td><td align=\"center\">100</td><td align=\"center\">6.6</td><td align=\"center\">1.18 (0.79, 1.77)</td></tr><tr><td align=\"left\"> Lead</td><td align=\"center\">65</td><td align=\"center\">12.7</td><td align=\"center\">182</td><td align=\"center\">12.1</td><td align=\"center\">1.03 (0.75, 1.42)</td></tr><tr><td align=\"left\"> Aluminum</td><td align=\"center\">71</td><td align=\"center\">13.8</td><td align=\"center\">220</td><td align=\"center\">14.6</td><td align=\"center\">1.03 (0.76, 1.40)</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Miscellaneous</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Asbestos</td><td align=\"center\">76</td><td align=\"center\">14.8</td><td align=\"center\">237</td><td align=\"center\">15.7</td><td align=\"center\">0.91 (0.68, 1.21)</td></tr><tr><td align=\"left\"> Used motor oil</td><td align=\"center\">117</td><td align=\"center\">22.8</td><td align=\"center\">400</td><td align=\"center\">26.6</td><td align=\"center\">0.89 (0.69, 1.15)</td></tr><tr><td align=\"left\"> Diesel exhaust fumes</td><td align=\"center\">183</td><td align=\"center\">35.7</td><td align=\"center\">464</td><td align=\"center\">30.8</td><td align=\"center\"><bold>1.33 (1.06,1.67)</bold>^c</td></tr><tr><td align=\"left\"> Cutting oils</td><td align=\"center\">74</td><td align=\"center\">14.4</td><td align=\"center\">277</td><td align=\"center\">18.4</td><td align=\"center\">0.81 (0.60, 1.08)</td></tr><tr><td align=\"left\"> Cleaning fluids</td><td align=\"center\">124</td><td align=\"center\">24.2</td><td align=\"center\">419</td><td align=\"center\">27.8</td><td align=\"center\">0.93 (0.72, 1.19)</td></tr><tr><td align=\"left\"> Preservatives</td><td align=\"center\">9</td><td align=\"center\">1.7</td><td align=\"center\">21</td><td align=\"center\">1.4</td><td align=\"center\">1.11 (0.49, 2.50)</td></tr><tr><td align=\"left\"> Chlorine</td><td align=\"center\">68</td><td align=\"center\">13.3</td><td align=\"center\">202</td><td align=\"center\">13.4</td><td align=\"center\">1.05 (0.77, 1.43)</td></tr><tr><td align=\"left\"> Hair permanent solutions</td><td align=\"center\">11</td><td align=\"center\">2.1</td><td align=\"center\">33</td><td align=\"center\">2.2</td><td align=\"center\">0.99 (0.48, 2.04)</td></tr><tr><td align=\"left\"> Sour gas</td><td align=\"center\">24</td><td align=\"center\">4.7</td><td align=\"center\">92</td><td align=\"center\">6.1</td><td align=\"center\">0.69 (0.42, 1.12)</td></tr><tr><td align=\"left\"> Wood smoke</td><td align=\"center\">121</td><td align=\"center\">23.6</td><td align=\"center\">371</td><td align=\"center\">24.6</td><td align=\"center\">0.95 (0.75, 1.22)</td></tr><tr><td align=\"left\"> Lubricants</td><td align=\"center\">152</td><td align=\"center\">29.6</td><td align=\"center\">477</td><td align=\"center\">31.7</td><td align=\"center\">0.99 (0.78, 1.25)</td></tr><tr><td align=\"left\"> Solvents</td><td align=\"center\">167</td><td align=\"center\">32.5</td><td align=\"center\">516</td><td align=\"center\">34.3</td><td align=\"center\">1.01 (0.80, 1.28)</td></tr><tr><td align=\"left\"> Ether</td><td align=\"center\">51</td><td align=\"center\">9.9</td><td align=\"center\">170</td><td align=\"center\">11.3</td><td align=\"center\">0.88 (0.62, 1.25)</td></tr><tr><td align=\"left\"> Mouldy grain/forage</td><td align=\"center\">61</td><td align=\"center\">11.9</td><td align=\"center\">176</td><td align=\"center\">11.7</td><td align=\"center\">1.09 (0.78, 1.53)</td></tr><tr><td align=\"left\"> Hair dyes</td><td align=\"center\">15</td><td align=\"center\">2.9</td><td align=\"center\">33</td><td align=\"center\">2.2</td><td align=\"center\">1.33 (0.69, 2.52)</td></tr><tr><td align=\"left\"> Cyanide</td><td align=\"center\">10</td><td align=\"center\">1.9</td><td align=\"center\">36</td><td align=\"center\">2.4</td><td align=\"center\">0.79 (0.38, 1.63)</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Non-ionizing radiation</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Ultra Violet Light</td><td align=\"center\">44</td><td align=\"center\">8.6</td><td align=\"center\">151</td><td align=\"center\">10.0</td><td align=\"center\">1.06 (0.73, 1.55)</td></tr><tr><td align=\"left\"> Horticultural Grow lights</td><td align=\"center\">12</td><td align=\"center\">2.3</td><td align=\"center\">39</td><td align=\"center\">2.59</td><td align=\"center\">0.91 (0.46, 1.79)</td></tr><tr><td align=\"left\"> Unshielded microwaves</td><td align=\"center\">3</td><td align=\"center\">0.6</td><td align=\"center\">25</td><td align=\"center\">1.7</td><td align=\"center\">0.39 (0.11, 1.32)</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Ionizing radiation</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Radium</td><td align=\"center\">12</td><td align=\"center\">2.34</td><td align=\"center\">12</td><td align=\"center\">0.80</td><td align=\"center\"><bold>3.26 (1.38, 7.73)</bold>^c</td></tr><tr><td align=\"left\"> Uranium</td><td align=\"center\">12</td><td align=\"center\">2.34</td><td align=\"center\">18</td><td align=\"center\">1.20</td><td align=\"center\">2.10 (0.97, 4.56)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Multivariate models of the important covariates associated with NHL for long held occupations.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Variable</td><td align=\"center\">Farmer</td><td align=\"center\">Machinist</td></tr><tr><td/><td colspan=\"2\"><hr/></td></tr><tr><td/><td align=\"center\">OR (95% CI)^a</td><td align=\"center\">OR (95% CI)^a</td></tr></thead><tbody><tr><td align=\"left\">Personal history of another cancer (yes)</td><td align=\"center\"><bold>2.60 (1.83, 3.69)</bold>^c</td><td align=\"center\"><bold>2.57 (1.82, 3.65)</bold>^c</td></tr><tr><td align=\"left\">Ever exposed to ionizing radiation (radium) (yes)</td><td align=\"center\"><bold>3.41 (1.44, 8.11)</bold>^c</td><td align=\"center\"><bold>3.21 (1.34, 7.67)</bold>^c</td></tr><tr><td align=\"left\">Ever exposed to diesel (yes)</td><td align=\"center\">1.23 (0.97, 1.56)</td><td align=\"center\"><bold>1.28 (1.02, 1.61)</bold>^c</td></tr><tr><td/><td/><td/></tr><tr><td align=\"left\">Duration (reference to no exposure)</td><td/><td/></tr><tr><td align=\"left\">&lt;10 years</td><td align=\"center\">0.77 (0.45, 1.30)</td><td align=\"center\">0.73 (0.29, 1.86)</td></tr><tr><td align=\"left\">10–20 years</td><td align=\"center\">1.34 (0.54, 3.34)</td><td align=\"center\">1.87 (0.33, 10.57)</td></tr><tr><td align=\"left\">&gt; 20 years</td><td align=\"center\">1.47 (0.95, 2.29)</td><td align=\"center\">2.34 (0.97, 5.68)</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>^a25 missing</p><p>^bAdjusted for age (5 year groups) and province</p><p>^cStatistically significant results are bold.</p></table-wrap-foot>", "<table-wrap-foot><p>^#Statistics Canada. Standard occupational classification. Ottawa: Minister of Supply and Services, 1980.</p><p>^a All odds ratios were adjusted for age and province of residence.</p><p>^cStatistically significant results are bold.</p></table-wrap-foot>", "<table-wrap-foot><p>^aall odds ratios were adjusted for age and province of residence.</p><p>^cStatistically significant results are bold.</p></table-wrap-foot>", "<table-wrap-foot><p>^aall odds ratios were adjusted for age and province of residence.</p><p>^bn and % are given for the \"yes\" responses.</p><p>^cStatistically significant results are bold.</p></table-wrap-foot>", "<table-wrap-foot><p>^aall odds ratios were adjusted for age and province of residence.</p><p>^cStatistically significant at 5% level results are bold.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Thoracoscopic sympathectomy has established itself as a procedure for a thoracic surgeon since it was first reported by Hughues in 1942[##UREF##0##1##] and popularized by Kux [##REF##13107399##2##]. This was first introduced in 1996 at our regional thoracic surgical unit for the treatment of 3 conditions namely hyperhidrosis, facial flushing and intractable angina. We report our experience of the first hundred patients who had resection of T2 – T4 sympathetic ganglia along with the rami communicantes performed by a single surgeon and their early and long term outcomes.</p>" ]

[ "<title>Methods</title>", "<p>A retrospective review of prospectively collected data was performed on Video Assisted Thoracoscopic Sympathectomies with histological proof of excision to review early and late outcomes. 100 patients underwent 200 VATS sympathectomy by a single surgeon in a tertiary thoracic centre between September 1996 and March 2007 There were 47 males(47%) with a mean age of 32 years (range 18–80) All patients had maximum medical therapy prior to surgery. They were classified into 3 groups based on indications, Group 1: hyperhidrosis, Group 2: facial flushing and Group 3: intractable angina. The distribution of the cases has been illustrated in figure ##FIG##0##1##. The study was approved by the department and discussed with the ethics committee which advised ethics approval was not required as this was an outcome audit. Diagnosis was made in all patients by history and examination. All patients who underwent the procedure had subjective symptoms affecting quality of life. The patients in the hyperhidrosis group had an unsuccessful trial of medical therapy. Their symptoms were scaled on the basis of symptoms and impact on quality of life (Grade 1: minimal impact on their QOL, Grade 2: caused significant impact on quality of life and Grade 3: severe impact on quality of life). Surgery was only offered to patients with grade 2 and grade 3 symptoms. In the angina group, patients were referred with angina refractory to maximal anti-anginal therapy and deemed unsuitable anatomically for coronary revascularisation. Initial assessment included a detailed history with regards to angina symptoms, degree of disability and effects on quality of life. Patients had a pre-study exercise tolerance test to assess exercise capacity and confirm objective evidence of ischaemia. MUGA scans or transthoracic echocardiogram was performed to determine the left ventricular function. Each patient's angiogram was reviewed by an independent cardiologist and cardiothoracic surgeon and confirmed to be unsuitable for coronary artery bypass grafting or percutaneous cardiological intervention.</p>", "<title>Procedure</title>", "<p>Bilateral VATS sympathectomy was performed under general anaesthesia with single lung ventilation starting with the left side followed by right. We found this approach helpful in avoiding arrhythmias in patients with intractable angina. All the patients had electrocardiogram, pulse oximetry and blood pressure monitored during the procedure. In addition the patients undergoing it for angina had invasive arterial monitoring as well (in our initial practice we routinely placed a pulmonary artery floatation catheter in all patients undergoing sympathectomy for angina). A slight degree of cranial elevation and the lateral thoracotomy position helps the lungs to drop away from the operating site exposing the sympathetic chain. The first port was placed in the 5th intercostal space below and anterior to inferior angle of scapula. A 10 mm zero degree telescope was passed through this port. Two further ports were placed for instrumentation at the level of the 3rd and 4th intercostal spaces in the anterior axillary line. The parietal pleura was then incised to expose the sympathetic chain. An extensive thoracic sympathectomy was performed using electrocautery and excision of thoracic sympathetic chain from 2nd to 4th ganglia along with associated rami communicantes. In patients with hyperhidrosis and facial flushing the parietal pleura was cleared for 2 cm lateral to the sympathetic chain in the 2nd intercostal space. This was performed to identify the accessory nerve of Kuntz which was then ablated. The excised ganglia are confirmed histologically in each case. A redivac drain was placed in the apex through the anterior port. The wound was closed in layers with 2'0' vicryl on a 'J-shaped' needle for the intercostal muscle layer, 2'0' vicryl for the subcutaneous tissue and 3'0' monocryl for the skin. Local anaesthetic was infiltrated to the port sites. The patient was then repositioned and the procedure was repeated on the contra lateral side.</p>", "<p>All the patients were extubated on table and were nursed in the thoracic surgical ward except the angina patients who were nursed in the Coronary Care Unit. Patient controlled morphine analgesia was provided for pain relief. The redivac drains were removed the day after the operation if the lungs were satisfactorily expanded. Follow-up was made by outpatient visit, medical notes review, and a telephone interview of patients who were discharged from our care. Patients were asked to rate their operative outcome of the procedure as 1 for no change, 2 for satisfactory and 3 to denote a significant improvement and its impact on their quality of life.</p>" ]

[ "<title>Results</title>", "<p>In the time frame study 100 patients underwent 200 VATS sympathectomies. Ninety nine patients had bilateral procedures (one unilateral re operation after 4 years) and one patient had unilateral procedure. There were no post-operative deaths. There was conversion to thoracotomy in the angina group in one patient who had a previous decortication. The median post operative length of stay was 2.4 days for the facial flushing and hyperhidrosis groups and 5.1 days for the angina group. Early complications included acute coronary syndrome, pneumothorax, seroma, transient Horner's syndrome, transient paraesthesia and wound infection as detailed in Table ##TAB##1##2##. Compensatory hyperhidrosis occurred in 18 patients and was not severe enough to affect their quality of life.</p>", "<p>The patients were subjectively assessed for their symptoms in the out patients clinic at 4 weeks, 3 months and 6 months and the care was transferred to the referring clinician. Late outcomes were obtained through telephone-conducted patient interviews. All patients were graded for their improvement in the symptoms as 1 for no change, 2 for better and 3 for satisfied. In the hyperhidrosis group 46 patients (96%) were satisfied with the procedure. In the facial flushing group 22(85%) were satisfied with the procedure (Figure ##FIG##1##2##).</p>", "<p>The angina group were assessed by their pre and post procedure Canadian Cardiovascular score for angina, subjective angina score out of 10 and frequency of anginal episodes. Of the twenty five patients who underwent the procedure for relief of refractory angina only one patient (4%) did not feel any difference all the rest had an immediate relief in their symptoms. One patient had atrial fibrillation and two had a post operative myocardial infarction in the early post operative phase.</p>", "<title>Follow up</title>", "<p>Full follow-up was available for a mean 67.8 months. In the angina group there were 5 deaths at a mean of 28.75 +/- 13.7 months after procedure. Of these, 2 deaths were cardiac related deaths, one patient died of lung cancer, one patient due to perforated bowel and one died of terminal colonic cancer. The patients who died had symptomatic relief and were satisfied with the operation. On late follow-up 1 patient continued to have anginal symptoms and there was recurrence of angina in two patients at a follow up of 3 and 36 months. Of these patients with recurrence one patient the angina symptoms were present only on the right side and one patient had a dorsal spinal cord stimulator fitted which offered him relief.</p>", "<p>In the hyperhidrosis group one patient was noted to have unilateral persistence of sweating and he underwent a re do right sympathectomy after four years which abolished his symptoms. In the original operation the T 2 sympathectomy was not performed on the right due to the presence of large veins this was remedied in the second operation. 4 (15%) patients of facial flushing group had residual facial flushing. 18 patients (18%) had compensatory hyperhidrosis but none found this complication to adversely affect their quality of life. Five patients had post thoracoscopic pain of which one had chronic pain needing referral to the pain clinic. The patient satisfaction results are tabulated in table ##TAB##0##1##/figure ##FIG##1##2##.</p>" ]

[ "<title>Discussion</title>", "<p>VATS sympathectomy is an established therapeutic option for hyperhidrosis [##REF##12678542##3##], facial flushing [##REF##16631687##4##], Raynaud's disease [##REF##9641380##5##] and ischaemic heart disease [##REF##10536958##6##].</p>", "<p>Primary or essential Hyperhidrosis is a functionally, professionally, and socially disabling condition. It is a pathological condition characterized by excessive secretion of the eccrine glands resulting in overpespiration disproportionate to the requirements of thermoregulation and dissipation of body heat [##REF##16829101##7##]. The current medical treatment modalities for hyperhidrosis include topical application of aluminium salts, tap water iontophoresis, botulinum toxin injections, behavioural and psychotherapy with limited success [##REF##17166108##8##]. The pathophysiology of facial flushing is uncertain, but it is thought to involve vasomotor and sudomotor imbalances [##REF##16631687##4##].</p>", "<p>In patients unsuitable for surgical intervention but with intractable angina therapeutic options included long-term intermittent urokinase, spinal cord stimulation and Trans Myocardial Revascularisation. Wettervik showed that VATS sympathectomy had encouraging early results in this group of patient [##REF##7815891##9##]. Benefit is thought to be related to pain anaesthesia of the upper thoracic sympathetic afferent and efferent fibres[##REF##3668072##10##] and vasodilatation following coronary blockade of alpha adreno receptor mediated sympathetic vasoconstriction [##REF##2240673##11##].</p>", "<p>Several groups have adopted a single or two port technique for VATS sympathectomy [##REF##16829101##7##,##REF##10921715##12##,##REF##11788259##13##]. In our series we performed the procedure with the 3-port technique, since it allows accurate, safe and reproducible dissection of the thoracic sympathetic chain. We use conventional Thoracoscopic instruments (i.e. no articulating instruments) and training residents is much easier with the three port technique.</p>", "<p>Excision of the appropriate sympathetic ganglia is essential for effective post operative outcomes. Excision of the lower third of the stellate ganglia increases the risk of Horner's syndrome [##REF##8664016##14##]. Removal of ganglia between T2 – T4 is optimal for sympathetic denervation of the upper limb [##REF##8024363##15##]. In the treatment of intractable angina we have shown that T2 – T4 sympathectomy was sufficient to alleviate symptoms without risking undue hypotension and Horner's syndrome associated with more extensive excision. Excision of the second to fourth sympathetic ganglia as opposed to clipping [##REF##11285952##16##] or electro coagulation [##REF##7601951##17##] may reduce the incidence of recurrence. In patients with hyperhidrosis and facial flushing, the nerve of Kuntz was excised to reduce recurrence[##REF##11882821##18##,##REF##14855081##19##].</p>", "<p>The cause for late recurrence after a successful sympathectomy involves sensitization produced by section of postganglionic fibres. In our study the recurrence of hyperhidrosis was 2% which is comparable to that reported in the literature 6.5% reported in literature [##REF##1555100##20##]. The most common complication of thoracic sympathectomy is transient compensatory sweating probably due to thermoregulatory imbalance. This occurred in 18% of the patients in our study which is consistent with the reported incidence of 20% to 86% [##REF##9218302##21##, ####REF##17234017##22##, ##REF##16008010##23####16008010##23##]. We feel the major adverse complications in our series are less due to the three port technique and better visualisation. We did not have any vascular injuries, reexplorations for bleeding, permanent Horner's syndrome or chylothorax which are reported in the literature [##REF##17234017##22##].</p>", "<p>We have demonstrated that patients with intractable angina treated with VATS sympathectomy, had a decrease in angina score and frequency of angina as well as an improvement in exercise tolerance [##REF##10536958##6##]. One of the concerns with this procedure was that the denervation of the heart might result in silent ischaemia and mortality, which has not been the case. The other concern was if denervation leads to symptom relief why this was not a complete abolition of symptoms. We feel the benefits are individualised because the sympathetic innervation is collateralised from the aortic plexus which derives its fibres from the cervical plexus. In this current study, we have studied the long term outcomes of the same and noted persistent benefit in symptom alleviation. VATS sympathectomy is a durable palliation for symptoms of angina in a group with few other reliable therapeutic options.</p>", "<p>It is our policy to send the excised sympathetic chain for histological confirmation as a quality control measure. We feel this is a good practice in the current medico legal climate so that the surgeon can demonstrate the chain was excised.</p>" ]

[ "<title>Conclusion</title>", "<p>Video assisted thoracoscopic sympathectomy with histological proof of excision is a safe and effective procedure attended by low complications. It is an effective treatment for hyperhidrosis and facial flushing with good long term benefits and patient satisfaction. It offers lasting relief from angina and improves quality of life in patients with intractable angina without options of conventional revascularisation. Histological confirmation of the excised sympathetic chain is valuable for medico-legal documentation.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Video-Assisted Thoracoscopic Sympathectomy (VATS) is an established minimally invasive procedure for thoracic sympathetic blockade in patients with hyperhidrosis, facial flushing and intractable angina. Various techniques using clips, diathermy and excision are used to perform sympathectomy. We present our technique of excision of the sympathetic chain with histological proof and the analysis of the early and late outcomes.</p>", "<title>Methods</title>", "<p>We evaluated 200 procedures in 100 consecutive patients, who underwent Video Assisted Thoracoscopic Sympathectomy by a single surgeon in our centre between September 1996 to March 2007. All patients had maximum medical therapy prior to surgery and were divided into 3 groups based on indications, Group 1(hyperhidrosis: 48 patients), Group 2 (facial flushing: 26 patients) and Group 3(intractable angina: 26 patients). The demography and severity of symptoms for each group were analysed. The endpoints were success rate, 30 day mortality, complications and patient's satisfaction.</p>", "<title>Results</title>", "<p>99 patients had bilateral VATS sympathectomy and 1 had unilateral sympathectomy. The conversion rate to open was 1(1%). All patients had successful removal of ganglia proven histologically with no perioperative mortality in our series. The complications included pneumothorax (5%), acute coronary syndrome (2%), transient Horner's syndrome (1%), transient paraesthesia (1%), wound infection (4%), compensatory hyperhidrosis (18%), residual flushing (3%) and wound pain (5%). There were five late deaths in the intractable angina group at a mean follow up of 36.7 months. Overall success rates of abolishing the symptoms were 96.3%, 87.5% and 95.2% for Group 1, 2 and 3 respectively.</p>", "<title>Conclusion</title>", "<p>Excision of the sympathetic chain with histological confirmation during VATS sympathectomy is a safe and effective method in treating hyperhidrosis, facial flushing and intractable angina with good long term results and satisfaction.</p>" ]

[ "<title>Abbreviations</title>", "<p>VATS: Video assisted Thoracoscopic surgery; QOL: Quality of Life</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>SR was involved with study design, performed the data analysis and authored the manuscript, PN was involved in data collection and follow up, SS designed the study, collected the data and performed data analysis and PBR devised the study, performed all the operations and co authored the manuscript. All authors have read and approved the manuscript.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>The Distribution of cases in the various groups.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>The patient satisfaction outcomes.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Patient Satisfaction Outcomes</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Group</td><td align=\"left\">No change</td><td align=\"left\">Better</td><td align=\"left\">Very satisfied</td><td align=\"left\">% improved</td></tr></thead><tbody><tr><td align=\"left\">Hyperhidrosis (48)</td><td align=\"left\">2</td><td align=\"left\">9</td><td align=\"left\">37</td><td align=\"left\">96%</td></tr><tr><td align=\"left\">Facial flushing (26)</td><td align=\"left\">4</td><td align=\"left\">9</td><td align=\"left\">13</td><td align=\"left\">85%</td></tr><tr><td align=\"left\">Intractable angina (26)</td><td align=\"left\">2</td><td align=\"left\">6</td><td align=\"left\">18</td><td align=\"left\">92%</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Early complications</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Complication</td><td align=\"left\">Number of patients</td><td align=\"left\">%</td></tr></thead><tbody><tr><td align=\"left\">Acute coronary syndrome</td><td align=\"left\">2</td><td align=\"left\">2%</td></tr><tr><td align=\"left\">Pneumothorax</td><td align=\"left\">5</td><td align=\"left\">5%</td></tr><tr><td align=\"left\">Transient Horner's Syndrome</td><td align=\"left\">1</td><td align=\"left\">1%</td></tr><tr><td align=\"left\">Transient Parasthesia</td><td align=\"left\">1</td><td align=\"left\">1%</td></tr><tr><td align=\"left\">Compensatory hyperhidrosis</td><td align=\"left\">18</td><td align=\"left\">18%</td></tr><tr><td align=\"left\">Wound infection</td><td align=\"left\">4</td><td align=\"left\">4%</td></tr><tr><td align=\"left\">Seroma</td><td align=\"left\">1</td><td align=\"left\">1%</td></tr></tbody></table></table-wrap>" ]

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{ "acronym": [], "definition": [] }

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[ "<title>Conclusion</title>", "<p>Although the roles of Runx in neural development have just begun to be investigated, studies in gene knockout mice indicate that the roles of Runx in the nervous system are as important as its roles in other, non-neuronal tissues. However, a number of open questions should be addressed in the future. First, upstream signalling cascades remain elusive. The mRNA expression and protein synthesis for <italic>Runx1</italic>/<italic>Runx3 </italic>are tightly regulated and DRG is one of the tissues in which <italic>Runx1</italic>/<italic>Runx3 </italic>display their highest protein levels among the entire body; how do DRG neurons achieve such a high protein level for <italic>Runx1</italic>/<italic>Runx3</italic>? Second, the molecular bases of tissue specificity are largely unknown. Runx1 and Runx3 are highly homologous but they control the development of distinct subpopulations of sensory neurons. In particular, Runx1⁺neurons and Runx3⁺neurons project axons into totally different target tissues; how is this specificity achieved? Third, transcriptional regulation is not the only determinant of DRG neurogenesis. Ectopic synthesis of TrkC receptor <italic>per se </italic>influences the lineage commitment of DRG neurons [##REF##15247919##44##], while Runx3 plays a crucial role in TrkB/TrkC status [##REF##16446142##25##,##REF##17584746##45##]. It is likely that Runx and neurotrophin status are closely related to each other. How this cross-regulation is carried out is a challenging question. Finally, since all three Runx proteins have common features, some of the knowledge about Runx function in oncology, haematology, immunology and bone biology is likely to be applicable to neuroscience as well, particularly at the molecular level [##REF##18171930##53##].</p>" ]

[ "<p>This is an open access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Runt-related (Runx) transcription factors control diverse aspects of embryonic development and are responsible for the pathogenesis of many human diseases. In recent years, the functions of this transcription factor family in the nervous system have just begun to be understood. In dorsal root ganglion neurons, Runx1 and Runx3 play pivotal roles in the development of nociceptive and proprioceptive sensory neurons, respectively. Runx appears to control the transcriptional regulation of neurotrophin receptors, numerous ion channels and neuropeptides. As a consequence, Runx contributes to diverse aspects of the sensory system in higher vertebrates. In this review, we summarize recent progress in determining the role of Runx in neuronal development.</p>" ]

[ "<title>History</title>", "<p><italic>Runt related </italic>(<italic>Runx</italic>) genes are evolutionarily conserved developmental regulators in metazoa, where they play diverse roles in several different biological systems, including cell differentiation. One of the <italic>Drosophila </italic>pair-rule genes, <italic>Runt</italic>, controls segmentation, sex-determination and neuronal development [##REF##8036992##1##]. The mammalian <italic>Runx </italic>gene was first identified as <italic>AML1</italic>, which is frequently involved in the chromosomal translocations associated with acute myeloid leukaemia (AML) [##REF##1720541##2##]. Both <italic>Runt </italic>and <italic>AML1 </italic>encode a DNA binding subunit of the heterodimeric transcription factor PEBP2/CBF. Polyomavirus enhancer binding complex (PEBP2/PEA2) was identified during the characterization of the cellular mechanisms involved in differentiation using embryonal carcinoma cells [##REF##18037406##3##]. CBF was first identified as a protein that binds to the core sequence of the murine retrovirus enhancer, which influences the tissue specificity of viral replication [##REF##8834230##4##].</p>", "<p>There are three mammalian <italic>RUNX </italic>genes, <italic>RUNX1 </italic>(<italic>AML1</italic>), <italic>RUNX2 </italic>(<italic>CBFA1</italic>) and <italic>RUNX3 </italic>[##REF##10620014##5##]. <italic>RUNX1 </italic>is essential for definitive hematopoiesis and frequently involved in human leukaemia [##REF##12094236##6##]. Runx2 is a master regulator of bone development [##REF##16795049##7##]. Moreover, haploinsufficiency of <italic>RUNX2 </italic>is one of the causes of the hereditary bone disease Cleidcranial displasia [##REF##10204840##8##]. <italic>RUNX3</italic>, the third member of the <italic>RUNX </italic>gene family, was the least characterized until gene targeting studies opened up new avenues of investigation into Runx function. First of all, <italic>RUNX3 </italic>is involved in many types of human cancer as a tumour suppressor [##REF##11955451##9##,##REF##15156173##10##]. Hypermethylation of the <italic>RUNX3 </italic>promoter and deletion of the <italic>RUNX3 </italic>gene are frequently observed in several cancers, and RUNX3 protein is now best considered as an apoptosis inducer [##REF##16140942##11##,##REF##16738314##12##]. Second, RUNX3 controls the generation of the T-cell sub-lineage [##REF##12464175##13##, ####REF##17195845##14##, ##REF##18258917##15####18258917##15##]. In particular, transcriptional regulation of <italic>CD4 </italic>silencer and <italic>Th-POK </italic>have been described in detail [##REF##12464175##13##,##REF##18258917##15##]. Finally, Runx3 controls the development of proprioceptive dorsal root ganglion (DRG) neurons [##REF##12352981##16##,##REF##12093746##17##]. The last discovery was particularly relevant to developmental neurobiology and, since then, several groups have characterized not only Runx3, but also Runx1 as a crucial regulator of DRG neurogenesis [##REF##16446135##18##,##REF##17237804##19##].</p>", "<title>Expression of Runx1 and Runx3 in the nervous system</title>", "<p>Earlier <italic>in situ </italic>hybridization studies indicated strong expression of <italic>Runx1 </italic>mRNA in spinal motor neurons, DRG, cranial ganglia and specialized sensory epithelial structures such as olfactory and gustatory mucosa, and follicles of the vibrissae [##REF##7647375##20##]. Subsequently, the generation of specific antibodies against Runx1 and Runx3 and the utilization of <italic>Runx1</italic>^{<italic>β-gal </italic>}or <italic>Runx3</italic>^{<italic>β-gal </italic>}mice revealed the expression of Runx1 and Runx3 in the nervous system in more detail [##REF##12352981##16##,##REF##11731260##21##,##REF##15240886##22##]. Runx1 is synthesized in both the central and peripheral nervous systems of mouse embryos. In the central nervous system, Runx1 is synthesized in selective populations of somatic motor neurons in the spinal cord and in cholinergic branchial and visceral motor neurons in the hindbrain, such as dorsal vagal nucleus and nucleus ambiguus [##REF##11731260##21##,##REF##15240886##22##]. In the peripheral nervous system, Runx1 is localized to DRG and selective cranial ganglia, including trigeminal (V) and vestibulocochlear (VIII) ganglia and the glossopharyngeal-vagal (IX-X) ganglia complex [##REF##11731260##21##,##REF##15240886##22##]. In contrast to Runx1, Runx3 is confined to the peripheral nervous system, specifically to DRG and cranial ganglia [##REF##12352981##16##,##REF##11731260##21##]. Although Runx1 and Runx3 are almost exclusively found in postmitotic neurons in the central nervous system and peripheral ganglia [##REF##12352981##16##,##REF##11731260##21##,##REF##15240886##22##], a rare exception is the expression of Runx1 in proliferating progenitors of the olfactory epithelium [##REF##15728845##23##]. These observations suggest Runx1 and Runx3 have extensive functions in the mammalian nervous system.</p>", "<title>Roles of Runx3 in the development of DRG neurons</title>", "<p>DRG neurons convey peripheral somatosensory stimuli to the spinal cord. There are three major subpopulations of DRG neurons – nociceptive, mechanoreceptive, and proprioceptive – which differ in their cell size, dependency on neurotrophins, and distinct axonal terminal fields in the spinal cord and peripheral tissues. Runx1 and Runx3 are synthesized initially in TrkA⁺nociceptive and TrkC⁺proprioceptive neurons, respectively (Figure ##FIG##0##1##) [##REF##12093746##17##,##REF##16446143##24##,##REF##16446142##25##]. This complementary expression pattern suggests specific roles for Runx1 and Runx3 in subtypes of DRG neurons. Indeed, the phenotype of Runx3 knockout mice is similar to that of NT3 and TrkC knockout mice [##REF##12352981##16##,##REF##12093746##17##,##REF##7514502##26##, ####REF##8208292##27##, ##REF##8145824##28##, ##REF##7991545##29####7991545##29##]. Namely, Ia/Ib type DRG neurons fail to form a stretch reflex circuit with motor neurons in the spinal cord, resulting in severe motor discoordination [##REF##12352981##16##,##REF##12093746##17##]. What is the molecular basis of the phenotype? Several elegant studies have been performed to answer this question.</p>", "<p>First, the role of Runx3 in the neurotrophin receptor phenotype was shown by Arber and her colleagues [##REF##16446142##25##], who thoroughly compared neurotrophin receptor synthesis in mouse strains in which <italic>Runx3 </italic>had been disrupted or expressed ectopically. In DRG neurogenesis, dynamic changes are observed during the synthesis of neurotrophin receptors (TrkB, TrkC) [##REF##16446142##25##]. At early developmental stages, most DRG neurons synthesize TrkC protein first before the onset of TrkB synthesis. Thus, some TrkC⁺DRG neurons co-synthesize TrkB (Figure ##FIG##0##1a##). Subsequently, the ratio of TrkB/TrkC-hybrid neurons declines to produce DRG neurons that synthesize either TrkC or TrkB (Figure ##FIG##0##1a##). During this segregation, Runx3 is observed in most TrkC⁺neurons but not in TrkB⁺neurons [##REF##16446142##25##]. One of the functions of Runx3 is to repress TrkB when DRG neurons acquire TrkC⁺identity (Figure ##FIG##0##1a##) [##REF##16446142##25##].</p>", "<p>Second, the axonal outgrowth and/or axonal guidance of propiroceptive DRG neurons are also regulated by Runx3. Two different interpretations were proposed for the phenotype of the <italic>Runx3</italic>-/- DRG. One group proposed that Runx3 controls the appropriate axon targeting of <italic>trkC</italic>-expressing proprioceptive DRG neurons to motor neurons [##REF##12352981##16##]. However, another group observed massive cell death of TrkC⁺neurons in <italic>Runx3</italic>-/- DRG in apparent contradiction to the previous proposition [##REF##12093746##17##]. A recent study with <italic>Runx3 </italic>and <italic>Bax</italic>-double knockout mouse revealed clearly that the axonal projection of propioceptive DRG neurons to motor neurons is still lost in the <italic>Runx3 </italic>mutant even in the absence of apoptosis [##REF##18385258##30##]. The study further clarified that the initial model 'Runx3 → TrkC and Runx1 → TrkA' might not apply to later developmental stages [##REF##18385258##30##]. They observed that Runx3 co-localizes not only with TrkC, but also TrkA and TrkB at postnatal day 0 (P0) [##REF##18385258##30##]. Of note, Runx1⁺and Runx3⁺neurons were clearly segregated at embryonic day 16.5 (E16.5) but almost all Runx3⁺neurons co-synthesize Runx1 at E18.5 and P0 [##REF##18385258##30##]. It is possible that Runx3 has some functions not only in proprioceptive neurons, but also in nociceptive neurons [##REF##18385258##30##]. Overall, the evidence obtained from <italic>Runx3 </italic>and <italic>Bax </italic>compound mutants support a role for Runx3 in the control of axonal projection, although the molecular mechanisms remain unknown [##REF##18385258##30##]. Prior studies showed that DRG explants from <italic>Runx3</italic>-knockout mouse embryos extended short neurites in the presence of NT3, a ligand for TrkC, but not in the presence of NGF, a ligand for TrkA [##REF##12352981##16##]. This suggests that Runx3 may regulate the axonal outgrowth of specific DRG neurons independently of the target tissue. On the other hand, Chen <italic>et al</italic>. [##REF##16446143##24##] revealed, using a <italic>tour de force </italic>method, that Runx3 activity determines the dorso-ventral position of axonal termination of DRG neurons in the spinal cord. DRG neurons with high Runx3 activity extended their axons far into the ventral spinal cord like proprioceptive neurons, whereas those neurons with low Runx3 activity extended their axons into the dorsal spinal cord. Ectopic expression of Runx3 is sufficient to drive axons from the dorsal to the ventral spinal cord, indicating that Runx3 <italic>per se </italic>has instructive roles in central axon targeting in DRG neurons.</p>", "<p>Thus, Runx3 controls the neurotrophin receptor phenotype as well as the axonal projection of proprioceptive DRG neurons. The two functions may not be mutually exclusive but closely related to each other. For example, NGF/TrkA signalling and NT3/TrkC signalling are required for proper axonal projection [##REF##10719890##31##,##REF##12741988##32##].</p>", "<title>Roles of Runx1 in the development of DRG neurons</title>", "<p>In contrast to Runx3, the study of Runx1 function in DRG development was delayed owing to the early embryonic lethality of the targeting mouse [##REF##15240886##22##,##REF##15728845##23##,##REF##17208218##33##]. Thus, Runx1 knockout mice die due to a lack of definitive hematopoiesis by E12.5, which is before the onset of major events in the development of TrkA⁺DRG neurons. However, recent studies have investigated the roles of Runx1 in DRG neurons using different experimental models.</p>", "<p>First of all, Runx1 controls the lineage diversification of nociceptive neurons [##REF##16446142##25##,##REF##17208218##33##,##REF##16446141##34##]. During late embryonic and early postnatal periods, <italic>trkA</italic>-expressing neurons differentiate into two subpopulations of nociceptive neurons; <italic>trkA</italic>-retaining peptidergic neurons, and non-peptidergic neurons that repress <italic>trkA </italic>and instead activate <italic>Ret</italic>, a receptor for glial-derived neurotrophic factor (GDNF; Figure ##FIG##0##1b##). During the late embryonic stages, most <italic>trkA</italic>-expressing DRG neurons coexpress <italic>Runx1 </italic>(Figure ##FIG##0##1b##). Postnatally, Runx1 disappears in <italic>trkA</italic>-retaining peptidergic neurons but continues to exist in <italic>Ret</italic>-inducing non-peptidergic neurons (Figure ##FIG##0##1b##). Using the <italic>Runx1</italic>-conditional knockout mouse, it was shown that Runx1 is dispensable for the <italic>de novo </italic>induction of TrkA [##REF##16446141##34##]. This was confirmed by Shiga and his colleagues [##REF##17208218##33##], who used a different gene-targeting method that relied on the rescuing of <italic>Runx1 </italic>expression in hematopoietic cells. However, Marmigere <italic>et al</italic>. [##REF##16429136##35##] showed that virally expressed <italic>Runx1 </italic>induced <italic>de novo </italic>synthesis of TrkA in the DRG and spinal cord of chick embryos. One possible explanation is that the minimal enhancer of <italic>trkA</italic>, which Runx1 regulates [##REF##16429136##35##], may not be required for the <italic>de novo </italic>induction of <italic>trkA </italic>expression [##REF##10934022##36##]. On the other hand, Runx1 is essential for the late repression of <italic>trkA </italic>and induction of <italic>Ret </italic>when TrkA⁺and Ret⁺neurons segregate (Figure ##FIG##0##1b##) [##REF##16446141##34##]. In addition to <italic>trkA</italic>, Runx1 also represses the neuropeptide, calcitonin-gene-related peptide (CGRP; Figure ##FIG##0##1b##) [##REF##16446142##25##,##REF##17208218##33##,##REF##16446141##34##]. More surprisingly, nearly all the known marker genes for nociception are under the control of Runx1. In the conditional <italic>Runx1 </italic>mutant DRG, expression of a number of nociceptor-specific G protein coupled receptors, ATP channels, and TRPV channels is attenuated (Figure ##FIG##0##1b##) [##REF##16446141##34##].</p>", "<p>Similar to Runx3, Runx1 also regulates the axonal outgrowth and guidance of nociceptive neurons. Marmigere <italic>et al</italic>. [##REF##16429136##35##] revealed that the transfection of Runx1 into boundary cap-derived neural crest stem cells increased neurite length and branching. In Runx1-knockout mice, the axonal projection to laminae IIi of the dorsal spinal cord was perturbed [##REF##17208218##33##,##REF##16446141##34##]. In the wild type, peptidergic nociceptive axons project to layer I/IIo in the superficial dorsal horn, whereas non-peptidergic nociceptive axons project to deeper layer IIi. In Runx1-knockout mouse, non-peptidergic axonal projection displays dorsal shift to layer I/IIo [##REF##16446141##34##].</p>", "<p>Thus, Runx1 controls a battery of genes that are associated with the generation of non-peptidergic nociceptive neurons. The findings that both Runx3 and Runx1 play critical roles in distinct sensory neurons suggest that Runx factors are involved in the evolution of sophisticated sensory systems in higher vertebrates.</p>", "<title>Upstream/downstream genes</title>", "<p>The upstream signals and transcriptional regulation of <italic>RUNX </italic>genes have been studied in non-neuronal tissues [##REF##15156175##37##]. However, only limited studies have addressed this issue in the nervous system. Both <italic>Runx3 </italic>and <italic>Runx1 </italic>genes contain Brn-3a binding sites in their 5'-upstream regions, suggesting that <italic>Runx3 </italic>and <italic>Runx1 </italic>are candidate downstream targets of Brn-3a, a well characterized transcription factor in sensory neurons [##REF##11733147##38##,##REF##11179664##39##]. Microarray studies have shown decreased levels of <italic>Runx1 </italic>and <italic>Runx3 </italic>transcripts in the sensory neurons of <italic>Brn-3a</italic>-knokout mice [##REF##15253936##40##,##REF##17239249##41##]. Kramer <italic>et al</italic>. [##REF##16446142##25##] investigated the putative upstream signal of <italic>Runx1</italic>/<italic>Runx3 </italic>in DRG neurons. Plausible candidates are TrkC/TrkA signalling and the basic helix-loop-helix transcription factors Ngn2/Ngn1; however, a genetic study has excluded these possibilities [##REF##16446142##25##]. Ginty and colleagues [##REF##17553423##42##] investigated the roles of NGF and the Ret receptor in DRG neurons. In <italic>Ngf-Bax </italic>compound knockout DRG, TrkA neurons are hypotrophic although <italic>de novo Runx1 </italic>expression is unaffected [##REF##17553423##42##]. However, <italic>Runx1 </italic>expression is not maintained to the neonate stage and the expression of all putative Runx1 target genes is altered [##REF##17553423##42##]. Thus, NGF signalling is essential for sustained expression of <italic>Runx1</italic>. In <italic>Ret </italic>conditional knockout DRG, <italic>Runx1 </italic>expression is normal but a part of Runx1 target genes are affected, suggesting the GFR/Ret dependent transcriptional regulation by Runx1 in DRG neurons [##REF##17553423##42##]. Although this study placed Runx in a pivotal position in developmental signalling cascades, the upstream signalling event(s) still remains elusive.</p>", "<p>On the other hand, how does Runx1/Runx3 regulate downstream transcriptional cascades? In DRG neurons, TrkC is a critical signalling receptor involved not only in the control of cell survival, but also in axon path-finding and fate determination of proprioceptive DRG neurons [##REF##12741988##32##,##REF##8205613##43##,##REF##15247919##44##]. Therefore, it is natural to infer that <italic>trkC </italic>is a transcriptional target of Runx3 [##REF##12093746##17##]. However, unbiased computational analysis suggested that a <italic>cis</italic>-regulatory element exists in the gene locus of TrkB, rather than in the gene locus of TrkC [##REF##17584746##45##]. This was unexpected because <italic>trkB </italic>is expressed in neurons of an alternative sensory fate, TrkB⁺TrkC^-neurons [##REF##8205613##43##]. The strategy \"to repress alternative traits\" appears to be a common feature in neuronal lineage commitment [##REF##11290324##46##]. At the molecular level, <italic>trkB </italic>possesses a conserved cluster of Runx binding sites that function as a silencer of the <italic>trkB </italic>promoter in cultured DRG neurons [##REF##17584746##45##]. In <italic>Runx3 </italic>knockout DRG, derepression of <italic>trkB </italic>seems to be a crucial event, influencing lineage commitment [##REF##16446142##25##,##REF##17584746##45##], and, eventually, resulting in drastic behavioural consequences [##REF##12352981##16##,##REF##12093746##17##].</p>", "<p>Runx protein works both as an activator and repressor, depending on the molecular context [##REF##16926135##47##]. The finding that Runx3 represses <italic>trkB </italic>raises a question as to the identities of its partner molecules in the transcriptional repressor complex. The function of Runx1 as a transcriptional repressor has been widely studied [##REF##15156176##48##,##REF##15156182##49##]. A plausible candidate in the context of DRG is the Groucho corepressor. In motoneuron fate specification, Groucho-mediated repression is a common mechanism for homeodomain proteins containing the EH1 domain [##REF##11290324##46##]. Runx proteins have the evolutionarily conserved VWRPY carboxy-terminal motif, which is considered to be critical for Groucho binding/function [##REF##9271433##50##,##REF##9837750##51##]. Yarmus <italic>et al</italic>. [##REF##16651517##52##] generated mice in which Runx3 lacks these amino acids. Surprisingly, VWRPY knockout mice displayed the normal development in DRG neurons, though they showed the phenocopy to <italic>Runx3 </italic>knockout mice in dendritic cells [##REF##16651517##52##]. The results suggest that Runx3 represses <italic>trkB </italic>through a Groucho independent mechanism. Recently, Ma and his colleagues [##REF##18171930##53##] investigated the significance of the VWRPY motif of Runx1 in DRG neurons. <italic>Runx1 </italic>cDNA, which lacks the VWRPY coding sequence, was knocked into the native <italic>Runx1 </italic>locus in <italic>delta446 </italic>mice [##REF##14504086##54##]. In the <italic>delta446 </italic>mice, derepression of Mrg class G-protein-coupled receptor genes was observed, suggesting that Mrg genes are repressed by a Groucho-dependent mechanism (Figure ##FIG##0##1c##) [##REF##18171930##53##]. Interestingly, two putative target genes that are repressed by Runx1, <italic>trkA </italic>and <italic>CGRP</italic>, were unaffected in the <italic>delta446 </italic>mice [##REF##18171930##53##]. These results suggest that Runx1 represses target genes through either a Groucho-dependent or an independent mechanism in DRG neurons.</p>", "<p>Chen <italic>et al</italic>. [##REF##16446141##34##] indicated that Runx1 controls nearly all known marker genes critical for nociceptive functions. Such global control by Runx over the transcription landscape is also observed in other physiological functions, such as hematopoietic stem cell formation (Runx1) and osteoblast maturation (Runx2). How this unique transcription factor has such a huge influence on many different transcriptional cascades remains a challenging question.</p>", "<title>Other neurological phenotypes of Runx1/Runx3 knockout mice</title>", "<p>Stifani and his colleagues [##REF##15240886##22##,##REF##15728845##23##] have worked on the neurological phenotypes of the Runx1 knockout mouse other than those arising from defects in DRG neurons. They analysed the cranial sensory neurons as well as cholinergic branchial and visceral motor neurons of hindbrain at an early embryonic stage [##REF##15240886##22##]. The expression of Runx1 was restricted to post-mitotic neurons, and disruption of Runx1 resulted in massive neuronal apoptosis [##REF##15240886##22##]. In contrast to this finding, <italic>Runx1 </italic>is expressed in the proliferating neuronal progenitors/precursors of olfactory receptor neurons (ORNs) [##REF##15728845##23##]. Runx1 drives the cell cycle in ORN progenitors through transcription repression of the cyclin dependent kinase inhibitor <italic>p21 </italic>[##REF##15728845##23##]. Unlike DRG, they did not observe any changes in the lineage markers in the neurons examined (cranial, hindbrain and olfactory), indicating that Runx1 has distinct functions in different types of neurons [##REF##15240886##22##,##REF##15728845##23##].</p>", "<p>The study of the neurological function of Runx3 other than in DRG is very limited. Levanon <italic>et al</italic>. [##REF##12093746##17##] reported that TrkC⁺neurons in the trigeminal ganglion survive in contrast to DRG neurons in <italic>Runx3</italic>-/- mouse. Most <italic>Runx3 </italic>knockout mice of the C57/B6 strain die within one day after birth [##REF##11955451##9##,##REF##12352981##16##]. The main cause of death may be starvation, as little milk is found in the stomachs of these mice [##REF##11955451##9##]. As this is probably related to the pups being unable to swallow milk, it is interesting to note that <italic>Runx3 </italic>is strongly expressed in cranial ganglia, including the glossopharyngeal ganglion [##REF##12352981##16##,##REF##12093746##17##]. It is possible that Runx3 is essential for the functional glossopharyngeal system (swallowing), suggesting the critical roles in developmental cranial neurons.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>The first draft of this review was written by KI together with TS, which was then complemented by YI. The figure was composed by KI.</p>" ]

[ "<title>Acknowledgements</title>", "<p>Work in the laboratory of YI is funded by A*STAR (Agency for Science, Technology and Research). TS is supported by Grant-in-Aid for Scientific Research from the 21^stCentury COE Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Runx proteins control the diversification of sensory neurons.</bold><bold>(a) </bold>Proprioceptive (TrkC⁺) and mechanoreceptive (TrkB⁺) DRG neurons are derived from the common precursors (TrkB⁺, TrkC⁺). During segregation of two complementary sensory populations, Runx3 represses <italic>trkB </italic>expression in TrkC⁺neurons. <bold>(b) </bold>During early postnatal periods, TrkA⁺DRG neurons differentiate into two nociceptive subpopulations; TrkA⁺peptidergic neurons, and Ret⁺non-peptidergic neurons that repress <italic>trkA</italic>. In Ret⁺non-peptidergic neurons, Runx1 represses <italic>trkA </italic>and neuropeptide <italic>CGRP</italic>. Runx1 also activates a number of nociceptor-specific G protein coupled receptors, ATP channels, and TRPV channels. <bold>(c) </bold>G protein coupled receptor MrgA, B and C are under dynamic transcriptional regulation in DRG neurons. A carboxy-terminal VWRPY motif of Runx proteins is critical for binding to Groucho corepressor. Runx1, which lacks VWRPY, fails to repress MrgA, B and C in DRG neurons.</p></caption></fig>" ]

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[ "<title>Introduction</title>", "<p>Chlorine (Cl₂) gas is a common inhalational irritant, encountered both occupationally and environmentally[##REF##8004329##1##,##REF##884993##2##]. The acute effects of Cl₂gas inhalation can range from mild respiratory mucus membrane irritation to marked denudation of the mucosa, pulmonary oedema, and even death. Recovery from Cl₂-induced lung injury requires repair and/or regeneration of the epithelial layer. The repair process after Cl₂exposure may not restore normal structure and function as cases of subepithelial fibrosis, mucous hyperplasia, and non-specific airway hyperresponsiveness have been reported in persons after recovery from Cl₂injury[##REF##8308178##3##,##REF##4028848##4##]. Repeated exposure to chlorine through swimming appears to be a significant risk factor for airway disease manifesting as asthma[##REF##17545376##5##].</p>", "<p>The airway epithelium is the first target of inhaled Cl₂gas. Although the exact mechanism of epithelial damage is unknown, oxidative injury is likely involved as Cl₂gas can combine with reactive oxygen species to form a variety of highly reactive oxidants [##REF##9065416##6##]. Direct oxidative injury to the epithelium may occur immediately with exposure to Cl₂, but further damage to the epithelium may occur with migration of inflammatory cells such as neutrophils into the airway epithelium and the subsequent release of oxidants and proteolytic enzymes.</p>", "<p>Limited information is available regarding the time course of injury and repair of the epithelium after acute Cl₂gas exposure. Bronchial biopsies from humans have shown epithelial desquamation from 3 to 15 days after accidental Cl₂exposure followed by epithelial regeneration, characterized by proliferation of basal cells at two months post-exposure[##REF##9032521##7##]. Animal studies of Cl₂exposure have furthered our understanding of the time course of injury and repair. However, these studies have been primarily descriptive in nature. Rats acutely exposed to high concentrations of Cl₂gas demonstrated bronchial epithelial sloughing 1 hour after exposure with epithelial regeneration occurring by 72 hrs after exposure[##REF##9623698##8##]. Recently, we have described the response of A/J mice to a single exposure to varying concentrations of Cl₂exposure[##REF##12724121##9##]. Exposure to the highest concentration of Cl₂gas (800 ppm for 5 minutes) resulted in marked epithelial loss and airway hyperresponsiveness to methacholine 24 hrs after exposure.</p>", "<p>Airway remodelling is a feature of asthma that has the potential to explain the induction and chronicity of the disease. Generally animal models have focussed on allergen-driven changes in airway structure which are of uncertain relevance to irritant-induced asthma. For this reason we wished to explore the injury and repair processes involved in irritant-induced asthma. To do this we characterized the time course of airway injury and repair after a single exposure to Cl₂gas in mice using quantitative measures of epithelial damage and repair. Markers of epithelial damage were apoptosis, assessed by terminal dUTP nick end labelling (TUNEL) staining, and the presence of protein and epithelial cells in the bronchoalveolar lavage fluid. Epithelial repair was assessed by quantifying cell proliferation using the proliferation marker proliferating cell nuclear antigen (PCNA). PCNA is a DNA polymerase-δ cofactor located in the nuclear compartment of proliferating cells [##REF##2882423##10##,##REF##2889739##11##]. Airway remodelling was assessed by quantification of airway smooth muscle mass using standard morphometric techniques on smooth muscle specific α-actin immunostained tissue sections and by scoring of airway fibrosis on Picrosirius red stained tissue sections. Goblet cell numbers were assessed by light microscopy and standard morphometric techniques. Airway histology was also used to qualitatively assess the time course of damage and repair to the airways. We wished to relate these markers of damage and repair to functional consequences of Cl₂-induced injury in terms of airway mechanics and airway responsiveness to methacholine.</p>" ]

[ "<title>Methods</title>", "<title>Animals and chlorine exposure</title>", "<p>Male A/J mice (23–27 g) were purchased from Harlan (Indianapolis, Indiana) and housed in a conventional animal facility at McGill University. Animals were treated according to guidelines of the Canadian Council for Animal Care and protocols were approved by the Animal Care Committee of McGill University.</p>", "<p>Forty-eight mice were exposed to either room air (control) or 800 ppm Cl₂gas diluted in room air for 5 minutes using a nose-only exposure chamber. This concentration of Cl₂gas was chosen as it was previously shown to result in severe airway damage but with minimal animal mortality[##REF##12724121##9##]. Mice exposed to Cl₂were studied at 12 hrs, 24 hrs, 48 hrs, 5 days (d), or 10 d after Cl₂exposure (n = 8 at each time point). The control mice were studied 24 hrs after exposure to room air (n = 8).</p>", "<title>Bronchoalveolar lavage, lung histology and morphometry</title>", "<p>The chest was opened, the left main bronchus clamped, and 0.3 ml of sterile saline followed by four separate 0.5 ml instillations were washed into the right lung. Fluid recovered from the first wash was centrifuged at 1500 rpm for 5 minutes at 4°C and the supernatant used for protein quantification. The cell pellet was pooled with the remaining lavage samples and total live and dead cells were counted using trypan blue exclusion. Cytospin slides were prepared using a cytocentrifuge (Shandon, Pittsburgh, PA) and stained with Dip Quick (Jorgensen Labs Inc., Loveland, CO). Differential cell counts, including epithelial cells, were determined on 300 cells/slide. Total protein in the BAL supernatant was quantified using a dye-binding colorimetric assay (Bio-Rad, Hercules, CA), and determined by spectrophotometry at 620 nm and quantified using a bovine serum albumin standard curve.</p>", "<title>Tissue preparation</title>", "<p>Following BAL, the lungs were removed and the left lung was fixed with an intratracheal perfusion of 10% buffered formalin at a constant pressure of 25 cmH₂O for a period of 24 hrs. Histology and immunohistochemistry were performed on 5 μm thick paraffin-embedded sections taken from the parahilar region. Adjacent sections were either stained with hematoxylin-eosin (H&amp;E), periodic acid Schiff (PAS), or processed for immunohistochemistry.</p>", "<title>Immunohistochemistry</title>", "<p>Cells undergoing proliferation were detected in tissue sections by immunostaining for proliferating cell associated nuclear antigen (PCNA. Following deparaffination in xylene and rehydration through graded ethanol solutions, the tissue sections underwent a high temperature epitope unmasking treatment by a modified version of the microwave boiling method. An acidic antigen retrieval buffer (Vector Laboratories, Burlingame, CA) was microwave pre-heated to 95°C, and the slides were incubated in it for 30 minutes using a pre-warmed coplin jar protected with styrofoam. After cooling for 20 minutes, a membrane permeabilization treatment was applied by immersing the slides for 20 minutes in a 0.2% dilution of Triton X-100 (Sigma Chemical Co., St. Louis, MO) in pH 7.6 Trizma base (Sigma) buffered saline. The tissues were then blocked for 1 hour using a blocking reagent designed for immunohistochemistry using mouse primary antibodies on mouse tissues (Vector Laboratories). Primary murine anti-PCNA antibody was applied at a concentration of 2.5 μg/ml and the sections were incubated for 30 min. at room temperature. A biotinylated anti-mouse antibody (1:250 dilution; Vector Laboratories) was applied for 10 min. followed by a 45-min. incubation with an avidin-biotin complex-alkaline phosphatase reagent (ABC-AP). Rat intestine was used as a positive control and mouse lung sections incubated with isotype control mouse IgG were used as a negative control. PCNA-positive cells were visualized with Vector Red chromogen (Vector Laboratories) and the tissue was counterstained using methyl green (Sigma). Finally, the sections were dehydrated and mounted under glass coverslips with VectaMount (Vector Laboratories).</p>", "<p>To determine the amount of airway smooth muscle by morphometry, airway smooth muscle was detected by immunostaining for smooth muscle α-actin. The lung sections were prepared as described above with the exception of high temperature antigen unmasking, and incubated with monoclonal antibody to smooth muscle α-actin (1A4, 1:1000 dilution; Sigma) for 30 minutes followed by biotinylated anti-mouse IgG antibody and ABC-AP steps as above.</p>", "<p>PCNA was colocalized with smooth muscle α-actin in order to detect cell proliferation in the airway smooth muscle. Immunohistochemistry for PCNA was done first as described above, and the signal developed with BCIP/NTB chromogen (Vector Laboratories) instead. The sections were then incubated with anti-smooth muscle α-actin antibody (1A4, 1:1000 dilution, Sigma) for 30 min. at 37°C, followed by the biotinylated anti-mouse antibody and ABC-AP steps as above. The smooth muscle α-actin signal was developed with Vector Red, and the tissues counterstained with methyl green.</p>", "<title>Detection of apoptotic cells <italic>in situ</italic></title>", "<p>To detect apoptotic cells in lung tissue sections we used a TUNEL technique (ApopTag peroxidase detection kit; Intergen, Purchase, NY). The sections were deparaffinized, pretreated with 20 μg/ml proteinase K (Intergen) for 15 min at 37°C, and endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 5 min This was followed by polymerization of digoxigenin-labeled UTP on nicked DNA ends and application of anti-digoxigenin peroxidase conjugate, using ApopTag kit components as per manufacturer's instructions. The signal was developed with DAB chromogen, and the tissues counterstained with methyl green.</p>", "<title>Quantitative morphology on airway sections</title>", "<p>Quantification of PCNA-positive cells was performed on parahilar lung sections. Cross-sectioned airways, with a major/minor diameter ratio &lt; 2.5, were selected for analysis. The number of PCNA⁺cells in the epithelium and sub-epithelial layers were quantified under a light microscope using a 40× objective. The airway basement membrane length was measured by superimposing the image of the airway onto a calibrated digitizing tablet (Jandel Scientific, Chicago, IL), with a microscope equipped with a <italic>camera lucida </italic>projection system (Leica Microsystems, Richmond Hill, ON, Canada). The numbers of proliferating cells corrected for airway size were expressed as PCNA⁺cells/mm of basement membrane perimeter (P_BM).</p>", "<title>Quantification of ASM mass and proliferation</title>", "<p>ASM mass was measured on control, 5 d, and 10 d post-exposure groups by tracing the ASM bundles, as defined by positive staining for smooth muscle α-actin, using a camera lucida and digitizing system. The sum of the ASM bundle areas was calculated for each airway and referenced to P_BM²for airway size correction. To determine if airway smooth muscle cells expressed PCNA, co-localization of PCNA with smooth muscle α-actin was done in a subset of animals. The number of PCNA+ cells in the epithelial and sub-epithelial layers of each airway with a major/minor diameter ratio &lt; 2.5 was quantified and expressed per mm of P_BMfor epithelium or P_BM²for subepithelial cells.</p>", "<title>Goblet cell quantification</title>", "<p>The number of goblet cells was assessed on PAS stained tissue sections. A total of 118 airways from 28 animals representing animals from the different exposure times was analyzed and cells were expressed as cell numbers per mm of P_BM.</p>", "<title>Semiquantitative assessment of collagen deposition</title>", "<p>To address whether chlorine exposure could affect the development of subepithelial fibrosis, lung sections were stained with Picrosirius red and collagen deposition scored in airways. Scoring by two blinded observers of collagen deposition in airways was performed independently using a scale from 1 to 3. The cumulative score for each mouse was averaged according to treatment group.</p>", "<p>The quantity of airway smooth muscle (ASM) was quantified by the <italic>camera lucida </italic>technique. Images of the airways were traced using a microscope side arm attachment and areas of the α-actin positive smooth muscle bundles were digitized using commercial software. The area of ASM was standardized for airway size using the P_BM, with the quantity of ASM expressed as ASM/P_BM²(mm²). Morphometric assessments were made on all airways in the tissue section that met the above criterion for its aspect ratio.</p>", "<title>Methacholine responsiveness</title>", "<p>In a separate group of sixty mice, airway responsiveness to methacholine was measured at similar time points after room air or Cl₂exposure (n = 10 at each time point). Animals were sedated with xylazine hydrochloride (10 mg/kg i.p.) and anaesthetized with sodium pentobarbital (40 mg/kg i.p). A flexible, saline-filled cannula (PE-10 tubing) was inserted into the jugular vein for administration of drugs and the trachea was cannulated with a snug-fitting metal cannula. Animals were connected to a computer-controlled small animal ventilator (flexiVent, Scireq, Montreal, PQ, Canada) and paralysed using pancuronium chloride (0.8 mg/kg i.v.). Mice were ventilated in a quasi-sinusoidal fashion with a tidal volume of 0.18 ml at a rate of 150 breaths/min. A positive end-expiratory pressure (PEEP) of 1.5 cmH₂O was used. Measurements of pulmonary mechanics were made using a 2.5 Hz sinusoidal forcing function with an amplitude of 0.18 ml. The perturbation was applied after cessation of regular ventilation and expiration by the animal to functional residual capacity. Respiratory system resistance (Rrs) and dynamic elastance (Ers) was derived from the relationship between airway opening pressure, tidal flow and volume After initial baseline measurements of Rrs and Ers, doubling doses of methacholine chloride (Sigma;10 μg/kg to 320 μg/kg i.v.) were administered. Rrs and Ers were measured every 15 seconds after methacholine infusion until peak Rrs was reached. Thirty seconds after peak Rrs was reached, the next highest dose of methacholine was administered. The peak Rrs and Ers at each methacholine dose were used to construct a dose-response curve. After completion of all methacholine doses, animals were euthanized by i.v. pentobarbital overdose. Airway responses were evaluated as the difference between the peak in Ers after 160 μg/kg methacholine and baseline Ers (ΔErs). Changes in Ers rather than Rrs were chosen to represent airway responsiveness because methacholine-induced changes in elastance are affected to a greater degree in mice after Cl₂exposure[##REF##12724121##9##].</p>", "<title>Statistical analysis</title>", "<p>One-way analysis of variance was used to determine the effect of time on the dependent variables except ASM/mm². The significance of the post-hoc comparisons was determined using Dunnett's test versus control at the p &lt; 0.05 level. The effect of Cl₂on ASM/P_BM²(in mm²) at different times after exposure was tested using the Kolmogorov-Smirnoff test.</p>" ]

[ "<title>Results</title>", "<title>Histological and immunohistochemical evaluation of airways</title>", "<p>Normal airway structure and basal levels of proliferation and apoptosis in airway epithelium are shown in Figures ##FIG##0##1A##, ##FIG##1##2A##, ##FIG##2##3A##. Histological examination from samples obtained 12 hrs after exposure showed severe injury to the bronchial epithelium with extensive detachment of the epithelium from the basement membrane and complete denudation of the epithelium in some airways (Figure ##FIG##0##1B##). Cell cycle was inhibited at this time point after chlorine exposure, as indicated by the virtual absence of positive staining for PCNA (Figure ##FIG##1##2B##). The TUNEL technique produced cytoplasmic staining of the injured epithelium, but not a signal conforming to usual histopathological criteria for the identification of apoptosis, suggesting that a mechanism other than apoptosis accounts for the rapid and massive epithelial disaggregation following Cl₂gas exposure (Figure ##FIG##2##3B##). At 24 hrs after Cl₂exposure, most of the detached airway epithelial cells were cleared and airway epithelial cell proliferation was re-established (Figure ##FIG##2##3C##). In this phase, some clusters of basal cells undergoing apoptosis alternated with proliferating cells, overlying a preserved basement membrane (Figure ##FIG##2##3D##). Epithelial regeneration was evident at 48 hrs with flattened cells with elongated nuclei lining the basement membrane and an increased frequency of PCNA positive cells. Co-localisation of PCNA and smooth muscle α-actin provided evidence of airway smooth muscle proliferation (Figure ##FIG##1##2F##). Five days following chlorine exposure, the airway epithelium was evenly re-populated with cells showing an intense proliferative activity, and the frequency of apoptotic cells was similar to baseline levels. Ten days after chlorine exposure, the epithelium was reconstituted and the airway wall was thickened (1 D). Cl₂exposure did not induce goblet cell metaplasia as determined by PAS staining at any time point (data not shown). Only 4 of 118 airways analyzed from 28 mice, sampled at all time points showed any PAS positive cells and these were very infrequent.</p>", "<p>Cl₂exposure did affect the quantity of ASM as determined by morphometry (Figure ##FIG##3##4##). 10 days after Cl₂exposure, a shift was observed in the distribution of airways with small amounts of ASM. For example, the proportion of airways with values of ASM area &gt; 0.0015 (ASM/mm²of BM) was approximately 50% for control animals, but &lt; 10% for the 10 day post-exposure group.</p>", "<title>Quantification of PCNA</title>", "<p>The number of PCNA+ cells in the airway epithelium and sub-epithelium is shown in Figure ##FIG##4##5##. A baseline frequency of epithelial and sub-epithelial proliferation was detectable in control animals. Twelve hours after Cl₂exposure, epithelial PCNA expression tended to be lower than control values although the difference did not reach statistical significance. Epithelial PCNA expression was significantly elevated by 48 hrs after chlorine exposure, increasing approximately 14-fold from control levels (p &lt; 0.05) and over 30-fold by 5 d post-exposure (p &lt; 0.05). Although the majority of the PCNA+ cells in the airways were epithelial cells, a significant amount of sub-epithelial PCNA expression was also observed after Cl₂exposure. Subepithelial PCNA expression was significantly elevated at 5 d post-exposure. By 10 d post-exposure, both epithelial and subepithelial PCNA immunoreactivity had returned to control levels. No significant correlation was found between airway size (as determined by basement membrane length) and PCNA index at any of the time points.</p>", "<title>Determination of airway fibrosis</title>", "<p>Assessment of collagen deposition using Picrosirius red staining demonstrated a significant increase in collagen in the airways 10 days following chlorine exposure (Figure ##FIG##5##6##). There was no significant difference in the amount of collagen at 24 hours or 5 days. Twenty nine animals were analyzed and assessed by two observers independently.</p>", "<title>Bronchoalveolar lavage</title>", "<p>The recovery of BALF averaged 90% and did not differ significantly among groups. Total cell counts were significantly elevated at 5 d and remained elevated at 10 d post-exposure relative to controls (Table ##TAB##0##1##). Differential cell counts showed no significant change in eosinophils or lymphocytes after Cl₂exposure (Figure ##FIG##6##7##), but neutrophils were significantly elevated relative to controls at 5 d post-exposure (0.02 ± 0.01 (SE) × 10⁴cells in controls, 4.76 ± 1.94 at 5 d post-exposure; p &lt; 0.05) and macrophages were significantly elevated at both 5 d and 10 d post-exposure (12.0 ± 1.9 × 10⁴in controls, 32.2 ± 7.7 at 5 d, 33.7 ± 3.3 at 10 d, p &lt; 0.05 versus controls). Dead cells in the BALF, identified by trypan blue, were markedly elevated from 12 hrs to 48 hrs post-exposure (Table ##TAB##0##1##); these cells were almost exclusively comprised of epithelial cells, identified by their cuboidal shape and cilia. Similarly, the number of epithelial cells counted during differential cell counting of cytospin slides was markedly elevated at 12 and 24 hr (p &lt; 0.05) but had returned to control levels by 48 hr (Figure ##FIG##6##7##). The amount of total protein in BALF supernatant, a marker of airway microvascular permeability and epithelial damage, was significantly elevated 12 hrs after chlorine exposure, and remained elevated up to 5 d post-exposure (Table ##TAB##0##1##).</p>", "<title>Airway mechanics and responsiveness to methacholine</title>", "<p>Cl₂exposure altered respiratory mechanics as reflected by changes in baseline Ers and Rrs. The initial response to Cl₂exposure was an elevation of Ers and Rrs, which persisted up to 48 hrs post-exposure (Ers = 51.1 ± 3.09 cmH₂O/ml in control mice vs. 70.9 ± 3.23, 67.5 ± 2.16, and 61.5 ± 1.67 cmH₂O/ml at 12, 24, and 48 hrs post-exposure respectively, p &lt; 0.05; Rrs = 0.98 ± 0.05 cmH₂O/ml/sec in control mice vs. 1.32 ± 0.06 and 1.23 ± 0.05 cmH₂O/ml/sec at 12 and 24 hrs post-exposure respectively, p &lt; 0.05) (Figure ##FIG##7##8##). Airway mechanics returned to baseline levels by 5 d, but at 10 d post-exposure, Ers levels fell significantly below control levels (Ers = 51.1 ± 3.09 cmH₂O/ml in control mice vs. 40.7 ± 0.97 cmH₂O/ml at 10 d post-exposure, p &lt; 0.05). Airway responsiveness to methacholine, as determined by ΔErs, increased after Cl₂exposure compared to control, and was significantly higher at 12 hrs and 5 d post exposure (ΔErs = 100 ± 19.7 in control mice vs. 257 ± 45.3 and 269 ± 34.0 at 12 hrs and 5 d post-exposure respectively, p &lt; 0.05) (Figure ##FIG##8##9##). ΔRrs was not significantly altered at any time point after Cl₂exposure, although a trend for ΔRrs to be lower 24 hrs after Cl₂exposure was observed (p = 0.055).</p>" ]

[ "<title>Discussion</title>", "<p>This study describes the time course of airway epithelial damage and repair in A/J mice following a single exposure to a high concentration of Cl₂gas. Cl₂exposure resulted in marked damage to the airways, as indicated by epithelial cell sloughing, increased protein in BALF, an inflammatory response with neutrophil and macrophage recruitment into the airways, and altered lung mechanics. Subsequent airway repair was characterized by increased epithelial and subepithelial cell proliferation, complete restoration of the epithelial layer, increases in the quantity of ASM and modest airway hyperresponsiveness. There was also evidence of airway fibrosis at 10 days after the Cl₂exposure.</p>", "<p>A pronounced feature of the acute injury phase after Cl₂exposure was extensive and synchronous loss of airway epithelial cells. Programmed cell death was not likely the mechanism of the generalized loss of epithelial cells, since the TUNEL technique did not produce a nuclear signal consistent with apoptosis. The explanation for the diffuse cytoplasmic staining observed in the detached epithelium is not clear but may have been caused by the highly reactive chlorine molecules. As opposed to apoptosis, disruption of the intercellular junctions and the attachments of the epithelial cells to the basement membrane by the Cl₂gas may have been the mechanism responsible for detachment of the epithelium. Other oxidants such as hypochlorous acid (HOCl) and ozone can disrupt cell adhesion via damage to extracellular matrix proteins and β-1 integrins[##REF##9730863##12##,##REF##9214576##13##], thus Cl₂gas may act via similar mechanisms.</p>", "<p>Acute loss of epithelial barrier function resulted from the extensive sloughing of the airway epithelium, as reflected by the increased protein concentration in BALF. Changes in baseline respiratory mechanics (resistance and dynamic elastance) paralleled the time course of BALF protein concentration with the most pronounced alterations occurring 12 hrs after exposure followed by resolution of these changes over the 10 d study period. Pulmonary edema and alveolar flooding may have contributed to the acute decreases in lung elastance in this model, as has been demonstrated in other species after Cl₂gas exposure[##REF##9163653##14##,##REF##7473069##15##]. However, heterogeneous airway narrowing may have also contributed.</p>", "<p>Exposure to chlorine gas exposure had a direct toxic effect on airway epithelium as severe airway damage was observed at early time points in the absence of an inflammatory response. When inhaled, chlorine gas combines with water to form hydrochloric and hypochlorous acids (Cl₂+ H₂O → HCl + HOCl). HOCl is unstable and breaks down into HCl and free oxygen. Oxidant injury due to this nascent oxygen is thought to be the primary mechanism of cytotoxicity, with the acid production being secondary. In a similar study from our laboratory, positive staining for 3-nitrotyrosine residues, a marker of oxidative stress, was observed in mouse airways 24 hrs after exposure to 800 ppm Cl₂gas, supporting oxidative injury as a mechanism in this model[##REF##12724121##9##].</p>", "<p>A modest neutrophil and macrophage inflammation did subsequently develop after Cl₂exposure and the inflammatory cells themselves could also have contributed to airway damage. Activated neutrophils can produce reactive oxygen species and myeloperoxidase, a neutrophil-specific enzyme that catalyses the formation of hypochlorous acid/hypochloride (HOCl/OCl^-) from hydrogen peroxide. Neutrophils can also release proteolytic enzymes such as collagenase and elastase which could also contribute to the airway damage.</p>", "<p>Following the acute airway injury induced by Cl₂gas exposure, tissue repair and restoration of the barrier function of the epithelium occurred. One mechanism by which an epithelial layer can be repaired is by migration of healthy epithelial cells from an area adjacent to the damaged epithelium. Studies of mechanical de-epithelialisation <italic>in vivo </italic>demonstrate that this is a quickly occurring process, with initial migration of adjacent epithelium to the wound site occurring within 8–15 hrs[##REF##7648624##16##]. The relevance of migration as a mechanism, however, is questionable in cases of near to complete denudation of the epithelium, as was observed in many airways in this study. In this instance, growth and differentiation of local progenitor cells is another mechanism by which the epithelial layer can be repopulated. In the trachea and bronchi, basal cells constitute a separate layer of cells attached to the airway basement membrane. In response to epithelial injury, these cells can turn into a highly proliferative cell phenotype and can become flattened and cover the basement membrane[##REF##7517145##17##]. In smaller bronchioles, Clara cells likely play the role of progenitor cell after injury[##REF##11415931##18##] Intriguing new evidence suggests a possible role for circulating bone marrow stem cells in bronchiolar repopulation after injury[##REF##12815096##19##]. Ortiz et al. [##REF##12815096##19##] have demonstrated that murine mesenchymal stem cells are able to home to the lung after injury and adopt an epithelium-like phenotype. It is uncertain at this time as to which specific cell population may have acted as progenitor cells for the airway epithelium in this study.</p>", "<p>The time course of epithelial repair after Cl₂gas exposure was assessed by quantifying the amount of cellular proliferation occurring in the airway. Increased levels of PCNA immunoreactivity were detectable by 48 hrs and maximal proliferative activity in the airways occurred 5 d post-exposure. Compared to other studies reporting dynamics of epithelial repair after acute airway injury, the recovery of murine airways from Cl₂damage was relatively prolonged. In rats, peak cell proliferation occurred 26 to 36 hrs after mechanical injury of tracheal epithelium[##REF##856476##20##,##REF##7392567##21##] and at 24 to 48 hrs after acute ozone exposure[##REF##9517619##22##]. In mice, epithelial cell proliferation after desquamation of airway epithelium by naphthalene treatment was maximal 2 to 7 days post-treatment depending on mouse strain[##REF##11786425##23##]. The time course of epithelial repair after damage is likely related to the severity of injury, and therefore is difficult to compare among these different models.</p>", "<p>Increased cellular proliferation after Cl₂exposure was not limited to the airway epithelium as significant PCNA immunoreactivity was also observed in the sub-epithelial layer of airways. Using immunohistochemical co-localization, we provide evidence of airway smooth muscle cell proliferation. This finding, together with the quantification of ASM mass, suggests that chlorine exposure in this model results in ASM hyperplasia. This is in agreement with the study of Demnati et al [##REF##9623698##8##] who reported an increase, albeit transient, in ASM quantity in rats after acute exposure to Cl₂gas.</p>", "<p>The signals involved in repair and in the repopulation of the epithelium after Cl₂-induced injury are unclear. Epidermal growth factor (EGF)-dependent mechanisms may be important as mediators such as epidermal growth factor (EGF) and TGF-α can bind to EGF receptors located on both basal cells and epithelial cells and stimulate cell migration, proliferation and differentiation[##REF##12101277##24##]. The absence of goblet cells is however somewhat surprising if indeed EGF receptor ligands are important in repair as stimulation of the EGF receptor has been repeatedly demonstrated to cause goblet cell differentiation in the airways[##REF##10077640##25##]. EGF-independent factors may also be important. Neutrophils, for example, may contribute to signalling of repair processes as neutrophil defensins, antimicrobial peptides present in the neutrophil, may also stimulate proliferation[##UREF##0##26##]. Interestingly, the maximal proliferative activity of the airway epithelium at 5 d corresponded to the time of maximal neutrophil influx in the BALF.</p>", "<p>Restoration of the airway epithelial layer, as assessed histologically, was complete by 10 days after Cl₂exposure. However, not all variables had returned to control levels after 10 days; inflammatory cells in the BALF were still elevated and baseline elastance was lower than control levels. Therefore complete resolution of the Cl₂-induced damage may not have occurred in the timeframe of this study. Also the timeframe of this study may not have been long enough to fully evaluate remodelling processes. As we only detected changes in ASM quantity at our latest time point, 10 days after exposure, the possibility remains that further remodelling may take place at even later time points. There was also an increase in collagen deposition in the airway wall at this same time point. The epithelium is a source of fibrogenic cytokines[##REF##15563691##27##] and it is potentially the cause of the collagen deposition. Although the changes were not significant there appeared to be a trend for a reduction in airway smooth muscle mass at 5 days after Cl₂exposure, suggesting that damage may have penetrated beyond the epithelium to the ASM layer.</p>", "<p>Persistent airway hyperresponsiveness occurs in a small percentage of people after acute Cl₂gas exposure[##REF##8404087##28##]. In this study, mice receiving a single exposure to a high concentration of Cl₂gas did display modest increase in dynamic elastance in response to methacholine but it was transient in nature. That responsiveness of pulmonary dynamic elastance to methacholine was affected to a greater degree by Cl₂gas exposure than was responsiveness of pulmonary resistance is consistent with results from a previous study[##REF##12724121##9##]. This suggests that changes in responsiveness to methacholine after Cl₂gas exposure in mice may be dominated by abnormalities in the peripheral lung, as opposed to central airways. Perhaps also the trend for a reduction in responsiveness to methacholine may reflect injury to the airway smooth muscle from the high levels of Cl₂used for exposure.</p>", "<p>In conclusion, this study describes the time-course of injury and repair after an acute exposure of mice to a high concentration of Cl₂gas. Severe epithelial injury was induced quickly after exposure with loss of the epithelial barrier function and acute alterations in respiratory mechanics. Epithelial repair processes were apparent by 24 hrs and restoration of the epithelium was complete by 10 d. Recovery from the Cl₂-induced damage was associated with modest airway hyperresponsiveness and alterations in airway smooth muscle mass. Whether comparable airway remodelling is associated with lesser degrees of repeated exposures remains to be explored.</p>" ]

[]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Accidental chlorine (Cl₂) gas inhalation is a common cause of acute airway injury. However, little is known about the kinetics of airway injury and repair after Cl₂exposure. We investigated the time course of airway epithelial damage and repair in mice after a single exposure to a high concentration of Cl₂gas. Mice were exposed to 800 ppm Cl₂gas for 5 minutes and studied from 12 hrs to 10 days post-exposure. The acute injury phase after Cl₂exposure (≤ 24 hrs post-exposure) was characterized by airway epithelial cell apoptosis (increased TUNEL staining) and sloughing, elevated protein in bronchoalveolar lavage fluid, and a modest increase in airway responses to methacholine. The repair phase after Cl₂exposure was characterized by increased airway epithelial cell proliferation, measured by immunoreactive proliferating cell nuclear antigen (PCNA), with maximal proliferation occurring 5 days after Cl₂exposure. At 10 days after Cl₂exposure the airway smooth muscle mass was increased relative to controls, suggestive of airway smooth muscle hyperplasia and there was evidence of airway fibrosis. No increase in goblet cells occurred at any time point. We conclude that a single exposure of mice to Cl₂gas causes acute changes in lung function, including pulmonary responsiveness to methacholine challenge, associated with airway damage, followed by subsequent repair and airway remodelling.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>ST was involved in the design and performance of the experiments and wrote the manuscript. DRB was responsible for the planning and oversight of all immunohistochemistry and contributed to the manuscript. HC assisted in the performance of measurements of airway responsiveness and tissue harvesting. TM performed histochemical staining for goblet cells and collagen and performed quantification of same. HKQ assisted in the analysis of histochemical images for goblet cells and collagen and assisted in editing the manuscript. JGM was responsible for the questions being tested and for the design of the experiments. He reviewed all phases of analysis and finalized the writing of the manuscript. All of the authors have read and approved the manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>Supported by grants from NIOSH (R01 OH004058-03) and l'Institut de recherche Robert Sauvé en santé et en sécurité du travail.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Effects of Cl₂exposure on lung histology. A: Normal mouse lung showing a large airway in cross section, an accompanying artery and two terminal bronchioles (Tb) that open into their respective alveolar ducts. B: Lung histology 12 h after a single 800 ppm Cl₂exposure. Partial or complete detachment of airway epithelium, as seen in this example, occurred in all airways. C: 10 d post-exposure, the epithelium is reconstituted and the airway wall is thickened. D: 10 d post-exposure, high magnification detail showing fully reconstituted airway epithelium. Stain: H&amp;E. Scale bars: 100 μm in A-C; 25 μm in D.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Effect of Cl₂exposure on cell proliferation as detected by PCNA immunostaining. A: Control mouse airway, showing baseline airway epithelial cell proliferation. PCNA positive cells are indicated by open arrowheads. B: 12 h post-exposure. There is an absence of PCNA positive events, suggesting inhibition of cell cycle. C and D: 24 h post-exposure. Proliferation of airway epithelial cells (C) is re-established. Endothelial cell proliferation (En) is also observed at this time point (D). E: 48 h post-exposure. An increase in PCNA positive epithelial cells is observed. F: Co-localisation of smooth muscle α-actin (red cytoplasmic signal) and PCNA (dark-violet nuclear signal), 48 h post-exposure. PCNA positive cells can be seen in the airway epithelium, smooth muscle layer, and adventitia. The inset shows an example of a PCNA positive airway myocyte at high magnification. G: 5 d post-exposure. The airway epithelium is evenly re-populated with cells undergoing intense proliferative activity. Scale bars: 50 μm (25 μ in F inset). Pn: Pneumocytes; SM: Smooth muscle.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Effect of Cl₂exposure on airway cell apoptosis; TUNEL technique. A: Control mouse airway, showing baseline airway epithelial cell apoptosis (arrowheads). B: 12 h post-exposure. Cytoplasmic TUNEL signal in damaged epithelium. The high magnification inset details the cytoplasmic localisation of the TUNEL stain on cells with methyl green counterstained nuclei. These cells lack a TUNEL signal attributable to apoptosis-related DNA fragmentation. The arrowheads indicate examples of cells that appear truly apoptotic. C: 24 h post-exposure. Some clusters of basal cells undergoing apoptosis are visible. Inset shows high magnification detail. D: 5 d post-exposure. The frequency of TUNEL positive cells at 5 d is back to baseline level. Scale bars: 100 μm in I; 50 μm in A, B, C inset and D.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Cumulative distribution of airway smooth muscle mass per mm²of basement membrane (ASM/mm²of P_BM). The values plotted are individual airway measurements. 2–8 airways were quantified per animal. The distribution of the 10 day group was significantly different from both the control and 5 day groups (p &lt; 0.05). n = 38, 40, and 31 for control, 5 days, and 10 days.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p>Time course of PCNA expression in the epithelium (A) and subepithelium (B) of airways in mice exposed to air (control) or Cl₂gas. Data is expressed as PCNA-positive cells/mm basement membrane. The number of airways evaluated at each time point ranged from 25 to 57. Values are means ± S.E. *significantly different from control (p &lt; 0.05).</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p>Illustrative photomicrograph showing collagen in the airway walls by Picrosirius red staining (two left panels). Quantitative analysis of degree of staining by semi-quantitative scoring at different time points after Cl₂gas exposure.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p>Time course of BALF differential cell counts after a single Cl₂gas exposure. At each time point, n = 8. Values are means ± S.E. * significantly different from control (p &lt; 0.05).</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p>Time course of baseline respiratory elastance (Ers) and resistance (Rrs) in mice exposed to Cl₂gas. Ers and Rrs were measured using a 2.5 Hz sine-wave perturbation with an amplitude of 0.18 ml. At each time point, n = 10. Values are means ± S.E. * significantly different from control (p &lt; 0.05).</p></caption></fig>", "<fig position=\"float\" id=\"F9\"><label>Figure 9</label><caption><p>Time course of airway responsiveness of elastance (Ers) and resistance (Rrs) to methacholine in mice exposed to Cl₂gas. Responsiveness is expressed as the peak Ers or Rrs after administration of 160 μg/kg methacholine minus baseline Ers or Rrs. Values are means ± S.E. * significantly different from control (p &lt; 0.05).</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Time course of protein, live and dead cell counts in BALF after Cl₂exposure.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\">Control</td><td align=\"center\">12 hr</td><td align=\"center\">24 hr</td><td align=\"center\">48 hr</td><td align=\"center\">5 d</td><td align=\"center\">10 d</td></tr></thead><tbody><tr><td align=\"left\">Live cells (×10⁴/ml BALF)</td><td align=\"center\">12.3 ± 1.9</td><td align=\"center\">9.4 ± 1.9</td><td align=\"center\">14.0 ± 1.1</td><td align=\"center\">33.1 ± 6.3</td><td align=\"center\">38.0 ± 9.8*</td><td align=\"center\">36.9 ± 3.4*</td></tr><tr><td align=\"left\">Dead cells (×10⁴/ml BALF)</td><td align=\"center\">1.3 ± 0.5</td><td align=\"center\">92.6 ± 11.2*</td><td align=\"center\">106.2 ± 9.7*</td><td align=\"center\">54.1 ± 17.0*</td><td align=\"center\">5.8 ± 0.7</td><td align=\"center\">3.5 ± 0.6</td></tr><tr><td align=\"left\">Protein (g/ml)</td><td align=\"center\">69.4 ± 8.1</td><td align=\"center\">612.8 ± 178.6*</td><td align=\"center\">391.7 ± 102.2*</td><td align=\"center\">251.5 ± 29.5*</td><td align=\"center\">221.0 ± 42.7*</td><td align=\"center\">116.7 ± 6.3</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>Values are means ± S.E. * p &lt; 0.05 versus control</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>β-Galactosidases (Bgals) (EC 3.2.1.23) catalyze the hydrolysis of the glycosidic bonds of terminal non-reducing β-D-galactosyl residues of oligosaccharides and β-D-galactopyranosides. This group of enzymes has a broad distribution, which encompasses plants, animals and microorganisms. The biological functions of these enzymes include degradation of structural polysaccharides in plant cell walls [##REF##10792826##1##,##REF##10852969##2##], thereby acting to control fruit softening during ripening [##REF##12867545##3##], hydrolysis of dietary lactose [##REF##5953213##4##,##REF##14246671##5##] and degradation of glycolipids and proteoglycans in mammals [##UREF##0##6##,##REF##3310533##7##], and metabolism of lactose and other galactosides in microorganisms [##REF##4124306##8##,##REF##14118##9##]. The extensive diversity of Bgals raises the question of what is the basis of their different substrate specificities, which help determine their functions.</p>", "<p>Glycosyl hydrolases (GH) have been grouped and classified as families on the basis of structural similarity [##REF##1747104##10##] and Bgals fall into glycosyl hydrolase (GH) families 1, 2, 35, and 42, which are part of superfamily A (or Clan A). Based on amino acid sequence similarities [##REF##8352747##11##], plant Bgals that have been described belong to GH family 35 (GH35). Like other glycosidase families, GH35 includes multiple genes in various plant species, including <italic>Arabidopsis </italic>[##REF##17466346##12##], tomato [##REF##10889266##13##], papaya [##REF##15694277##14##], apple [##REF##7991682##15##], <italic>Vigna unguiculata </italic>[##UREF##1##16##] and barley [##UREF##2##17##], suggesting that GH35 gene multiplicity is ubiquitous in plants.</p>", "<p>Analysis of plant Bgal activities led to the proposal of two classes, I and II [##REF##15980190##18##]. Class I is made up of enzymes with well characterized exo-β-(1→4)-galactanase activities that can specifically act on pectic β-(1→4)-galactan. Class II has hydrolytic activity on the β-(1→3)- and β-(1→6)-galactosyl linkages of arabinogalactan proteins (AGPs), but lacks activity toward pectic β-(1→4)-galactan [##REF##16666809##19##,##UREF##3##20##], as reported in spinach leaves [##UREF##3##20##] and hypocotyls and young leaves of radish [##REF##15980190##18##]. Class I OsBgals can specifically act on β-(1→4)-galactosyl residues in pectin and xyloglucan. Therefore, they could play an important role in modification of the architecture of the cell wall and intercellular attachment [##REF##10792826##1##,##REF##10852969##2##]. The apparent involvement of Bgals in pectin disassembly during fruit ripening has been reported in various plant species, including tomato [##REF##12867545##3##,##REF##16662771##21##,##REF##7630937##22##], muskmelon [##REF##16653123##23##], kiwifruit [##UREF##4##24##], persimmon [##REF##8058842##25##], sweet cherry [##UREF##5##26##], mango [##REF##7766393##27##], and peach [##REF##8058842##25##]. Bgals expression also accompanies many stages of plant development in other tissues, for example, spinach leaves [##UREF##3##20##], mungbean seedlings [##REF##11393513##29##], radish hypocotyls and young leaves [##REF##15980190##18##], and meristem zones of roots, cotyledons, vascular tissues, trichomes, and pollen of tobacco [##REF##16704113##30##,##REF##15517348##31##]. Bgals that appear to catalyze galactose removal from xyloglucans during their disassembly were observed in cotyledons of nasturtium (<italic>Tropueolum mujus </italic>L.) seed [##REF##3126187##32##], <italic>Copaifera langsdorffii </italic>seed [##UREF##6##33##], and <italic>Hymenaea courbaril </italic>seed, where they were reported to act in cooperation with α-xylosidase, β-glucosidase, and other enzymes to achieve xyloglucan degradation [##REF##10729610##34##,##UREF##7##35##]. Therefore, Bgals appear to play a role in cell wall remodeling in many plant processes.</p>", "<p>In rice, GH35 has not yet been well characterized, and the biological function of rice Bgals remain mysterious. Konno and Tsumuki [##UREF##8##36##] identified both soluble and cell-wall-bound Bgal, of which a 42 kDa soluble Bgal was purified and shown to release galactose from larch wood and rice cell wall arabinogalactans. Likewise, Kaneko and Kobayashi [##REF##12723614##37##] isolated a Bgal with 40 and 47 kDa subunits from the medium of rice suspension cells. Recently, Chantarangsee et al. [##UREF##9##38##] characterized two recombinant Bgal isozymes, including the 90 kDa OsBgal1 and 72 kDa OsBgal2, which had different expression patterns, though both are found throughout plant growth. The complete rice genome sequence allows extensive study of rice GH35, so the gene structures, encoded protein sequences and phylogenetic relationships with other Bgals from rice and other organisms, which may provide clues to their evolution and possible functions, were determined. In addition, the transcript expression patterns of all rice GH35 <italic>Bgals </italic>have been determined and OsBgal13 was expressed in <italic>Escherichia coli </italic>to gain a clue to OsBgal physiological functions.</p>" ]

[ "<title>Methods</title>", "<title>Sequence data and database search</title>", "<p>To find all GH35 genes in rice, tBLASTn searches [##REF##9254694##57##] were performed in the National Center for Biotechnology Information (NCBI) Genbank nr, indica rice genome, and expressed sequence tags (dbEST) databases with <italic>OsBgal1 </italic>(AK102192) as the query. The retrieved <italic>OsBgal </italic>genes were used to identify their Unigene cluster, Gene Locus, conserved domains, gene position on the 12 <italic>Oryza sativa </italic>(rice) chromosomes, homolog genes, EST-based expression profiles and GEO profile in the NCBI databases (please see Availability &amp; requirements for more information). The number and positions of exons and introns for each individual gene were determined by manually comparing the cDNA and predicted cDNA sequences with their corresponding genomic DNA sequences. Homologous proteins from other organisms were retrieved by links from the CAZY website (please see Availability &amp; requirements for more information) or by BLASTp searches at NCBI. Putative signal peptides were predicted with the SignalP program (please see Availability &amp; requirements for more information) [##REF##15223320##58##]; and putative N-glycosylation sites were identified with the NetNGlyc [##UREF##13##59##]; (please see Availability &amp; requirements for more information) and manually inspected to remove NPS/T sites.</p>", "<p>Chromosomal locations of genes were identified on and the map drawn with the NCBI map viewer (please see Availability &amp; requirements for more information). Segmental duplication analysis was done with DAGchainer [##REF##15247098##60##] and the TIGR rice segmental duplication database (please see Availability &amp; requirements for more information) with the maximum length distance permitted between collinear gene pairs set to be 500 kb. LTR-retrotransposon elements, interspersed repeats and low complexity DNA sequences were identified with RepeatMasker (please see Availability &amp; requirements for more information).</p>", "<title>Construction of phylogenetic trees</title>", "<p>The multiple sequence alignment of β-galactosidase protein sequences from rice and other organisms was made with ClustalW [##REF##7984417##61##] and manually adjusted and edited to remove unconserved N- and C-terminal regions with Genedoc (please see Availability &amp; requirements for more information) [##UREF##14##62##]. Phylogenetic trees were constructed by the neighbor-joining facility in ClustalX 1.83 [##REF##9810230##63##] and the branching order verified for maximum parsimony with the Protpars program in the PHYLIP software suite [##UREF##15##64##].</p>", "<title>Plant material</title>", "<p>Indica rice (<italic>Oryza sativa </italic>L., cv. KDML 105) seeds were soaked under sterile conditions on tissue paper moistened with sterile distilled water at 28°C until germination. Germinated rice was moved to soil-filled plastic pots and grown until 15 days, then moved to large clay pots. Samples were collected every 3 days from seeding until 15 days after seeding, then sampling was continued every month. To attain more materials at the flowering stage, samples were collected from KDML105 fields at the Pathum thani Rice Research Center in November, 2005. The samples were immediately frozen in liquid nitrogen and stored at -80°C until use.</p>", "<title>Semi-quantitative reverse transcription-polymerase chain reaction (qRT-PCR)</title>", "<p>Total RNA was isolated from various tissues: germinated seed, root, shoot, leaf blade, leaf sheath, node, internode, initiating panicle, developing panicle, emerging panicle, anther, flower, milk grain, and grain during dry down by the following procedure. Tissues (100 mg) were ground to powder under liquid nitrogen, then RNA was extracted using RNeasy Plant Mini (QIAGEN GmbH, Hilden, Germany) or Sigma Spectrum RNA extraction (Sigma-Aldrich, St Louis, USA) kits. For starchy tissues: germinated seed and grain during dry down, RNA extractions were first done with Trizol reagent (Invitrogen, Carlsbad, CA, USA [##REF##2440339##65##]), and the RNA further purified from the extracts by the spin column procedure of the above RNA extraction kits. The RNA was quantified in a spectrophotometer at 260 nm.</p>", "<p>For RT-PCR, approximately 5 μg of total RNA was treated with RQ1 RNase-free DNase I (Promega Corporation, Madison, WI, USA). First stand cDNA was synthesized from RNA template primed with Oligo (dT)₂₀with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). The gene-specific primers used for semi-quantitative PCR were designed from the 3'UTR or 3' coding sequence of each β-galactosidase gene (Table ##TAB##4##5##). The constitutively expressed <italic>β-Actin </italic>and <italic>Ubiquitin-6 </italic>genes were used for normalization. The PCR (10 μl total volume) was done with a 10 ng or 100 ng aliquot of the first stand cDNA as template and 0.5 units of <italic>Taq </italic>DNA polymerase (Promega). The relative expression abundances were obtained by dividing the total densimetric intensities measured on a Fluor-S gel documentation system with Quantity 1 software (Bio-RAD, Hercules, CA, USA) for each gene by those for the control gene. All samples were assayed 3 times in separate reactions to give means and standard deviations for the relative abundances.</p>", "<title>Amplification of cDNA clones and sequencing</title>", "<p>The first strand cDNA reverse-transcribed from RNA of developing and emerging panicle was used as a template to amplify <italic>OsBgal6</italic>, <italic>OsBgal11</italic>, and <italic>OsBgal15 </italic>cDNA. The set of primers for amplification of partial or full-length cDNA of each gene was designed from its genomic sequence. The PCR was catalyzed by <italic>Pfu </italic>polymerase (Promega) with heating at 94°C for 5 min, followed by 30 cycles of 1 min at 94°C, 30 s at the appropriate annealing temperature, (58°C for most primers) and 5 min at 72°C, and a final extension for 7 min at 72°C. The final product was gel purified with the QIAquick Gel Extraction Kit (QIAGEN), then sequenced by automated sequencing at Macrogen Inc. (Seoul, Korea).</p>", "<p>Two pairs of primers were used to amplify <italic>OsBgal6 </italic>cDNA: (1), bgal6_startF (TCAGTCAGTAGTCAGACATG) and bgal6_QIENEY_R (AATGCAGGCTCAATCATCAG); and (2), bgal6_QIENEY_F (GATGATTGAGCCTGCATTTG) and bgal6_stopR (AGTTTCCTGTGTTGCATCAC). The full-length sequence of <italic>OsBgal6 </italic>was determined with the above primer sets and additional primers, including bgal6_1163r (GGTGTGTTATGCTGATCGAAG), bgal6_1650f (GGATTCTGGCGCCTACATG), bgal6_1143F (CTTCGATCAGCATAACACACC), and bgal6_1671r (CCATGTAGGCG CCAGAATC).</p>", "<p>The oligonucleotide primers used for amplification of a partial <italic>OsBgal11 </italic>cDNA were: bgal11_Seq_r1 (TCATCGCATGTGCAGTG) and bgal11_STOP_R (CCTTCTTCCTAAGCCGCCTG).</p>", "<p>The <italic>Osbgal15 </italic>cDNA was amplified with 2 pairs of primers. First, the bgal15_START_F (CGCGTGCCGGCGATGAAG) and bgal15_QIENE_Rev (CTCGTTTTCAATCTGTGCCAG) primers were used to amplify a 5' cDNA fragment, and the bgal15_QIENEY_F (CTGGCACAGATTGAAAACGAG) and bgal15_STOP_R (TATCAACATGAAGCCTGAACGGTG) primers were used to amplify an overlapping 3' cDNA fragment. The products of these first two PCR were mixed and a sequential PCR was performed with the bgal15_START_F and bgal15_STOP_R primers. The full-length CDS cDNA was cloned into the <italic>EcoR</italic>Isite of pBluescript KS(+) by standard methods [##UREF##16##66##]. The recombinant plasmid was sequenced with the T7, M13, bgal15QIENE_F, bgal15QIENE_REV, bgal15_1138_F (ACTCATCTTCTGCCTGCTTG), and Bgal15_1797_R (TACGGTGCCATTGTTG TTG) primers.</p>", "<title>Expression of OsBgal13</title>", "<p>A cDNA encoding the predicted mature OsBgal13 protein was amplified from the full-length cDNA clone of Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"AK065546\">AK065546</ext-link>[##REF##12869764##67##], obtained from the Rice Genome Resource Center, Tsukuba, Japan. The plasmid clone was used as template for amplification with the bgal13matN (CACCGCCGCCGCGTATGC) and bgal13stopR (GATCACATCTCACCGCGAGGCTC) primers and <italic>Pfu </italic>polymerase (Promega), according the supplier's instructions. The PCR product was gel-purified and cloned into the pENTR/D-TOPO plasmid (Invitrogen), according to the supplier's instructions, and sequenced. The cDNA was transferred into the pET32/DEST vector [##REF##17196101##68##] by an LR clonase reaction (Invitrogen). The thioredoxin fusion protein was expressed from this plasmid and expressed as described for OsBgal1 by Chantarangsee et al. [##UREF##9##38##]. The protein was extracted and purified on Ni-NTA IMAC resin (QIAGEN), as with OsBgal1 [##UREF##9##38##], except that 0.1 mg/mL soybean trypsin inhibitor was added to the extraction buffer, and the protein was eluted from the IMAC column with 5–20 mM imidazole. The OsBgal13 protein was further purified by gel-filtration on a Superdex S200 column. The purified OsBgal13 was tested for glycone specificity by incubation of enzyme with 1 mM <italic>p</italic>-nitrophenyl (<italic>p</italic>NP) glycosides for 30 min at 30°C, followed by stopping the reaction with 2 volumes 0.4 M Na₂CO₃and measuring the 405 nm absorbance on a microtiter plate. Action on galactose-containing oligosaccharides and polysaccharides was determined by incubating with 1 mM oligosaccharide or 0.5% polysaccharide at 30°C 24 h, then separating the products on TLC and detecting carbohydrates, as previously described [##UREF##9##38##].</p>" ]

[ "<title>Results and Discussion</title>", "<title>Identification of rice GH35 genes and their protein products</title>", "<p>A total of 15 GH35 genes (defined <italic>OsBgal</italic>) were identified in rice genome databases (Table ##TAB##0##1##), each of which encodes a protein that contains the GH35 β-galactosidase active site consensus sequence G-G-P- [LIVM]-x-Q-x-E-N-E- [FY] [##REF##9649738##39##]. Most of the encoded proteins contain a GH35 domain at the N-terminus and a Gal-lectin-like domain at the C-terminus. Although the speculated carbohydrate-binding function of these Gal-lectin domains is not yet proven, their existence and conserved nature has led to the suggestion that they may increase the catalytic efficiency on polysaccharide substrates [##REF##17466346##12##]. The proteins from <italic>OsBgal</italic>2, <italic>OsBgal</italic>7 and <italic>OsBgal9 </italic>lack the Gal-lectin-like domain, suggesting that it is not necessary for the functions of these rice GH35 members.</p>", "<p>The deduced amino acid sequences of the OsBgal proteins were used to predict their putative signal peptides, protein lengths, molecular masses, pI values, possible N-glycosylation sites, and cellular destinations (Table ##TAB##1##2##). All OsBgals contain putative signal peptides, which range in length from 20 to 36 amino acids, except for that of OsBgal8 which is predicted to have 62 amino acids. The mature OsBgal proteins were predicted to contain from 653 (OsBgal9) to 894 (OsBgal8) amino acids, corresponding to molecular masses of 73.5 to 97.9 kDa. Most of the proteins were predicted to have pI in the acidic range (5.56–6.76), except those of OsBgal2, OsBgal7 and OsBgal10 are in the basic range (7.65–9.1) (Table ##TAB##1##2##). Only OsBgal2 and OsBgal7 lacked putative N-glycosylation sites, while the other enzymes were predicted to contain from 2 to 10 sites. Most OsBgal members are predicted to localize to the organelles of the secretory pathway, for instance, the Golgi apparatus, endoplasmic reticulum, or vacuole, or to be secreted. Only 3 OsBgals, including OsBgal9, OsBgal10 and OsBgal11, were predicted to be localized in the lysosome-like vacuole, which correlates to the location of mammalian β-galactosidases in the lysosome (Table ##TAB##1##2##). Although OsBgal10 and OsBgal11 are very similar in predicted mature protein length and possible cellular destinations, their predicted pI values are quite different. OsBgal5, OsBgal12, OsBgal14 and OsBgal15 all have high numbers of glycosylation sites, similar MW, pI and predicted possible destinations, which suggests that they may have redundant or overlapping functions.</p>", "<title>Phylogenetic analysis</title>", "<p>The multiple alignment of full-length protein sequences was used to construct an unrooted phylogenetic tree. The phylogenetic tree, which includes all the GH35 genes identified in the rice (monocot), <italic>A. thaliana </italic>(dicot) and <italic>Physcomitrella patens </italic>(a bryophyte moss) genomes, as well as representatives of animals, fungi, protists, archaea, and eubacteria, has three major branches, one of which is nearly plant specific (except for one <italic>Dictyostelium discoideum </italic>gene product), one of which contains representatives of animals, eubacteria and plants, and one of which contains archaeal, eubacterial and fungal enzymes (Figure ##FIG##0##1##). Almost all the rice, <italic>A. thaliana</italic>, and <italic>P. patens </italic>enzymes fall within the plant-specific cluster, except <italic>OsBgal9</italic>, <italic>AtBGAL</italic>17 and <italic>P. patens </italic>EDQ62875, which fall in the cluster with animal β-galactosidases. Previously, it was suggested that the plant enzymes that cluster with animals might have been transferred to plants by horizontal gene transfer [##REF##17466346##12##], but the fact that the bryophyte, which is thought to have diverged from the vascular plants early in plant evolution, has this type of β-galactosidase and the broad distribution of organisms with this type of β-galactosidase suggest that plants maintained a copy of this gene when plants and animals diverged. Perhaps the more relevant question is how the plant specific Bgal ancestor came to be, and how the C-terminal Gal-lectin-like domain was acquired by this lineage. It appears that the fungal-type and plant-type lineages may have diverged from the animal-type β-galactosidases early in GH35 evolution, but plants retained the animal-type β-galactosidase, as well.</p>", "<p>Within the plant-type β-galactosidases, OsBgals can be divided into three distinct groups (a, b and c in Figure ##FIG##0##1##). The major group (group a) contains 5 clusters with total 7 OsBgal members. The cluster a1 contains 4 rice genes, including OsBgal1, OsBgal2, OsBgal3, and OsBgal7, along with 6 AtBGALs, the <italic>Asparagus officinalis </italic>Bgal, and 4 tomato and 5 chickpea Bgals that are not shown, some of which contain C-terminal galactose-binding lectin-like domains and some of which do not. Another cluster, a2, contains OsBgal13, AtBGAL9 and two putative <italic>P. patens </italic>Bgals (EDQ59880 and EDQ81397), which suggests this cluster is of ancient origin. Two other putative <italic>P. patens </italic>Bgals fall in cluster a3, which appears to be closely related to cluster a2. OsBgal8 groups with AtBGAL8 and RsBgal1 of radish, which specifically hydrolyzes the β-(1→3)- and β-(1→6)-linked oligogalactans of AGPs [##REF##15980190##18##], in subsgroup a4. OsBgal4 groups with AtBGAL10 in cluster a5. The second biggest group, b, contains 4 rice Bgals with high amino acid sequence similarity, namely OsBgal5, OsBgal12, OsBgal14, and OsBgal15. These isozymes have nearly the same protein lengths and gene structures, as described below. Within group c, OsBgal10 and OsBgal11 (cluster c1) are closely related, while OsBgal6 (cluster c2) is less clearly associated, though both distance-based and maximum parsimony trees back this model.</p>", "<title>Chromosomal locations of OsBgals</title>", "<p>As shown in the map of their chromosomal locations and directions of transcription in Figure ##FIG##1##2##, the <italic>OsBgals </italic>are distributed over all chromosomes, except chromosomes 4, 7 and 11. Three <italic>OsBgals </italic>are located on chromosome 1, while 2 <italic>OsBgal </italic>each are found on chromosomes 3, 5, 6, and 10, and one <italic>OsBgal </italic>each is present on chromosomes 2, 8, 9, and 12. Similar to other gene families, <italic>OsBgals </italic>appear to have undergone gene duplication, as the rice genome has undergone genome-wide duplication events, including polyploidy, which promote the amplification of gene family members. Segmental duplication analysis identified four <italic>OsBgals </italic>located on the duplicated segmental regions in rice chromosomes. <italic>OsBgal7 </italic>(Rice genome project locus Os02g0219200) and <italic>OsBgal2 </italic>(Os06g0573600) are located on a chromosomal segment duplicated between chromosomes 2 and 6, while <italic>OsBgal10 </italic>(Os08g0549200) and <italic>OsBgal11 </italic>(Os09g0539200) are located on segments duplicated between chromosomes 8 and 9. These duplications are supported by the close phylogenetic relationships of these genes. Therefore, chromosomal segment duplication appears to have played a role in multiplication of <italic>OsBgals </italic>within the rice genome. In contrast, <italic>OsBgal12 </italic>and <italic>OsBgal14</italic>, which have high sequence similarity, are located close together on the same chromosome. Thus, these two genes may have diverged by tandem duplication.</p>", "<title>Exon-intron organization and primary gene structure analysis</title>", "<p>Automated annotations for all <italic>OsBgal </italic>genes were available in the public databases, except for <italic>OsBgal6</italic>, <italic>OsBgal14 </italic>and <italic>OsBgal15</italic>. However, we confirmed and corrected exon-intron organization manually by comparing the corresponding full length cDNA and EST sequences, which were either already available in publicly accessible databases or were obtained during this study, with genomic sequences.</p>", "<p>Gene structure comparisons showed eight different splicing patterns. The sizes of the majority of the coding exons are conserved, but some appear to have had intron-loss events (Figure ##FIG##2##3##). The pattern with the highest number of exons, found in <italic>OsBgal1, OsBgal3, OsBgal4</italic>, <italic>OsBgal6 and OsBgal13</italic>, contains 19 exons with 18 introns, with exons 1 through 10 encoding the GH35 domain, and exons 18 and 19 encoding the Gal-lectin-like domain. Ahn et al [##REF##17466346##12##] observed this same pattern in <italic>A. thaliana Bgals </italic>and surmised from parsimony that it is likely to be the ancestral gene pattern and other patterns were derived from it, mainly by intron loss. The presence of this pattern in diverse rice genes and in a <italic>P. patens </italic>gene (EDQ59880) supports this conclusion. The second most similar pattern is 18 exons separated by 17 introns with the loss of intron 1, which occurred in <italic>OsBgal11</italic>. A pattern of two-intron loss was found in <italic>OsBgal5, OsBgal12</italic>, <italic>OsBgal14</italic>, and <italic>OsBgal15</italic>, which contain 17 exons with the loss of introns 17 and 18.</p>", "<p>The <italic>OsBgal2</italic>, <italic>OsBgal7 </italic>and <italic>OsBgal9 </italic>genes all lack the galactose-binding, lectin-like domain at the C-terminus, however, their splicing patterns were different. <italic>OsBgal2 </italic>and <italic>OsBgal7 </italic>have patterns similar to other rice GH35 genes and retain the 5' end of exon 18, but <italic>OsBgal2 </italic>contains 16 exons with the loss of introns 11 and 16, while intron 15 has been lost in <italic>OsBgal7</italic>, leaving 17 exons (Figure ##FIG##2##3##). In contrast, <italic>OsBgal9 </italic>appeared to be distantly diverged from other rice GH35 members since its exon/intron organization does not correspond to the other <italic>OsBgals</italic>, except that an intron is found in the same position as intron 1 in the other genes.</p>", "<p>Although the pattern and lengths of exons in rice GH35 genes are similar, <italic>OsBgal13 </italic>spans approximately 20.8 kb, because it contains over three-fold longer introns than the other genes in the family. Long terminal repeat (LTR)-retrotransposons elements and other repeated sequences were found within <italic>OsBgal13 </italic>gene introns 1, 2, 8, 9, 10, 13, 14 and 16, which contain one GC rice region, three AT rich regions and eight transposable elements (Table ##TAB##2##3##). Likewise, <italic>OsBgal6</italic>, the second largest <italic>OsBgal </italic>gene due to its long (4 kb) intron 1, had one AT rich region and seven transposable elements, all of which were located within intron 1. In <italic>OsBgal </italic>genes of normal length, such as <italic>OsBgal1</italic>, no transposable elements were observed. These results may reflect the observation that rice genome expansion is forced by transposable element amplification [##REF##16819716##40##].</p>", "<p>Comparison of gene splice patterns and phylogenetic relationships between plant-type Bgals from rice, <italic>A. thaliana</italic>, and <italic>P. patens </italic>reveals that, although the ancestral gene pattern is the same, no other splice patterns are shared within a phylogenetic lineage. Therefore, though most introns appear to have been inserted in the common ancestor gene for this lineage (an extra exon was inserted in <italic>P. patens </italic>gene EDQ878340), intron losses appear to have occurred independently in rice, <italic>Arabidopsis </italic>and <italic>P. patens</italic>. The same may be true for the C-terminal domain, which appears to have been lost only once in rice (in the putative ancestor of <italic>OsBgal2 </italic>and <italic>OsBgal7</italic>), though it appears to have been lost at least 4 times in <italic>Arabidopsis </italic>GH35 <italic>Bgals </italic>[##REF##17466346##12##].</p>", "<title>Analysis of expression by RT-PCR</title>", "<p><italic>OsBgal </italic>transcript expression in various tissues and stages of growth was analyzed by RT-PCR with primers specific to the 3'UTR of each gene to gain further insight into possible functions. The relative expression levels derived from normalization with the <italic>UBQ6 </italic>polyubiquitin gene gave the same patterns as those shown for normalization with the <italic>β-Actin </italic>gene in Figure ##FIG##3##4## and Additional file ##SUPPL##0##1##, indicating the patterns likely reflect the expression of the <italic>OsBgal </italic>genes, rather than that of the control.</p>", "<p>The rice tissues/organs and developmental stages chosen for RT-PCR analysis are shown in Figure ##FIG##3##4##. All 15 <italic>OsBgal </italic>genes were found to be expressed with different but overlapping expression patterns, confirming that all genes are active. These relative expression levels were used to define genes as having expression specific to a given tissue, if the relative transcript levels of the gene at that stage are significantly higher (color scale above 7, where 10 is the level in the tissue with the highest expression of each gene) over the levels at all other stages. The expression profiles revealed that at least 9 genes were highly expressed in one of the stages of vegetative tissues and 10 were highly expressed in reproductive tissues. Only <italic>OsBgal13 </italic>appears to be expressed in nearly every organ and developmental stage, though at different levels (Figure ##FIG##3##4##).</p>", "<p>Nine <italic>OsBgals </italic>(<italic>OsBgal1, OsBgal2, OsBgal3, OsBgal4, OsBgal6, OsBgal7, OsBgal8 and OsBgal9</italic>) displayed their highest transcript levels in young and early-mature stages of shoot, root, and leaf sheath. Among these genes, <italic>OsBgal6, OsBgal7, OsBgal8 and OsBgal9 </italic>showed highest intensity in leaf sheath at different times, especially, 15-day and 1-month old, which are both fast cell elongation stages. The high accumulation of OsBgals in this stage may act in sugar remodeling, since the carbohydrate remobilized from the leaf sheath and culm to grain can contribute as much as 38% to rice yield [##UREF##10##41##]. In contrast, low levels of most gene transcripts were detected in leaf blade, except for <italic>OsBgal13 </italic>transcripts, which were detected throughout the growth of the blade.</p>", "<p>Six genes, <italic>OsBgal5, OsBgal10, OsBgal11, OsBgal12, OsBgal14</italic>, and <italic>OsBgal15 </italic>appear to be expressed primarily in reproductive tissues. The relative transcript levels for these genes were at least five times higher in reproductive tissues than in vegetative tissues (Figure ##FIG##3##4##). In addition, no expression was detected at the initiating panicle stage, but it was high in later panicle development and flowering stages. Only <italic>OsBgal10, OsBgal11 </italic>and <italic>OsBgal15 </italic>transcripts were found to accumulate in anther. These genes appear to have diverged from two ancestor genes, since <italic>OsBgal5</italic>, <italic>OsBgal12</italic>, <italic>OsBgal14</italic>, and <italic>OsBgal15 </italic>have sequences and gene structures that are similar to each other, as do <italic>OsBgal10 </italic>and <italic>OsBgal11 </italic>(Figures ##FIG##0##1## and ##FIG##2##3##). Interestingly, <italic>AtBGAL7 </italic>and <italic>AtBGAL15</italic>, which fall into the group b with <italic>OsBgal5</italic>, <italic>OsBgal12</italic>, <italic>OsBgal14</italic>, and <italic>OsBgal15 </italic>(Figure ##FIG##0##1##), were reported to function in the early stages of microspore and pollen development [##REF##15517348##31##]. Likewise, <italic>AtBGAL11 </italic>and <italic>AtBGAL13</italic>, which fall in cluster c1 with <italic>OsBgal10 </italic>and <italic>OsBgal11 </italic>(Figures ##FIG##0##1##), show maximum expression in mature pollen [##REF##15517348##31##]. So, related rice and <italic>Arabidopsis </italic>Bgals function in reproductive tissues, suggesting their roles may be somewhat conserved in the two plants.</p>", "<p>Only the <italic>OsBgal4 </italic>transcript was expressed abundantly in seed at the imbibition stage, while <italic>OsBgal2 </italic>and <italic>OsBgal13 </italic>were also present at low levels. Five <italic>OsBgals </italic>were expressed in milk grain and six in grain during dry down. A high abundance of <italic>OsBgal2 </italic>and <italic>OsBgal13 </italic>transcripts was observed in grain during dry down. This evidence implies that they may function in grain development and senescence or are stored for roles in germination, such as cell wall remodeling. <italic>OsBgal1</italic>, <italic>OsBgal2, OsBgal6, OsBgal8</italic>, and <italic>OsBgal13 </italic>transcripts were also found in shoots and roots of seedlings after germination. Chantarangsee et al. [##UREF##9##38##] reported β-galactosidase activity in rice seeds, roots and shoots at 0–7 days after seed-soaking, and detected OsBgal1 and OsBgal2 proteins in embryos and seedling roots and shoots. In barley (<italic>Hordeum vulgare</italic>), Giannakouros and colleagues [##UREF##2##17##] were able to separate 4 isozymes of β-galactosidase from germinated grain. Thus, Bgals are present and may play roles in the growth and emergence of root and shoot primordia from the hull.</p>", "<p>To complement the RT-PCR expression profiles of the <italic>OsBals</italic>, ESTs retrieved from the UniGene database (UniGene December, 2007, NCBI) were compared with our findings. Full-length or partial cDNA and ESTs for most <italic>OsBgals </italic>are available in the database, except <italic>OsBgal6</italic>, for which only one EST from unspecified tissues is available, and <italic>OsBgal15</italic>, for which no corresponding ESTs or cDNA are available (Table ##TAB##0##1##). However, the other <italic>OsBgal </italic>genes had cDNA and ESTs present in the database, with numbers of ESTs that varied from 1 to 253.</p>", "<p>The <italic>OsBgal8 </italic>(Unigene cluster Os.22360, locus <italic>Os03g0255100</italic>) has the highest number of ESTs in the database: 253 ESTs from various tissues, including flower, stem, leaf, panicle, root, callus, seed, whole plant and unspecified tissues. The Unigene profiles indicate relatively abundant accumulation in flower, leaf, panicle, root, seed and stem with 761, 133, 120, 190, 216 and 228 transcripts per million (TPM), respectively (Table ##TAB##0##1##). The Unigene profile is somewhat similar to the RT-PCR pattern of <italic>OsBgal8</italic>, in which the transcripts accumulated significantly throughout rice plant, including in seed during dry-down, but not in seedling and milk grain.</p>", "<p><italic>OsBgal10 </italic>(Os.18310), <italic>OsBgal11</italic>, <italic>OsBgal12 </italic>(Os.46702) and <italic>OsBgal14 </italic>(Os.22528) expression data included mainly flower and panicle clones, or had relatively high expression in these tissues, which correlates to our RT-PCR analysis. For instance,<italic>OsBgal11 </italic>shows 249 TPM in flower and 158 TPM in panicle, while <italic>OsBgal14 </italic>shows 358 TPM in flower (Table ##TAB##0##1##).</p>", "<p>The EST and cDNA clones for the remaining <italic>OsBgals </italic>had diverse origins, which is consistent with the evaluation of <italic>OsBgals </italic>by RT-PCR, though the frequencies of expression derived from these two forms of expression data were not exactly the same. Therefore, Unigene database analysis could be used for an initial guideline of gene expression, though the expression patterns based on ESTs are fragmentary, since the tissues are not equally or completely represented and the source tissue annotation of many EST libraries is unclear or incomplete. Microarray experiments, such as that by Ma et al. [##REF##16140994##42##], who assessed expression of 37,132 non cross-hybridizing gene models in rice seedling shoots, tillering-stage shoots and roots, heading and filling stages of panicles and suspension cells, offer a more comprehensive picture of gene expression within a tissue. Although the tissue coverage in public microarray databases is as yet limited, the GEO expression data in Table ##TAB##0##1## shows that OsBgal expression levels may be affected by many treatments.</p>", "<title>Rice β-galactosidase cDNAs sequencing and identification of alternative splicing</title>", "<p>Although the expression of all <italic>OsBgals </italic>can be observed by RT-PCR and most have corresponding cDNAs and ESTs in the database, complete full-length cDNA clones were not available for <italic>OsBgal6</italic>, <italic>OsBgal11 </italic>and <italic>OsBgal15</italic>. Therefore, to confirm the mRNA sequence and gene structure, these genes were amplified by PCR and sequenced. The accession numbers for these sequences are listed in Table ##TAB##0##1##.</p>", "<p>The full-length cDNA of <italic>OsBgal</italic>6 was amplified by a set of primers corresponding to the transcribed locus <ext-link ext-link-type=\"gen\" xlink:href=\"XM_475258\">XM_475258</ext-link>. The cDNA was isolated from 15-day leaf sheaths, a tissue with abundant expression, and exerting panicles, a tissue with poor expression, based on RT-PCR expression analysis. Sequencing of the cDNA from the highly expressing tissue revealed an open reading frame (ORF) of 2,436 nucleotides encoding a protein of 812 amino acids (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU602310\">EU602310</ext-link>), which confirmed the our predicted β-<italic>galactosidase </italic>sequence from chromosome 5 (Genbank accession no. <ext-link ext-link-type=\"gen\" xlink:href=\"AC135419\">AC135419</ext-link> and <ext-link ext-link-type=\"gen\" xlink:href=\"AC135429\">AC135429</ext-link>). A search through the dbEST database of <italic>O. sativa </italic>found 99% identity with a 520 bp EST from fertile panicle (<ext-link ext-link-type=\"gen\" xlink:href=\"CK048087\">CK048087</ext-link>). On the contrary, the Os <italic>Bgal</italic>6 cDNA amplified from exerting panicle, where expression is low, contains only 2,338 bp (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU603285\">EU603285</ext-link>) which is 101 bp shorter than the cDNA from high expression tissues. The open reading frame beginning at the initiation codon of this transcript covers only exons 1 to 7 and encodes only 244 amino acids. Alternative splicing of this transcript occurred to utilize an alternative acceptor site (CAG) 80 nt downstream of functional acceptor site for intron 7, which introduced a deletion of 27 amino acid and a frameshift that leads to an in-frame stop codon (Figure ##FIG##4##5##). A second missplicing of this gene occurred at the intron 10/exon 11 junction, which again used an alternate acceptor site (CAG) located 20 nt downstream of the regular acceptor site. So, alternative splicing is likely to be one of the gene regulation mechanisms, since the transcript would be subject to nonsense mediated decay (nmd) [##REF##17194304##43##] and the protein produced would be useless as a β-galactosidase.</p>", "<p><italic>OsBgal11 </italic>(locus Os09g0539200) is annotated as a pseudogene in GenBank, because of its apparent inability to produce full-length protein due to a frameshift and subsequent stop codon in the AK119414 cDNA. We hypothesized that such phenomenon probably resulted from missplicing, as seen in <italic>OsBgal6</italic>. A 1343-nucleotide fragment from the 3' half of the <italic>OsBgal11 </italic>cDNA was amplified from exerting panicle (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU603286\">EU603286</ext-link>), in which its expression is high based on RT-PCR. This cDNA shares 98% identity with the AK119414 cDNA, but had a substitution of 75 nucleotides in place of 95 nucleotides at positions 1282 to 1377 of AK119414. These substituted sequences allow the full-length translation of the predicted 839 amino acids of OsBgal11, whereas only 426 amino acids are translated from AK119414. Comparison with the genomic sequence and our amplified cDNA sequence indicates that the AK119414 transcript results from retention of intron 10, which extends exon 10 by 101 nt, thereby introducing an in-frame stop codon. In addition, an alternative donor site located at the functional acceptor site of intron 10 and the first nucleotide of the functional exon 11 produces an alternate intron 10 with the acceptor site located 79 nt downstream, within the functional exon 11, thereby reducing the size of exon 11. The shortened (456-residue) protein, includes all of the catalytic domain, based on the <italic>Penicillium </italic>sp. β-galactosidase structure [##REF##15491613##44##], so it is possible that it could still function as a Bgal.</p>", "<p>No experimentally determined cDNA or ESTs corresponding to <italic>OsBgal15 </italic>were available in public databases, despite the fact that our RT-PCR demonstrated its expression. Therefore, the full-length <italic>OsBgal15 </italic>cDNA was amplified, cloned and sequenced. The <italic>OsBgal15 </italic>cDNA sequence (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU051629\">EU051629</ext-link>) contains a 2487-nucleotide ORF, which encodes 829 amino acids. The encoded protein shares over 99% identity with a predicted β-galactosidase of <italic>Oryza sativa </italic>(japonica cultivar-group) (accession no. <ext-link ext-link-type=\"gen\" xlink:href=\"AP004733\">AP004733</ext-link>), except that 7 extra amino acid residues were inserted at residue 293, compared to the predicted sequence from <ext-link ext-link-type=\"gen\" xlink:href=\"AP004733\">AP004733</ext-link>. Therefore, the correct splice donor site of exon 8 is located 21 nucleotides downstream of that predicted for the mRNA of <ext-link ext-link-type=\"gen\" xlink:href=\"AP004733\">AP004733</ext-link>.</p>", "<p>The alternative splicing observed in this study introduces in-frame stop codons into the mRNA of <italic>OsBgal6 </italic>in the low expression tissue, but not for the high expression tissues, while it can also introduce a premature stop codon in <italic>OsBgal11</italic>, based on the AK119414 cDNA sequence. In vertebrates, there are a number of well-known alternatively splicing genes, for instance the fibronectin gene, which can generate over 20 different proteins, some of which have different patterns of localization and slightly different functions in human cells [##REF##7226224##45##]. Until relatively recently, there were very few examples of alternatively spliced genes in higher plants and still fewer examples known to generate functionally distinct proteins. Generally, alternative splicing yields two polypeptides of different size that are identical, except for the presence of a number of additional amino acids at the C-terminus of the larger isoform, eg., ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase [##REF##10430961##46##]; diacylglycerol kinase in tomato [##REF##11069705##47##]; and chloroplast ascorbate peroxidase in spinach [##REF##9931296##48##]. Missplicing events were observed in maize (<italic>Zea mays</italic>) <italic>Viviparous 1 </italic>(<italic>Vp1</italic>) transcripts, the majority of which are spliced incorrectly and contain insertions of intron sequences or deletions of coding region, so that they do not encode full-length proteins [##REF##12119408##49##]. Although alternative splicing is a widespread process used in higher eukaryotes to regulate gene expression and functional diversification of proteins [##REF##11173120##50##,##REF##12600935##51##], the putative control of tissue-specific expression by missplicing we observed has not been well characterized in rice.</p>", "<title>Preliminary Characterization of OsBgal13</title>", "<p>OsBgal13 is in cluster a2 of group a of the phyloenetic tree in Figure ##FIG##0##1##, along with AtBGAL9 and two <italic>P. patens </italic>Bgals. This and its general expression pattern suggest it may have a conserved maintenance function in plants. To assess what its function might be, OsBgal13 was expressed as a thioredoxin fusion protein in <italic>Escherichia coli </italic>strain OrigamiB (DE3), which we previously found to have little background β-galactosidase acitivity [##UREF##9##38##]. The protein was purified by immobilized metal affinity chromatography on nickel resin, and most of the OsBgal13 activity eluted in 5–20 mM imidazole. Further purification was achieved by gel filtration chromatography and the activity of the resulting protein was assessed. As shown in Table ##TAB##3##4##, among <italic>p</italic>-nitrophenol (<italic>p</italic>NP) glycosides, the OsBgal13 enzyme had highest activity toward <italic>p</italic>NP-β-D-galactoside (<italic>p</italic>NPGal), but also had 30% of this activity toward <italic>p</italic>NP-α-L-arabinoside (<italic>p</italic>NPAra), and low activity toward <italic>p</italic>NP-β-D-fucoside and <italic>p</italic>NP-β-D-mannoside, but no detectable activity toward <italic>p</italic>NP-β-D-glucoside and <italic>p</italic>NP-β-D-xyloside and negligible activity toward <italic>p</italic>NP-α-D-galactoside. OsBgal13 also hydrolyzed β-(1→3)-, β-(1→4)- and β-(1→6)-linked galactobiose and galactotriose, but no release of galactose or arabinose could be detected when the protein was incubated for 24 h with rice seedling alcohol insoluble residue (AIR), larchwood arabinogalactan, galactan, apple pectin, citrus pectin or oat xylan. So, despite the intriguing observation that OsBgal13 can hydrolyze both β-D-galactosyl residues and α-L-arabinosyl residues, components of type I and II arabinogalactans and xyloglucan and glucuronoarabinoxylan side chains [##REF##15012297##52##], only short galacto-oligosaccharides can be identified as possible natural substrates to date.</p>", "<title>Possible Functions of Other Isozymes</title>", "<p>The activity of mammal β-galactosidase specifically releases terminal β-galactosyl residues from glycosaminoglycans and the glycolipid GM1 ganglioside. Similarly, the digestion of the galactolipid monogalactosyldiacylglyerol by vacuolar and chloroplast β-galactosidases was reported in wheat [##REF##16663831##53##]. Likewise, β-galacosidase of tomato was reported to act in galactolipid turnover and degradation, which occurs in chloroplasts and chromoplasts during tomato fruit development [##UREF##11##54##,##UREF##12##55##]. However, relatively little is known about metabolism of other glycolipids, glycopoteins and proteoglycans involving plant β-galactosidases, though it is well characterized in mammals. The fact that OsBgal9 is similar to animal β-galactosidases and is predicted to localize to a lysosome-like vacuole, suggests it may play a similar role in rice.</p>", "<p>Among the plant-like OsBgals, OsBgal10 and OsBgal11 are also predicted to localize to lysosome-like vacuoles, so they could also play a role similar to animal-type β-galactosidases. However, these closely related isozymes have reproductive tissue specific expression, as are the isozymes of cluster b, OsBgal5, OsBgal12, OsBgal14 and OsBgal15. Similarly, AtBGAL11 and AtBGAL13, which clustered in group c1 with OsBgal10 and OsBgal11, and AtBGAL7 and AtBGAL15, which clustered in group b, were found to be expressed in flower [##REF##17466346##12##] and pollen [##REF##15517348##31##]. It may be that the ancestral genes for these enzymes developed reproductive-tissue specific roles before the ancestors of monocots and dicots diverged. If these functions are conserved, these isozymes may have similar roles in the two plants, though these functions remain to be determined.</p>", "<p>Among the other plant-like genes, it is likely many of them act in cell wall metabolism, but these roles are yet to be discerned. OsBgal1 was shown to hydrolyze β-(1→3)-, β-(1→4)- and β-(1→6)-linked oligosaccharides and, at a low level, arabinogalactan and be expressed in a range of vegetative and reproductive tissues [##UREF##9##38##], so it may have a general role on similar substrates in the cell wall. OsBgal2, OsBgal3 and OsBgal7 are closely related to it, as are 4 tomato Bgals involved in fruit ripening [##REF##10889266##13##] and an asparagus Bgal thought to act in senescence [##REF##12228457##56##], so a cell wall function seems likely for these other cluster c1 isozymes as well, though such a role must be proven. Kaneko and Kobayashi [##REF##12723614##37##] showed that OsBgal8, which is secreted from rice tissue culture cells, could release galactose from galactoxyloglucans and pectic galactans. So, a role in cell wall metabolism is quite likely for this group a isozyme.</p>", "<p>The role of OsBgal6 remains obscure, as no closely related enzyme has been characterized, however it seems to be specific to vegetative tissues of the young plant. The fact that the transcript found in panicle was misspliced to prevent production of active enzyme is intriguing, but the functional implications of the alternative splicing of both OsBgal6 and OsBgal11 remain to be clarified.</p>" ]

[ "<title>Results and Discussion</title>", "<title>Identification of rice GH35 genes and their protein products</title>", "<p>A total of 15 GH35 genes (defined <italic>OsBgal</italic>) were identified in rice genome databases (Table ##TAB##0##1##), each of which encodes a protein that contains the GH35 β-galactosidase active site consensus sequence G-G-P- [LIVM]-x-Q-x-E-N-E- [FY] [##REF##9649738##39##]. Most of the encoded proteins contain a GH35 domain at the N-terminus and a Gal-lectin-like domain at the C-terminus. Although the speculated carbohydrate-binding function of these Gal-lectin domains is not yet proven, their existence and conserved nature has led to the suggestion that they may increase the catalytic efficiency on polysaccharide substrates [##REF##17466346##12##]. The proteins from <italic>OsBgal</italic>2, <italic>OsBgal</italic>7 and <italic>OsBgal9 </italic>lack the Gal-lectin-like domain, suggesting that it is not necessary for the functions of these rice GH35 members.</p>", "<p>The deduced amino acid sequences of the OsBgal proteins were used to predict their putative signal peptides, protein lengths, molecular masses, pI values, possible N-glycosylation sites, and cellular destinations (Table ##TAB##1##2##). All OsBgals contain putative signal peptides, which range in length from 20 to 36 amino acids, except for that of OsBgal8 which is predicted to have 62 amino acids. The mature OsBgal proteins were predicted to contain from 653 (OsBgal9) to 894 (OsBgal8) amino acids, corresponding to molecular masses of 73.5 to 97.9 kDa. Most of the proteins were predicted to have pI in the acidic range (5.56–6.76), except those of OsBgal2, OsBgal7 and OsBgal10 are in the basic range (7.65–9.1) (Table ##TAB##1##2##). Only OsBgal2 and OsBgal7 lacked putative N-glycosylation sites, while the other enzymes were predicted to contain from 2 to 10 sites. Most OsBgal members are predicted to localize to the organelles of the secretory pathway, for instance, the Golgi apparatus, endoplasmic reticulum, or vacuole, or to be secreted. Only 3 OsBgals, including OsBgal9, OsBgal10 and OsBgal11, were predicted to be localized in the lysosome-like vacuole, which correlates to the location of mammalian β-galactosidases in the lysosome (Table ##TAB##1##2##). Although OsBgal10 and OsBgal11 are very similar in predicted mature protein length and possible cellular destinations, their predicted pI values are quite different. OsBgal5, OsBgal12, OsBgal14 and OsBgal15 all have high numbers of glycosylation sites, similar MW, pI and predicted possible destinations, which suggests that they may have redundant or overlapping functions.</p>", "<title>Phylogenetic analysis</title>", "<p>The multiple alignment of full-length protein sequences was used to construct an unrooted phylogenetic tree. The phylogenetic tree, which includes all the GH35 genes identified in the rice (monocot), <italic>A. thaliana </italic>(dicot) and <italic>Physcomitrella patens </italic>(a bryophyte moss) genomes, as well as representatives of animals, fungi, protists, archaea, and eubacteria, has three major branches, one of which is nearly plant specific (except for one <italic>Dictyostelium discoideum </italic>gene product), one of which contains representatives of animals, eubacteria and plants, and one of which contains archaeal, eubacterial and fungal enzymes (Figure ##FIG##0##1##). Almost all the rice, <italic>A. thaliana</italic>, and <italic>P. patens </italic>enzymes fall within the plant-specific cluster, except <italic>OsBgal9</italic>, <italic>AtBGAL</italic>17 and <italic>P. patens </italic>EDQ62875, which fall in the cluster with animal β-galactosidases. Previously, it was suggested that the plant enzymes that cluster with animals might have been transferred to plants by horizontal gene transfer [##REF##17466346##12##], but the fact that the bryophyte, which is thought to have diverged from the vascular plants early in plant evolution, has this type of β-galactosidase and the broad distribution of organisms with this type of β-galactosidase suggest that plants maintained a copy of this gene when plants and animals diverged. Perhaps the more relevant question is how the plant specific Bgal ancestor came to be, and how the C-terminal Gal-lectin-like domain was acquired by this lineage. It appears that the fungal-type and plant-type lineages may have diverged from the animal-type β-galactosidases early in GH35 evolution, but plants retained the animal-type β-galactosidase, as well.</p>", "<p>Within the plant-type β-galactosidases, OsBgals can be divided into three distinct groups (a, b and c in Figure ##FIG##0##1##). The major group (group a) contains 5 clusters with total 7 OsBgal members. The cluster a1 contains 4 rice genes, including OsBgal1, OsBgal2, OsBgal3, and OsBgal7, along with 6 AtBGALs, the <italic>Asparagus officinalis </italic>Bgal, and 4 tomato and 5 chickpea Bgals that are not shown, some of which contain C-terminal galactose-binding lectin-like domains and some of which do not. Another cluster, a2, contains OsBgal13, AtBGAL9 and two putative <italic>P. patens </italic>Bgals (EDQ59880 and EDQ81397), which suggests this cluster is of ancient origin. Two other putative <italic>P. patens </italic>Bgals fall in cluster a3, which appears to be closely related to cluster a2. OsBgal8 groups with AtBGAL8 and RsBgal1 of radish, which specifically hydrolyzes the β-(1→3)- and β-(1→6)-linked oligogalactans of AGPs [##REF##15980190##18##], in subsgroup a4. OsBgal4 groups with AtBGAL10 in cluster a5. The second biggest group, b, contains 4 rice Bgals with high amino acid sequence similarity, namely OsBgal5, OsBgal12, OsBgal14, and OsBgal15. These isozymes have nearly the same protein lengths and gene structures, as described below. Within group c, OsBgal10 and OsBgal11 (cluster c1) are closely related, while OsBgal6 (cluster c2) is less clearly associated, though both distance-based and maximum parsimony trees back this model.</p>", "<title>Chromosomal locations of OsBgals</title>", "<p>As shown in the map of their chromosomal locations and directions of transcription in Figure ##FIG##1##2##, the <italic>OsBgals </italic>are distributed over all chromosomes, except chromosomes 4, 7 and 11. Three <italic>OsBgals </italic>are located on chromosome 1, while 2 <italic>OsBgal </italic>each are found on chromosomes 3, 5, 6, and 10, and one <italic>OsBgal </italic>each is present on chromosomes 2, 8, 9, and 12. Similar to other gene families, <italic>OsBgals </italic>appear to have undergone gene duplication, as the rice genome has undergone genome-wide duplication events, including polyploidy, which promote the amplification of gene family members. Segmental duplication analysis identified four <italic>OsBgals </italic>located on the duplicated segmental regions in rice chromosomes. <italic>OsBgal7 </italic>(Rice genome project locus Os02g0219200) and <italic>OsBgal2 </italic>(Os06g0573600) are located on a chromosomal segment duplicated between chromosomes 2 and 6, while <italic>OsBgal10 </italic>(Os08g0549200) and <italic>OsBgal11 </italic>(Os09g0539200) are located on segments duplicated between chromosomes 8 and 9. These duplications are supported by the close phylogenetic relationships of these genes. Therefore, chromosomal segment duplication appears to have played a role in multiplication of <italic>OsBgals </italic>within the rice genome. In contrast, <italic>OsBgal12 </italic>and <italic>OsBgal14</italic>, which have high sequence similarity, are located close together on the same chromosome. Thus, these two genes may have diverged by tandem duplication.</p>", "<title>Exon-intron organization and primary gene structure analysis</title>", "<p>Automated annotations for all <italic>OsBgal </italic>genes were available in the public databases, except for <italic>OsBgal6</italic>, <italic>OsBgal14 </italic>and <italic>OsBgal15</italic>. However, we confirmed and corrected exon-intron organization manually by comparing the corresponding full length cDNA and EST sequences, which were either already available in publicly accessible databases or were obtained during this study, with genomic sequences.</p>", "<p>Gene structure comparisons showed eight different splicing patterns. The sizes of the majority of the coding exons are conserved, but some appear to have had intron-loss events (Figure ##FIG##2##3##). The pattern with the highest number of exons, found in <italic>OsBgal1, OsBgal3, OsBgal4</italic>, <italic>OsBgal6 and OsBgal13</italic>, contains 19 exons with 18 introns, with exons 1 through 10 encoding the GH35 domain, and exons 18 and 19 encoding the Gal-lectin-like domain. Ahn et al [##REF##17466346##12##] observed this same pattern in <italic>A. thaliana Bgals </italic>and surmised from parsimony that it is likely to be the ancestral gene pattern and other patterns were derived from it, mainly by intron loss. The presence of this pattern in diverse rice genes and in a <italic>P. patens </italic>gene (EDQ59880) supports this conclusion. The second most similar pattern is 18 exons separated by 17 introns with the loss of intron 1, which occurred in <italic>OsBgal11</italic>. A pattern of two-intron loss was found in <italic>OsBgal5, OsBgal12</italic>, <italic>OsBgal14</italic>, and <italic>OsBgal15</italic>, which contain 17 exons with the loss of introns 17 and 18.</p>", "<p>The <italic>OsBgal2</italic>, <italic>OsBgal7 </italic>and <italic>OsBgal9 </italic>genes all lack the galactose-binding, lectin-like domain at the C-terminus, however, their splicing patterns were different. <italic>OsBgal2 </italic>and <italic>OsBgal7 </italic>have patterns similar to other rice GH35 genes and retain the 5' end of exon 18, but <italic>OsBgal2 </italic>contains 16 exons with the loss of introns 11 and 16, while intron 15 has been lost in <italic>OsBgal7</italic>, leaving 17 exons (Figure ##FIG##2##3##). In contrast, <italic>OsBgal9 </italic>appeared to be distantly diverged from other rice GH35 members since its exon/intron organization does not correspond to the other <italic>OsBgals</italic>, except that an intron is found in the same position as intron 1 in the other genes.</p>", "<p>Although the pattern and lengths of exons in rice GH35 genes are similar, <italic>OsBgal13 </italic>spans approximately 20.8 kb, because it contains over three-fold longer introns than the other genes in the family. Long terminal repeat (LTR)-retrotransposons elements and other repeated sequences were found within <italic>OsBgal13 </italic>gene introns 1, 2, 8, 9, 10, 13, 14 and 16, which contain one GC rice region, three AT rich regions and eight transposable elements (Table ##TAB##2##3##). Likewise, <italic>OsBgal6</italic>, the second largest <italic>OsBgal </italic>gene due to its long (4 kb) intron 1, had one AT rich region and seven transposable elements, all of which were located within intron 1. In <italic>OsBgal </italic>genes of normal length, such as <italic>OsBgal1</italic>, no transposable elements were observed. These results may reflect the observation that rice genome expansion is forced by transposable element amplification [##REF##16819716##40##].</p>", "<p>Comparison of gene splice patterns and phylogenetic relationships between plant-type Bgals from rice, <italic>A. thaliana</italic>, and <italic>P. patens </italic>reveals that, although the ancestral gene pattern is the same, no other splice patterns are shared within a phylogenetic lineage. Therefore, though most introns appear to have been inserted in the common ancestor gene for this lineage (an extra exon was inserted in <italic>P. patens </italic>gene EDQ878340), intron losses appear to have occurred independently in rice, <italic>Arabidopsis </italic>and <italic>P. patens</italic>. The same may be true for the C-terminal domain, which appears to have been lost only once in rice (in the putative ancestor of <italic>OsBgal2 </italic>and <italic>OsBgal7</italic>), though it appears to have been lost at least 4 times in <italic>Arabidopsis </italic>GH35 <italic>Bgals </italic>[##REF##17466346##12##].</p>", "<title>Analysis of expression by RT-PCR</title>", "<p><italic>OsBgal </italic>transcript expression in various tissues and stages of growth was analyzed by RT-PCR with primers specific to the 3'UTR of each gene to gain further insight into possible functions. The relative expression levels derived from normalization with the <italic>UBQ6 </italic>polyubiquitin gene gave the same patterns as those shown for normalization with the <italic>β-Actin </italic>gene in Figure ##FIG##3##4## and Additional file ##SUPPL##0##1##, indicating the patterns likely reflect the expression of the <italic>OsBgal </italic>genes, rather than that of the control.</p>", "<p>The rice tissues/organs and developmental stages chosen for RT-PCR analysis are shown in Figure ##FIG##3##4##. All 15 <italic>OsBgal </italic>genes were found to be expressed with different but overlapping expression patterns, confirming that all genes are active. These relative expression levels were used to define genes as having expression specific to a given tissue, if the relative transcript levels of the gene at that stage are significantly higher (color scale above 7, where 10 is the level in the tissue with the highest expression of each gene) over the levels at all other stages. The expression profiles revealed that at least 9 genes were highly expressed in one of the stages of vegetative tissues and 10 were highly expressed in reproductive tissues. Only <italic>OsBgal13 </italic>appears to be expressed in nearly every organ and developmental stage, though at different levels (Figure ##FIG##3##4##).</p>", "<p>Nine <italic>OsBgals </italic>(<italic>OsBgal1, OsBgal2, OsBgal3, OsBgal4, OsBgal6, OsBgal7, OsBgal8 and OsBgal9</italic>) displayed their highest transcript levels in young and early-mature stages of shoot, root, and leaf sheath. Among these genes, <italic>OsBgal6, OsBgal7, OsBgal8 and OsBgal9 </italic>showed highest intensity in leaf sheath at different times, especially, 15-day and 1-month old, which are both fast cell elongation stages. The high accumulation of OsBgals in this stage may act in sugar remodeling, since the carbohydrate remobilized from the leaf sheath and culm to grain can contribute as much as 38% to rice yield [##UREF##10##41##]. In contrast, low levels of most gene transcripts were detected in leaf blade, except for <italic>OsBgal13 </italic>transcripts, which were detected throughout the growth of the blade.</p>", "<p>Six genes, <italic>OsBgal5, OsBgal10, OsBgal11, OsBgal12, OsBgal14</italic>, and <italic>OsBgal15 </italic>appear to be expressed primarily in reproductive tissues. The relative transcript levels for these genes were at least five times higher in reproductive tissues than in vegetative tissues (Figure ##FIG##3##4##). In addition, no expression was detected at the initiating panicle stage, but it was high in later panicle development and flowering stages. Only <italic>OsBgal10, OsBgal11 </italic>and <italic>OsBgal15 </italic>transcripts were found to accumulate in anther. These genes appear to have diverged from two ancestor genes, since <italic>OsBgal5</italic>, <italic>OsBgal12</italic>, <italic>OsBgal14</italic>, and <italic>OsBgal15 </italic>have sequences and gene structures that are similar to each other, as do <italic>OsBgal10 </italic>and <italic>OsBgal11 </italic>(Figures ##FIG##0##1## and ##FIG##2##3##). Interestingly, <italic>AtBGAL7 </italic>and <italic>AtBGAL15</italic>, which fall into the group b with <italic>OsBgal5</italic>, <italic>OsBgal12</italic>, <italic>OsBgal14</italic>, and <italic>OsBgal15 </italic>(Figure ##FIG##0##1##), were reported to function in the early stages of microspore and pollen development [##REF##15517348##31##]. Likewise, <italic>AtBGAL11 </italic>and <italic>AtBGAL13</italic>, which fall in cluster c1 with <italic>OsBgal10 </italic>and <italic>OsBgal11 </italic>(Figures ##FIG##0##1##), show maximum expression in mature pollen [##REF##15517348##31##]. So, related rice and <italic>Arabidopsis </italic>Bgals function in reproductive tissues, suggesting their roles may be somewhat conserved in the two plants.</p>", "<p>Only the <italic>OsBgal4 </italic>transcript was expressed abundantly in seed at the imbibition stage, while <italic>OsBgal2 </italic>and <italic>OsBgal13 </italic>were also present at low levels. Five <italic>OsBgals </italic>were expressed in milk grain and six in grain during dry down. A high abundance of <italic>OsBgal2 </italic>and <italic>OsBgal13 </italic>transcripts was observed in grain during dry down. This evidence implies that they may function in grain development and senescence or are stored for roles in germination, such as cell wall remodeling. <italic>OsBgal1</italic>, <italic>OsBgal2, OsBgal6, OsBgal8</italic>, and <italic>OsBgal13 </italic>transcripts were also found in shoots and roots of seedlings after germination. Chantarangsee et al. [##UREF##9##38##] reported β-galactosidase activity in rice seeds, roots and shoots at 0–7 days after seed-soaking, and detected OsBgal1 and OsBgal2 proteins in embryos and seedling roots and shoots. In barley (<italic>Hordeum vulgare</italic>), Giannakouros and colleagues [##UREF##2##17##] were able to separate 4 isozymes of β-galactosidase from germinated grain. Thus, Bgals are present and may play roles in the growth and emergence of root and shoot primordia from the hull.</p>", "<p>To complement the RT-PCR expression profiles of the <italic>OsBals</italic>, ESTs retrieved from the UniGene database (UniGene December, 2007, NCBI) were compared with our findings. Full-length or partial cDNA and ESTs for most <italic>OsBgals </italic>are available in the database, except <italic>OsBgal6</italic>, for which only one EST from unspecified tissues is available, and <italic>OsBgal15</italic>, for which no corresponding ESTs or cDNA are available (Table ##TAB##0##1##). However, the other <italic>OsBgal </italic>genes had cDNA and ESTs present in the database, with numbers of ESTs that varied from 1 to 253.</p>", "<p>The <italic>OsBgal8 </italic>(Unigene cluster Os.22360, locus <italic>Os03g0255100</italic>) has the highest number of ESTs in the database: 253 ESTs from various tissues, including flower, stem, leaf, panicle, root, callus, seed, whole plant and unspecified tissues. The Unigene profiles indicate relatively abundant accumulation in flower, leaf, panicle, root, seed and stem with 761, 133, 120, 190, 216 and 228 transcripts per million (TPM), respectively (Table ##TAB##0##1##). The Unigene profile is somewhat similar to the RT-PCR pattern of <italic>OsBgal8</italic>, in which the transcripts accumulated significantly throughout rice plant, including in seed during dry-down, but not in seedling and milk grain.</p>", "<p><italic>OsBgal10 </italic>(Os.18310), <italic>OsBgal11</italic>, <italic>OsBgal12 </italic>(Os.46702) and <italic>OsBgal14 </italic>(Os.22528) expression data included mainly flower and panicle clones, or had relatively high expression in these tissues, which correlates to our RT-PCR analysis. For instance,<italic>OsBgal11 </italic>shows 249 TPM in flower and 158 TPM in panicle, while <italic>OsBgal14 </italic>shows 358 TPM in flower (Table ##TAB##0##1##).</p>", "<p>The EST and cDNA clones for the remaining <italic>OsBgals </italic>had diverse origins, which is consistent with the evaluation of <italic>OsBgals </italic>by RT-PCR, though the frequencies of expression derived from these two forms of expression data were not exactly the same. Therefore, Unigene database analysis could be used for an initial guideline of gene expression, though the expression patterns based on ESTs are fragmentary, since the tissues are not equally or completely represented and the source tissue annotation of many EST libraries is unclear or incomplete. Microarray experiments, such as that by Ma et al. [##REF##16140994##42##], who assessed expression of 37,132 non cross-hybridizing gene models in rice seedling shoots, tillering-stage shoots and roots, heading and filling stages of panicles and suspension cells, offer a more comprehensive picture of gene expression within a tissue. Although the tissue coverage in public microarray databases is as yet limited, the GEO expression data in Table ##TAB##0##1## shows that OsBgal expression levels may be affected by many treatments.</p>", "<title>Rice β-galactosidase cDNAs sequencing and identification of alternative splicing</title>", "<p>Although the expression of all <italic>OsBgals </italic>can be observed by RT-PCR and most have corresponding cDNAs and ESTs in the database, complete full-length cDNA clones were not available for <italic>OsBgal6</italic>, <italic>OsBgal11 </italic>and <italic>OsBgal15</italic>. Therefore, to confirm the mRNA sequence and gene structure, these genes were amplified by PCR and sequenced. The accession numbers for these sequences are listed in Table ##TAB##0##1##.</p>", "<p>The full-length cDNA of <italic>OsBgal</italic>6 was amplified by a set of primers corresponding to the transcribed locus <ext-link ext-link-type=\"gen\" xlink:href=\"XM_475258\">XM_475258</ext-link>. The cDNA was isolated from 15-day leaf sheaths, a tissue with abundant expression, and exerting panicles, a tissue with poor expression, based on RT-PCR expression analysis. Sequencing of the cDNA from the highly expressing tissue revealed an open reading frame (ORF) of 2,436 nucleotides encoding a protein of 812 amino acids (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU602310\">EU602310</ext-link>), which confirmed the our predicted β-<italic>galactosidase </italic>sequence from chromosome 5 (Genbank accession no. <ext-link ext-link-type=\"gen\" xlink:href=\"AC135419\">AC135419</ext-link> and <ext-link ext-link-type=\"gen\" xlink:href=\"AC135429\">AC135429</ext-link>). A search through the dbEST database of <italic>O. sativa </italic>found 99% identity with a 520 bp EST from fertile panicle (<ext-link ext-link-type=\"gen\" xlink:href=\"CK048087\">CK048087</ext-link>). On the contrary, the Os <italic>Bgal</italic>6 cDNA amplified from exerting panicle, where expression is low, contains only 2,338 bp (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU603285\">EU603285</ext-link>) which is 101 bp shorter than the cDNA from high expression tissues. The open reading frame beginning at the initiation codon of this transcript covers only exons 1 to 7 and encodes only 244 amino acids. Alternative splicing of this transcript occurred to utilize an alternative acceptor site (CAG) 80 nt downstream of functional acceptor site for intron 7, which introduced a deletion of 27 amino acid and a frameshift that leads to an in-frame stop codon (Figure ##FIG##4##5##). A second missplicing of this gene occurred at the intron 10/exon 11 junction, which again used an alternate acceptor site (CAG) located 20 nt downstream of the regular acceptor site. So, alternative splicing is likely to be one of the gene regulation mechanisms, since the transcript would be subject to nonsense mediated decay (nmd) [##REF##17194304##43##] and the protein produced would be useless as a β-galactosidase.</p>", "<p><italic>OsBgal11 </italic>(locus Os09g0539200) is annotated as a pseudogene in GenBank, because of its apparent inability to produce full-length protein due to a frameshift and subsequent stop codon in the AK119414 cDNA. We hypothesized that such phenomenon probably resulted from missplicing, as seen in <italic>OsBgal6</italic>. A 1343-nucleotide fragment from the 3' half of the <italic>OsBgal11 </italic>cDNA was amplified from exerting panicle (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU603286\">EU603286</ext-link>), in which its expression is high based on RT-PCR. This cDNA shares 98% identity with the AK119414 cDNA, but had a substitution of 75 nucleotides in place of 95 nucleotides at positions 1282 to 1377 of AK119414. These substituted sequences allow the full-length translation of the predicted 839 amino acids of OsBgal11, whereas only 426 amino acids are translated from AK119414. Comparison with the genomic sequence and our amplified cDNA sequence indicates that the AK119414 transcript results from retention of intron 10, which extends exon 10 by 101 nt, thereby introducing an in-frame stop codon. In addition, an alternative donor site located at the functional acceptor site of intron 10 and the first nucleotide of the functional exon 11 produces an alternate intron 10 with the acceptor site located 79 nt downstream, within the functional exon 11, thereby reducing the size of exon 11. The shortened (456-residue) protein, includes all of the catalytic domain, based on the <italic>Penicillium </italic>sp. β-galactosidase structure [##REF##15491613##44##], so it is possible that it could still function as a Bgal.</p>", "<p>No experimentally determined cDNA or ESTs corresponding to <italic>OsBgal15 </italic>were available in public databases, despite the fact that our RT-PCR demonstrated its expression. Therefore, the full-length <italic>OsBgal15 </italic>cDNA was amplified, cloned and sequenced. The <italic>OsBgal15 </italic>cDNA sequence (Genbank Accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU051629\">EU051629</ext-link>) contains a 2487-nucleotide ORF, which encodes 829 amino acids. The encoded protein shares over 99% identity with a predicted β-galactosidase of <italic>Oryza sativa </italic>(japonica cultivar-group) (accession no. <ext-link ext-link-type=\"gen\" xlink:href=\"AP004733\">AP004733</ext-link>), except that 7 extra amino acid residues were inserted at residue 293, compared to the predicted sequence from <ext-link ext-link-type=\"gen\" xlink:href=\"AP004733\">AP004733</ext-link>. Therefore, the correct splice donor site of exon 8 is located 21 nucleotides downstream of that predicted for the mRNA of <ext-link ext-link-type=\"gen\" xlink:href=\"AP004733\">AP004733</ext-link>.</p>", "<p>The alternative splicing observed in this study introduces in-frame stop codons into the mRNA of <italic>OsBgal6 </italic>in the low expression tissue, but not for the high expression tissues, while it can also introduce a premature stop codon in <italic>OsBgal11</italic>, based on the AK119414 cDNA sequence. In vertebrates, there are a number of well-known alternatively splicing genes, for instance the fibronectin gene, which can generate over 20 different proteins, some of which have different patterns of localization and slightly different functions in human cells [##REF##7226224##45##]. Until relatively recently, there were very few examples of alternatively spliced genes in higher plants and still fewer examples known to generate functionally distinct proteins. Generally, alternative splicing yields two polypeptides of different size that are identical, except for the presence of a number of additional amino acids at the C-terminus of the larger isoform, eg., ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase [##REF##10430961##46##]; diacylglycerol kinase in tomato [##REF##11069705##47##]; and chloroplast ascorbate peroxidase in spinach [##REF##9931296##48##]. Missplicing events were observed in maize (<italic>Zea mays</italic>) <italic>Viviparous 1 </italic>(<italic>Vp1</italic>) transcripts, the majority of which are spliced incorrectly and contain insertions of intron sequences or deletions of coding region, so that they do not encode full-length proteins [##REF##12119408##49##]. Although alternative splicing is a widespread process used in higher eukaryotes to regulate gene expression and functional diversification of proteins [##REF##11173120##50##,##REF##12600935##51##], the putative control of tissue-specific expression by missplicing we observed has not been well characterized in rice.</p>", "<title>Preliminary Characterization of OsBgal13</title>", "<p>OsBgal13 is in cluster a2 of group a of the phyloenetic tree in Figure ##FIG##0##1##, along with AtBGAL9 and two <italic>P. patens </italic>Bgals. This and its general expression pattern suggest it may have a conserved maintenance function in plants. To assess what its function might be, OsBgal13 was expressed as a thioredoxin fusion protein in <italic>Escherichia coli </italic>strain OrigamiB (DE3), which we previously found to have little background β-galactosidase acitivity [##UREF##9##38##]. The protein was purified by immobilized metal affinity chromatography on nickel resin, and most of the OsBgal13 activity eluted in 5–20 mM imidazole. Further purification was achieved by gel filtration chromatography and the activity of the resulting protein was assessed. As shown in Table ##TAB##3##4##, among <italic>p</italic>-nitrophenol (<italic>p</italic>NP) glycosides, the OsBgal13 enzyme had highest activity toward <italic>p</italic>NP-β-D-galactoside (<italic>p</italic>NPGal), but also had 30% of this activity toward <italic>p</italic>NP-α-L-arabinoside (<italic>p</italic>NPAra), and low activity toward <italic>p</italic>NP-β-D-fucoside and <italic>p</italic>NP-β-D-mannoside, but no detectable activity toward <italic>p</italic>NP-β-D-glucoside and <italic>p</italic>NP-β-D-xyloside and negligible activity toward <italic>p</italic>NP-α-D-galactoside. OsBgal13 also hydrolyzed β-(1→3)-, β-(1→4)- and β-(1→6)-linked galactobiose and galactotriose, but no release of galactose or arabinose could be detected when the protein was incubated for 24 h with rice seedling alcohol insoluble residue (AIR), larchwood arabinogalactan, galactan, apple pectin, citrus pectin or oat xylan. So, despite the intriguing observation that OsBgal13 can hydrolyze both β-D-galactosyl residues and α-L-arabinosyl residues, components of type I and II arabinogalactans and xyloglucan and glucuronoarabinoxylan side chains [##REF##15012297##52##], only short galacto-oligosaccharides can be identified as possible natural substrates to date.</p>", "<title>Possible Functions of Other Isozymes</title>", "<p>The activity of mammal β-galactosidase specifically releases terminal β-galactosyl residues from glycosaminoglycans and the glycolipid GM1 ganglioside. Similarly, the digestion of the galactolipid monogalactosyldiacylglyerol by vacuolar and chloroplast β-galactosidases was reported in wheat [##REF##16663831##53##]. Likewise, β-galacosidase of tomato was reported to act in galactolipid turnover and degradation, which occurs in chloroplasts and chromoplasts during tomato fruit development [##UREF##11##54##,##UREF##12##55##]. However, relatively little is known about metabolism of other glycolipids, glycopoteins and proteoglycans involving plant β-galactosidases, though it is well characterized in mammals. The fact that OsBgal9 is similar to animal β-galactosidases and is predicted to localize to a lysosome-like vacuole, suggests it may play a similar role in rice.</p>", "<p>Among the plant-like OsBgals, OsBgal10 and OsBgal11 are also predicted to localize to lysosome-like vacuoles, so they could also play a role similar to animal-type β-galactosidases. However, these closely related isozymes have reproductive tissue specific expression, as are the isozymes of cluster b, OsBgal5, OsBgal12, OsBgal14 and OsBgal15. Similarly, AtBGAL11 and AtBGAL13, which clustered in group c1 with OsBgal10 and OsBgal11, and AtBGAL7 and AtBGAL15, which clustered in group b, were found to be expressed in flower [##REF##17466346##12##] and pollen [##REF##15517348##31##]. It may be that the ancestral genes for these enzymes developed reproductive-tissue specific roles before the ancestors of monocots and dicots diverged. If these functions are conserved, these isozymes may have similar roles in the two plants, though these functions remain to be determined.</p>", "<p>Among the other plant-like genes, it is likely many of them act in cell wall metabolism, but these roles are yet to be discerned. OsBgal1 was shown to hydrolyze β-(1→3)-, β-(1→4)- and β-(1→6)-linked oligosaccharides and, at a low level, arabinogalactan and be expressed in a range of vegetative and reproductive tissues [##UREF##9##38##], so it may have a general role on similar substrates in the cell wall. OsBgal2, OsBgal3 and OsBgal7 are closely related to it, as are 4 tomato Bgals involved in fruit ripening [##REF##10889266##13##] and an asparagus Bgal thought to act in senescence [##REF##12228457##56##], so a cell wall function seems likely for these other cluster c1 isozymes as well, though such a role must be proven. Kaneko and Kobayashi [##REF##12723614##37##] showed that OsBgal8, which is secreted from rice tissue culture cells, could release galactose from galactoxyloglucans and pectic galactans. So, a role in cell wall metabolism is quite likely for this group a isozyme.</p>", "<p>The role of OsBgal6 remains obscure, as no closely related enzyme has been characterized, however it seems to be specific to vegetative tissues of the young plant. The fact that the transcript found in panicle was misspliced to prevent production of active enzyme is intriguing, but the functional implications of the alternative splicing of both OsBgal6 and OsBgal11 remain to be clarified.</p>" ]

[ "<title>Conclusion</title>", "<p>Fifteen rice glycosyl hydrolase family 35 genes encoding putative β-galactosidases were identified in this study. This number is similar to the 17 seen in <italic>Arabidopsis </italic>and these plants appear to contain 9 common gene lineages present in their ancestors before they diverged [##REF##17466346##12##]. OsBgal9 was clustered into the same group as Bgals from animal species, such as <italic>H. sapiens </italic>and <italic>D. melanogaster </italic>in the phylogenetic tree, while the other rice BGals fall in a nearly plant-specific subfamily of Bgals, most of which contain a C-terminal lectin-like domain. The presence of both types of β-galactosidase in the moss <italic>P. patens</italic>, a nonvascular plant, suggests that both types of genes were present early in plant evolution. Within the plant-type Bgals, group a (Figure ##FIG##0##1##), contains 7 OsBgals and many Bgals that have been characterized from other plants (for example, 9 of <italic>Arabidopsis</italic>, 4 of <italic>C. arietinum </italic>and 3 of <italic>L. esculentum</italic>), including some with and some without C-terminal galactose-binding-lectin-like domains. Many of these proteins, including OsBgal8 [##REF##12723614##37##], appear to act on the cell-wall-derived substrates. The isozymes of phylogenetic groups b and c1, OsBgal5, OsBgal10, OsBgal11, OsBgal14, and OsBgal15, appear to have reproductive-tissue specific functions, while OsBgal6 (group c2) is predominantly in young leaves and roots. <italic>OsBgal6 </italic>appeared to undergo alternative splicing to prevent its production in panicle, and alternative splicing was also found for <italic>OsBgal11</italic>. Understanding the functions of this gene family and significance of alternative splicing in this will require further functional investigation.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Many plant β-galactosidases (Bgals) have been well characterized and their deduced biological functions mainly involve degradation of structural pectins, xyloglucans or arabinogalactoproteins in plant cell walls. However, gene multiplicity in glycosyl hydrolase family 35 (GH35), to which these proteins belong, implies diverse functions. In this study, the gene multiplicity, apparent evolutionary relationships and transcript expression of rice Bgal genes were examined, in order to predict their biological functions.</p>", "<title>Results</title>", "<p>Fifteen rice Bgal genes were identified in the plant genome, one of which encodes a protein similar to animal Bgals (<italic>OsBgal9</italic>), and the remaining 14 fall in a nearly plant-specific subfamily of Bgals. The presence of both classes of Bgals in bryophytes, as well as vascular plants, suggests both gene lineages were present early in plant evolution. All 15 proteins were predicted to contain secretory signal sequences, suggesting they have secretory pathway or external roles. RT-PCR and database analysis found two distinct lineages to be expressed nearly exclusively in reproductive tissues and to be closely related to <italic>Arabidopsis </italic>Bgals expressed most highly in flower and pollen. On the other hand, <italic>OsBgal6 </italic>is expressed primarily in young vegetative tissues, and alternative splicing in panicle prevents its production of full-length protein in this reproductive tissue. <italic>OsBgal11 </italic>also showed alternative splicing to produce different length proteins. OsBgal13 produced by recombinant expression in <italic>Escherichia coli </italic>hydrolyzed α-L-arabinoside in addition to β-D-galactoside and β-(1→3)-, β-(1→4)- and β-(1→6)- linked galacto-oligosaccharides.</p>", "<title>Conclusion</title>", "<p>Rice <italic>GH35 </italic>contains fifteen genes with a diversity of protein sequences, predicted locations and expression and splicing patterns that suggest that OsBgals enzymes may play a variety of roles in metabolism of cell wall polysaccharides, glycoproteins and glycolipids.</p>" ]

[ "<title>Availability &amp; requirements</title>", "<p>NCBI databases: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov/sites/entrez\"/></p>", "<p>CAZY website: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cazy.org/\"/></p>", "<p>SignalP program: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cbs.dtu.dk/services/SignalP/\"/></p>", "<p>NetNGlyc: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cbs.dtu.dk/services/NetNGlyc/\"/></p>", "<p>NCBI map viewer: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov/projects/mapview\"/></p>", "<p>PHYLIP: <ext-link ext-link-type=\"uri\" xlink:href=\"http://evolution.genetics.washington.edu/phylip.html\"/></p>", "<p>TIGR rice segmental duplication database: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.tigr.org/tdb/e2k1/osa1/segmental_dup/500kb/segdup_500kb.shtml\"/></p>", "<p> RepeatMasker: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker\"/></p>", "<p>Genedoc: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.nrbsc.org/gfx/genedoc/index.html\"/></p>", "<title>Authors' contributions</title>", "<p>WT performed rice plant cultivation, RT-PCR analysis, cDNA cloning and sequencing, and drafted the manuscript. WT, JK–C and MC carried out gene structural analysis. WT and JK–C participated in data analysis, and gene and protein analysis. JM expressed, purified and characterized the recombinant OsBgal13 protein. JK–C organized and directed the project and helped to draft the manuscript. All authors read and approved the final manuscript.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>Tassanee Onkoksong is gratefully acknowledged for OsBgal13 expression vector construction and Rodjana Opassiri for helpful comments and discussion. Prof. Yoichi Tsumuraya and Prof. Toshihisa Kotake are thanked for providing galacto-oligosaccharides. Funding for this work was provided by the National Center for Genetic Engineering and Biotechnology of the National Science and Technology Development Agency, Thailand, grant number BT-B-06-RG-19-4608. WT was supported by postdoctoral grant of the same Agency.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Phylogenetic relationship among the rice and other glycosyl hydrolase family 35 proteins.</bold> Deduced amino acid sequences were aligned using ClustalX program [##REF##9810230##63##] and edited with Gendoc [##UREF##14##62##], then the tree were created using neighbor joining method and analyzed with 1000 bootstrap replicates, for which the percent reproducibility is shown on the branches that gave higher than 50% reproducibility. A tree was also made by maximum parsimony with the PHYLIP Protpars program, and branches supported by this analysis are shown with thicker lines. The rice sequences are named Os and a number according to the OsBgal numbers in Tables 1 and 2. <italic>Arabidopsis thaliana </italic>sequences are indicated as At, followed by the AtBGAL numbers of Ahn et al. [##REF##17466346##12##]. Sequences derived from the genome of the bryophyte <italic>Physcomitrella patens subsp. patens are given as Pp and the Genbank accession number without the initial \"EDQ.\" Other β-galactosidase or related sequences (and their accession numbers) shown are: Ao, Asparagus officinalis </italic>(CAA54525); Aso, <italic>Aspergillus oryzae </italic>(BAE58662); Bac, <italic>Bacillus circulans </italic>(O31341); Baf, <italic>Bacteroides fragilis </italic>(CAH09349 and CAH06328); Cac, <italic>Caulobacter crescentus </italic>(Q9A4M9); Cam, <italic>Caldivirga_maquiingensis </italic>(ABW01734); Ce, <italic>Caenorhabditis elegans </italic>(O76632); Dd (<italic>Dictyostelium discoideum</italic>) (EAL64656 and XP_635837); Dm, <italic>Drosophila melanogaster </italic>(Q9VMJ5); Enf, <italic>Enterococcus faecalis </italic>(AAO81613); Hs, human (<italic>Homo sapiens) b-galactosidase </italic>(P16278); HsBglk1, human b-galactosidase-1-like protein, (Q6UWU2); Hs Bglk 3, human b-galactosidase-1-like protein 3, (Q8NCI6); Hs_4, human locus 89944 (Q8IW92); Nv, sea anemone (<italic>Nematostella vectensis</italic>, XP_001631933); Pensp, <italic>Penicillium </italic>sp. (CAF32457); Rs, radish (<italic>Raphanus sativus</italic>) (BAD20774); Xac, <italic>Xanthomonas campestris </italic>(AAM41682); Xao, <italic>Xanthomonas oryzae </italic>(BAE68425); Xl African three-toed frog (<italic>Xenopus laevis</italic>): Xl 1 (AAI24928), Xl 2 (AAH74351), Xl 3 (AAH46858).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Distribution and direction of <italic>OsBgal </italic>genes on rice chromosomes.</bold> The chromosome numbers are indicated at the bottom of each bar. Genes lying on duplicated segments of the genome have been joined by dashed lines. Os is short for OsBgals. The gene map was generated with the NCBI map viewer.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>The exon-intron organization of <italic>OsBgal </italic>genes.</bold> Exons are shown as boxes with corresponding exons having the same pattern. Introns are represented by lines. Groupings of the genes with the same splice pattern are enclosed by red boxes.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Relative expression levels of 15 <italic>OsBgal </italic>genes in different tissues determined by semiquantitative RT-PCR.</bold> Signals were quantified and normalized to the expression of <italic>β-Actin</italic>, and the highest observed expression level of each gene was designated as 10 (black), with other tissues expression set relative to this maximal level. See Additional file ##SUPPL##0##1## for actual values and standard deviations. IP, initiating panicle; DP, differentiating panicle; EP, exerting panicle; F, flower; A, Anther; MG, milk grain; GD, grain during dry down; d, day(s); m, month(s).</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Alternative splicing of <italic>OsBgal6 </italic>(A) and <italic>OsBgal11 </italic>(B).</bold> The high expression tissue for <italic>OsBgal6 </italic>is 15-day-old rice leaf sheath, while the low expression tissue is exerting panicle. For OsBgl11, the splicing pattern of the cDNA amplified from exerting panicle of KDML105 indica rice (Genbank accession <ext-link ext-link-type=\"gen\" xlink:href=\"EU603286\">EU603286</ext-link>) is shown above, while the database sequence from Nipponbare japonica rice (<ext-link ext-link-type=\"gen\" xlink:href=\"AK119414\">AK119414</ext-link>) is shown below. Abbreviations include AAS: alternative acceptor site, E: Exon. AAS1: alternative acceptor site (CAG) located 80 nt downstream of the functional acceptor site; AAS2: alternative acceptor site (CAG) located 20 nt downstream of the functional acceptor site; AAS3: intron retention and alternative splice site within exon 11. The data for <italic>OsBgal1 </italic>and <italic>OsBgal2 </italic>were previously reported by Chantarangsee et al. [##UREF##9##38##].</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Summary of identified genes homologous to glycosyl hydrolase family 35 galactosidase</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Gene name</td><td align=\"center\">BGI entries (AAAA...)^a</td><td align=\"center\">Genomic Contig.</td><td align=\"center\">Chr.^b</td><td align=\"center\">UniGene</td><td align=\"center\">Gene Locus (NCBI, RGP)</td><td align=\"center\">Gene Locus (TIGR)</td><td align=\"center\">Corresponding cDNA^c</td><td align=\"center\" colspan=\"2\">Number^d</td><td align=\"center\">Tissue librarys^e</td><td align=\"center\" colspan=\"8\">Expression profile (TPM) Transcript per millon^f</td><td align=\"center\">GEO profile^g</td></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td colspan=\"2\"><hr/></td><td/><td colspan=\"8\"><hr/></td><td/></tr><tr><td/><td/><td/><td/><td/><td/><td/><td/><td align=\"center\">mRNA</td><td align=\"center\">ESTs</td><td/><td align=\"center\">Cl.</td><td align=\"center\">Fw.</td><td align=\"center\">Lf.</td><td align=\"center\">P</td><td align=\"center\">Rt.</td><td align=\"center\">Sd.</td><td align=\"center\">St.</td><td align=\"center\">Vm</td><td/></tr></thead><tbody><tr><td align=\"center\"><italic>OsBgal1</italic></td><td align=\"center\">1002951</td><td align=\"center\">AP008209.1</td><td align=\"center\">3</td><td align=\"center\">Os.13559</td><td align=\"center\">Os03g0165400</td><td align=\"center\">LOC_Os03g06940</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AF499737\">AF499737</ext-link></td><td align=\"center\">5</td><td align=\"center\">99</td><td align=\"center\">Fw, Cl, P, Mx, Wp, UnT, Vm, Sd, St, Rt</td><td align=\"center\">121</td><td align=\"center\">241</td><td align=\"center\">46</td><td align=\"center\">120</td><td align=\"center\">14</td><td align=\"center\">92</td><td align=\"center\">23</td><td align=\"center\">217</td><td align=\"center\">Up: CKl, AB&amp;GBd, AnX, Dw: AB&amp;GB, NaCl, Na₃AsO₄, CKrl</td></tr><tr><td align=\"center\"><italic>OsBgal2</italic></td><td align=\"center\">1003471</td><td align=\"center\">AP004729.3, AP008212.1</td><td align=\"center\">6</td><td align=\"center\">Os.87715</td><td align=\"center\">Os06g0573600</td><td align=\"center\">LOC_Os06g37560</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK102756\">AK102756</ext-link></td><td align=\"center\">4</td><td align=\"center\">201</td><td align=\"center\">P, Lf, Mx, St, Rt, Cl, Fw, Wp, UnT</td><td align=\"center\">66</td><td align=\"center\">80</td><td align=\"center\">291</td><td align=\"center\">391</td><td align=\"center\">205</td><td align=\"center\">0</td><td align=\"center\">118</td><td align=\"center\">0</td><td align=\"center\">Up: CKl, AB&amp;GBd, NaCl Dw: AnX, AB&amp;GB, Na₃AsO₄, CKrl</td></tr><tr><td align=\"center\"><italic>OsBgal3</italic></td><td align=\"center\">01012445, 01022298</td><td align=\"center\">AP008207.1, AP003546.4</td><td align=\"center\">1</td><td align=\"center\">Os.18562</td><td align=\"center\">Os01g0580200</td><td align=\"center\">LOC_Os01g39830</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK103045\">AK103045</ext-link></td><td align=\"center\">2</td><td align=\"center\">44</td><td align=\"center\">Cl, Wp, P, St, Fw, Mx, UnT, Vm</td><td align=\"center\">115</td><td align=\"center\">43</td><td align=\"center\">0</td><td align=\"center\">37</td><td align=\"center\">0</td><td align=\"center\">30</td><td align=\"center\">47</td><td align=\"center\">217</td><td align=\"center\">Up: CKl, AB&amp;GBd, AB&amp;GB, Na₃AsO₄, NaCl Dw: AnX, CKrl</td></tr><tr><td align=\"center\"><italic>OsBgal4</italic></td><td align=\"center\">ND</td><td align=\"center\">AP003297.3, AP008207.1</td><td align=\"center\">1</td><td align=\"center\">Os.9892</td><td align=\"center\">Os01g0875500</td><td align=\"center\">LOC_Os01g65460</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK101399\">AK101399</ext-link></td><td align=\"center\">3</td><td align=\"center\">107</td><td align=\"center\">Cl, Fw, Mx, P, Rt, UnT, St, Wp</td><td align=\"center\">279</td><td align=\"center\">263</td><td align=\"center\">0</td><td align=\"center\">45</td><td align=\"center\">29</td><td align=\"center\">30</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">Up: CKl, AnX, AB&amp;GBd, Na3AsO4, NaCl Dw: AB&amp;GB</td></tr><tr><td align=\"center\"><italic>OsBgal5</italic></td><td align=\"center\">1000473.1</td><td align=\"center\">AP003447.2, AP008207.1, AP003445.2</td><td align=\"center\">1</td><td align=\"center\">Os.41043</td><td align=\"center\">Os01g0533400</td><td align=\"center\">LOC_Os01g34920</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK119447\">AK119447</ext-link></td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">Mx, St</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">Up: Na₃AsO₄, CKrl Dw: CKl, AnX, NaCl</td></tr><tr><td align=\"center\"><italic>OsBgal6</italic></td><td align=\"center\">101013714.1</td><td align=\"center\">AC135419.2, AC135429.2</td><td align=\"center\">5</td><td align=\"center\">Os.82841</td><td align=\"center\">P0636F09.15</td><td align=\"center\">LOC_Os05g35360</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"EU602310\">EU602310</ext-link></td><td align=\"center\">ND</td><td align=\"center\">1</td><td align=\"center\">UnT</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">-</td></tr><tr><td align=\"center\"><italic>OsBgal7</italic></td><td align=\"center\">01011567, 01017443</td><td align=\"center\">AP008208.1</td><td align=\"center\">2</td><td align=\"center\">Os.14358</td><td align=\"center\">Os02g0219200</td><td align=\"center\">LOC_Os02g12730</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK059059\">AK059059</ext-link></td><td align=\"center\">7</td><td align=\"center\">57</td><td align=\"center\">P, Mx, St, Cl, UnT, Fw, Wp, Rt</td><td align=\"center\">24</td><td align=\"center\">21</td><td align=\"center\">0</td><td align=\"center\">135</td><td align=\"center\">14</td><td align=\"center\">92</td><td align=\"center\">78</td><td align=\"center\">0</td><td align=\"center\">Up: AnX, CKl, AB&amp;GB, AB&amp;GBd, NaCl, Na3AsO4 Dw: CKrl</td></tr><tr><td align=\"center\"><italic>OsBgal8</italic></td><td align=\"center\">1001024</td><td align=\"center\">AP008209.1, DP000009.2</td><td align=\"center\">3</td><td align=\"center\">Os.22360</td><td align=\"center\">Os03g0255100</td><td align=\"center\">LOC_Os03g15020</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK067479\">AK067479</ext-link></td><td align=\"center\">3</td><td align=\"center\">253</td><td align=\"center\">Fw, Mx, St, Lf, P, Rt, Cl, UnT, Sd, Wp</td><td align=\"center\">42</td><td align=\"center\">761</td><td align=\"center\">133</td><td align=\"center\">120</td><td align=\"center\">190</td><td align=\"center\">216</td><td align=\"center\">228</td><td align=\"center\">0</td><td align=\"center\">Up: AnX, CKl, AB&amp;GBd, NaCl Dw: Na₃AsO₄</td></tr><tr><td align=\"center\"><italic>OsBgal9</italic></td><td align=\"center\">ND</td><td align=\"center\">AP008211.1</td><td align=\"center\">5</td><td align=\"center\">Os.14570</td><td align=\"center\">Os05g0539400</td><td align=\"center\">LOC_Os05g46200</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK068572\">AK068572</ext-link></td><td align=\"center\">2</td><td align=\"center\">66</td><td align=\"center\">Lf, St, P, Mx, Cl, UnT, Wp, Sd, Rt</td><td align=\"center\">12</td><td align=\"center\">0</td><td align=\"center\">122</td><td align=\"center\">52</td><td align=\"center\">14</td><td align=\"center\">247</td><td align=\"center\">110</td><td align=\"center\">0</td><td align=\"center\">Up: CKl, AB&amp;GBd, AB&amp;GB, NaCl Dw: AnX, Na₃AsO₄</td></tr><tr><td align=\"center\"><italic>OsBgal10</italic></td><td align=\"center\">1005991</td><td align=\"center\">AP008214.1, AP003912.3</td><td align=\"center\">8</td><td align=\"center\">Os.18310</td><td align=\"center\">Os08g0549200</td><td align=\"center\">LOC_Os08g43570</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK069066\">AK069066</ext-link></td><td align=\"center\">3</td><td align=\"center\">137</td><td align=\"center\">Mx, Fw, P, St, UnT</td><td align=\"center\">0</td><td align=\"center\">249</td><td align=\"center\">0</td><td align=\"center\">158</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">Up: AB&amp;GBd, AB&amp;GB, AnX Dw: Na₃AsO₄, CKrl</td></tr><tr><td align=\"center\"><italic>OsBgal11</italic></td><td align=\"center\">1013958</td><td align=\"center\">AP008215.1</td><td align=\"center\">9</td><td align=\"center\">Os.49945</td><td align=\"center\">Os09g0539200</td><td align=\"center\">LOC_Os09g36810</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"EU603286\">EU603286</ext-link></td><td align=\"center\">2</td><td align=\"center\">7</td><td align=\"center\">Mx, Sd</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">61</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">Up: AnX, Na₃AsO₄, NaCl Dw: CKl</td></tr><tr><td align=\"center\"><italic>OsBgal12</italic></td><td align=\"center\">10005757.1</td><td align=\"center\">AP008216.1, DP000086.1</td><td align=\"center\">10</td><td align=\"center\">Os.46702</td><td align=\"center\">Os10g0330600</td><td align=\"center\">LOC_Os10g18400</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK119350\">AK119350</ext-link></td><td align=\"center\">3</td><td align=\"center\">3</td><td align=\"center\">Mx, Fw</td><td align=\"center\">0</td><td align=\"center\">7</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">Up: AnX</td></tr><tr><td align=\"center\"><italic>OsBgal13</italic></td><td align=\"center\">ND</td><td align=\"center\">AP008218.1</td><td align=\"center\">12</td><td align=\"center\">Os.52193</td><td align=\"center\">Os12g0429200</td><td align=\"center\">LOC_Os12g24170</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK065546\">AK065546</ext-link></td><td align=\"center\">2</td><td align=\"center\">36</td><td align=\"center\">Mx, St, Lf, Fw, UnT, P, Wp, Cl</td><td align=\"center\">6</td><td align=\"center\">21</td><td align=\"center\">34</td><td align=\"center\">15</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">55</td><td align=\"center\">0</td><td align=\"center\">Up: CKl, AB&amp;GBd, AB&amp;GB Dw: AnX, Na₃AsO₄, NaCl</td></tr><tr><td align=\"center\"><italic>OsBgal14</italic></td><td align=\"center\">10006090</td><td align=\"center\">ND</td><td align=\"center\">10</td><td align=\"center\">Os.22528</td><td align=\"center\">J090043H02</td><td align=\"center\">LOC_Os10g19960</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK242960\">AK242960</ext-link></td><td align=\"center\">2</td><td align=\"center\">58</td><td align=\"center\">Fw, P, UnT, Sd, Wp</td><td align=\"center\">0</td><td align=\"center\">358</td><td align=\"center\">0</td><td align=\"center\">7</td><td align=\"center\">0</td><td align=\"center\">61</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">-</td></tr><tr><td align=\"center\"><italic>OsBgal15</italic></td><td align=\"center\">1003798.1</td><td align=\"center\">AP004733</td><td align=\"center\">6</td><td align=\"center\">NF</td><td align=\"center\">AP004733</td><td align=\"center\">LOC_Os06g42310</td><td align=\"center\"><ext-link ext-link-type=\"gen\" xlink:href=\"EU051629\">EU051629</ext-link></td><td align=\"center\">ND</td><td/><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">ND</td><td align=\"center\">-</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Predicted rice GH family 35 β-galactosidase protein properties and locations.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Gene Name</td><td align=\"left\">Gene Locus <break/>(NCBI, RGP)</td><td align=\"center\" colspan=\"3\">Pre-protein</td><td align=\"center\" colspan=\"5\">Mature Protein</td></tr><tr><td/><td/><td colspan=\"3\"><hr/></td><td colspan=\"5\"><hr/></td></tr><tr><td/><td/><td align=\"left\">MW^a(kDa)</td><td align=\"left\">AA^b</td><td align=\"left\">Cleavage site^c</td><td align=\"left\">MW^a</td><td align=\"left\">AA^b</td><td align=\"left\">pI^a</td><td align=\"center\">N-gly site^d</td><td align=\"left\">Possible destination^e</td></tr></thead><tbody><tr><td align=\"left\"><italic>OsBgal1</italic></td><td align=\"left\">Os03g0165400</td><td align=\"left\">92.2</td><td align=\"left\">841</td><td align=\"left\">25–26</td><td align=\"left\">89.8</td><td align=\"left\">816</td><td align=\"left\">5.78</td><td align=\"center\">2</td><td align=\"left\">Gol, Vac, ER ext</td></tr><tr><td align=\"left\"><italic>OsBgal2</italic></td><td align=\"left\">Os06g0573600</td><td align=\"left\">78.3</td><td align=\"left\">715</td><td align=\"left\">20–21</td><td align=\"left\">76.5</td><td align=\"left\">695</td><td align=\"left\">7.65</td><td align=\"center\">0</td><td align=\"left\">Gol, PM, IMP, MII, ER ext</td></tr><tr><td align=\"left\"><italic>OsBgal3</italic></td><td align=\"left\">Os01g0580200</td><td align=\"left\">91.5</td><td align=\"left\">827</td><td align=\"left\">24–25</td><td align=\"left\">89.5</td><td align=\"left\">803</td><td align=\"left\">5.59</td><td align=\"center\">2</td><td align=\"left\">Gol, PM, IMP, MII, ER, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal4</italic></td><td align=\"left\">Os01g0875500</td><td align=\"left\">94.7</td><td align=\"left\">851</td><td align=\"left\">29–30</td><td align=\"left\">91.7</td><td align=\"left\">822</td><td align=\"left\">6.76</td><td align=\"center\">6</td><td align=\"left\">Gol, Cis-Gol, MII, ER, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal5</italic></td><td align=\"left\">Os01g0533400</td><td align=\"left\">91.7</td><td align=\"left\">827</td><td align=\"left\">25–26</td><td align=\"left\">88.9</td><td align=\"left\">802</td><td align=\"left\">5.66</td><td align=\"center\">8</td><td align=\"left\">Gol, MII, ER</td></tr><tr><td align=\"left\"><italic>OsBgal6</italic></td><td align=\"left\">P0636F09.15</td><td align=\"left\">90.6</td><td align=\"left\">811</td><td align=\"left\">17–18</td><td align=\"left\">88.9</td><td align=\"left\">794</td><td align=\"left\">6.61</td><td align=\"center\">5</td><td align=\"left\">IMP, MC in, Sec, Cy, N</td></tr><tr><td align=\"left\"><italic>OsBgal7</italic></td><td align=\"left\">Os02g0219200</td><td align=\"left\">78</td><td align=\"left\">729</td><td align=\"left\">36–37</td><td align=\"left\">76.8</td><td align=\"left\">693</td><td align=\"left\">8.71</td><td align=\"center\">0</td><td align=\"left\">Gol, MII, ER, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal8</italic></td><td align=\"left\">Os03g0255100</td><td align=\"left\">103.7</td><td align=\"left\">956</td><td align=\"left\">62–63</td><td align=\"left\">96.5</td><td align=\"left\">894</td><td align=\"left\">5.62</td><td align=\"center\">7</td><td align=\"left\">PM, IMP, ER, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal9</italic></td><td align=\"left\">Os05g0539400</td><td align=\"left\">75.7</td><td align=\"left\">673</td><td align=\"left\">20–21</td><td align=\"left\">73.5</td><td align=\"left\">653</td><td align=\"left\">5.57</td><td align=\"center\">6</td><td align=\"left\">Vac-Lys, IMP, MC in, Per, ERl, N, Sec, Cy</td></tr><tr><td align=\"left\"><italic>OsBgal10</italic></td><td align=\"left\">Os08g0549200</td><td align=\"left\">94.7</td><td align=\"left\">848</td><td align=\"left\">23–24</td><td align=\"left\">92.8</td><td align=\"left\">825</td><td align=\"left\">9.1</td><td align=\"center\">5</td><td align=\"left\">Vac-Lys, PM, IMP, MC in, ER, Cy, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal11</italic></td><td align=\"left\">Os09g0539200</td><td align=\"left\">94.11</td><td align=\"left\">838</td><td align=\"left\">25–26</td><td align=\"left\">91.9</td><td align=\"left\">813</td><td align=\"left\">6.52</td><td align=\"center\">3</td><td align=\"left\">Vac-Lys, Gol, PM, IMP, MII, Mic, ER, MC in, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal12</italic></td><td align=\"left\">Os10g0330600</td><td align=\"left\">92</td><td align=\"left\">828</td><td align=\"left\">23–24</td><td align=\"left\">89.7</td><td align=\"left\">805</td><td align=\"left\">6.07</td><td align=\"center\">10</td><td align=\"left\">Gol, MII, ER, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal13</italic></td><td align=\"left\">Os12g0429200</td><td align=\"left\">101.0</td><td align=\"left\">919</td><td align=\"left\">31–32</td><td align=\"left\">97.9</td><td align=\"left\">888</td><td align=\"left\">5.56</td><td align=\"center\">5</td><td align=\"left\">IMP, ER, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal14</italic></td><td align=\"left\">J090043H02</td><td align=\"left\">92.2</td><td align=\"left\">828</td><td align=\"left\">28–29</td><td align=\"left\">89.6</td><td align=\"left\">800</td><td align=\"left\">5.99</td><td align=\"center\">9</td><td align=\"left\">PM, IMP, MII, ER, Sec</td></tr><tr><td align=\"left\"><italic>OsBgal15</italic></td><td align=\"left\">AP004733</td><td align=\"left\">89.6</td><td align=\"left\">809</td><td align=\"left\">23–24</td><td align=\"left\">87.3</td><td align=\"left\">789</td><td align=\"left\">6.17</td><td align=\"center\">8</td><td align=\"left\">Gol, MII, ER, Sec</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Identification of LTR-retrotransposon elements and DNA sequences for interspersed repeats and low complexity DNA sequences within <italic>OsBgal6 </italic>and <italic>OsBgal13</italic>.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Genes</td><td align=\"center\" colspan=\"7\">LTR-retrotransposon elements and DNA sequences for interspersed repeats</td></tr><tr><td/><td colspan=\"7\"><hr/></td></tr><tr><td/><td align=\"left\">intron 1</td><td align=\"left\">intron 3</td><td align=\"left\">intron 8</td><td align=\"left\">intron 10</td><td align=\"left\">intron 13</td><td align=\"left\">intron 14</td><td align=\"left\">intron 16</td></tr></thead><tbody><tr><td align=\"left\"><italic>OsBgal6</italic></td><td align=\"left\">Stowaway41_OS, OLO24C, OLO24B, RPO_OS, AT rich Low Complexity, AMYLTP, TA rich Low Complexity, OSTE15</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"><italic>OsBgal13</italic></td><td align=\"left\">GC rich Low complexity</td><td align=\"left\">AT rich Low complexity</td><td align=\"left\">MUDRN2</td><td align=\"left\">OSTE28, OSTE33, CRM-I_OS LTR/Gypsy,</td><td align=\"left\">OSLINE1-5, AT rich Low complexity</td><td align=\"left\">AT rich Low complexity</td><td align=\"left\">RdSpm875A_3.1, ID-2 DNA/Tourist</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Hydrolysis of <italic>p</italic>-nitrophenyl glycosides, oligosaccharides and polysaccharides by OsBgal13.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Substrate</td><td align=\"center\">Activity</td></tr></thead><tbody><tr><td align=\"left\"><italic>p</italic>NP-glycosides</td><td align=\"center\">(Percent Relative Activity)</td></tr><tr><td align=\"left\"><italic>p</italic>NP-β-D-galactopyranoside</td><td align=\"center\">100</td></tr><tr><td align=\"left\"><italic>p</italic>NP-β-D-glucopyranoside</td><td align=\"center\">N.D.</td></tr><tr><td align=\"left\"><italic>p</italic>NP-β-D-fucopyranoside</td><td align=\"center\">7.4</td></tr><tr><td align=\"left\"><italic>p</italic>NP-β-D-mannopyranoside</td><td align=\"center\">4.5</td></tr><tr><td align=\"left\"><italic>p</italic>NP-β-D-xylopyranoside</td><td align=\"center\">N.D.</td></tr><tr><td align=\"left\"><italic>p</italic>NP-β-L-arabinopyranoside</td><td align=\"center\">30</td></tr><tr><td align=\"left\"><italic>p</italic>NP-β-D-galactopyranoside</td><td align=\"center\">1.5</td></tr><tr><td colspan=\"2\"><hr/></td></tr><tr><td align=\"left\">Oligosaccharides</td><td align=\"center\">(estimated relative activity)</td></tr><tr><td align=\"left\">β-1,3-galactobiose</td><td align=\"center\">+++</td></tr><tr><td align=\"left\">β-1,3-galactotriose</td><td align=\"center\">++</td></tr><tr><td align=\"left\">β-1,4-galactobiose</td><td align=\"center\">++ *</td></tr><tr><td align=\"left\">β-1,4-galactotriose</td><td align=\"center\">++ *</td></tr><tr><td align=\"left\">β-1,6-galactobiose</td><td align=\"center\">+++</td></tr><tr><td align=\"left\">β-1,6-galactotriose</td><td align=\"center\">++</td></tr><tr><td align=\"left\">Arabinogalactan, xylan from oat spelts, birchwood xylan, polygalacturonic acid, apple pectin, citric pectin, galactan</td><td align=\"center\">N.D.</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Sequences of primers used for semi-quantitative RT-PCR analysis of β-galactosidase gene expression.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Gene name</td><td align=\"left\">GenBank ID</td><td align=\"left\">Sense primer</td><td align=\"left\">Antisense primer</td><td align=\"left\">Annealing Tm (°C)</td></tr></thead><tbody><tr><td align=\"left\">Actin</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK100267\">AK100267</ext-link></td><td align=\"left\">ACTCTGGTGATGGTGTCAGCC</td><td align=\"left\">GTCAGCAATGCCAGGGAACATA</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>UBQ6</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"XM464194\">XM464194</ext-link></td><td align=\"left\">TCCTCCGTCTCAGGGGAG</td><td align=\"left\">CTTGCCAGCGAAGATCAGAC</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal1</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK102192\">AK102192</ext-link></td><td align=\"left\">CAAAGCACACAGAAAGCGAT</td><td align=\"left\">TGCTCACCGCACAATCAACGA</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal2</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK102756\">AK102756</ext-link></td><td align=\"left\">AGGAAAGTGGGGGCGTATAG</td><td align=\"left\">TCCCATTTACAACTCAACGT</td><td align=\"left\">56</td></tr><tr><td align=\"left\"><italic>OsBgal3</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK103045\">AK103045</ext-link></td><td align=\"left\">CCAAGGGGCTGTATGTATGGTC</td><td align=\"left\">CCTGAGAGAATTCATTCACATACGG</td><td align=\"left\">56</td></tr><tr><td align=\"left\"><italic>OsBgal4</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK102715\">AK102715</ext-link></td><td align=\"left\">TCCATCGCTACAGATTCGCTC</td><td align=\"left\">TCCAGAAATATCATGACGCGAC</td><td align=\"left\">56</td></tr><tr><td align=\"left\"><italic>OsBgal5</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK119447\">AK119447</ext-link></td><td align=\"left\">CTCATCTGCTTGCTTCATC</td><td align=\"left\">CTAAAGTTGCCCTTCTCATC</td><td align=\"left\">50</td></tr><tr><td align=\"left\"><italic>OsBgal6</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"CR292731\">CR292731</ext-link></td><td align=\"left\">AGTCTTGCATAGGCAGGAG</td><td align=\"left\">TCTGAACGAAGGTATCGCAC</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal7</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK059059\">AK059059</ext-link></td><td align=\"left\">CCACCATTTGATACAGTCGTCG</td><td align=\"left\">TTCCCGAGCAACGCAAAC</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal8</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK067479\">AK067479</ext-link></td><td align=\"left\">CCTGACAGGTTTGATAGTGCTCG</td><td align=\"left\">TGCTTTTCTTCACACAAGTGCATC</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal9</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK068572\">AK068572</ext-link></td><td align=\"left\">GAAGGATCCAGATTTCACATGC</td><td align=\"left\">TGCTGTTCATGTCATCATGTGC</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal10</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK069066\">AK069066</ext-link></td><td align=\"left\">TCCAAGAGGCCTCCTCGTTC</td><td align=\"left\">CTACATATAAAACCATGGACGATGGTG</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal11</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK119414\">AK119414</ext-link></td><td align=\"left\">GTGCAGGTGAGATGCAAGGTATC</td><td align=\"left\">CAAACTGTCTGTCAACCTGTGATGG</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal12</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK119350\">AK119350</ext-link></td><td align=\"left\">CAACACAGCAAAACCATCT</td><td align=\"left\">TTCCACGAAACAAAGTAAAGACA</td><td align=\"left\">56</td></tr><tr><td align=\"left\"><italic>OsBgal13</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK065546\">AK065546</ext-link></td><td align=\"left\">CCGAGGAGTCCTCAAAGATTTAGC</td><td align=\"left\">GGAAATCTCCTTTGCATTTTTATTCAC</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal14</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AK242960\">AK242960</ext-link></td><td align=\"left\">GTCGAAGGTGGCTTATGACG</td><td align=\"left\">AATCGACAGTGCGGTATCTC</td><td align=\"left\">54</td></tr><tr><td align=\"left\"><italic>OsBgal15</italic></td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AP004733\">AP004733</ext-link></td><td align=\"left\">CGTACAAGGCTTTCACAGAAG</td><td align=\"left\">CTACGATTACTTGATCACACTC</td><td align=\"left\">54</td></tr></tbody></table></table-wrap>" ]

[]

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[]

[]

[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p><bold>Relative expression levels of 15 <italic>OsBgal </italic>genes in different tissues determined by semiquantitative RT-PCR</bold>. Signals were quantified and normalized to the expression of β-actin. The values given are means with standard deviations from triplicate experiments.</p></caption></supplementary-material>" ]

[ "<table-wrap-foot><p>^aContig number in Beijing Genome Institute (the numbers given are the Genbank Accession without the initial 'AAAA').</p><p>^bChromosome locations from mapping of the corresponding genes on the 12 rice chromosomes in GenBank.</p><p>^cAccessions of full-length cDNA clones of japonica rice databases [##UREF##15##64##] or indica clones from this study (<italic>OsBgal6</italic>, <italic>OsBgal11 </italic>and <italic>OsBgal15</italic>).</p><p>^dThe number of mRNA and EST identified by UniGene from the NCBI Genbank database.</p><p>^eTissue libraries means the source tissue from which the corresponding ESTs were isolated. Tissue abbreviations are: Cl: callus, Fw: Flower, Lf: leaf, P: Panicle, Rt: root, Sd: Seed, St: Stem, Mx: Mixed Tissues, Vm: Vegetative meristem, Wp: whole plant, UnT: unspecified tissue.</p><p>^fExpression profile means transcript frequency per million calculated from the number of ESTs in a certain library. ND: not determined.</p><p>^gGEO (Gene Expression Omnibus public repository of microarray and other forms of high-throughput data submitted by the scientific community). Abbreviations are: Up: up-regulated, Dw:down-regulate, CKrl: Cytokinin effect on rice roots and leaves: time course, CKl: Cytokinin-inducible type-A response regulator OsRR6 overexpression effect on rice leaves, AB&amp;GB: Abscisic acid and gibberellin effect on calluses, gene expression array-based, log ratio, AB&amp;GBd: Abscisic acid and gibberellin effect on calluses (dye-swap), AnX: Anoxia effect on rice coleoptiles, NaCl: Salinity stress response, Na₃AsO₄: Sodium arsenate effect on Azucena and Bala varieties.</p></table-wrap-foot>", "<table-wrap-foot><p>^adetermined by ProtParam. ^bAA means number of amino acids. ^cpredicted by SignalP [##UREF##7##35##]. ^dpredicted by NetNGlyc at the Expasy proteomics server <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.expasy.org\"/> and manually curated to remove NPS/T sites, ^ecellular locations predicted by PSORT [##REF##2440339##65##], abbreviated as PM: plasma membrane, MII: Type II membrane protein, Gol: Golgi, IMP: Integral Membrane Protein, Sec: Secreted, ER: Endoplasmic reticulum; ext: extracellular; l: lumen, MC: Mitochondrial; in: inner membrane, Cy: Cytoplasmic, N: nuclear, Vac: vacuoles, Vac-Lys: Lysosome-like vacuoles, Per: Peroxisomal, and Mic: microsomal.</p></table-wrap-foot>", "<table-wrap-foot><p>RepeatMasker <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker\"/> was used to identify these sequences.</p></table-wrap-foot>", "<table-wrap-foot><p>N.D. means not detectable. * For β-1,4-galactotriose and β-1,4-galactobiose transglycosylation was observed as much or more than hydrolysis.</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1471-2229-8-84-1\"/>", "<graphic xlink:href=\"1471-2229-8-84-2\"/>", "<graphic xlink:href=\"1471-2229-8-84-3\"/>", "<graphic xlink:href=\"1471-2229-8-84-4\"/>", "<graphic xlink:href=\"1471-2229-8-84-5\"/>" ]

[ "<media xlink:href=\"1471-2229-8-84-S1.xls\" mimetype=\"application\" mime-subtype=\"vnd.ms-excel\"><caption><p>Click here for file</p></caption></media>" ]

{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Insufficient nutritional intake and consequent weight loss during disease remains a serious problem for many elderly inpatients. There is ample reason for countering under nutrition during hospitalisation, among others because weight loss resulting from disease is harmful to all elderly people, whether over- or underweight [##UREF##0##1##]. Many patients eat and drink insufficiently during hospitalisation, and 30 to 50% of elderly patients are undernourished [##REF##16457988##2##, ####REF##10459070##3##, ##REF##10895110##4##, ##REF##16280442##5####16280442##5##], and the protein and energy requirements of most of these patients are not met [##REF##10459070##3##,##REF##15363101##6##, ####REF##12468365##7##, ##REF##17608883##8##, ##REF##17419798##9####17419798##9##]. The clinical consequences include lassitude, difficulty in mobilising, prolonged convalescence [##REF##10459070##3##,##REF##11884007##10##, ####REF##16698132##11##, ##REF##12765661##12##, ##REF##18195138##13##, ##REF##17635309##14####17635309##14##] and an increased risk of pressure wounds [##REF##17662510##15##], phlebitis and infections [##UREF##1##16##,##REF##8666762##17##].</p>", "<p>Intervention studies have shown that patients' protein and energy intake can be increased significantly through optimisation of the nutritional care using the available hospital food [##REF##11104597##18##, ####REF##15980933##19##, ##REF##12171454##20##, ##REF##8676539##21##, ##REF##17662509##22####17662509##22##].</p>", "<p>To combat under nutrition and its clinical consequences, it appears relevant to incorporate the nutritional care as a priority area in the daily practice of nursing care. Normally, in Danish hospitals the nursing team is jointly responsible for the patients' nutritional care. Nevertheless, several studies suggest that the nutritional care in daily practice is given a low priority in the nursing care.</p>", "<p>Some of the identified factors that inhibit optimal nutritional care at the wards are lack of time and interest in nutrition care among the nursing staff, and the collective nature of the responsibility for the practical implementation of nutritional care. Even if responsibility is collective, only few staff members are actively engaged [##REF##15363101##6##,##REF##16701921##23##, ####REF##17036740##24##, ##REF##16835596##25##, ##REF##16457707##26##, ##REF##17383776##27##, ##REF##17882130##28####17882130##28##]. So the question is \"How do we in daily practice redress this situation?\". An organisational model in which a proffessional is hired exclusively to take care of tasks related to nutrition at the bed wards meets the criteria required to raise nutritional standards at the departmental level; the assistant's time is devoted to provide individual nutritional care to nibblers, she has intimate knowledge of and takes an interest in nutrition, and she has general knowledge of the food range offered by the production kitchen with which she is in close contact [##REF##16457707##26##]. In this study we will focus on these aspects; and the aim is to implement and evaluate an organisational model which has a clear division of responsibilities and competencies in the nutritional care with a view to eliminating factors inhibiting optimal nutritional care.</p>" ]

[ "<title>Methods</title>", "<title>Setting</title>", "<p>Three medical wards specialised in lung, gastric and liver disease, and endocrinology at the Medical Centre at Bispebjerg Hospital (Copenhagen Hospital Co-operation) participate in the study. The number of beds at the wards is 20, 22 and 20, respectively. The monthly bed occupancy rate is 95 to 103%. Patients are hospitalised for eight days on average and are usually admitted acutely (data printout from the Office of Economic Affairs, Bispebjerg Hospital).</p>", "<p>The hospital food is produced in the central hospital kitchen and transported daily to the wards. At fixed times on a weekly basis, groceries, dairy products, single-portion frozen meals and the like are delivered when ordered by the wards. Each ward has a kitchen with facilities for limited cooking, like baking or preparation of small dishes.</p>", "<title>Design of the intervention study</title>", "<p>The pre-intervention parts of data acquisition include:</p>", "<p>1) To interview medical inpatients at nutritional risk about the nutritional care (Patient group A)</p>", "<p>2) To interview occupational groups about the nutritional care in daily practise</p>", "<p>3) To provide supplementary training to three social and healthcare assistants to upgrade them to nutritional and healthcare assistants</p>", "<p>The elements in the intervention at three wards are:</p>", "<p>4) The nutritional and healthcare assistant at each ward provides nutritional care to patients who are assessed as being at nutritional risk (Patient group C) and has the responsibility for ordering food, recording food wastage, etc.</p>", "<p>Data on the effect of the intervention are obtained by:</p>", "<p>5) Interviewing medical inpatients about the nutritional care (Patient group C at the ward receiving nutritional care and patient group B at the ward not receiving nutritional care from the nutritional and healthcare assistant)</p>", "<p>6) Interviewing occupational groups about the nutritional care in daily practise in the intervention</p>", "<p>7) Investigating the effect on the food supply resources.</p>", "<p>At the participating wards, no other organisational changes in the nutritional care are carried out during the intervention period. The patients and staff receive oral information about the investigation, underlining the voluntary and anonymous nature of their participation. The study fulfills the Declaration of Helsinki II. The quantitative methods, use of factual and describing data fall outside the framework of the Local Scientific Ethics Committee. The management at the Medical Centre at Bispebjerg Hospital has approved the study.</p>", "<title>Training programme</title>", "<p>The educational background of social and healthcare assistants in Denmark is two years of education and ten months of training with a focus on nursing and care of elderly, ill and handicapped persons in relation to personal care, nursing, cooking, etc. The three participating social and healthcare assistants are chosen because they have much interest in and experience from previous jobs with regard to nursing care of nibblers, elderly and patients with senile dementia and lung disease. In this study the assistants receive supplementary education and training in a one-month training programme specified in Table ##TAB##0##1##. Different professionals such as clinical dietician, psychologist, nurses and a catering officer serve as teachers in the disciplines related to clinical expertise and organisational pedagogy. A nursing officer has the responsibility for the training in practical skills and organisation at the wards. The senior staff nurses are taught how to fill the function of co-operator for the assistants focusing on the improvement of the nutritional care through behavioural changes [##UREF##2##29##]. At meetings the nursing officer informe the nursing staff at the wards about the new job function, which is illustrated by a video film [##UREF##3##30##].</p>", "<title>Patient screening</title>", "<p>Using the Nutritional Risk Screening 2002, a registered nurse conducts a nutrition screening of adult patients within 24 hours after admission to assess the patient's nutritional risk [##REF##12765673##31##]. If the registered nurse assesses the patient as being at nutritional risk, nutritional therapy is to be initiated. The following parameters are assessed: the patient's energy and protein requirements, three-day dietary records, the patient's protein and energy intake; and individual nutritional care is planned to meet the patient's nutritional needs [##UREF##4##32##]. In the intervention the registered nurse has the possibility of delegating the responsibility for the nutritional therapy to the nutritional and healthcare assistant working in day duty. The registered nurses select the patients entering the study. The assistant is professionally trained to work actively with nutritional care and has the necessary time to do so in practice. The patients included are typical for the wards.</p>", "<title>The job function of the nutritional and healthcare assistant</title>", "<p>The work of the nutritional and healthcare assistant is a full time job to ensure that the patients who are assessed as being at nutritional risk have their nutritional and fluid requirements met; she is responsible for the following:</p>", "<p>- The patient is neat and clean at meal times and receives medication with the meal</p>", "<p>- The patient sits or lies in the best possible position when eating the meal</p>", "<p>- The patient eats the meal in a comfortable and aesthetic environment and receives the required help to eat the meals</p>", "<p>- The patient is informed about nutritional and dietary options available from the production kitchen</p>", "<p>- The patient is offered appetising small dishes, in-between meals and home baking, fluid and supplements that meet the individual patient's protein, energy and fluid requirements.</p>", "<p>In relation to her colleagues she is responsible for</p>", "<p>- Preparing individual meals, in-between meals, home baking etc.</p>", "<p>- Planning and assigning priorities to her own work time, ensuring that the nutritional and fluid requirements of undernourished patients are met as far as possible</p>", "<p>- Working together with the senior staff nurse to implement the new job function</p>", "<p>- Working together with the nursing staff and the kitchen assistant on the tasks related to the nutritional care</p>", "<p>- Ordering food from the production kitchen and adjusting the orders on a daily basis according to the patients' requirements and wishes</p>", "<p>- Co-operation between the ward and the production kitchen in regard to food service</p>", "<p>The nutritional and healthcare assistant and the senior staff nurse are jointly responsible for ensuring that all nursing team members have updated knowledge of nutrition and adhere to the relevant hygiene and nutritional guidelines, cf. the accreditation [##UREF##4##32##]. The nutritional and healthcare assistant and the nursing staff are jointly responsible for documenting the patient's nutritional status, nutritional and fluid requirements and intake and to keep records of the amount of food ordered and food wastage.</p>", "<title>The effect of the intervention</title>", "<p>Before the intervention is started and one month before it is terminated, the investigator assesses the effect on the basis of the data collected from a) structured interviews with 75 patients, b) structured interviews with the ward and production kitchen staff, c) the records of the amounts of food ordered and food waste at the wards (data was obtained from the nutritional and healthcare assistants).</p>", "<title>Evaluation by the patients</title>", "<title>The patients</title>", "<p>Medical patients of all ages participate. The inclusion criteria are defined as: 1) the patient is hospitalised for at least five days, 2) the patient is capable of communicating, and 3) the patient eats and drinks a normal diet. The exclusion criteria are defined as: 1) patients with dementia and 2) patients who are severely mentally or physically impaired. These exclusion criteria are based on ethical considerations. The registrered nurses select the patients who meet the criteria, and the patients are included consecutively. The patients receive oral information about the investigation, underlining the voluntary and anonymous nature of their participation. Verbal consent are given to investigator by the study participants.</p>", "<title>The questionnaire</title>", "<p>The questionnaire is based on a questionnaire developed and used in [##REF##16457707##26##]. The questions are designed to focus on issues believed to be related to the patients' nutritional intake and to reflect their experience with the nutritional care they receive. The questionnaire has been validated through interviews with five medical patients to secure that the questions are unambiguous; that the patients understand the questions and that the order of the questions is correct. The questionnaire is used for the structured interviews of the patients. The patient has the option of elaborating on all answers through comments [##UREF##5##33##], and the comments are written down word-by-word. All interviews are conducted by the same interviewer.</p>", "<title>Structured interviews</title>", "<p>Before the intervention, a total of 30 patients take part in individual structured interviews (group A). The investigator, a research scientist, contacts the patient who receives oral information about the study, and the voluntary nature of the participation is emphasised. No drop-out analysis is made when patients are selected and contacted. In the interview, the investigator reads the questions aloud to the patient, ticks the appropriate boxes according to the patient's answers and writes down any comments the patient has. After five months' intervention, structured interviews are conducted with 25 patients not receiving nutritional care from the nutritional and healthcare assistant (patient group B), and with 20 patients receiving nutritional care from the nutritional and healthcare assistant (patient group C). The criteria for inclusion are unchanged.</p>", "<title>Analysing data</title>", "<p>The distribution of the quantitative data is analysed. Patient comments are printed and analysed as text [##UREF##6##34##]. The presentation of the quantitative data is supplemented by qualitative data from patient comments.</p>", "<title>Evaluation by the staff</title>", "<p>Before and after five months of intervention, the proffesional groups are interviwed in eight focus groups about their work with nutritional care, their responsibilities and their experience in nutritional care as practiced [##UREF##7##35##]. These groups include senior staff nurses, managers and representatives from the production kitchen, nutritional and healthcare assistants, registered nurses, social and healthcare helpers and assistants. A total of 34 and 30 informants participate before and after five months intervention, respectively. The focus in the interviews concerns to identify where in the system changes related to optimum nutritional care are taking place. The interviews are tape-recorded with the informant's permission. The transcribed interviews are analysed as a text with focus on central and opinion-forming topics relating to the intervention study [##UREF##7##35##].</p>", "<title>The amount of food ordered</title>", "<p>Quantitative data are collected about 1) deliveries from the production kitchen to the participating wards during October and November of the calendar years before and during the intervention, 2) the number of patients and 3) bed days during these periods. For all wards at the Medical Centre, data are collected about food budgets and food consumption in the calendar year before and during the employment of the nutritional and healthcare assistants (data printout from the production kitchen, Bispebjerg Hospital).</p>", "<p>The total monthly food supply and daily food supply per patient on the participating wards before and during the intervention are calculated. The corresponding values are calculated for four non-participating medical wards at the Medical Centre at Bispebjerg Hospital.</p>", "<title>The amount of food waste</title>", "<p>Before the intervention, the amount of food waste in the food containers is recorded for three randomly chosen days: two investigators do the visual assessment in regard to how large a share of the ordered food is left after the staff has served the patients. During the intervention period, the nutritional and healthcare assistants daily as a part of her job records the following: 1) number and type of delivered portions, 2) visual assessment of the number of left-over portions and 3) where relevant they estimate the number of missing portions. On weekday day duty these recordings are carried out by the nutritional and healthcare assistant. On evening, night and weekend duty they are carried out by the other nursing staff. The collected data do not take into account any plate waste or food eaten by staff members.</p>", "<title>Data processing</title>", "<p>The amount of food waste at breakfast, lunch, supper and \"late evening\" (meal served at 8 pm) is calculated as percentages. Data from the first 10 weekdays of June, October and December are presented.</p>" ]

[ "<title>Results</title>", "<title>Evaluation by the patients</title>", "<title>Patient characteristics</title>", "<p>Participants are elderly medical patients with chronic disease. Three out of five are mobility-impaired (Table ##TAB##1##2##). Typical patient diagnoses include acute or chronic lung disease, infectious disease and metabolic disorders. Five to eight out of ten patients in the three groups have experienced unwanted weight loss recently before admission to hospital. To estimate the unwanted weight loss before admission, the patients are asked to estimate their weight loss and the number of days during which they have experienced a weight loss. The patient-reported average daily weight loss is 160 grams. Although this figure has to be judged with caution, it does give an indication of the level of weight loss in this group of patients.</p>", "<title>The patient's dialogue with staff</title>", "<p>The results from the structured interviews with the patients show that during their hospitalisation, one to two thirds of the patients who do not receive nutritional care from the nutritional and healthcare assistants (patient groups A and B) have spoken with other staff members about weight loss (Table ##TAB##1##2##). Patients receiving nutritional care from the nutritional and healthcare assistants (patient group C) have all spoken with the nutritional and healthcare assistant about weight loss. More than two thirds of the respondents have eaten less than usual for short or long periods of time (Table ##TAB##2##3a##). Many gave as a reason that they have less or no appetite. A number of patients have had diarrhoea, emesis, pain or low spirits, which have caused them to eat scantily or not at all. Many patients in groups A and B express that they <italic>\"don't feel like eating the food. If I had other choices, I would eat more\"</italic>. Some patients state that they do not have enough time at meal times: <italic>\"Everything has to go so fast at meal times – I can't keep up\"</italic>.</p>", "<p>Patients with reduced food and fluid intake are asked whether the staff has attempted to ensure that their nutritional intakes are improved. Half of the patients in group A, one third in group B and two thirds in group C answer \"yes\" to this question (Table ##TAB##2##3b##). Patients in groups A and B generally answered, <italic>\"The staff has asked whether I would like other things\", \"They give me water\"</italic>, and <italic>\"They give me protein drinks\"</italic>. However, the patients find that the offers made by the nursing staff are insufficient.</p>", "<p>Several of these patients have tried themselves to make an effort to eat more, for instance by asking for ice cream or having family members to bring them food. One patient express it as follows, <italic>\"I must do something to be able to go home. I asked for a protein drink today – it wasn't cold – but I hope it was fattening\"</italic>. Several patients notice this lack of focus on nutrition: one patient finds it <italic>\"rather odd for a hospital not to be leading the way, showing us how to eat</italic>. <italic>They have the opportunity\"</italic>. Several patients have specific suggestions for the nutritional care, like <italic>\"richer food\"</italic>, <italic>\"food I can chew\" </italic>and <italic>\"more soup, for instance split pea soup\"</italic>.</p>", "<p>Patients in group C who have had days with reduced nutritional intake experience that the nutritional and healthcare assistant takes special care of them: <italic>\"It seems to me she does everything\"</italic>. <italic>\"She comes in later and offers me a second serving. Half an hour before the meal, she brings me an anti-nausea tablet\"</italic>. <italic>\"She takes special care of us. I missed her at the weekend\"</italic>. The patients find that <italic>\"the food looks more delicious when the portions are smaller. It makes it more palatable – otherwise you give up from the start\"</italic>. The nutritional and healthcare assistant is also reported to be <italic>\"good at enticing me: she keeps coming back, reminding me how important proteins are. I feel that she's genuinely interested in each individual person's need for food\"</italic>. Patients in group C are also aware that the nutritional and healthcare assistant and the staff enter into records what and how much they eat and drink, and <italic>\"if I have eaten or drunk too little, she discusses it with me\"</italic>. The knowledge acquired by the patients through their dialogue with the nutritional and healthcare assistant is translated directly into action. For instance the patients deliberately chose certain kinds of foods after a conversation with the staff: \"<italic>I have been told a lot about how I should eat more so my weight will be OK and I can get out of here\" </italic>and <italic>\"She tells me that milk is better for me than fruit juice and that those mashed potatoes are better than boiled potatoes\"</italic>. The data suggest that there is a difference between the contents of the patients' dialogue about nutrition with the staff and the contents of their dialogue with the nutritional and healthcare assistant. An example is that the nutritional and healthcare assistant gives the patients specific advice on how to get optimum nutrition.</p>", "<title>The importance of nutrition for health</title>", "<p>The staff has communicated the importance of food to health to two thirds of the patients in group A, in group B one third and more than two thirds in group C (Table ##TAB##2##3c##). In groups A and B the patients express that the staff keeps telling them to eat and drink because <italic>\"if you want to get better you need to eat\"</italic>, and the nursing staff often tells them, <italic>\"You must eat now\"</italic>. Some patients feel pressured to eat, since <italic>\"if you reject the food they insist\"</italic>. Some patients in group B have been given the impression that food is important for their recovery because of <italic>\"the way fellow patients are treated by the nutritional and healthcare assistant\"</italic>; <italic>\"It is clearly an inspiration to everyone on the ward that the nutritional and healthcare assistant comes here. It makes us think about what we eat, too\"</italic>. In group C some patients say more specifically that <italic>\"the nutritional and healthcare assistant has discussed with me my lack of strength and has told me I need to eat more\"</italic>.</p>", "<p>Several patients have noticed that the nutritional and healthcare assistant and the other staff do not always share the same view of the importance of food for the care and treatment: <italic>\"the nutritional and healthcare assistant says that food is important, but the others only say that it's important to drink plenty\"</italic>. One patient has noticed a difference between the nutritional cares on day and night duty: <italic>\"Only the nutritional and healthcare assistant cares about it – the others don't really care. In particular, the staff on evening duty is really tired of keeping dietary records: they say, 'oh, what a load of rubbish\"'</italic>.</p>", "<p>The majority of all respondents believe that food is important for the speed of their recovery (Table ##TAB##2##3d##). Many patients express that they know that food is crucial for their recovery: <italic>\"I realise that if I don't eat and drink, I won't make it\"</italic>. Some patients notice that food also has much impact on their mood.</p>", "<title>Evaluation by the staff</title>", "<title>Evaluation by the nutritional and healthcare assistant</title>", "<p>It is a demanding task for the nutritional and healthcare assistants to build acceptance among their colleagues and to establish a new form of cooperation with a focus on providing nutritional care and managing resources in the nutritional care. The assistants find that their job function require significant dedication, good interpersonal skills, and a strong will to create room for themselves to fulfil their job function.</p>", "<p>The assistants find that nutritional care for the patient is the easiest task as <italic>\"the patients have been very open and receptive to my advice\"</italic>. The very contact with the patients is the best part of the new work function, because <italic>\"it's nice to have the possibility and the time to provide this sort of care. Normally, you simply don't have the time for it when you're running around cleaning and doing all sorts of other things\"</italic>.</p>", "<p>The number of inpatients receiving nutritional care from the nutritional and healthcare assistants varies between the three wards: on one ward 10 to 12 patients will usually receive help whereas on the other two wards the number will be four to eight patients. The assistants organise their daily work in accordance with the number of patients and the patients' condition. The assistants are generally very busy, attending to the nutritional care of six to eight patients. As a result of their work they experince that the majority of the patients at nutritional risk gain weight. The assistants receive mush positive feedback from patients expressing their gratitude for the care the assistants have provided.</p>", "<title>Division of responsibilities and tasks in the nutritional care</title>", "<p>The nursing staff on the three participating wards express that they have accepted the fact that the nutritional and healthcare assistants attend only to nursing tasks related to the nutritional care. For example, the nutritional and healthcare assistants do not respond to patient call bells, assist patients to the toilet or wash patients. Given that the nutritional and healthcare assistants have taken over the nutritional care of the weakest patients, the nursing staff finds that they have more time for their nursing work.</p>", "<p>According to the nursing staff, nutritional care tasks are in fierce competition with other nursing tasks for time and, sometimes, interest. As a result, nutritional care tasks are given a low priority. Very often no nursing team member wishes to take over the responsibility of the nutritional and healthcare assistant. Some nutritional care tasks, like keeping dietary records for patients who are nibblers, or recording 24-hour energy and protein intake of patients, are regarded by several nursing team members as boring work, which means that data in the dietary records will be missing or that there will be no continuity, and no decisions are made as to actions that can and shall be taken [##UREF##4##32##]. Similarly, it is difficult to ensure that the nutritional initiatives implemented and carried out by the nutritional and healthcare assistant on day duty are continued during evening and weekend duty.</p>", "<p>The nutritional and healthcare assistants and some senior staff nurses state that the senior staff nurse plays a key role in the successful implementation of the new work function, and that they need her support to further acceptance of the nutritional care and to place it on the ward agenda.</p>", "<title>Cooperation in the nutritional care</title>", "<p>The qualifications, dedication and knowledge of the nutritional and healthcare assistant are appreciated by the majority of the colleagues: <italic>\"it's very obvious that after she started in that project everyone pays a lot of attention to the patients' diet and we discuss it more\"</italic>. A nurse elaborates, <italic>\"I suppose we used to see eating as a necessity and never really thought about it as something that could improve their </italic>[the patients'] <italic>stay here. Meal times have always simply been something to be dispensed with quickly\"</italic>. For the nursing staff, and particularly for the newly employed, she serves as a resource person in the nutritional care; she knows the choices offered by the production kitchen and knows how to use these choices to meet patients' individual food requests. Many nursing team members are relieved that they no longer feel guilty about not giving patients sufficient nutrition, because they know that the nutritional and healthcare assistant on day duty will attend to the nibblers' nutritional care.</p>", "<p>The nutritional and healthcare assistant reminds the staff to discuss, plan and carry out nutritional tasks in the nursing care; <italic>\"Now action is taken much sooner whenever it's clear that things </italic>[nutritional care] <italic>are going in the wrong direction – because she </italic>[the nutritional and healthcare assistant] <italic>is there to point it out to us. If we were to be responsible for noticing it ourselves it might take longer\"</italic>. The assistant's attendance at morning conferences may strengthen the focus on nutritional issues and tasks and involve other nursing and medical staff. Thus, through her presence, knowledge and dedication the nutritional and healthcare assistant brings attention to the patients' nutritional care – and to the issue of overweight patients, too, because <italic>\"you have a tendency to do nothing, which is wrong, really. For, of course, patients loose the wrong things when they're in here. And that's when she steps in and tells us what we should do\"</italic>.</p>", "<p>The work of the nutritional and healthcare assistant make visible unnecessary tasks in the nutritional care, like for instance continuous dietary records that are not used in the nutritional care after all. The nursing staff has the experience that \"<italic>the patients feel taken special care of\" </italic>by the nutritional and healthcare assistant. For instance, <italic>\"when she bakes and they can smell fresh bread or cake, it gives them something to look forward to</italic>.</p>", "<title>Opposition to the organisation model</title>", "<p>A few nurses (14% of the senior staff nurses, registered nurses and social and healthcare helpers and assistants) show no appreciation of the importance of nutrition for the care and treatment of the patients, nor do they appreciate the fact that agreements to provide the nutritional care according to the accreditation requirements shall be observed. They are sceptical about the separation of the nutritional care of undernourished patients from the nursing care.</p>", "<p>In general, the nursing teams' experience that the nutritional care on evening and weekend duty is going <italic>\"downhill\" </italic>as compared with day duty, as <italic>\"it's the fundamental things that are dealt with: washing and changing – whatever's urgent at the time\"</italic>. Most staff members find that on evening and weekend duty <italic>\"placing a tray in front of the patient is no problem. But if you're supposed to give them special treatment – well, you can't. The most important thing is that they're washed and wear clean clothes, and that they get food – that is, if they get any. But, at least, you put a tray in front of them ...\"</italic>.</p>", "<title>The ordering of food</title>", "<p>Before the intervention, food is usually ordered from the production kitchen by a secretary, who orders a number of portions equivalent to the number of inpatients, but <italic>\"no one focused on what we needed on the ward to enable patients to get what they need\"</italic>. The nutritional and healthcare assistants' job function is to adjust the food orders to patients' wishes, needs and consumption of food and drink. As a result, according to the senior staff nurses, <italic>\"the new system ensures the ward gets the right things </italic>[food and drinks]<italic>\"</italic>. A nurse points out that <italic>there's always been this wide choice; we just haven't used it that effectively\"</italic>. After the employment of the nutritional and healthcare assistant, the nursing staff has <italic>\"realised that there are many options of special diets: vegetarian diet, soft diet, and the patient's foods of choice – I didn't know there were that many options. Before, we offered them something. If the patient doesn't like it? Then what? Should we ring the kitchen or what should we do?\"</italic>.</p>", "<p>The nursing staff accepts the fact that the intervention involves control of the consumption of food supplies, because <italic>\"it's quite nice that she sees to it that the standard is kept. All of the things prescribed by the accreditation, she knows all about that. That's the sort of thing you might usually compromise on, you know\"</italic>. For the nutritional and healthcare assistants, on the other hand, it is no easy task to <italic>\"come here and change routines. Like, for instance they weren't allowed to eat leftovers. I still sense that some staff members think all this is really odd\"</italic>. According to the assistants, it is unpleasant <italic>\"hitting barriers from colleagues\" </italic>in their work to optimise the use of food supply resources.</p>", "<title>The perspective of the staff in the production kitchen</title>", "<p>In the changed organisation, the staff in the production kitchen communicates directly with the nutritional and healthcare assistants who are responsible for the nutritional care and the ordering of food on the ward and who knows the patients well and the products and services from the production kitchen. The manager in the production kitchen and his staff regard the nutritional and healthcare assistant as their <italic>\"ambassador\" </italic>on the ward. This is expressed by the manager in the following way: <italic>\"It is essential that someone takes the patients' food seriously and that someone cares about their diet so that they'll get what they like. There's no point in us calculating that they </italic>[the patients] <italic>can have 9,000 kJ a day if that's not what they want to eat!\"</italic>. When the production kitchen has no such contact, the staff in the kitchen has difficulties getting feedback from the patients and making the wards aware of and use the range of products they offered.</p>", "<title>The amount of food ordered</title>", "<p>Table ##TAB##3##4## shows key figures during the control and intervention periods of food supply expenses and the total number of inpatients (bed days) on the three participating wards. The average food supply expenses per patient per day amount to EURO 7.0 during the control period and EURO 5.5 during the intervention period. The food supply expenses of the wards decrease by 17 to 27% (20% on average) during the intervention compared with the control period.</p>", "<p>The food and drink ordering pattern changes during the intervention; hence variety in orders increase, for instance more types of bread or cold cuts are ordered, and a larger number of various foods is ordered, like desserts, soups, stewed fruit, bread and cold cuts.</p>", "<p>Data from the Office of Financial Affairs at Bispebjerg Hospital (Table ##TAB##4##5##) show that in the calendar year before the intervention, the three participating wards experience a 10 to 24% increase in food supplies (20% on average). This increase in food supply changes 6 to 10% (7.6% on average) during the first 11 months of the calendar year of the intervention. The fact that the increase in food supply is nearly halved on an annual basis can be explained by the six-month intervention period. These data are supported by production kitchen statements (Table ##TAB##3##4##). In overall terms, these results suggest that the work of the nutritional and healthcare assistants reduce food supply expenses per patient.</p>", "<p>As a further control, the data are compared with data from the four non-participating wards at the Medical Centre, Bispebjerg Hospital. These four wards experience a 19.3% increase in food supply in relation to the food budget (Table ##TAB##5##6##). This corresponds with the average 20% increase in food supply of the three participating wards in the year before the intervention (Table ##TAB##4##5##).</p>", "<title>The amount of food waste</title>", "<p>The food waste is calculated as a percentage for four meal types for one control period before the intervention and during the intervention. Towards the end of the intervention, the relative amount of food wastage is approximately reduced by two thirds (specified in Table ##TAB##6##7##).</p>" ]

[ "<title>Discussion</title>", "<p>Data from interviews with patients not being nursed by the nutritional and healthcare assistants show that the nursing staff often does not talk to the patients about their reduced food and fluid intake and weight loss. The patients, who all can be characterised as nibblers, do not experience that they have a dialogue with the nursing staff about nutritional issues, nor that they are guided in what to eat and drink in order to recover. The patients generally experience that the nutritional care is not a priority in the nursing care; a finding that is supported by similar studies [##REF##16280442##5##,##REF##17383776##27##,##REF##17882130##28##,##REF##16101854##36##]. Most of the patients find themselves that nutrition is very important for their convalescence.</p>", "<p>Before the intervention, few patients have experienced any interest in nutritional care on the part of the nursing staff. But during the intervention study the nutritional and healthcare assistants have close communication with the patients and strengthened the focus on preventing undernourishment and weight loss. Patients who are at nutritional risk experience a considerable improvement in the quality of the nursing care in this respect: the assistant ensures that the patient's energy and protein requirements are met by serving small and appetising portions, choosing appropriate foods and keeping the patient focused on the importance of nutrition for his or her recovery. Through this care she imparts knowledge of optimum nutrition to the patient – knowledge that the patient and his/hers family can use after discharge. The patient's own focus on the importance of nutrition for his/hers recovery and efforts to eat sufficiently is likely to be strengthened when a professionally trained person provides care and shows interest in and dedication to nutritional issues [##REF##15669934##37##]. Patients express sincere gratitude, as they have often themselves tried to deal with the problem of reduced appetite and underweight.</p>", "<p>All relevant professional groups on the wards experience the job function of the nutritional and healthcare assistant as meaningful. Many nursing team members express their relief that they no longer feel guilty for not providing nibblers with sufficient nutrition. The knowledge and dedication of the nutritional and healthcare assistant carries over to – and strengthened the focus on – the role of nutrition in the nursing care and treatment. Her work renders visible how resources can be used more effectively and how continuity can be established in the nutritional care. The factors that inhibit optimal nutritional care are eliminated. Such factors include lack of time and interest in nutritional care, and that the nursing staff is unable to provide food outside the fixed mealtimes [##REF##16701921##23##,##REF##16457707##26##]. Both patients and staff members express that the intervention is most successful on the day shift, when the nutritional and healthcare assistant is present. This result points out the importance of having a specific staff to provide optimal nutritional care.</p>", "<p>A few nurses express a fear that the nutritional care is being separated from the nursing care. They believe that nurses have the required knowledge to attend to this part of the nursing care. Nevertheless, training in nutritional care is often given a low priority in the Danish nursing education [##REF##16457707##26##]. The job function of the nutritional and healthcare assistants may redress the imbalance in daily practice of nutritional care in relation to more technical and medical fields of nursing care. The overall results of her job are an improvement in the quality of the nursing care experienced by the patients and optimisation of the food ordering management. This optimisation and a better utilisation of food supplies can, however, cause disagreement between colleagues, as it tinkers with \"old routines\" in the food supplies management, for instance by making it clear that the food is for the patients. Yet, the opportunity to build a positive cooperation with the rest of the nursing staff is a crucial factor. In this respect, the management and the senior staff nurse are playing important roles in creating room for the work of the assistant.</p>", "<p>The management in the production kitchen experience that with the traditional organisation of the nutritional care, the wards do not have the required competencies, knowledge and work effort to utilise optimally the products offered by the production kitchen. The nutritional and healthcare assistant is as a desired partner ensuring a direct contact between the patients at the ward and the production kitchen.</p>", "<p>Before the intervention, the amount of ordered food is based on the number of inpatients. During the intervention, the ordered food is adjusted according to the patients' wishes and requirements. This change produces a surplus of 20% compared with the food budget despite the fact that the nutritional and healthcare assistants order a considerably wider range of food and drink from the production kitchen. The non-participating medical wards at the Medical Centre have a 20% increase in ordered food in relation to the food budget. Besides, the job of the nutritional and healthcare assistants results in a relative reduction in the amount of food wastage from 55% to 18%. Other studies conclude that more than 40% of hospital food is wasted [##REF##12468365##7##,##REF##11104596##38##].</p>", "<p>The data related to the resources used in the food supply are compared in Figure ##FIG##0##1##; Before the intervention there is a 20% increase in food ordered (120% in total), 66% of which ends up as food waste. The remaining part of the food budget (54%) is presumed to have been served to patients (Plate wastage and food eaten by staff members are not taken into account). During the last part of the intervention period, 18% of the food budget (100% in total) ends up as food waste, producing a 20% surplus in relation to the food budget. The employment of the nutritional and healthcare assistants substantially optimise food supply resources: food waste is reduced by two thirds, and the food budget is kept – even with a surplus of 20%.</p>", "<p>Nutritional care is given a low priority in the hospital sector, the purchase of raw products accounting for no more than a few per cent of the total hospitalisation expenses [##REF##16457707##26##]. Considering the fact that raw materials in the form of food and drink are a vital fuel enabling the patients to combat disease and recover, it is crucial that sufficient financial resources are used for the purchase of raw products for patient food supplies. The financial results obtained with this model support the assumption that there is a significant potential for optimising the use of resources in the nutritional care. Other studies suggest that a reorganisation of the nutritional care is required, with certain staff members being in charge of the nutritional care, if the optimisation of resources is to produce a positive result [##REF##12553949##39##]. Hence, the results from this intervention study provide evidence that the present organisational model is usable. Further investigation at a larger scale is recommended to evaluate the benefit of this organisation of the nutritional care in relation to length of stay, complication rate and readmission.</p>" ]

[ "<title>Conclusion</title>", "<p>Retrained social and healthcare assistants working on wards as nutritional and healthcare assistants strengthen the focus on and raise significantly the quality of the nutritional care of under-resourced patients who are at nutritional risk. Patients give great value to the work of the nutritional and healthcare assistant, as she provides nutritional care to each individual patient and offers them guidance and support in their struggle against weight loss. In contrast, inpatients not attended to by a nutritional and healthcare assistant experience that nutrition is not a priority in the nursing care.</p>", "<p>The dedication, knowledge and comprehensive view of the nutritional and healthcare assistants have a positive influence on how their colleagues view and work with the nutritional care. The nutritional and healthcare assistants have a comprehensive view of food orders and adjust them according to the wishes and requirements of the patients. As a result, the food supply resources are used more effectively. The relative amount of food wastage is reduced by two thirds.</p>", "<p>The general picture of Danish hospital wards is that no specialists are employed with the same range of competence and field of work as the nutritional and healthcare assistants. In Danish hospitals, quality enhancement and quality measurement are in focus. Hence, the implementation of this organisational model, which is innovative in its field, can be used with great advantage; it has been documented that it raises the quality of the nutritional care for the benefit and delight of patients and staff, and optimises the utilisation of resources. A program describing a specific staff to do the tasks in the day, evening and weekend duty is recommended if undernourishment of patients during hospitalisation shall be eliminated.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Many patients are undernourished during hospitalisation. The clinical consequences of this include lassitude, an increased risk of complications and prolonged convalescence. The aim of the study is 1) to implement a new organisation with a focus on improving the quality of the nutritional care of medical inpatients at risk of undernutrition, and 2) to investigate the effect of the intervention.</p>", "<title>Methods</title>", "<p>Social and healthcare assistants are educated to the higher level of nutritional and healthcare assistants to provide nutritional care in daily practice to undernourished medical inpatients. The effect of the intervention is investigated before and five months after the employment of the nutritional and healthcare assistants. Data are obtained from structured interviews with patients and staff, and the amount of ordered and wasted food is recorded.</p>", "<title>Results</title>", "<p>Patients regard the work of the nutritional and healthcare assistant as very important for their recovery and weight gain: the assistant takes care of the individual patient's nutritional requirements and wishes, and she imparts knowledge to the patient about optimum nutrition. Staff members benefit from the knowledge and dedication of the nutritional and healthcare assistant and from her work; the staff is often too busy with other nursing tasks to make it a priority to ensure that patients who are nibblers get sufficient nutrition. The choices of food from the production kitchen are utilised to a higher degree, and more of the food is eaten by the patients. Before the intervention, a 20% increase in ordered food in relation to the food budget is found. During the intervention a 20% decrease in ordered food in relation to the food budget is found, and food wastage decreases from 55% to 18% owing to the intervention.</p>", "<title>Conclusion</title>", "<p>The job function of the nutritional and healthcare assistants on the medical wards is of great value to patients, nursing staff members and the production kitchen. The quality of the nutritional care of undernourished patients increases significantly, and a considerable optimisation of resources in the production and ordering of food takes place. Hospitals can benefit from implementation of the present organisational model if they focus on improving the quality of the nutritional care of weak and elderly inpatients and on optimisating the use of resources.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>KOL carried out research design and fundraising, designed focus group interviews and questionnaire, interviewed patients and staff, collected data related to the use and wastage of food and performed the analysis of this data, drafted the manuscript and is the guarantor of the manuscript. EG carried out research design, fundraising and coordination of organisational communication and implementation, designed focus group interviews, questionnaire and the implementation of the new organisational structure on the wards, and participated in the data analysis and in the discussion of the manuscript as an author of the manuscript. RN carried out research design, fundraising, and coordination of organisational communication, planning and implementation of the employment of the social and healthcare assistants, employment of the nutritional and healthcare assistants, and implementation of the new organisational structure on the wards and participated in the discussion of the manuscript as an author of the manuscript. All authors have read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6963/8/168/prepub\"/></p>" ]

[ "<title>Acknowledgements</title>", "<p>Thanks are due to patients and staff on the participating wards, the production kitchen and the office of Economic Affairs, Bispebjerg Hospital, Denmark, and the Foundation <italic>IMK Almene Fond </italic>for sponsorship. Thanks are also due to Ulrikke van Kuppelfelt and Susanne H. Dalskjaer for their professional assistance.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>The use of food supply resources</bold>. The use of food supply resources with and without the employment of nutritional and healthcare assistants. This figure recapitulates Tables 4 to 7. The left-hand column shows how the use of financial resources is distributed on control wards and participating wards before the employment of the nutritional and healthcare assistants. The right-hand column shows how the use of financial resources is distributed on the participating wards after five months' employment of the nutritional and healthcare assistants. The data related to surplus in the food budget are shown in Tables 4 to 6. The data related to the amount of food waste are shown in Table 3. The part of the budget spent on food served to the patients is estimated on the basis of these figures. Plate waste and food eaten by staff members are not taken into account. In the calculations, it is presumed that food waste of the various types of food is weighted equally in terms of expenses. The employment of the nutritional and healthcare assistants results in a surplus of 20% in relation to the food budget and 18% food wastage. Without the assistants the amount in ordered food increases about 20% with 66% food wastage.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>The training program</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Field of expertise</td><td align=\"left\">Disciplines</td></tr></thead><tbody><tr><td align=\"left\">Clinical expertise</td><td align=\"left\">Physiology, pathology, vitamins and minerals, product orientation, nutrition and nutritional therapy.</td></tr><tr><td align=\"left\">Practical skills</td><td align=\"left\">Hygiene, cooking, baking, aesthetic presentation and serving of food, and feeding tube care.</td></tr><tr><td align=\"left\">Organisation</td><td align=\"left\">Cooperation forms (cross-functional, network, etc.), division of responsibilities in the nutritional care, documentation in the nursing care, use of the information and IT system for the ordering of food and the recording of food wastage.</td></tr><tr><td align=\"left\">Pedagogy</td><td align=\"left\">Philosophy of man, provision of care, communication, patient's perspective, the art of motivating and providing information, personal appearance, self-insight and being present.</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Characteristics of patients participating in the questionnaire survey</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Variable\\Patient group</td><td align=\"center\">A <break/>n = 30</td><td align=\"center\">B <break/>n = 25</td><td align=\"center\">C <break/>n = 20</td></tr></thead><tbody><tr><td align=\"left\">Sex, female/male</td><td align=\"center\">18/12</td><td align=\"center\">14/11</td><td align=\"center\">10/10</td></tr><tr><td align=\"left\">Age, years on average (min.; max.)</td><td align=\"center\">70(46;89)</td><td align=\"center\">76(55;91)</td><td align=\"center\">69(55;87)</td></tr><tr><td align=\"left\">Length of stay as of the date of the interview*, days on average</td><td align=\"center\">11.6</td><td align=\"center\">13.2</td><td align=\"center\">13.1</td></tr><tr><td align=\"left\">Mobility-impaired patients**, (%)</td><td align=\"center\">19 (63)</td><td align=\"center\">11 (44)</td><td align=\"center\">15 (75)</td></tr><tr><td align=\"left\">Patients who believe to know their own body weight, (%)</td><td align=\"center\">25 (83)</td><td align=\"center\">22 (88)</td><td align=\"center\">17 (85)</td></tr><tr><td align=\"left\">- Patients who have experienced unwanted weight loss recently, (%)</td><td align=\"center\">18 (60)</td><td align=\"center\">12 (48)</td><td align=\"center\">16 (80)</td></tr><tr><td align=\"left\">- Patients who remember having experienced weight loss over a period of time, (%)</td><td align=\"center\">12 (40)</td><td align=\"center\">5 (20)</td><td align=\"center\">12 (60)</td></tr><tr><td align=\"left\">- Average weight loss according to the patients' own assessments, grams/day***</td><td align=\"center\">197</td><td align=\"center\">168</td><td align=\"center\">134</td></tr><tr><td align=\"left\">Patients who have spoken with other staff members about weight loss, (%)</td><td align=\"center\">11 (61)</td><td align=\"center\">5 (42)</td><td align=\"center\">6 (38)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Questions and distribution of quantitative answers</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Questions and answers\\Patient group</td><td align=\"center\">A <break/>n = 30</td><td align=\"center\">B <break/>n = 25</td><td align=\"center\">C <break/>n = 20</td></tr></thead><tbody><tr><td align=\"left\">a) Have there been days during your stay when you have not eaten much?</td><td/><td/><td/></tr><tr><td align=\"left\"> Yes/No/Don't know</td><td align=\"center\">24/6/0</td><td align=\"center\">17/7/1</td><td align=\"center\">17/3/0</td></tr><tr><td align=\"left\">b) If yes, has the staff done anything to ensure that you have something to eat and drink?</td><td/><td/><td/></tr><tr><td align=\"left\"> Yes/No/Don't know</td><td align=\"center\">12/9/3</td><td align=\"center\">6/6/5</td><td align=\"center\">11/3/3</td></tr><tr><td align=\"left\">c) Has the staff given you the impression that food is important for your health?</td><td/><td/><td/></tr><tr><td align=\"left\"> Yes/No/Don't know</td><td align=\"center\">18/11/1</td><td align=\"center\">7/16/2</td><td align=\"center\">14/3/3</td></tr><tr><td align=\"left\">d) Do you believe food is important for the speed of your recovery?</td><td/><td/><td/></tr><tr><td align=\"left\"> Yes/No/Don't know</td><td align=\"center\">25/3/2</td><td align=\"center\">19/5/1</td><td align=\"center\">16/2/1</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Food supply expenses of the participating wards</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"3\">Control period</td><td align=\"center\" colspan=\"3\">Intervention</td></tr></thead><tbody><tr><td align=\"left\">Category\\Ward</td><td align=\"center\">I</td><td align=\"center\">II</td><td align=\"center\">III</td><td align=\"center\">I</td><td align=\"center\">II</td><td align=\"center\">III</td></tr><tr><td colspan=\"7\"><hr/></td></tr><tr><td align=\"left\">Monthly food consumption, EURO</td><td align=\"center\">7188</td><td align=\"center\">2840</td><td align=\"center\">4014</td><td align=\"center\">3709</td><td align=\"center\">4529</td><td align=\"center\">3606</td></tr><tr><td align=\"left\">Monthly number of bed days</td><td align=\"center\">1,168</td><td align=\"center\">367</td><td align=\"center\">578</td><td align=\"center\">726</td><td align=\"center\">726</td><td align=\"center\">718</td></tr><tr><td align=\"left\">Expenses per bed day, EURO</td><td align=\"center\">6.16</td><td align=\"center\">7.85</td><td align=\"center\">6.95</td><td align=\"center\">5.11</td><td align=\"center\">6.24</td><td align=\"center\">5.03</td></tr><tr><td align=\"left\">Change in expenses, %,</td><td/><td/><td/><td align=\"center\">- 17</td><td align=\"center\">- 17</td><td align=\"center\">- 27</td></tr><tr><td align=\"left\">Expenses per bed day on average, EURO</td><td align=\"center\" colspan=\"3\">7.0</td><td align=\"center\" colspan=\"3\">5.5</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Changes in food ordered for participating wards</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\">Food budget*</td><td align=\"center\">Food ordered</td><td align=\"center\">Increase in food ordered or surplus</td><td align=\"center\">Increase in food ordered or surplus</td></tr></thead><tbody><tr><td align=\"left\">Ward, year</td><td align=\"center\" colspan=\"3\">EURO</td><td align=\"center\">%</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">I, 2003 (control)</td><td align=\"center\">27069</td><td align=\"center\">33200</td><td align=\"center\">6131</td><td align=\"center\">+22.7</td></tr><tr><td align=\"left\">I, 1 Jan – 30 Nov 2004</td><td align=\"center\">24085</td><td align=\"center\">22738</td><td align=\"center\">-1347</td><td align=\"center\">-5.6</td></tr><tr><td align=\"left\">II, 2003 (control)</td><td align=\"center\">16825</td><td align=\"center\">18484</td><td align=\"center\">1659</td><td align=\"center\">+9.9</td></tr><tr><td align=\"left\">II, 1 Jan – 30 Nov 2004</td><td align=\"center\">26302</td><td align=\"center\">24584</td><td align=\"center\">-1718</td><td align=\"center\">-6.5</td></tr><tr><td align=\"left\">III, 2003 (control)</td><td align=\"center\">25717</td><td align=\"center\">31864</td><td align=\"center\">6147</td><td align=\"center\">+23.9</td></tr><tr><td align=\"left\">III, 1 Jan – 30 Nov 2004</td><td align=\"center\">29274</td><td align=\"center\">26301</td><td align=\"center\">-2973</td><td align=\"center\">-10.2</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T6\"><label>Table 6</label><caption><p>Food ordered for the non-participating wards</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\">Food budget*</td><td align=\"center\">Food ordered</td><td align=\"center\">Increase in food ordered</td><td align=\"center\">Increase in food ordered</td></tr></thead><tbody><tr><td align=\"left\">Ward</td><td align=\"center\" colspan=\"3\">EURO</td><td align=\"center\">%</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">IV</td><td align=\"center\">30 099</td><td align=\"center\">33 059</td><td align=\"center\">2 960</td><td align=\"center\">+9.8</td></tr><tr><td align=\"left\">V</td><td align=\"center\">90 077</td><td align=\"center\">108 489</td><td align=\"center\">18 412</td><td align=\"center\">+20.4</td></tr><tr><td align=\"left\">VI</td><td align=\"center\">71 435</td><td align=\"center\">81 681</td><td align=\"center\">10 246</td><td align=\"center\">+14.3</td></tr><tr><td align=\"left\">VII</td><td align=\"center\">70 387</td><td align=\"center\">89 222</td><td align=\"center\">18 835</td><td align=\"center\">+26.8</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">In total</td><td align=\"center\">261 998</td><td align=\"center\">312 451</td><td align=\"center\">50 453</td><td align=\"center\">+19.3</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T7\"><label>Table 7</label><caption><p>Food wastage at the participating wards</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\">Control</td><td align=\"center\" colspan=\"3\">Intervention</td></tr><tr><td/><td/><td colspan=\"3\"><hr/></td></tr><tr><td/><td/><td align=\"center\">Beginning</td><td align=\"center\">Middle</td><td align=\"center\">End</td></tr></thead><tbody><tr><td/><td align=\"center\" colspan=\"4\">Wastage rate,%</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">Warm breakfast*</td><td align=\"center\">49</td><td align=\"center\">56</td><td align=\"center\">36</td><td align=\"center\">17</td></tr><tr><td align=\"left\">Lunch</td><td align=\"center\">55</td><td align=\"center\">37</td><td align=\"center\">24</td><td align=\"center\">20</td></tr><tr><td align=\"left\">Supper</td><td align=\"center\">60</td><td align=\"center\">34</td><td align=\"center\">17</td><td align=\"center\">19</td></tr><tr><td align=\"left\">\"Late evening\"**</td><td align=\"center\">Not recorded</td><td align=\"center\">68</td><td align=\"center\">31</td><td align=\"center\">17</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">Average wastage for all meals recorded</td><td align=\"center\">55</td><td align=\"center\">49</td><td align=\"center\">27</td><td align=\"center\">18</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>Fields of expertise and disciplines in the in-service training programme for the social and healthcare assistants are described in this table. The four fields of expertise are weighted equally and outside instructors are attached as teachers. In total 90 lessons.</p></table-wrap-foot>", "<table-wrap-foot><p>* The total length of stay is not known, as the patients were interviewed during their stay.</p><p>** Patients who are bedfast, confined to a wheelchair or walking-impaired.</p><p>***If the patient has had unwanted weight loss, the patient is asked how many kilograms over the number of days. </p><p>Before and during the intervention the following patients participate; Group A: patients who are admitted before the nutritional and health assistant is employed on the ward. Group B: patients who are admitted during the employment period of the nutritional and healthcare assistant but not receiving her nutritional care. Group C: patients who are receiving nutritional care from the nutritional and healthcare assistant during their entire or part of their stay at the hospital.</p></table-wrap-foot>", "<table-wrap-foot><p>Before and during the intervention the following patients answer the questions; Group A: patients who are admitted before the nutritional and health assistant is employed on the ward. Group B: patients who are admitted during the employment period of the nutritional and healthcare assistant but not receiving her nutritional care. Group C: patients who are receiving nutritional care from the nutritional and healthcare assistant during their entire or part of their stay at the hospital.</p></table-wrap-foot>", "<table-wrap-foot><p>Food supply expenses of participating wards I, II and III before and during the intervention. The control period before the intervention data is an average of October and November 2003. During the intervention data is an average of October and November 2004. Source: Data printout from the Office of Economic Affairs and Production Kitchen, Bispebjerg Hospital.</p></table-wrap-foot>", "<table-wrap-foot><p>* Ward budgets are prepared on the basis of the number of inpatients per day recorded in the hospital's computer system, the number of inpatients being calculated at the beginning of each day.</p><p>Food budget, food ordered, and changes in food ordered for participating wards I, II and III before and with intervention. The control period is the calendar year 2003. The following year includes the intervention period (from 1 July to 31 December 2004). The collecting of data is done one month before termination of the intervention, as the effect is assumed to be at its peak at this time. The increase in consumption has been calculated as a percentage, \"+\" meaning increase in consumption and \"-\" meaning surplus. (Source: Data printout from the Office of Economic Affairs, Bispebjerg Hospital)</p></table-wrap-foot>", "<table-wrap-foot><p>* Ward budgets are prepared on the basis of the number of inpatients per day recorded in the hospital's computer system, the number of inpatients being calculated at the beginning of each day.</p><p>Food budget, food ordered, and increases in food ordered for the non-participating wards IV-VII at the Medical Centre in 2004. Increase in food ordered is \"+\"; surplus is \"-\". (Source: Data printout from the Office of Economic Affairs, Bispebjerg Hospital).</p></table-wrap-foot>", "<table-wrap-foot><p>* Porridge etc.</p><p>** Meal served at 8 pm; includes small dishes such as soup, milk beverages, porridge, stewed fruit and cocoa.</p><p>Average food wastage at the participating wards I, II and III before and during the employment of the nutritional and healthcare assistant. The control data are collected on three weekdays. The data from the intervention period are collected on a daily basis by the nutritional and healthcare assistant, and represent for 10 days during start-up of the job function (beginning) and after 3 months' (middle) and 5 months' (end) intervention.</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1472-6963-8-168-1\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Neck/shoulder pain (NSP) may affect up to half of adolescents [##REF##15105072##1##], leading to significant loss of function [##REF##11568698##2##]. Up to 25% of adolescents with NSP experience some degree of disability [##REF##15275795##3##] and 11% may require prescription drugs to manage pain [##REF##2934780##4##]. Some risk factors for adolescent NSP have been identified, including high levels of computer use [##REF##16131744##5##], employment [##REF##11880838##6##], negative psychosocial factors [##REF##11880838##6##, ####REF##11045747##7##, ##REF##15284513##8####15284513##8##], female gender [##REF##15284513##8##], and sustained postures [##REF##15105072##1##]. Very low and high levels of physical activity [##REF##11045747##7##] are also associated with adolescent NSP. Activity levels may influence NSP directly, or via other factors such as physical characteristics.</p>", "<p>Physical characteristics such as muscle strength, flexibility, endurance or motor competence may be associated with spinal posture [##REF##15105072##1##,##REF##11045747##7##,##UREF##0##9##] or spinal stability [##REF##16239098##10##], both of which may have an association with spinal pain [##REF##15105072##1##,##REF##2934780##4##,##REF##16915076##11##]. There is some evidence that physical characteristics and adult NSP are related. Adult studies have reported that decreases in neck flexibility [##REF##14699273##12##], neck endurance [##REF##14699273##12##], neck muscle motor control [##REF##15245710##13##], grip strength [##REF##9474732##14##] and high body mass index (BMI) [##REF##12782992##15##] are associated with NSP.</p>", "<p>However the evidence for a link in adolescents is less clear. It has been noted [##REF##16431995##16##] that low levels of flexibility in male adolescents and low levels of trunk endurance in female adolescents have been associated with a greater risk of \"tension neck syndrome\" 25 years later. Similarly, lower arm endurance in males during adolescence has been associated with more NSP in adulthood [##REF##9474732##14##]. With respect to body composition, one study noted no associations between BMI at age 14 and NSP in early adulthood [##REF##15843973##17##]. However, only one study to our knowledge has investigated the association of adolescent physical characteristics with NSP experienced <italic>during </italic>adolescence [##REF##11880838##6##] and it reported no association between NSP and BMI. Salminen [##REF##6241979##18##] investigated the relationship between flexibility and adolescent NSP, but this was in conjunction with LBP, and so a specific relationship with NSP was not defined. No adolescent studies have investigated the links between NSP and aerobic capacity or motor competence.</p>", "<p>There is therefore a need for an initial exploratory study to examine the suspected links between adolescent NSP and certain physical characteristics. If it can be shown that any of these physical characteristics are related to adolescent NSP, this will provide the basis for further longitudinal work, which may in turn inform the development of specific strategies to prevent this common problem. The research question was whether lower and/or higher levels of fitness, motor competence and body composition were related to increased risk of NSP in adolescents. The physical variables used in this study reflect generalized motor performance and characteristics, rather than specific neck muscle performance, as the more general variables bear a greater relation to performance measures customarily used in schools and clinics.</p>" ]

[ "<title>Methods</title>", "<title>Participants</title>", "<p>Data were collected from 1608 adolescents (783 females, 825 males) of mean (SD) age 14.06 (0.20) yrs, who were participating in the Western Australian Pregnancy Cohort \"Raine\" Study <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.rainestudy.org.au\"/>. This project began with a cohort of women attending antenatal clinics at King Edward Memorial Hospital for Women, Perth, Australia between 1989 and 1991. The children have been followed at birth, 1, 2, 3, 5, 8, 10, and now 14 years of age. Inclusion criteria for the women were gestational age of 16–20 weeks, adequate English language to understand the implications of participation, and an intention to remain in Western Australia throughout follow-up. 2337 adolescents were eligible for the 14 year follow-up, and 1704 (72.9%) of these agreed to participate in some aspect of the follow-up. 1608 (68.8%) completed the data collection requirements for the analysis reported in this paper. There were no exclusion criteria for this part of the cohort. A comparison of the cohort with the Western Australian general population showed a higher proportion of high risk births, as would be expected for a major specialist hospital [##UREF##1##19##].</p>", "<title>Procedure</title>", "<p>With the assistance of a research assistant, participants completed a laptop questionnaire at an assessment centre. The questionnaire contained 130 questions concerning a broad range of issues, many of which were not relevant to this study. Adolescents were asked about their experience of NSP, described as pain in the area of the posterior neck and upper trapezius, as diagrammatically defined by Kourinka et al. [##REF##15676628##20##]. The relevant NSP questions were: Have you ever had neck/shoulder pain? (\"yes\" or \"no\"), Has your neck/shoulder been painful in the last month? (\"yes\" or \"no\"), and Did your neck/shoulder pain last for more than 3 months? (\"yes\" or \"no\"). The full questionnaire took about 1 hour to complete, and the NSP questions occurred in the first half. The life prevalence question is very similar to that used by Chiu and Leung [##UREF##2##21##], which was shown to be reliable and valid.</p>", "<p>Information on diagnosed neck pain was obtained from a paper questionnaire given to the primary carer, which included the question, \"Does your child have now, or has your child had in the past, any of the following health professional diagnosed medical conditions or health problems?\". The primary carer had to indicate which medical diagnoses their child had experienced from a short list of general medical problems, which included \"neck pain\". This question was part of a questionnaire given to the primary carer, covering many other factors that are not relevant to this study.</p>", "<p>A physical assessment of the child was carried out after the laptop questionnaire, and parts relevant to this study are described below. Height (m), body mass (kg), waist girth (cm) and arm girth (cm) were measured without shoes. Several physical performance tests were then carried out. Maximal aerobic capacity was estimated from heart rate recordings during sub-maximal cycle ergometry using the Physical Work Capacity 170 protocol [##REF##2384936##22##]. The sustained back extension test [##REF##6233709##23##] and the number of abdominal curls performed in 3 minutes [##UREF##3##24##,##UREF##4##25##] were used to measure trunk muscle performance. Limb muscle performance was evaluated by standing long jump [##UREF##3##24##,##UREF##5##26##], seated basketball throw [##UREF##3##24##,##REF##16118308##27##] and grip strength [##UREF##5##26##,##REF##16118308##27##]. Flexibility was tested using the shoulder stretch [##REF##12466694##28##]. Motor competence was evaluated using the McCarron Assessment of Neuromuscular Development (MAND) [##UREF##5##26##]. The Neurodevelopmental Index (NDI) was derived from summing gender and age corrected scores from 10 tests of fine and gross motor skills, and then converting to a scale with 100 as the mean and a SD of 15. Four sub indices (muscle power, kinaesthetic integration, bimanual dexterity and persistent control) [##UREF##5##26##], each comprising two of the items, were calculated. Details of the 10 items used to generate the overall NDI and 4 sub indices are shown in table ##TAB##0##1##.</p>", "<p>All of these physical performance tests have been previously validated in very similar forms [##UREF##4##25##, ####UREF##5##26##, ##REF##16118308##27####16118308##27##,##UREF##6##29##, ####REF##15320660##30##, ##REF##11208223##31####11208223##31##] except the shoulder stretch, which has acceptable face validity. Reliability of the same or similar versions of the tests is also good [##UREF##5##26##,##REF##15320660##30##, ####REF##11208223##31##, ##REF##4648576##32####4648576##32##], although there are no reports on the shoulder stretch or the basketball throw.</p>", "<title>Data analysis</title>", "<p>Gender differences were analysed using independent t tests for each of the continuous variables, and Chi squared tests for the categorical variables. To facilitate the interpretation of non-linear relationships, continuous variables were banded into the bottom 25%, inter-quartile range and top 25%, and the proportion of subjects with neck pain in each segment were compared. Univariate logistic regression models predicting lifetime, last month, chronic (lasting more than 3 months) and diagnosed neck pain from each physical characteristic were calculated separately for males and females, with statistical significance set at p &lt; 0.05, and the interquartile range of each continuous variable defined as the reference category. For the only binary variable (shoulder stretch), being able to perform the stretch was the reference category. Corrections for multiple univariate tests were not carried out as the multivariate results were the end point of the study.</p>", "<p>Backwards stepwise likelihood ratio multivariate logistic regression models were used to evaluate the combined associations of performance factors for males and for females, with the probability for entry and removal of the likelihood ratio score statistic being p = 0.05 and 0.10 respectively. For each gender, 4 multivariate analyses for each of neck pain ever, last month, chronic and diagnosed were performed. Height and weight were included in an initial step, with abdominal curls, basketball throw, jump, back muscle endurance, PWC170, hand strength and shoulder stretch included in a second step, along with one of the body composition variables and either the NDI score or the 4 motor competence factor scores. The choice of body composition variables or motor competence variables was determined by the strength of univariate relationships with pain, and was determined for each of the eight multivariate analyses separately. The strength of the predictive ability of the model was estimated by Nagelkerke R². All statistical analysis was performed using SPSS version 13.</p>", "<title>Ethics</title>", "<p>Adolescents provided written informed assent and their parent/guardian provided written informed consent prior to participation. The study was approved by the Human Research Ethics Committees of Curtin University of Technology and Princess Margaret Hospital.</p>" ]

[ "<title>Results</title>", "<title>Neck/shoulder pain</title>", "<p>NSP ever was experienced by 46.8% of the participants, NSP in the past month by 28.7%, 'chronic' (lasting more than 3 months) NSP by 8.2% and diagnosed NSP by 7.1%. Females had a significantly higher prevalence of NSP ever, month and chronic, but not diagnosed NSP (see table ##TAB##1##2##). 64.9% of those with chronic NSP also had experienced NSP within the last month and 20.9% of those with chronic NSP also had diagnosed NSP according to parental report.</p>", "<title>Physical characteristics</title>", "<p>Descriptive statistics for physical characteristics are given in table ##TAB##1##2##. Females obtained significantly higher mean scores for BMI, back endurance and the motor competence factors of Persistent Control and Bimanual Dexterity. A greater proportion of females could perform the shoulder stretch test. Males obtained significantly higher mean scores for waist girth, aerobic capacity, abdominal curl number, standing long jump, basketball throw, grip strength and the motor competence factor of muscle power. Males were also taller and heavier, with a lower BMI. There were no gender differences in arm circumference, NDI or Kinaesthetic integration.</p>", "<title>Associations between NSP and physical performance</title>", "<title>Males</title>", "<p>On univariate analysis, there was an increase in risk of NSP in the past month for male subjects with greatest height, an increase in risk of chronic NSP for male subjects with greatest basketball throw and NDI, and a decreased risk for chronic NSP for male subjects with the lowest weight and arm circumference. There were no significant effects on the risk of diagnosed NSP (Table ##TAB##2##3##).</p>", "<p>For multivariate analysis, the common variables described in the methods section were entered, together with BMI and NDI for the NSP ever analysis, arm circumference and the four MAND factors for the NSP month analysis, arm circumference and NDI for the NSP chronic analysis, and waist circumference and the four MAND factors for the NSP diagnosed analysis, according to the strength of univariate relationships. Males in the lowest quartile of back muscle endurance were less likely to have NSP ever, and there was a similar trend for those in the highest quartile of NDI. There were no multivariate associations between physical characteristics and male NSP in the past month. Males in the highest quartile of basketball throw distance were more likely to have chronic NSP and there was a trend for those unable to do the shoulder stretch to be less likely to have chronic NSP. Males in the highest quartile of jump distance were more likely to have diagnosed NSP, but those in the highest quartile of muscle power were less likely to have diagnosed NSP. Those in the lowest quartile of persistent control were less likely to have NSP. Nagelkerke R²of logistic regression models ranged from 0.019 to 0.085 (Table ##TAB##3##4##).</p>", "<title>Females</title>", "<p>On univariate analysis there was an increase in risk for NSP in the past month for females with the highest bimanual dexterity, and an increase in risk of chronic NSP for females with the lowest hand strength. There was a decreased risk of NSP ever for females with the highest basketball throw, and a decreased risk of NSP in the past month for females with highest PWC170, and lowest and highest jump distance. There were no significant effects on the risk of diagnosed NSP (Table ##TAB##4##5##).</p>", "<p>For multivariate analysis, the common variables described in the methods section were entered, together with arm circumference and the four MAND factors for the NSP ever analysis and the NSP month analysis, and BMI and the four MAND factors for the NSP chronic and diagnosed analysis, according to the strength of univariate relationships. Females in the highest quartile of basketball throw were less likely to have NSP ever, and females in the highest quartile of abdominal curls were more likely to have NSP. Females unable to do the shoulder stretch were less likely to have NSP. Females in the lowest quartile of jump distance were less likely to have NSP in the past month. Females in the highest quartile of basketball throw, bimanual dexterity and PWC170 were less likely to have NSP in the past month. There was also a trend for those in the highest quartile of jump distance to be less likely to have NSP in the past month. There were no multivariate associations between physical characteristics and chronic NSP in females. Females in the lowest quartile of back endurance were more likely to have diagnosed NSP, and those in the highest quartile of back endurance were more likely to have diagnosed NSP. The Nagelkerke R²of logistic regression models ranged from 0.001 to 0.064 (Table ##TAB##3##4##)</p>" ]

[ "<title>Discussion</title>", "<p>NSP is clearly a common problem in adolescents, with this study showing a prevalence of pain similar to that reported in other adolescent studies [##REF##15105072##1##,##REF##11568698##2##]. That almost one in ten adolescents have experienced NSP of at least 3 months duration is a strong indicator that adolescent NSP is a significant problem. The search for adolescent risk factors is therefore of great importance, so that effective prevention and management can be implemented. This study is the first to suggest that some physical characteristics are associated with adolescent NSP, although the strength of these associations was weaker than anticipated.</p>", "<title>Cross-sectional data</title>", "<p>This study analysed cross-sectional data only, so relationships identified could be the result of causality in both directions: NSP could be influenced by physical characteristics or vice versa. There is evidence that adults with back pain may experience a 'deconditioning' effect associated with pain inhibiting and restricting participation in work, leisure and household activities [##REF##16395183##33##]. In contrast, there is evidence that poor back muscle endurance increases the risk of back pain episodes in manual workers [##REF##11426154##34##].</p>", "<p>Longitudinal data (currently being collected on this cohort) is required to elucidate the direction of any relationship. The remainder of this section discusses the cross-sectional results and suggests potential mechanisms for observed relationships.</p>", "<title>Body composition</title>", "<p>Although a weak univariate relationship between low arm circumference and a lower risk of chronic NSP was observed in males, body composition was not associated with any form of NSP in either gender after multivariate analysis. This concurs with previous adolescent findings [##REF##11427402##35##] and underlines the importance of multivariate analysis with a comprehensive range of covariates.</p>", "<title>Aerobic capacity</title>", "<p>Higher aerobic capacity, after correction for other variables including body weight, was associated with a lower risk of NSP in the last month for females only, with a similar trend in NSP ever. The lower risk of NSP with improved aerobic capacity for NSP in females may be associated with increased levels of physical activity which is known to sometimes have a beneficial effect on spinal pain disorders [##REF##11045747##7##]. This may also relate to a deconditioning mechanism, where females with NSP reduce their participation in physical activity and lose aerobic capacity. The lack of any relationships for males may indicate a differing mechanism or response to neck pain based on gender.</p>", "<title>Muscle performance</title>", "<p>There were inconsistent associations between arm muscle performance and NSP after multivariate analysis. Greater upper body power, as measured by the basketball throw, was protective in females for both NSP ever and in the past month, but a risk factor for chronic NSP in males. The reason for this gender difference is unclear although other factors such as specific sport participation may influence these findings. Females (but not males) engaging in more dynamic arm activities have less pain [##REF##15284513##8##,##REF##8807538##36##], and given that greater amounts of dynamic arm activities may increase upper body power, this may explain the pattern in females. The opposite pattern in males, with increased risk of chronic NSP in the most powerful quartile, may partly relate to greater arm activity not having a protective effect in males [##REF##15284513##8##,##REF##16395183##33##], and also because their high arm power may be a proxy for greater overall physical activity levels (not just upper limb activity), which relates to greater NSP in males [##UREF##7##37##]. In contrast, Barnekow-Bergvist et al. [##REF##9474732##14##] reported that greater arm endurance in adolescent males was related to a reduced risk of NSP in adulthood, which may relate to a deconditioning effect secondary to NSP.</p>", "<p>Multivariate associations between NSP and leg power were very different to those with arm power. In females, a low jump performance decreased risk of NSP in the past month, effectively the opposite effect seen with upper body power. Aurvinen et al. [##REF##17450080##38##] reported that higher overall activity levels may increase NSP risk in females. Since it is possible that higher overall activity may be associated with greater leg power, this may explain our finding of low leg power reducing risk. Although differing effects on NSP from arm activity levels and overall activity levels may initially appear paradoxical, it is possible that the relationship between overall activity levels and NSP is not direct but mediated by performance in sports that may increase risk of NSP. Similarly, diagnosed neck pain was associated with greater jump distance in males, although this was not observed for the other pain variables. This result may indicate a similar mechanism to that described in females.</p>", "<p>A very similar pattern was observed between abdominal endurance and NSP ever after multivariate analysis, with better performance associated with greater risk of pain in females only. Mechanisms may be similar to those described for leg power. In contrast, Mikkelson et al. [##REF##16431995##16##] reported that poorer female adolescent abdominal endurance was a risk factor. However, Mikkelson et al. [##REF##16431995##16##] reported these outcomes in adulthood.</p>", "<p>Less back muscle endurance was associated with a decreased risk of NSP ever in males after multivariate analysis, which was analogous to the findings for leg power and abdominal endurance in females, and may again relate to the males being involved in more vigorous physical activity [##UREF##7##37##]. Similarly, females with a diagnosis of NSP were more likely to have high back endurance, and this could relate to greater overall activity levels, as previously described. However, females with low back endurance also had a higher risk of diagnosed NSP. It is possible that these females were below a threshold of endurance at which any effects on spinal stability became important, or alternatively were experiencing a deconditioning effect as a result of the pain. However, this effect was not seen in males, who had lower back endurance overall.</p>", "<title>Flexibility</title>", "<p>One surprising multivariate finding was that poorer shoulder girdle flexibility, as measured by the shoulder stretch, was related to a significantly decreased risk of NSP in the past month in females. There was also a strong trend for the same effect on chronic NSP in males. Though counter-intuitive, there are reports of a relationship between lower shoulder rotational flexibility and greater upper limb activity levels in elite adult water polo [##UREF##8##39##] and volleyball players [##REF##10690449##40##]. Greater amounts of dynamic upper limb activity have also been shown to reduce the risk of female adolescent NSP [##REF##15284513##8##,##REF##8807538##36##] and so these separate findings may explain the overall association of reduced flexibility and lower risk of NSP observed in this study.</p>", "<title>Motor competence</title>", "<p>Males with higher levels of the motor competence factor of muscle power had a reduced risk of diagnosed NSP, and there was a trend for higher overall motor competence (NDI) to be associated with lower risk of NSP ever in males after multivariate analysis. This was expected, given that higher motor competence might have a protective effective on the musculoskeletal system [##REF##1490035##41##]. However this relationship may be weakened by males with better motor competence being more involved in vigorous activities, as suggested by evidence that pre-pubescent children with higher motor competence engage in more vigorous play [##UREF##9##42##], and thus more likely to develop NSP [##UREF##7##37##]. This potential confounding may possibly explain the contradictory finding of lower persistent control being associated with a lower risk of diagnosed NSP. In contrast, poorer coordination may be a result of reduced motor practice as part of a reduction of activities associated with NSP.</p>", "<p>In females, higher bimanual dexterity significantly increased risk of NSP in the past month. Bimanual dexterity relates to the co-ordination of fine motor skills across both arms, and might be developed by activities such as playing musical instruments, needlework, computer work or craftwork, which are known risk factors for female adolescent NSP [##REF##8807538##36##].</p>", "<title>Strength of associations</title>", "<p>Evidence from longitudinal studies [##REF##9474732##14##,##REF##16431995##16##] demonstrates that physical performance in adolescence can influence the development of NSP in adulthood, although a deconditioning response to the presence of NSP is also possible. The predictive utility of the models in the current study was very low however, with Nagelkerke R²ranging from only 0.001 to 0.085. The lack of stronger relationships was not due to missed curvilinear relationships as these were accounted for in the analysis, and the study was not underpowered as weak relationships were detected.</p>", "<p>This may indicate that either physical performance is not strongly related to NSP during adolescence or that the direction and mechanisms are more complex and other factors need to be considered. One of the strengths of our study was the broad range of physical variables adjusted for in the analyses, but certain possible confounders such as activity levels and sport participation were not included in the current analyses. Consideration of these may have either reduced or strengthened observed relationships, and should be attempted in further work. In addition, NSP was treated as a homogenous entity, but in reality it may have several sub-groups with different aetiologies. Real, but differing, associations between physical performance and each sub-group may thus have been lost in the current analysis. Further work towards subgroup identification is intended.</p>", "<p>NSP is not a simple construct and thus four measures were used, including a parental report of health professional diagnosed NSP. Whilst parental report of diagnosed neck pain has limited detail and accuracy, it reinforced the self-report measure of NSP. Further, strong relationships could be expected to be more consistent across the different measures.</p>", "<p>Associations were very different across genders, with no common effects seen. These differing gender effects may be the result of differences in the type and vigour of sporting activities [##UREF##10##43##], as well as anthropometric differences, and possible variation in underlying pain mechanisms and psychosocial effects. Whatever the cause, these differences emphasise the need to continue to consider gender in future work, as gender will be a possible confounder of many pain/physical characteristics relationships.</p>" ]

[ "<title>Conclusion</title>", "<p>NSP is clearly an important health issue for adolescents. Some aspects of physical performance are associated with adolescent NSP. Interestingly, better performance sometimes increased rather than decreased risk, suggesting that the direction and mechanisms are complex. Associations differed between genders, suggesting that NSP in males and females may have different mechanisms, or that these factors may interact differently. However, despite the large sample and examination of curvilinear relationships, multiple physical factors and gender specific effects, the associations were weak suggesting complex mechanisms for NSP development.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Adolescent neck/shoulder pain (NSP) is a common and sometimes debilitating problem. Several risk factors for this condition have been investigated, but no studies have previously evaluated associations between fitness, motor competence, body composition and adolescent NSP.</p>", "<title>Methods</title>", "<p>1608 males and females of mean age 14 years answered questions on their history of NSP (4 measures), and were tested for aerobic fitness, upper and lower limb power, trunk endurance, grip strength, shoulder flexibility, motor competence and anthropometric factors. Univariate and multivariate logistic regressions were used to test for associations between NSP and physical variables.</p>", "<title>Results</title>", "<p>There were significant gender differences for most physical and pain variables. After multivariate analysis, males had lower odds of NSP if they had reduced back endurance [OR: 0.66 (95% CI: 0.46–0.97)], reduced persistent control [0.42 (0.19–0.95], and increased muscle power [0.33 (0.12–0.94)], and higher odds of NSP if they had a higher basketball throw [2.47 (1.22–5.00)] and jump performance [3.47 (1.55–7.74)]. Females had lower odds for NSP if they had a reduced jump performance [0.61(0.41–0.92)], a better basketball throw [0.60(0.40–0.90)], lower shoulder flexibility [0.54 (0.30–0.98)] and a higher aerobic capacity [0.61 (0.40–0.93)], and higher odds for NSP if they had greater abdominal endurance [1.57(1.07–2.31)] and greater bimanual dexterity [1.77(1.18–2.65)]. Females showed a U shaped relationship between NSP and back endurance [low: 2.12 (1.20–3.74); high 2.12 (1.18–3.83)].</p>", "<title>Conclusion</title>", "<p>Adolescent NSP was associated with fitness and motor competence, although the associations varied with gender, and their strength was limited.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>MCP analysed data, drafted the manuscript and assisted with final approval. LMS designed the study, designed and revised the manuscript and assisted with final approval. PBO designed the study, revised the manuscript and assisted with final approval. AJS analysed data, revised the manuscript and assisted with final approval. BH designed the study, revised the manuscript and assisted with final approval.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2458/8/290/prepub\"/></p>" ]

[ "<title>Acknowledgements</title>", "<p>The authors would like to thank Rosemary Austin, Lee Clohessy, Jemma Coleman, Alex D'Vauz, Clare Haselgrove, Monique Robinson, Nick Sloan and Diane Wood for collection and/or processing of data.</p>", "<p>The authors acknowledge funding from the Australian National Health and Medical Research Council (project # 323200), the Raine Foundation at the University of Western Australia, Healthway, the Arthritis Foundation of Australia, and the Arthritis Foundation of Western Australia.</p>" ]

[]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Summary of MAND tests</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Test</td><td align=\"left\">Measurement</td><td align=\"left\">Sub-indices</td></tr></thead><tbody><tr><td align=\"left\">Rod slide</td><td align=\"left\">Smoothness and slowness of moving handle along a metre long rod, repeated both hands.</td><td align=\"left\">PC</td></tr><tr><td align=\"left\">Finger/nose finger</td><td align=\"left\">Accuracy and smoothness of index finger from nose to opposite hand's index finger, repeated both sides</td><td align=\"left\">PC</td></tr><tr><td align=\"left\">Hand strength</td><td align=\"left\">Hand grip strength with a hand dynamometer, repeated both sides</td><td align=\"left\">MP</td></tr><tr><td align=\"left\">Standing long jump</td><td align=\"left\">Distance and quality of two footed jump</td><td align=\"left\">MP</td></tr><tr><td align=\"left\">Heel toe walk</td><td align=\"left\">Quality of walking forwards and backwards along a 10 foot line</td><td align=\"left\">KI</td></tr><tr><td align=\"left\">Standing one leg</td><td align=\"left\">Time of balance on each leg with eyes open, then eyes closed.</td><td align=\"left\">KI</td></tr><tr><td align=\"left\">Beads on rod</td><td align=\"left\">Number of beads placed on rod held in non-dominant hand in 30 seconds, repeated with eyes open and closed</td><td align=\"left\">BD</td></tr><tr><td align=\"left\">Nut and bolt</td><td align=\"left\">Time to turn a large bolt, held in the dominant hand, fully onto a nut, repeated with a small bolt.</td><td align=\"left\">BD</td></tr><tr><td align=\"left\">Finger tapping</td><td align=\"left\">Number and quality of taps of index finger in 10 seconds, repeated both hands</td><td align=\"left\">-</td></tr><tr><td align=\"left\">Beads in box</td><td align=\"left\">Number of beads moved from one box to an adjacent box in 30 seconds, repeated both hands.</td><td align=\"left\">-</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Pain prevalence and physical test performance for males and females</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Pain variable</bold></td><td align=\"right\"><bold>All Participants % (count) with history of pain</bold></td><td align=\"right\"><bold>Male % (count) with history of pain</bold></td><td align=\"right\"><bold>Female % (count) with history of pain</bold></td><td align=\"center\" colspan=\"2\"><bold>Gender difference</bold></td></tr></thead><tbody><tr><td/><td/><td/><td/><td align=\"right\"><bold>χ²</bold></td><td align=\"right\"><bold>P</bold></td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\">NSP ever</td><td align=\"right\">46.8</td><td align=\"right\">41.9</td><td align=\"right\">52.0</td><td align=\"right\">16.3</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">NSP in last month</td><td align=\"right\">28.7</td><td align=\"right\">22.9</td><td align=\"right\">34.7</td><td align=\"right\">27.1</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Chronic NSP</td><td align=\"right\">8.2</td><td align=\"right\">6.8</td><td align=\"right\">9.8</td><td align=\"right\">4.8</td><td align=\"right\">0.029</td></tr><tr><td align=\"left\">Diagnosed NSP</td><td align=\"right\">7.1</td><td align=\"right\">6.9</td><td align=\"right\">7.2</td><td align=\"right\">0.05</td><td align=\"right\">0.828</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Physical variable</bold></td><td align=\"right\"><bold>All Participants mean (sd)</bold></td><td align=\"right\"><bold>Males mean (sd)</bold></td><td align=\"right\"><bold>Females mean (sd)</bold></td><td align=\"center\" colspan=\"2\"><bold>Gender difference</bold></td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td/><td/><td/><td/><td align=\"right\"><bold>t</bold>_{<bold>df(unless stated otherwise)</bold>}</td><td align=\"right\"><bold>P</bold></td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\">Height</td><td align=\"right\">1.64 (0.08)</td><td align=\"right\">1.66 (0.09)</td><td align=\"right\">1.62 (0.06)</td><td align=\"right\">-0.42₁₅₉₈</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Weight</td><td align=\"right\">57.7 (13.2)</td><td align=\"right\">58.6 (14.1)</td><td align=\"right\">56.7 (12.1)</td><td align=\"right\">-1.92₁₅₉₉</td><td align=\"right\">0.004</td></tr><tr><td align=\"left\">BMI</td><td align=\"right\">21.29 (4.15)</td><td align=\"right\">21.05 (4.14)</td><td align=\"right\">21.53 (4.16)</td><td align=\"right\">2.30₁₅₉₈</td><td align=\"right\">0.022</td></tr><tr><td align=\"left\">Waist girth (cm)</td><td align=\"right\">75.5 (10.8)</td><td align=\"right\">76.3 (11.4)</td><td align=\"right\">74.6 (10.1)</td><td align=\"right\">-3.15₁₅₇₉</td><td align=\"right\">0.002</td></tr><tr><td align=\"left\">Arm circumference (cm)</td><td align=\"right\">25.2 (3.3)</td><td align=\"right\">25.3 (3.4)</td><td align=\"right\">25.1 (3.3)</td><td align=\"right\">-1.17₁₅₉₉</td><td align=\"right\">0.244</td></tr><tr><td align=\"left\">PWC 170 score (W)</td><td align=\"right\">111.2 (29.9)</td><td align=\"right\">124.3 (31.7)</td><td align=\"right\">97.2 (19.9)</td><td align=\"right\">-19.60₁₅₀₁</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Back muscle endurance (seconds)</td><td align=\"right\">80.9 (60.4)</td><td align=\"right\">77.8 (60.1)</td><td align=\"right\">84.2 (60.5)</td><td align=\"right\">2.12₁₅₇₄</td><td align=\"right\">0.034</td></tr><tr><td align=\"left\">Abdominal muscle endurance (number of curls in 3 min)</td><td align=\"right\">21.4 (17.4)</td><td align=\"right\">25.4 (18.8)</td><td align=\"right\">17.2 (14.6)</td><td align=\"right\">-9.60₁₅₆₉</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Standing long jump distance (metres)</td><td align=\"right\">1.46 (0.29)</td><td align=\"right\">1.59 (0.28)</td><td align=\"right\">1.32 (0.23)</td><td align=\"right\">-20.90₁₅₈₈</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Basketball throw (metres)</td><td align=\"right\">5.3 (1.0)</td><td align=\"right\">5.7 (1.0)</td><td align=\"right\">4.8 (0.7)</td><td align=\"right\">-21.72₁₅₈₃</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Total Hand strength – right and left combined (kg)</td><td align=\"right\">51.8 (13.5)</td><td align=\"right\">57.0 (14.8)</td><td align=\"right\">46.3 (9.1)</td><td align=\"right\">-17.20₁₅₉₇</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Shoulder stretch (L) (%able)</td><td align=\"right\">88.9%</td><td align=\"right\">84.7%</td><td align=\"right\">92.8%</td><td align=\"right\">χ²= 26.1</td><td align=\"right\">¹&lt;0.001</td></tr><tr><td align=\"left\">NDI score</td><td align=\"right\">97.2 (17.4)</td><td align=\"right\">97.3 (18.1)</td><td align=\"right\">97.0 (16.6)</td><td align=\"right\">-0.31₁₅₇₆</td><td align=\"right\">0.741</td></tr><tr><td align=\"left\">Persistent control factor score</td><td align=\"right\">103.3 (25.4)</td><td align=\"right\">99.9 (26.4)</td><td align=\"right\">106.8 (23.7)</td><td align=\"right\">5.44₁₅₉₄</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Muscle Power factor score</td><td align=\"right\">95.9 (20.2)</td><td align=\"right\">102.4 (19.8)</td><td align=\"right\">89.2 (18.5)</td><td align=\"right\">-13.79₁₅₈₃</td><td align=\"right\">&lt;0.001</td></tr><tr><td align=\"left\">Kinaesthetic Integration factor score</td><td align=\"right\">96.9 (15.2)</td><td align=\"right\">96.7 (15.7)</td><td align=\"right\">97.2 (14.7)</td><td align=\"right\">0.68₁₅₉₆</td><td align=\"right\">0.501</td></tr><tr><td align=\"left\">Bimanual Dexterity factor score</td><td align=\"right\">97.1 (19.3)</td><td align=\"right\">95.1 (19.3)</td><td align=\"right\">99.1 (19.1)</td><td align=\"right\">4.09₁₅₉₈</td><td align=\"right\">&lt;0.001</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Univariate relationship between physical characteristics and neck/shoulder pain in males.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">Variable group</td><td align=\"left\">Physical variable</td><td align=\"left\" colspan=\"3\">% with pain in each group</td><td align=\"center\" colspan=\"4\">Log Regression Lowest 25% relative to IQR</td><td align=\"center\" colspan=\"4\">Log Regression Highest 25% relative to IQR</td></tr><tr><td/><td/><td/><td align=\"left\">low Q</td><td align=\"left\">IQR</td><td align=\"left\">high Q</td><td align=\"right\">p</td><td align=\"left\">OR</td><td align=\"left\">CI</td><td/><td align=\"right\">p</td><td align=\"left\">OR</td><td align=\"left\">CI</td><td/></tr></thead><tbody><tr><td align=\"left\">NSP ever</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">42%</td><td align=\"left\">40%</td><td align=\"left\">45%</td><td align=\"right\">0.635</td><td align=\"left\">1.08</td><td align=\"left\">0.78</td><td align=\"left\">1.52</td><td align=\"right\">0.235</td><td align=\"left\">1.24</td><td align=\"left\">0.87</td><td align=\"left\">1.76</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">42%</td><td align=\"left\">41%</td><td align=\"left\">44%</td><td align=\"right\">0.870</td><td align=\"left\">1.03</td><td align=\"left\">0.73</td><td align=\"left\">1.45</td><td align=\"right\">0.567</td><td align=\"left\">1.14</td><td align=\"left\">0.81</td><td align=\"left\">1.60</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">45%</td><td align=\"left\">40%</td><td align=\"left\">43%</td><td align=\"right\">0.198</td><td align=\"left\">1.25</td><td align=\"left\">0.89</td><td align=\"left\">1.76</td><td align=\"right\">0.472</td><td align=\"left\">1.13</td><td align=\"left\">0.81</td><td align=\"left\">1.60</td></tr><tr><td/><td/><td align=\"left\">waist circ.</td><td align=\"left\">43%</td><td align=\"left\">42%</td><td align=\"left\">42%</td><td align=\"right\">0.808</td><td align=\"left\">1.04</td><td align=\"left\">0.74</td><td align=\"left\">1.46</td><td align=\"right\">0.910</td><td align=\"left\">1.02</td><td align=\"left\">0.72</td><td align=\"left\">1.44</td></tr><tr><td/><td/><td align=\"left\">arm circ.</td><td align=\"left\">42%</td><td align=\"left\">41%</td><td align=\"left\">44%</td><td align=\"right\">0.792</td><td align=\"left\">1.05</td><td align=\"left\">0.75</td><td align=\"left\">1.46</td><td align=\"right\">0.409</td><td align=\"left\">1.16</td><td align=\"left\">0.82</td><td align=\"left\">1.63</td></tr><tr><td/><td align=\"left\">Aerobic</td><td align=\"left\">PWC 170</td><td align=\"left\">42%</td><td align=\"left\">42%</td><td align=\"left\">42%</td><td align=\"right\">0.893</td><td align=\"left\">1.02</td><td align=\"left\">0.72</td><td align=\"left\">1.45</td><td align=\"right\">0.972</td><td align=\"left\">1.01</td><td align=\"left\">0.71</td><td align=\"left\">1.43</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">back end.</td><td align=\"left\">38%</td><td align=\"left\">45%</td><td align=\"left\">41%</td><td align=\"right\">0.127</td><td align=\"left\">0.77</td><td align=\"left\">0.54</td><td align=\"left\">1.08</td><td align=\"right\">0.390</td><td align=\"left\">0.86</td><td align=\"left\">0.61</td><td align=\"left\">1.22</td></tr><tr><td/><td/><td align=\"left\">curls</td><td align=\"left\">43%</td><td align=\"left\">41%</td><td align=\"left\">42%</td><td align=\"right\">0.513</td><td align=\"left\">1.12</td><td align=\"left\">0.80</td><td align=\"left\">1.58</td><td align=\"right\">0.690</td><td align=\"left\">1.07</td><td align=\"left\">0.76</td><td align=\"left\">1.52</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"left\">44%</td><td align=\"left\">41%</td><td align=\"left\">41%</td><td align=\"right\">0.509</td><td align=\"left\">1.12</td><td align=\"left\">0.80</td><td align=\"left\">1.58</td><td align=\"right\">0.935</td><td align=\"left\">1.02</td><td align=\"left\">0.72</td><td align=\"left\">1.44</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"left\">40%</td><td align=\"left\">43%</td><td align=\"left\">40%</td><td align=\"right\">0.497</td><td align=\"left\">0.89</td><td align=\"left\">0.63</td><td align=\"left\">1.25</td><td align=\"right\">0.388</td><td align=\"left\">0.86</td><td align=\"left\">0.61</td><td align=\"left\">1.21</td></tr><tr><td/><td/><td align=\"left\">hand strength</td><td align=\"left\">39%</td><td align=\"left\">43%</td><td align=\"left\">43%</td><td align=\"right\">0.370</td><td align=\"left\">0.86</td><td align=\"left\">0.61</td><td align=\"left\">1.20</td><td align=\"right\">0.925</td><td align=\"left\">1.02</td><td align=\"left\">0.72</td><td align=\"left\">1.44</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">sh stretch ¹</td><td align=\"left\">34%</td><td align=\"left\">43%</td><td/><td align=\"right\">0.083</td><td align=\"left\">0.70</td><td align=\"left\">0.47</td><td align=\"left\">1.04</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">45%</td><td align=\"left\">43%</td><td align=\"left\">36%</td><td align=\"right\">0.523</td><td align=\"left\">1.12</td><td align=\"left\">0.80</td><td align=\"left\">1.57</td><td align=\"right\">0.148</td><td align=\"left\">0.77</td><td align=\"left\">0.54</td><td align=\"left\">1.10</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">43%</td><td align=\"left\">42%</td><td align=\"left\">40%</td><td align=\"right\">0.759</td><td align=\"left\">1.05</td><td align=\"left\">0.75</td><td align=\"left\">1.48</td><td align=\"right\">0.661</td><td align=\"left\">0.93</td><td align=\"left\">0.65</td><td align=\"left\">1.31</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">41%</td><td align=\"left\">44%</td><td align=\"left\">37%</td><td align=\"right\">0.427</td><td align=\"left\">0.87</td><td align=\"left\">0.62</td><td align=\"left\">1.22</td><td align=\"right\">0.179</td><td align=\"left\">0.76</td><td align=\"left\">0.51</td><td align=\"left\">1.13</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">45%</td><td align=\"left\">40%</td><td align=\"left\">42%</td><td align=\"right\">0.208</td><td align=\"left\">1.23</td><td align=\"left\">0.89</td><td align=\"left\">1.71</td><td align=\"right\">0.751</td><td align=\"left\">1.06</td><td align=\"left\">0.73</td><td align=\"left\">1.56</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">44%</td><td align=\"left\">42%</td><td align=\"left\">38%</td><td align=\"right\">0.603</td><td align=\"left\">1.08</td><td align=\"left\">0.80</td><td align=\"left\">1.47</td><td align=\"right\">0.504</td><td align=\"left\">0.87</td><td align=\"left\">0.58</td><td align=\"left\">1.31</td></tr><tr><td colspan=\"14\"><hr/></td></tr><tr><td align=\"left\">NSP month</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">21%</td><td align=\"left\">21%</td><td align=\"left\">30%</td><td align=\"right\">0.894</td><td align=\"left\">1.03</td><td align=\"left\">0.69</td><td align=\"left\">1.54</td><td align=\"right\">0.010**</td><td align=\"left\">1.69</td><td align=\"left\">1.13</td><td align=\"left\">2.50</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">19%</td><td align=\"left\">23%</td><td align=\"left\">27%</td><td align=\"right\">0.217</td><td align=\"left\">0.77</td><td align=\"left\">0.50</td><td align=\"left\">1.17</td><td align=\"right\">0.251</td><td align=\"left\">1.25</td><td align=\"left\">0.85</td><td align=\"left\">1.85</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">22%</td><td align=\"left\">23%</td><td align=\"left\">24%</td><td align=\"right\">0.850</td><td align=\"left\">0.96</td><td align=\"left\">0.65</td><td align=\"left\">1.44</td><td align=\"right\">0.709</td><td align=\"left\">0.92</td><td align=\"left\">0.58</td><td align=\"left\">1.46</td></tr><tr><td/><td/><td align=\"left\">waist circ.</td><td align=\"left\">20%</td><td align=\"left\">23%</td><td align=\"left\">25%</td><td align=\"right\">0.356</td><td align=\"left\">0.83</td><td align=\"left\">0.55</td><td align=\"left\">1.24</td><td align=\"right\">0.656</td><td align=\"left\">1.09</td><td align=\"left\">0.74</td><td align=\"left\">1.63</td></tr><tr><td/><td/><td align=\"left\">arm circ.</td><td align=\"left\">19%</td><td align=\"left\">25%</td><td align=\"left\">24%</td><td align=\"right\">0.100</td><td align=\"left\">0.71</td><td align=\"left\">0.48</td><td align=\"left\">1.07</td><td align=\"right\">0.805</td><td align=\"left\">0.95</td><td align=\"left\">0.64</td><td align=\"left\">1.42</td></tr><tr><td/><td align=\"left\">Aerobic</td><td align=\"left\">PWC 170</td><td align=\"left\">21%</td><td align=\"left\">23%</td><td align=\"left\">24%</td><td align=\"right\">0.747</td><td align=\"left\">0.93</td><td align=\"left\">0.61</td><td align=\"left\">1.42</td><td align=\"right\">0.751</td><td align=\"left\">1.07</td><td align=\"left\">0.71</td><td align=\"left\">1.61</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">back end.</td><td align=\"left\">20%</td><td align=\"left\">25%</td><td align=\"left\">21%</td><td align=\"right\">0.104</td><td align=\"left\">0.71</td><td align=\"left\">0.47</td><td align=\"left\">1.07</td><td align=\"right\">0.225</td><td align=\"left\">0.78</td><td align=\"left\">0.51</td><td align=\"left\">1.17</td></tr><tr><td/><td/><td align=\"left\">curls</td><td align=\"left\">25%</td><td align=\"left\">20%</td><td align=\"left\">25%</td><td align=\"right\">0.139</td><td align=\"left\">1.36</td><td align=\"left\">0.91</td><td align=\"left\">0.20</td><td align=\"right\">0.150</td><td align=\"left\">1.35</td><td align=\"left\">0.90</td><td align=\"left\">2.02</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"left\">23%</td><td align=\"left\">23%</td><td align=\"left\">23%</td><td align=\"right\">0.882</td><td align=\"left\">1.03</td><td align=\"left\">0.69</td><td align=\"left\">1.54</td><td align=\"right\">0.935</td><td align=\"left\">1.02</td><td align=\"left\">0.68</td><td align=\"left\">1.53</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"left\">21%</td><td align=\"left\">24%</td><td align=\"left\">22%</td><td align=\"right\">0.335</td><td align=\"left\">0.82</td><td align=\"left\">0.54</td><td align=\"left\">1.23</td><td align=\"right\">0.602</td><td align=\"left\">0.90</td><td align=\"left\">0.60</td><td align=\"left\">1.34</td></tr><tr><td/><td/><td align=\"left\">hand strength</td><td align=\"left\">18%</td><td align=\"left\">24%</td><td align=\"left\">26%</td><td align=\"right\">0.112</td><td align=\"left\">0.72</td><td align=\"left\">0.47</td><td align=\"left\">1.08</td><td align=\"right\">0.638</td><td align=\"left\">1.10</td><td align=\"left\">0.74</td><td align=\"left\">1.63</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">sh stretch ¹</td><td align=\"left\">19%</td><td align=\"left\">23%</td><td/><td align=\"right\">0.275</td><td align=\"left\">0.76</td><td align=\"left\">0.47</td><td align=\"left\">1.23</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">25%</td><td align=\"left\">23%</td><td align=\"left\">20%</td><td align=\"right\">0.598</td><td align=\"left\">1.11</td><td align=\"left\">0.75</td><td align=\"left\">1.65</td><td align=\"right\">0.447</td><td align=\"left\">0.85</td><td align=\"left\">0.56</td><td align=\"left\">1.29</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">21%</td><td align=\"left\">25%</td><td align=\"left\">20%</td><td align=\"right\">0.268</td><td align=\"left\">0.80</td><td align=\"left\">0.53</td><td align=\"left\">1.19</td><td align=\"right\">0.152</td><td align=\"left\">0.74</td><td align=\"left\">0.49</td><td align=\"left\">1.12</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">20%</td><td align=\"left\">25%</td><td align=\"left\">20%</td><td align=\"right\">0.163</td><td align=\"left\">0.75</td><td align=\"left\">0.50</td><td align=\"left\">1.12</td><td align=\"right\">0.254</td><td align=\"left\">0.76</td><td align=\"left\">0.47</td><td align=\"left\">1.22</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">24%</td><td align=\"left\">23%</td><td align=\"left\">22%</td><td align=\"right\">0.765</td><td align=\"left\">1.06</td><td align=\"left\">0.72</td><td align=\"left\">1.56</td><td align=\"right\">0.753</td><td align=\"left\">0.93</td><td align=\"left\">0.59</td><td align=\"left\">1.46</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">24%</td><td align=\"left\">23%</td><td align=\"left\">22%</td><td align=\"right\">0.835</td><td align=\"left\">1.04</td><td align=\"left\">0.73</td><td align=\"left\">1.49</td><td align=\"right\">0.805</td><td align=\"left\">0.94</td><td align=\"left\">0.58</td><td align=\"left\">1.52</td></tr><tr><td colspan=\"14\"><hr/></td></tr><tr><td align=\"left\">NSP chronic</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">6%</td><td align=\"left\">6%</td><td align=\"left\">9%</td><td align=\"right\">0.937</td><td align=\"left\">0.97</td><td align=\"left\">0.49</td><td align=\"left\">1.93</td><td align=\"right\">0.254</td><td align=\"left\">1.46</td><td align=\"left\">0.76</td><td align=\"left\">2.79</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">3%</td><td align=\"left\">8%</td><td align=\"left\">8%</td><td align=\"right\">0.050*</td><td align=\"left\">0.43</td><td align=\"left\">0.19</td><td align=\"left\">1.00</td><td align=\"right\">0.724</td><td align=\"left\">1.12</td><td align=\"left\">0.60</td><td align=\"left\">2.07</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">6%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.740</td><td align=\"left\">0.89</td><td align=\"left\">0.45</td><td align=\"left\">1.76</td><td align=\"right\">0.799</td><td align=\"left\">0.92</td><td align=\"left\">0.47</td><td align=\"left\">1.80</td></tr><tr><td/><td/><td align=\"left\">waist circ.</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.958</td><td align=\"left\">1.02</td><td align=\"left\">0.52</td><td align=\"left\">1.99</td><td align=\"right\">0.798</td><td align=\"left\">1.09</td><td align=\"left\">0.56</td><td align=\"left\">2.13</td></tr><tr><td/><td/><td align=\"left\">arm circ.</td><td align=\"left\">4%</td><td align=\"left\">9%</td><td align=\"left\">7%</td><td align=\"right\">0.028*</td><td align=\"left\">0.43</td><td align=\"left\">0.20</td><td align=\"left\">0.91</td><td align=\"right\">0.357</td><td align=\"left\">0.73</td><td align=\"left\">0.38</td><td align=\"left\">1.42</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">PWC 170</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"left\">6%</td><td align=\"right\">0.369</td><td align=\"left\">0.72</td><td align=\"left\">0.35</td><td align=\"left\">1.47</td><td align=\"right\">0.361</td><td align=\"left\">0.72</td><td align=\"left\">0.35</td><td align=\"left\">1.46</td></tr><tr><td/><td/><td align=\"left\">back end.</td><td align=\"left\">7%</td><td align=\"left\">6%</td><td align=\"left\">9%</td><td align=\"right\">0.453</td><td align=\"left\">1.29</td><td align=\"left\">0.66</td><td align=\"left\">2.54</td><td align=\"right\">0.183</td><td align=\"left\">1.56</td><td align=\"left\">0.81</td><td align=\"left\">2.99</td></tr><tr><td/><td/><td align=\"left\">curls</td><td align=\"left\">7%</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"right\">0.759</td><td align=\"left\">1.12</td><td align=\"left\">0.56</td><td align=\"left\">2.24</td><td align=\"right\">0.297</td><td align=\"left\">1.42</td><td align=\"left\">0.74</td><td align=\"left\">2.74</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.893</td><td align=\"left\">0.96</td><td align=\"left\">0.49</td><td align=\"left\">1.88</td><td align=\"right\">0.952</td><td align=\"left\">1.02</td><td align=\"left\">0.52</td><td align=\"left\">2.01</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"left\">6%</td><td align=\"left\">5%</td><td align=\"left\">10%</td><td align=\"right\">0.394</td><td align=\"left\">1.37</td><td align=\"left\">0.66</td><td align=\"left\">2.84</td><td align=\"right\">0.010**</td><td align=\"left\">2.33</td><td align=\"left\">1.22</td><td align=\"left\">4.44</td></tr><tr><td/><td/><td align=\"left\">hand strength</td><td align=\"left\">5%</td><td align=\"left\">6%</td><td align=\"left\">10%</td><td align=\"right\">0.398</td><td align=\"left\">0.72</td><td align=\"left\">0.34</td><td align=\"left\">1.53</td><td align=\"right\">0.084</td><td align=\"left\">1.72</td><td align=\"left\">0.93</td><td align=\"left\">3.18</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">sh stretch ¹</td><td align=\"left\">3%</td><td align=\"left\">8%</td><td/><td align=\"right\">0.054</td><td align=\"left\">0.31</td><td align=\"left\">0.09</td><td align=\"left\">1.02</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">8%</td><td align=\"left\">5%</td><td align=\"left\">10%</td><td align=\"right\">0.117</td><td align=\"left\">1.73</td><td align=\"left\">0.87</td><td align=\"left\">3.45</td><td align=\"right\">0.025*</td><td align=\"left\">2.13</td><td align=\"left\">1.10</td><td align=\"left\">4.12</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"left\">6%</td><td align=\"right\">0.384</td><td align=\"left\">0.74</td><td align=\"left\">0.37</td><td align=\"left\">1.47</td><td align=\"right\">0.491</td><td align=\"left\">0.79</td><td align=\"left\">0.39</td><td align=\"left\">1.56</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">6%</td><td align=\"left\">6%</td><td align=\"left\">10%</td><td align=\"right\">0.877</td><td align=\"left\">0.95</td><td align=\"left\">0.48</td><td align=\"left\">1.89</td><td align=\"right\">0.144</td><td align=\"left\">1.66</td><td align=\"left\">0.84</td><td align=\"left\">3.29</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">7%</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"right\">0.956</td><td align=\"left\">1.02</td><td align=\"left\">0.53</td><td align=\"left\">1.97</td><td align=\"right\">0.614</td><td align=\"left\">1.20</td><td align=\"left\">0.59</td><td align=\"left\">2.48</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">8%</td><td align=\"left\">6%</td><td align=\"left\">4%</td><td align=\"right\">0.317</td><td align=\"left\">1.34</td><td align=\"left\">0.75</td><td align=\"left\">2.39</td><td align=\"right\">0.255</td><td align=\"left\">0.57</td><td align=\"left\">0.21</td><td align=\"left\">1.51</td></tr><tr><td colspan=\"14\"><hr/></td></tr><tr><td align=\"left\">NSP Diag</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.690</td><td align=\"left\">1.14</td><td align=\"left\">0.60</td><td align=\"left\">2.17</td><td align=\"right\">0.991</td><td align=\"left\">1.00</td><td align=\"left\">0.50</td><td align=\"left\">2.03</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">4%</td><td align=\"left\">7%</td><td align=\"left\">9%</td><td align=\"right\">0.110</td><td align=\"left\">0.52</td><td align=\"left\">0.23</td><td align=\"left\">1.16</td><td align=\"right\">0.587</td><td align=\"left\">1.19</td><td align=\"left\">0.64</td><td align=\"left\">2.21</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">6%</td><td align=\"left\">7%</td><td align=\"left\">8%</td><td align=\"right\">0.628</td><td align=\"left\">0.84</td><td align=\"left\">0.42</td><td align=\"left\">1.69</td><td align=\"right\">0.765</td><td align=\"left\">1.10</td><td align=\"left\">0.58</td><td align=\"left\">2.12</td></tr><tr><td/><td/><td align=\"left\">waist</td><td align=\"left\">7%</td><td align=\"left\">6%</td><td align=\"left\">9%</td><td align=\"right\">0.870</td><td align=\"left\">1.06</td><td align=\"left\">0.53</td><td align=\"left\">2.12</td><td align=\"right\">0.210</td><td align=\"left\">1.51</td><td align=\"left\">0.79</td><td align=\"left\">2.86</td></tr><tr><td/><td/><td align=\"left\">arm</td><td align=\"left\">5%</td><td align=\"left\">7%</td><td align=\"left\">9%</td><td align=\"right\">0.433</td><td align=\"left\">0.75</td><td align=\"left\">0.37</td><td align=\"left\">1.53</td><td align=\"right\">0.440</td><td align=\"left\">1.29</td><td align=\"left\">0.68</td><td align=\"left\">2.43</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">PWC 170</td><td align=\"left\">9%</td><td align=\"left\">6%</td><td align=\"left\">6%</td><td align=\"right\">0.300</td><td align=\"left\">1.41</td><td align=\"left\">0.74</td><td align=\"left\">2.69</td><td align=\"right\">0.793</td><td align=\"left\">0.91</td><td align=\"left\">0.43</td><td align=\"left\">1.89</td></tr><tr><td/><td/><td align=\"left\">BE</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.985</td><td align=\"left\">1.01</td><td align=\"left\">0.52</td><td align=\"left\">1.97</td><td align=\"right\">0.920</td><td align=\"left\">0.97</td><td align=\"left\">0.49</td><td align=\"left\">1.92</td></tr><tr><td/><td/><td align=\"left\">Curls</td><td align=\"left\">6%</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"right\">0.934</td><td align=\"left\">0.97</td><td align=\"left\">0.48</td><td align=\"left\">1.98</td><td align=\"right\">0.462</td><td align=\"left\">1.28</td><td align=\"left\">0.66</td><td align=\"left\">2.50</td></tr><tr><td/><td/><td align=\"left\">Jump</td><td align=\"left\">8%</td><td align=\"left\">6%</td><td align=\"left\">9%</td><td align=\"right\">0.338</td><td align=\"left\">1.39</td><td align=\"left\">0.71</td><td align=\"left\">2.73</td><td align=\"right\">0.087</td><td align=\"left\">1.77</td><td align=\"left\">0.92</td><td align=\"left\">3.40</td></tr><tr><td/><td/><td align=\"left\">BT</td><td align=\"left\">5%</td><td align=\"left\">6%</td><td align=\"left\">9%</td><td align=\"right\">0.647</td><td align=\"left\">0.84</td><td align=\"left\">0.41</td><td align=\"left\">1.75</td><td align=\"right\">0.314</td><td align=\"left\">1.39</td><td align=\"left\">0.73</td><td align=\"left\">2.64</td></tr><tr><td/><td/><td align=\"left\">HS</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"left\">5%</td><td align=\"right\">0.319</td><td align=\"left\">0.71</td><td align=\"left\">0.37</td><td align=\"left\">1.39</td><td align=\"right\">0.226</td><td align=\"left\">0.64</td><td align=\"left\">0.31</td><td align=\"left\">1.32</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">sh stretch ¹</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td/><td align=\"right\">0.489</td><td align=\"left\">1.29</td><td align=\"left\">0.63</td><td align=\"left\">2.63</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">9%</td><td align=\"left\">6%</td><td align=\"left\">7%</td><td align=\"right\">0.230</td><td align=\"left\">1.49</td><td align=\"left\">0.78</td><td align=\"left\">2.86</td><td align=\"right\">0.497</td><td align=\"left\">1.27</td><td align=\"left\">0.64</td><td align=\"left\">2.53</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">5%</td><td align=\"left\">8%</td><td align=\"left\">5%</td><td align=\"right\">0.150</td><td align=\"left\">0.60</td><td align=\"left\">0.30</td><td align=\"left\">1.21</td><td align=\"right\">0.174</td><td align=\"left\">0.60</td><td align=\"left\">0.29</td><td align=\"left\">1.25</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">9%</td><td align=\"left\">7%</td><td align=\"left\">5%</td><td align=\"right\">0.444</td><td align=\"left\">1.27</td><td align=\"left\">0.69</td><td align=\"left\">2.35</td><td align=\"right\">0.446</td><td align=\"left\">0.71</td><td align=\"left\">0.29</td><td align=\"left\">1.73</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">7%</td><td align=\"left\">6%</td><td align=\"left\">9%</td><td align=\"right\">0.663</td><td align=\"left\">1.16</td><td align=\"left\">0.60</td><td align=\"left\">2.22</td><td align=\"right\">0.318</td><td align=\"left\">1.44</td><td align=\"left\">0.71</td><td align=\"left\">2.91</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"left\">3%</td><td align=\"right\">0.572</td><td align=\"left\">1.18</td><td align=\"left\">0.67</td><td align=\"left\">2.09</td><td align=\"right\">0.110</td><td align=\"left\">0.42</td><td align=\"left\">0.14</td><td align=\"left\">1.22</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Multivariate relationships between physical characteristics and each type of neck/shoulder pain in males and females.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Gender</bold></td><td align=\"left\"><bold>NSP variable</bold></td><td align=\"left\"><bold>Physical characteristics associating with NSP (at P &lt; 0.1)</bold></td><td align=\"right\"><bold>Lowest 25% relative to IQR</bold><bold>OR, (95% CI)</bold></td><td align=\"right\"><bold>Highest 25% relative to IQR,</bold><bold>OR (95% CI)</bold></td></tr></thead><tbody><tr><td align=\"center\">Male</td><td align=\"left\">NSP ever</td><td align=\"left\">NDI</td><td align=\"right\">1.26 (0.87–1.83)</td><td align=\"right\">0.73 (0.50–1.1)*</td></tr><tr><td/><td/><td align=\"left\">back endurance</td><td align=\"right\">0.66 (0.46–0.97)**</td><td align=\"right\">0.82 (0.57–1.18)</td></tr><tr><td/><td align=\"left\">NSP month</td><td align=\"left\">-</td><td align=\"right\">-</td><td align=\"right\">-</td></tr><tr><td/><td align=\"left\">NSP chronic</td><td align=\"left\">throw</td><td align=\"right\">1.96 (0.87–4.45)</td><td align=\"right\">2.47 (1.22–5.00)**</td></tr><tr><td/><td/><td align=\"left\">shoulder stretch</td><td align=\"right\">¹0.30 (0.09–1.01)*</td><td align=\"right\">NA</td></tr><tr><td/><td align=\"left\">NSP diagnosed</td><td align=\"left\">jump</td><td align=\"right\">0.75 ((0.27–2.07)</td><td align=\"right\">3.47 (1.55–7.74)***</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"right\">1.92 (0.70–5.30)</td><td align=\"right\">0.33 (0.12–0.94)**</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"right\">0.42 (0.19–0.95)**</td><td align=\"right\">0.69 (0.33–1.46)</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"center\">Female</td><td align=\"left\">NSP ever</td><td align=\"left\">Abdominal curls</td><td align=\"right\">1.36 (0.94–1.97)</td><td align=\"right\">1.57 (1.07–2.311)**</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"right\">0.97 (0.66–1.42)</td><td align=\"right\">0.60 (0.40–0.90)**</td></tr><tr><td/><td/><td align=\"left\">shoulder stretch</td><td align=\"right\">¹0.54 (0.30–0.98)**</td><td align=\"right\">NA</td></tr><tr><td/><td align=\"left\">NSP month</td><td align=\"left\">throw</td><td align=\"right\">1.27 (0.84–1.90)</td><td align=\"right\">0.53 (0.34–0.84)***</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"right\">0.61 (0.41–0.92)**</td><td align=\"right\">0.70 (0.46–1.06)*</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"right\">0.86 (0.58–1.26)</td><td align=\"right\">1.77 (1.18–2.65)***</td></tr><tr><td/><td/><td align=\"left\">PWC</td><td align=\"right\">0.751 (0.50–1.13)</td><td align=\"right\">0.61 (0.40–0.93)**</td></tr><tr><td/><td align=\"left\">NSP chronic</td><td align=\"left\">-</td><td align=\"right\">-</td><td align=\"right\">-</td></tr><tr><td/><td align=\"left\">NSP diagnosed</td><td align=\"left\">back endurance</td><td align=\"right\">2.12 (1.20–3.74)**</td><td align=\"right\">2.12 (1.18–3.83)**</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Univariate relationship between physical characteristics and neck/shoulder pain in females.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">Variable group</td><td align=\"left\">Physical variable</td><td align=\"left\" colspan=\"3\">% with pain in each group</td><td align=\"center\" colspan=\"4\">Log Regression Lowest 25% relative to IQR</td><td align=\"center\" colspan=\"4\">Log Regression Highest 25% relative to IQR</td></tr><tr><td/><td/><td/><td align=\"left\">low Q</td><td align=\"left\">IQR</td><td align=\"left\">high Q</td><td align=\"right\">p</td><td align=\"left\">OR</td><td align=\"left\">CI</td><td/><td align=\"right\">p</td><td align=\"left\">OR</td><td align=\"left\">CI</td><td/></tr></thead><tbody><tr><td align=\"left\">NSP ever</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">50%</td><td align=\"left\">52%</td><td align=\"left\">52%</td><td align=\"right\">0.658</td><td align=\"left\">0.93</td><td align=\"left\">0.66</td><td align=\"left\">1.3</td><td align=\"right\">0.955</td><td align=\"left\">0.99</td><td align=\"left\">0.7</td><td align=\"left\">1.41</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">51%</td><td align=\"left\">53%</td><td align=\"left\">51%</td><td align=\"right\">0.681</td><td align=\"left\">0.93</td><td align=\"left\">0.66</td><td align=\"left\">1.31</td><td align=\"right\">0.564</td><td align=\"left\">0.9</td><td align=\"left\">0.64</td><td align=\"left\">1.28</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">54%</td><td align=\"left\">52%</td><td align=\"left\">49%</td><td align=\"right\">0.617</td><td align=\"left\">1.09</td><td align=\"left\">0.77</td><td align=\"left\">1.54</td><td align=\"right\">0.501</td><td align=\"left\">0.89</td><td align=\"left\">0.63</td><td align=\"left\">1.26</td></tr><tr><td/><td/><td align=\"left\">waist circ.</td><td align=\"left\">51%</td><td align=\"left\">52%</td><td align=\"left\">52%</td><td align=\"right\">0.779</td><td align=\"left\">0.95</td><td align=\"left\">0.68</td><td align=\"left\">1.34</td><td align=\"right\">0.979</td><td align=\"left\">1</td><td align=\"left\">0.7</td><td align=\"left\">1.41</td></tr><tr><td/><td/><td align=\"left\">arm circ.</td><td align=\"left\">52%</td><td align=\"left\">53%</td><td align=\"left\">48%</td><td align=\"right\">0.800</td><td align=\"left\">0.96</td><td align=\"left\">0.69</td><td align=\"left\">1.33</td><td align=\"right\">0.302</td><td align=\"left\">0.82</td><td align=\"left\">0.57</td><td align=\"left\">1.19</td></tr><tr><td/><td align=\"left\">Aerobic</td><td align=\"left\">PWC 170</td><td align=\"left\">50%</td><td align=\"left\">55%</td><td align=\"left\">48%</td><td align=\"right\">0.314</td><td align=\"left\">0.83</td><td align=\"left\">0.58</td><td align=\"left\">1.19</td><td align=\"right\">0.136</td><td align=\"left\">0.76</td><td align=\"left\">0.53</td><td align=\"left\">1.09</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">back end.</td><td align=\"left\">51%</td><td align=\"left\">51%</td><td align=\"left\">55%</td><td align=\"right\">0.878</td><td align=\"left\">0.97</td><td align=\"left\">0.69</td><td align=\"left\">1.38</td><td align=\"right\">0.389</td><td align=\"left\">1.17</td><td align=\"left\">0.82</td><td align=\"left\">1.65</td></tr><tr><td/><td/><td align=\"left\">curls</td><td align=\"left\">54%</td><td align=\"left\">48%</td><td align=\"left\">56%</td><td align=\"right\">0.181</td><td align=\"left\">1.26</td><td align=\"left\">0.9</td><td align=\"left\">1.78</td><td align=\"right\">0.091</td><td align=\"left\">1.36</td><td align=\"left\">0.95</td><td align=\"left\">1.94</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"left\">47%</td><td align=\"left\">55%</td><td align=\"left\">51%</td><td align=\"right\">0.069</td><td align=\"left\">0.73</td><td align=\"left\">0.52</td><td align=\"left\">1.03</td><td align=\"right\">0.351</td><td align=\"left\">0.85</td><td align=\"left\">0.6</td><td align=\"left\">1.2</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"left\">54%</td><td align=\"left\">54%</td><td align=\"left\">44%</td><td align=\"right\">0.988</td><td align=\"left\">1</td><td align=\"left\">0.71</td><td align=\"left\">1.41</td><td align=\"right\">0.039*</td><td align=\"left\">0.68</td><td align=\"left\">0.47</td><td align=\"left\">0.98</td></tr><tr><td/><td/><td align=\"left\">hand strength</td><td align=\"left\">51%</td><td align=\"left\">51%</td><td align=\"left\">55%</td><td align=\"right\">0.975</td><td align=\"left\">1.01</td><td align=\"left\">0.72</td><td align=\"left\">1.41</td><td align=\"right\">0.312</td><td align=\"left\">1.2</td><td align=\"left\">0.84</td><td align=\"left\">1.71</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">sh stretch ¹</td><td align=\"left\">40%</td><td align=\"left\">53%</td><td/><td align=\"right\">0.074</td><td align=\"left\">0.60</td><td align=\"left\">0.34</td><td align=\"left\">1.05</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">48%</td><td align=\"left\">54%</td><td align=\"left\">53%</td><td align=\"right\">0.137</td><td align=\"left\">0.77</td><td align=\"left\">0.55</td><td align=\"left\">1.09</td><td align=\"right\">0.848</td><td align=\"left\">0.97</td><td align=\"left\">0.68</td><td align=\"left\">1.37</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">47%</td><td align=\"left\">55%</td><td align=\"left\">52%</td><td align=\"right\">0.079</td><td align=\"left\">0.75</td><td align=\"left\">0.54</td><td align=\"left\">1.03</td><td align=\"right\">0.617</td><td align=\"left\">0.9</td><td align=\"left\">0.6</td><td align=\"left\">1.35</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">49%</td><td align=\"left\">56%</td><td align=\"left\">48%</td><td align=\"right\">0.118</td><td align=\"left\">0.77</td><td align=\"left\">0.55</td><td align=\"left\">1.07</td><td align=\"right\">0.080</td><td align=\"left\">0.73</td><td align=\"left\">0.51</td><td align=\"left\">1.04</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">51%</td><td align=\"left\">52%</td><td align=\"left\">55%</td><td align=\"right\">0.840</td><td align=\"left\">0.97</td><td align=\"left\">0.69</td><td align=\"left\">1.35</td><td align=\"right\">0.583</td><td align=\"left\">1.12</td><td align=\"left\">0.75</td><td align=\"left\">1.65</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">50%</td><td align=\"left\">51%</td><td align=\"left\">57%</td><td align=\"right\">0.975</td><td align=\"left\">1</td><td align=\"left\">0.72</td><td align=\"left\">1.38</td><td align=\"right\">0.163</td><td align=\"left\">1.29</td><td align=\"left\">0.9</td><td align=\"left\">1.85</td></tr><tr><td colspan=\"14\"><hr/></td></tr><tr><td align=\"left\">NSP month</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">35%</td><td align=\"left\">35%</td><td align=\"left\">34%</td><td align=\"right\">0.951</td><td align=\"left\">0.99</td><td align=\"left\">0.69</td><td align=\"left\">1.41</td><td align=\"right\">0.723</td><td align=\"left\">0.94</td><td align=\"left\">0.65</td><td align=\"left\">1.35</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">36%</td><td align=\"left\">34%</td><td align=\"left\">34%</td><td align=\"right\">0.701</td><td align=\"left\">1.07</td><td align=\"left\">0.75</td><td align=\"left\">1.53</td><td align=\"right\">0.923</td><td align=\"left\">0.98</td><td align=\"left\">0.68</td><td align=\"left\">1.41</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">37%</td><td align=\"left\">35%</td><td align=\"left\">31%</td><td align=\"right\">0.567</td><td align=\"left\">1.11</td><td align=\"left\">0.78</td><td align=\"left\">1.59</td><td align=\"right\">0.431</td><td align=\"left\">0.86</td><td align=\"left\">0.6</td><td align=\"left\">1.25</td></tr><tr><td/><td/><td align=\"left\">waist circ.</td><td align=\"left\">37%</td><td align=\"left\">34%</td><td align=\"left\">32%</td><td align=\"right\">0.419</td><td align=\"left\">1.16</td><td align=\"left\">0.81</td><td align=\"left\">1.66</td><td align=\"right\">0.723</td><td align=\"left\">0.94</td><td align=\"left\">0.65</td><td align=\"left\">1.36</td></tr><tr><td/><td/><td align=\"left\">Arm circ.</td><td align=\"left\">38%</td><td align=\"left\">25%</td><td align=\"left\">31%</td><td align=\"right\">0.476</td><td align=\"left\">1.13</td><td align=\"left\">0.81</td><td align=\"left\">1.59</td><td align=\"right\">0.347</td><td align=\"left\">0.83</td><td align=\"left\">0.56</td><td align=\"left\">1.23</td></tr><tr><td/><td align=\"left\">Aerobic</td><td align=\"left\">PWC 170</td><td align=\"left\">34%</td><td align=\"left\">38%</td><td align=\"left\">29%</td><td align=\"right\">0.331</td><td align=\"left\">0.83</td><td align=\"left\">0.57</td><td align=\"left\">1.21</td><td align=\"right\">0.031*</td><td align=\"left\">0.65</td><td align=\"left\">0.44</td><td align=\"left\">0.96</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">back end.</td><td align=\"left\">34%</td><td align=\"left\">35%</td><td align=\"left\">34%</td><td align=\"right\">0.798</td><td align=\"left\">0.95</td><td align=\"left\">0.66</td><td align=\"left\">1.37</td><td align=\"right\">0.711</td><td align=\"left\">0.93</td><td align=\"left\">0.65</td><td align=\"left\">1.35</td></tr><tr><td/><td/><td align=\"left\">curls</td><td align=\"left\">33%</td><td align=\"left\">34%</td><td align=\"left\">37%</td><td align=\"right\">0.833</td><td align=\"left\">0.96</td><td align=\"left\">0.67</td><td align=\"left\">1.38</td><td align=\"right\">0.464</td><td align=\"left\">1.15</td><td align=\"left\">0.79</td><td align=\"left\">1.66</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"left\">31%</td><td align=\"left\">40%</td><td align=\"left\">30%</td><td align=\"right\">0.033*</td><td align=\"left\">0.68</td><td align=\"left\">0.47</td><td align=\"left\">0.97</td><td align=\"right\">0.024*</td><td align=\"left\">0.65</td><td align=\"left\">0.45</td><td align=\"left\">0.95</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"left\">38%</td><td align=\"left\">36%</td><td align=\"left\">28%</td><td align=\"right\">0.605</td><td align=\"left\">1.1</td><td align=\"left\">0.77</td><td align=\"left\">1.56</td><td align=\"right\">0.052</td><td align=\"left\">0.67</td><td align=\"left\">0.45</td><td align=\"left\">1</td></tr><tr><td/><td/><td align=\"left\">hand strength</td><td align=\"left\">33%</td><td align=\"left\">36%</td><td align=\"left\">35%</td><td align=\"right\">0.445</td><td align=\"left\">0.87</td><td align=\"left\">0.61</td><td align=\"left\">1.24</td><td align=\"right\">0.803</td><td align=\"left\">0.95</td><td align=\"left\">0.66</td><td align=\"left\">1.38</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">Sh stretch ¹</td><td align=\"left\">31%</td><td align=\"left\">35%</td><td/><td align=\"right\">0.574</td><td align=\"left\">0.85</td><td align=\"left\">0.47</td><td align=\"left\">1.66</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">30%</td><td align=\"left\">35%</td><td align=\"left\">39%</td><td align=\"right\">0.285</td><td align=\"left\">0.82</td><td align=\"left\">0.57</td><td align=\"left\">1.18</td><td align=\"right\">0.272</td><td align=\"left\">1.22</td><td align=\"left\">0.85</td><td align=\"left\">1.76</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">30%</td><td align=\"left\">36%</td><td align=\"left\">38%</td><td align=\"right\">0.075</td><td align=\"left\">0.73</td><td align=\"left\">0.52</td><td align=\"left\">1.03</td><td align=\"right\">0.725</td><td align=\"left\">1.08</td><td align=\"left\">0.71</td><td align=\"left\">1.63</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">35%</td><td align=\"left\">36%</td><td align=\"left\">31%</td><td align=\"right\">0.689</td><td align=\"left\">0.93</td><td align=\"left\">0.66</td><td align=\"left\">1.32</td><td align=\"right\">0.240</td><td align=\"left\">0.8</td><td align=\"left\">0.54</td><td align=\"left\">1.17</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">31%</td><td align=\"left\">36%</td><td align=\"left\">38%</td><td align=\"right\">0.275</td><td align=\"left\">0.82</td><td align=\"left\">0.57</td><td align=\"left\">1.17</td><td align=\"right\">0.655</td><td align=\"left\">1.1</td><td align=\"left\">0.73</td><td align=\"left\">1.64</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">31%</td><td align=\"left\">33%</td><td align=\"left\">44%</td><td align=\"right\">0.594</td><td align=\"left\">0.91</td><td align=\"left\">0.64</td><td align=\"left\">1.29</td><td align=\"right\">0.008**</td><td align=\"left\">1.64</td><td align=\"left\">1.14</td><td align=\"left\">2.37</td></tr><tr><td colspan=\"14\"><hr/></td></tr><tr><td align=\"left\">NSP chronic</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">11%</td><td align=\"left\">9%</td><td align=\"left\">9%</td><td align=\"right\">0.517</td><td align=\"left\">1.2</td><td align=\"left\">0.69</td><td align=\"left\">2.1</td><td align=\"right\">0.941</td><td align=\"left\">0.98</td><td align=\"left\">0.53</td><td align=\"left\">1.8</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">12%</td><td align=\"left\">9%</td><td align=\"left\">9%</td><td align=\"right\">0.183</td><td align=\"left\">1.46</td><td align=\"left\">0.84</td><td align=\"left\">2.53</td><td align=\"right\">0.772</td><td align=\"left\">1.09</td><td align=\"left\">0.6</td><td align=\"left\">1.99</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">12%</td><td align=\"left\">8%</td><td align=\"left\">10%</td><td align=\"right\">0.091</td><td align=\"left\">1.63</td><td align=\"left\">0.93</td><td align=\"left\">2.86</td><td align=\"right\">0.323</td><td align=\"left\">1.35</td><td align=\"left\">0.75</td><td align=\"left\">2.43</td></tr><tr><td/><td/><td align=\"left\">waist circ.</td><td align=\"left\">11%</td><td align=\"left\">9%</td><td align=\"left\">11%</td><td align=\"right\">0.350</td><td align=\"left\">1.32</td><td align=\"left\">0.74</td><td align=\"left\">2.33</td><td align=\"right\">0.282</td><td align=\"left\">1.37</td><td align=\"left\">0.77</td><td align=\"left\">2.44</td></tr><tr><td/><td/><td align=\"left\">arm circ.</td><td align=\"left\">12%</td><td align=\"left\">9%</td><td align=\"left\">10%</td><td align=\"right\">0.237</td><td align=\"left\">1.38</td><td align=\"left\">0.81</td><td align=\"left\">2.37</td><td align=\"right\">0.553</td><td align=\"left\">1.21</td><td align=\"left\">0.65</td><td align=\"left\">2.26</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">PWC 170</td><td align=\"left\">9%</td><td align=\"left\">10%</td><td align=\"left\">11%</td><td align=\"right\">0.576</td><td align=\"left\">0.84</td><td align=\"left\">0.45</td><td align=\"left\">1.55</td><td align=\"right\">0.929</td><td align=\"left\">1.03</td><td align=\"left\">0.57</td><td align=\"left\">1.84</td></tr><tr><td/><td/><td align=\"left\">back end.</td><td align=\"left\">10%</td><td align=\"left\">9%</td><td align=\"left\">10%</td><td align=\"right\">0.604</td><td align=\"left\">1.17</td><td align=\"left\">0.65</td><td align=\"left\">2.09</td><td align=\"right\">0.800</td><td align=\"left\">1.08</td><td align=\"left\">0.59</td><td align=\"left\">1.97</td></tr><tr><td/><td/><td align=\"left\">curls</td><td align=\"left\">12%</td><td align=\"left\">8%</td><td align=\"left\">9%</td><td align=\"right\">0.177</td><td align=\"left\">1.48</td><td align=\"left\">0.84</td><td align=\"left\">2.61</td><td align=\"right\">0.785</td><td align=\"left\">1.09</td><td align=\"left\">0.58</td><td align=\"left\">2.05</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"left\">9%</td><td align=\"left\">11%</td><td align=\"left\">9%</td><td align=\"right\">0.410</td><td align=\"left\">0.78</td><td align=\"left\">0.44</td><td align=\"left\">1.4</td><td align=\"right\">0.415</td><td align=\"left\">0.78</td><td align=\"left\">0.42</td><td align=\"left\">1.43</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"left\">10%</td><td align=\"left\">10%</td><td align=\"left\">10%</td><td align=\"right\">0.943</td><td align=\"left\">1.02</td><td align=\"left\">0.57</td><td align=\"left\">1.82</td><td align=\"right\">0.842</td><td align=\"left\">1.06</td><td align=\"left\">0.58</td><td align=\"left\">1.97</td></tr><tr><td/><td/><td align=\"left\">hand strength</td><td align=\"left\">13%</td><td align=\"left\">7%</td><td align=\"left\">11%</td><td align=\"right\">0.032*</td><td align=\"left\">1.85</td><td align=\"left\">1.05</td><td align=\"left\">3.25</td><td align=\"right\">0.114</td><td align=\"left\">1.62</td><td align=\"left\">0.89</td><td align=\"left\">2.95</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">sh stretch ¹</td><td align=\"left\">7%</td><td align=\"left\">10%</td><td/><td align=\"right\">0.572</td><td align=\"left\">0.74</td><td align=\"left\">0.26</td><td align=\"left\">2.13</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">11%</td><td align=\"left\">10%</td><td align=\"left\">8%</td><td align=\"right\">0.617</td><td align=\"left\">1.15</td><td align=\"left\">0.66</td><td align=\"left\">2.02</td><td align=\"right\">0.654</td><td align=\"left\">0.87</td><td align=\"left\">0.47</td><td align=\"left\">1.61</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">8%</td><td align=\"left\">10%</td><td align=\"left\">11%</td><td align=\"right\">0.362</td><td align=\"left\">0.77</td><td align=\"left\">0.43</td><td align=\"left\">1.36</td><td align=\"right\">0.665</td><td align=\"left\">1.15</td><td align=\"left\">0.61</td><td align=\"left\">2.18</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">11%</td><td align=\"left\">10%</td><td align=\"left\">8%</td><td align=\"right\">0.770</td><td align=\"left\">1.09</td><td align=\"left\">0.63</td><td align=\"left\">1.88</td><td align=\"right\">0.477</td><td align=\"left\">0.79</td><td align=\"left\">0.42</td><td align=\"left\">1.51</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">9%</td><td align=\"left\">11%</td><td align=\"left\">7%</td><td align=\"right\">0.389</td><td align=\"left\">0.78</td><td align=\"left\">0.43</td><td align=\"left\">1.38</td><td align=\"right\">0.186</td><td align=\"left\">0.61</td><td align=\"left\">0.29</td><td align=\"left\">1.27</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">11%</td><td align=\"left\">9%</td><td align=\"left\">9%</td><td align=\"right\">0.398</td><td align=\"left\">1.27</td><td align=\"left\">0.73</td><td align=\"left\">2.18</td><td align=\"right\">0.840</td><td align=\"left\">1.07</td><td align=\"left\">0.58</td><td align=\"left\">1.98</td></tr><tr><td colspan=\"14\"><hr/></td></tr><tr><td align=\"left\">NSP diagnosed</td><td align=\"left\">Anthropom</td><td align=\"left\">height</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"left\">8%</td><td align=\"right\">0.429</td><td align=\"left\">0.76</td><td align=\"left\">0.38</td><td align=\"left\">1.52</td><td align=\"right\">0.994</td><td align=\"left\">1.00</td><td align=\"left\">0.51</td><td align=\"left\">1.94</td></tr><tr><td/><td/><td align=\"left\">weight</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.800</td><td align=\"left\">0.92</td><td align=\"left\">0.46</td><td align=\"left\">1.81</td><td align=\"right\">0.980</td><td align=\"left\">0.99</td><td align=\"left\">0.51</td><td align=\"left\">1.93</td></tr><tr><td/><td align=\"left\">Body comp</td><td align=\"left\">BMI</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.551</td><td align=\"left\">1.22</td><td align=\"left\">0.63</td><td align=\"left\">2.38</td><td align=\"right\">0.725</td><td align=\"left\">1.13</td><td align=\"left\">0.57</td><td align=\"left\">2.23</td></tr><tr><td/><td/><td align=\"left\">waist circ.</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.854</td><td align=\"left\">1.07</td><td align=\"left\">0.55</td><td align=\"left\">2.07</td><td align=\"right\">0.785</td><td align=\"left\">0.91</td><td align=\"left\">0.45</td><td align=\"left\">1.83</td></tr><tr><td/><td/><td align=\"left\">arm circ.</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.516</td><td align=\"left\">1.23</td><td align=\"left\">0.66</td><td align=\"left\">2.30</td><td align=\"right\">0.901</td><td align=\"left\">1.05</td><td align=\"left\">0.50</td><td align=\"left\">2.18</td></tr><tr><td/><td align=\"left\">Muscle performance</td><td align=\"left\">PWC 170</td><td align=\"left\">5%</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"right\">0.226</td><td align=\"left\">0.62</td><td align=\"left\">0.29</td><td align=\"left\">1.34</td><td align=\"right\">0.791</td><td align=\"left\">0.91</td><td align=\"left\">0.46</td><td align=\"left\">1.81</td></tr><tr><td/><td/><td align=\"left\">back end.</td><td align=\"left\">9%</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"right\">0.109</td><td align=\"left\">1.71</td><td align=\"left\">0.89</td><td align=\"left\">3.29</td><td align=\"right\">0.275</td><td align=\"left\">1.47</td><td align=\"left\">0.74</td><td align=\"left\">2.92</td></tr><tr><td/><td/><td align=\"left\">curls</td><td align=\"left\">5%</td><td align=\"left\">6%</td><td align=\"left\">10%</td><td align=\"right\">0.520</td><td align=\"left\">0.78</td><td align=\"left\">0.37</td><td align=\"left\">1.67</td><td align=\"right\">0.167</td><td align=\"left\">1.58</td><td align=\"left\">0.83</td><td align=\"left\">3.03</td></tr><tr><td/><td/><td align=\"left\">jump</td><td align=\"left\">5%</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"right\">0.196</td><td align=\"left\">0.63</td><td align=\"left\">0.31</td><td align=\"left\">1.27</td><td align=\"right\">0.433</td><td align=\"left\">0.76</td><td align=\"left\">0.38</td><td align=\"left\">1.51</td></tr><tr><td/><td/><td align=\"left\">throw</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"left\">8%</td><td align=\"right\">0.695</td><td align=\"left\">1.14</td><td align=\"left\">0.58</td><td align=\"left\">2.25</td><td align=\"right\">0.460</td><td align=\"left\">1.30</td><td align=\"left\">0.65</td><td align=\"left\">2.60</td></tr><tr><td/><td/><td align=\"left\">hand strength</td><td align=\"left\">6%</td><td align=\"left\">7%</td><td align=\"left\">8%</td><td align=\"right\">0.467</td><td align=\"left\">0.77</td><td align=\"left\">0.38</td><td align=\"left\">1.56</td><td align=\"right\">0.754</td><td align=\"left\">1.11</td><td align=\"left\">0.58</td><td align=\"left\">2.15</td></tr><tr><td/><td align=\"left\">Flexibility</td><td align=\"left\">sh stretch ¹</td><td align=\"left\">11%</td><td align=\"left\">7%</td><td/><td align=\"right\">0.220</td><td align=\"left\">1.78</td><td align=\"left\">0.71</td><td align=\"left\">4.32</td><td/><td/><td/><td/></tr><tr><td/><td align=\"left\">Motor competence</td><td align=\"left\">NDI</td><td align=\"left\">7%</td><td align=\"left\">8%</td><td align=\"left\">6%</td><td align=\"right\">0.547</td><td align=\"left\">0.81</td><td align=\"left\">0.41</td><td align=\"left\">1.60</td><td align=\"right\">0.400</td><td align=\"left\">0.74</td><td align=\"left\">0.36</td><td align=\"left\">1.51</td></tr><tr><td/><td/><td align=\"left\">PC</td><td align=\"left\">6%</td><td align=\"left\">7%</td><td align=\"left\">10%</td><td align=\"right\">0.555</td><td align=\"left\">0.82</td><td align=\"left\">0.42</td><td align=\"left\">1.60</td><td align=\"right\">0.272</td><td align=\"left\">1.49</td><td align=\"left\">0.73</td><td align=\"left\">3.01</td></tr><tr><td/><td/><td align=\"left\">MP</td><td align=\"left\">6%</td><td align=\"left\">7%</td><td align=\"left\">8%</td><td align=\"right\">0.675</td><td align=\"left\">0.86</td><td align=\"left\">0.43</td><td align=\"left\">1.72</td><td align=\"right\">0.656</td><td align=\"left\">1.17</td><td align=\"left\">0.59</td><td align=\"left\">2.29</td></tr><tr><td/><td/><td align=\"left\">KI</td><td align=\"left\">6%</td><td align=\"left\">8%</td><td align=\"left\">6%</td><td align=\"right\">0.441</td><td align=\"left\">0.77</td><td align=\"left\">0.39</td><td align=\"left\">1.51</td><td align=\"right\">0.510</td><td align=\"left\">0.77</td><td align=\"left\">0.35</td><td align=\"left\">1.70</td></tr><tr><td/><td/><td align=\"left\">BD</td><td align=\"left\">8%</td><td align=\"left\">7%</td><td align=\"left\">7%</td><td align=\"right\">0.536</td><td align=\"left\">1.22</td><td align=\"left\">0.65</td><td align=\"left\">2.32</td><td align=\"right\">0.695</td><td align=\"left\">1.15</td><td align=\"left\">0.57</td><td align=\"left\">2.33</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>PC = Persistent Control, MP = Muscle Power, KI = Kinaesthetic Integration, BD = Bimanual Dexterity</p><p>PC = persistent control, MP = muscle power, KI = kinaesthetic integration, BD = bimanual dexterity</p></table-wrap-foot>", "<table-wrap-foot><p>Females had more NSP ever (P &lt; 0.001, χ²= 16.26), more NSP in the past month (P &lt; 0.001, χ²= 27.11) and more chronic NSP (P = 0.029, χ²= 4.75). There were significant gender differences for height and weight and all physical characteristics (P &lt; 0.05) except arm circumference, kinaesthetic integration and the NDI score.</p><p>*P &lt; 0.1, **P &lt; 0.05, ***P &lt; 0.01. OR = Odds Ratio, 95%CI = 95% confidence interval, IQR = interquartile range, NDI = neurodevelopmental index, MP = muscle power, PC = persistent control, BD = bimanual dexterity, PWC = Physical Work Capacity.</p><p>¹group unable to do stretch is compared to group able to do stretch.</p></table-wrap-foot>", "<table-wrap-foot><p>OR = Odds Ratio, 95%CI = 95% confidence interval, IQR = interquartile range, BMI = body mass index, PWC = physical work capacity, SS = shoulder stretch (L), NDI = neurodevelopmental index, PC = persistent control, MP = muscle power, KI = kinesthetic integration, BD = bimanual dexterity. *P &lt; 0.05, **P &lt; 0.01</p><p>*P &lt; 0.1, **P &lt; 0.05, ***P &lt; 0.01. OR = Odds Ratio, 95%CI = 95% confidence interval, IQR = interquartile range, NDI = neurodevelopmental index, MP = muscle power, PC = persistent control, BD = bimanual dexterity, PWC = Physical Work Capacity.</p><p>¹group unable to do stretch is compared to group able to do stretch.</p></table-wrap-foot>", "<table-wrap-foot><p>*P &lt; 0.1, **P &lt; 0.05, ***P &lt; 0.01. OR = Odds Ratio, 95%CI = 95% confidence interval, IQR = interquartile range, NDI = neurodevelopmental index, MP = muscle power, PC = persistent control, BD = bimanual dexterity, PWC = Physical Work Capacity.</p><p>¹group unable to do stretch is compared to group able to do stretch.</p></table-wrap-foot>", "<table-wrap-foot><p>OR = Odds Ratio, 95%CI = 95% confidence interval, IQR = interquartile range, BMI = body mass index, PWC = physical work capacity, SS = shoulder stretch (L), NDI = neurodevelopmental index, PC = persistent control, MP = muscle power, KI = kinesthetic integration, BD = bimanual dexterity.</p><p>*P &lt; 0.05, **P &lt; 0.01 *P &lt; 0.1, **P &lt; 0.05, ***P &lt; 0.01. OR = Odds Ratio, 95%CI = 95% confidence interval, IQR = interquartile range, NDI = neurodevelopmental index, MP = muscle power, PC = persistent control, BD = bimanual dexterity, PWC = Physical Work Capacity.</p><p>¹group unable to do stretch is compared to group able to do stretch.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Almost four out of five Australians have at least one long-term health condition [##UREF##0##1##]. National expenditure on chronic disease and associated care now accounts for over two thirds of the entire health care budget [##UREF##1##2##]. Many of these diseases are preventable through the modification of the risk factors that contribute to their development [##UREF##2##3##]. In Australia, the National Chronic Disease Strategy (NCDS) has driven a renewed focus on the determinants and settings that promote the development of chronic conditions, particularly those that relate to the designated National Priority Areas of asthma, cancer, cardiovascular disease, diabetes and musculoskeletal conditions [##UREF##3##4##]. Much of this focus has centred on eliminating many of the health inequalities that persist in our population. To date however, the disproportionate disease burden carried by men, particularly the ageing male, has received little attention.</p>", "<p>In Australia, as elsewhere, men display poorer health outcomes when compared with women [##UREF##4##5##,##REF##17137425##6##]. The problems of male health in Australia reflect those increasingly noted in other developed nations. Men in Australia have a lower life expectancy than women (76.6 years compared with 82.0 years) with higher rates of mortality at all ages, a discrepancy that begins from birth [##UREF##5##7##]. There is a disproportionate level of chronic physical and psychological disease in Australian men [##UREF##5##7##] and higher rates of illness-related disability [##UREF##0##1##]. In addition, men display a higher prevalence of the major risk factors – smoking, lack of physical activity, poor nutrition and alcohol abuse – linked to the development of most major chronic diseases [##UREF##2##3##,##REF##11731817##8##]. When combined with a reduced likelihood of adopting a healthy lifestyle [##REF##17602933##9##] and a resistance to public health messages [##UREF##6##10##], the health and behaviour of ageing males in Australia should be an urgent concern.</p>", "<p>Of the disease groups targeted by the NCDS, only musculoskeletal conditions have a lower prevalence in men at all ages as compared to Australian women [##UREF##0##1##] (with current indications that the incidence of rheumatoid- and osteo-arthritis in men aged over 60 is increasing faster than that of age-matched females [##UREF##7##11##]). Cardiovascular disease (CVD), the leading cause of death and disability in Australia, strikes more men than women across the entire age spectrum, with death rates among males aged 25–74 years two to three times that of females [##UREF##8##12##]. The rates of asthma in Australia are amongst the highest in the world and whilst the prevalence is greater in males than females during childhood, this reverses through the adult years. Men, however, aged 65 years and over in Australia are more likely to suffer disability and death as a result of their asthma [##UREF##9##13##]. Three times as many men as women over the age of 65 report some type of cancer [##UREF##2##3##], including a higher prevalence of those sex-independent cancers nominated as national priorities (colorectal cancer, lung cancer, melanoma, non-melanoma skin cancers and non-Hodgkin's lymphoma [##UREF##0##1##,##UREF##10##14##]). Men also present a significant challenge in the global diabetes epidemic, showing a higher prevalence of diabetes overall and an increased rate of mortality and complications arising from the condition [##UREF##2##3##,##UREF##11##15##].</p>", "<p>Despite all these disparities, the health of men and the related changes in biological, psychological and social settings through ageing remains one of the most understudied areas of health research in Australia. Recognition of this is feeding a groundswell of support for men's health issues and policy initiatives in Australia, including a call by a number of peak and government bodies for a comprehensive men's health longitudinal study [##UREF##12##16##, ####UREF##13##17##, ##UREF##14##18####14##18##]. The Florey Adelaide Male Ageing Study (FAMAS) is a multi-disciplinary population cohort study of 1195 men, aged 35–80 years at recruitment and living in the north-west regions of Adelaide, Australia. We report here, in an analysis of the baseline cross-sectional data of the men in the cohort, the relationships between biological, social and demographic factors and the presence a number of chronic conditions considered to be of national priority.</p>" ]

[ "<title>Methods</title>", "<title>The Florey Adelaide Male Ageing Study (FAMAS)</title>", "<p>Details of the FAMAS design, procedures and participants have been published elsewhere [##REF##17594505##19##]. Briefly, subjects were recruited at random from the Electronic White Pages (EWP) using six digits of the standard eight digit telephone number in addition to prefixes and exchanges for the North Western Suburbs of Adelaide. Randomly selected households were sent an introductory letter and brochure. Approximately two weeks later a call was made to the household and the male person aged between 35 and 80 years to last have his birthday was invited for a Computer-Assisted Telephone Interview (CATI) lasting no more than 15 minutes, together with an invitation to participate in the study. A series of questions relating to age, other socio-demographic variables, history of disease and presence of risk factors enabled comparison of responders and non-responders. Of those eligible to participate, 70.7% agreed to be interviewed (Participation Rate) and 45.1% ultimately attended a clinic (Final Response Rate). Non-responders were more likely to live alone, be current smokers, and had a higher prevalence of self-reported diabetes and stroke and lower prevalence of hypercholesterolemia. A comparison with 2001 Census data showed that participants matched the local and national population for most key demographics, but younger age groups and never married men under-represented and married men and elderly participants were over-represented [##REF##17594505##19##].</p>", "<p>Recruitment of participants occurred during two phases-from August 2002 to July 2003 and from June 2004 to May 2005 and complete clinical follow-up is scheduled to reoccur every five years from baseline throughout the life of the cohort. Follow-up questionnaires are designed by a multi-disciplinary investigative team using standard measures where available and are completed annually.</p>", "<p>All protocols and procedures were approved by the Royal Adelaide Hospital Research Ethics committee and, where appropriate, the Aboriginal Health Research Ethics Committee of South Australia.</p>", "<title>Demographic and lifestyle measures</title>", "<p>Demographic and lifestyle information were obtained by self-report questionnaires [##REF##17594505##19##]. Socio-economic status was assessed using the relative advantage/disadvantage index from the Socio-Economic Indexes for Areas (SEIFA) by the Australian Bureau of Statistics. Higher scores indicate community-dwelling areas with a relatively high proportion of people with high incomes or a skilled workforce, as well as a relatively low proportion of people with low incomes and relatively few unskilled people in the workforce. Medical conditions were assessed by the following question item, 'Have you ever been told by a doctor that you have any of the following conditions?' Smoking status was determined using question items from recent Australian National Health Surveys [##UREF##15##20##]. Leisure-time physical activity was determined using items from the National Physical Activity Survey 1999 [##UREF##16##21##]. The semi-quantitative food frequency questionnaire (FFQ) developed by the Cancer Council of Victoria was used to estimate usual energy-providing macronutrient intakes [##UREF##17##22##].</p>", "<title>Anthropometric measures</title>", "<p>Anthropometry was performed using standard protocols [##UREF##18##23##] in the morning, prior to breaking an overnight fast with participants barefoot and in light clothing. Height was measured using a wall-mounted stadiometer (Seca Model No. 220, Humberg, Germany). Body weight was obtained using digital platform scales (Wedderburn UW OFWB, Taiwan, CN) and maintained by on-site engineering services. Waist circumference was assessed using a fiberglass tape measure (Gulik II, Country Technology, Wisconsin, USA). Waist circumference was measured in triplicate, taken at the level of the narrowest point (or midway) between the lower costal border and the top of the iliac crest and read in the mid-axillary line, and the mean of the three measurements was used in analyses. Coefficients of variation for triplicate waist circumference measurements were less than 2.2% for 99.7% of the sample. Standard BMI cut-offs were used to classify participants in normal/underweight (&lt;25 kg/m2), overweight (25–29.9 kg/m2), and obesity (≥ 30 kg/m2) categories.</p>", "<title>Hormone measures</title>", "<p>Blood samples were drawn between 8 and 11 am after a 12-hour overnight fast. Fasting plasma glucose and lipids (triglyceride, total cholesterol, HDL, LDL) were measured on auto-analysers in the Diagnostic Services laboratory (IMVS) on a 24-hour basis. Determination of serum lipids was done enzymatically using a Hitachi 911 (Boehringer, Germany). The inter-assay CV's for the measurement of serum lipids are as follows; triglyceride 3%, total cholesterol 2.3%, HDL 6.7% and LDL 3.7%. Glucose was determined using an automated chemistry analyser system (Olympus AU5400, Olympus Optical Co Ltd. Japan). The inter-assay CV's for this assay are 2.5% at 3.5 mmol/L and 3.0% at 19.6 mmol/L. Glycated haemoglobin (HbA1c) was measured by high-pressure liquid chromatography (HPLC) using a spherical cation exchange gel (CV 2% at 6% of total haemoglobin).</p>", "<title>Chronic disease modelling</title>", "<p>The conditions examined were based on those National Health Priority Areas covered in the recent National Chronic Disease Strategy [##UREF##3##4##], namely: cardiovascular disease, asthma, cancer, T2DM and musculoskeletal conditions. In the case of cardiovascular disease, angina was specifically selected for this model (as per [##UREF##0##1##]), given the significant burden of this condition in ageing men. The use of the term 'angina' refers to the presence (current or past) of any chronic stable angina or unstable angina as diagnosed by physician. Osteoarthritis and rheumatoid arthritis were chosen as representative of musculoskeletal conditions in this cohort, given the relatively low proportion of men with osteoporosis. Exposure variables were selected from established or suspected associations with the relevant outcome. Descriptive and tabular analysis was initially conducted to examine data distributions and observe patterns in exposure-outcome relationships.</p>", "<p>Relative risks and 95% confidence intervals were estimated by binomial regression with the log link function using the GENMOD procedure in SAS (SAS Institute, Cary, NC). Firstly, the relative risk of each potential confounder on the outcome was examined to determine individual main effects. Next, bivariate regression models were fitted with the exposure variable and age to get age-adjusted relative risks. Age was then checked as a modifier for each of the exposure variables by fitting exposure, age and the age-exposure interaction. Age was considered a modifier for an exposure variable if the interaction had a p-value &lt; 0.005 (to account for the multiple testing).</p>", "<p>Those variables whose age adjusted relative risks had a <italic>p </italic>value &lt; 0.25 were included in the final multivariate model [##UREF##20##25##]. This initial higher <italic>p </italic>value was selected to account for the confounding effect of age in the model (i.e. inclusion of age in any model of this type would decrease the overall probability of other variables reaching significance).</p>", "<p>For all conditions, the socio-demographic variables investigated were: age, income, employment status, pension status, current smoker/ever smoked, physical activity, SEIFA (Index of Disadvantage), body mass index (BMI), and waist circumference. The choice of family history and co-morbidity variables varied according to outcome.</p>" ]

[ "<title>Results</title>", "<title>Angina</title>", "<p>Of the men examined, 6.5% (n = 78) reported to having been diagnosed with angina by a physician. The majority of cases were detected in men over 55 years (n = 60). Age was found to strongly associate with the condition, peaking in 55–64 year old men (Additional file ##SUPPL##0##1##). There was a reduced risk observed for men with higher gross household incomes (most notably in the $80, 000+ category), although this relationship was largely lost when controlled for age (Additional file ##SUPPL##0##1##). Widowed men were found to have an increased risk of angina when compared with men currently married or living with a partner, again this effect was not seen in the age-standardized model (Additional file ##SUPPL##0##1##). Not being in the workforce strongly increased the risk of developing angina in both models and those currently on a pension displayed an increased risk of angina (Additional file ##SUPPL##0##1##). When factored for the age of participants, there was a notable increase in the risk of angina for those with a family history of obesity and hypertension (although the latter was fractionally outside of the nominal significance range). There was a strong interaction observed between the presence of angina and other conditions. Men that had either diabetes, hypercholesterolaemia or hypertension all displayed an increased risk of also developing angina. With the exception of elevated cholesterol, this observation held when responses were age-adjusted (Additional file ##SUPPL##0##1##).</p>", "<p>When all qualifying variables were included in the final model, age proved an extremely robust determinant of angina. When compared with men from the youngest bracket, the risk of developing angina near doubled in successive age groups (55–64 years: RR = 9.75 (3.35, 28.37); 65–80 years: RR = 18.79 (6.44, 54.79)). Having a family history of obesity also increased the likelihood of developing angina (RR = 1.84 (1.12, 3.01)). Again, a strong risk was observed if men were also hypercholesterolemic (RR = 2.97 (1.44, 6.10)) (Additional file ##SUPPL##2##3##).</p>", "<title>Asthma</title>", "<p>The prevalence of men diagnosed with asthma in the cohort was 9.5% (n = 113). The majority of respondents (51.4%) with asthma were in the youngest age group (35–44 years).</p>", "<p>A binomial regression model was applied to all selected exposure variables (Additional file. ##SUPPL##0##1##). When controlled for age, only waist circumference was found to associate with asthma. Being born in regions other than Australia or New Zealand, and being separated/divorced, were the only other factors nearing significance. All age-adjusted exposure variables with a p-value lower than 0.25, were included in the final model, in which the prevalence of asthma was associated with larger waist circumference (RR = 1.01 (1.00, 1.03)) and a history of separation/divorce (RR = 1.75 (1.18, 2.60)) (Fig. 3).</p>", "<title>Cancer</title>", "<p>Within the cohort, 10.3% of men (n = 123) reported having some form of cancer. A variety of factors was shown to associate with cancer status following the application of regression models (Additional file ##SUPPL##0##1##). In the unadjusted binomial model, the strongest association was observed for age, with older age groups demonstrating increased cancer prevalence. Also, men not in the work force, currently receiving some form of pension and with hypertension were all found to have increased risk of all-types of cancer. When controlled for age, none of these associations held significance, with only men born outside of Australia/New Zealand showing a trend towards a lowered cancer risk (p = .03). Men from the highest SEIFA quartile were at increased risk of being diagnosed with some type of cancer, an observation that held after adjustment for age. In the converged model (Additional file ##SUPPL##2##3##), an increasing age (55–64 years: RR = 2.35 (1.24, 4.44); 65–80 years: RR = 3.88 (1.93, 7.79), and being unemployed (RR = 3.47 (1.29, 9.36), <italic>p </italic>= .014) were associated with cancer risk.</p>", "<title>Type 2 Diabetes Mellitus (T2DM)</title>", "<p>Within the cohort, 15.6% of men (n = 187) either self-reported being diagnosed with T2DM or were diabetic by established clinical indicators (see Methods). There were numerous associations with diabetic status. Age showed a clear positive relationship with T2DM, with higher age groups showing elevated risk of disease. Income levels showed a particularly strong linear relationship with risk of T2DM, with lower risks observed for higher incomes in both the full and age-adjusted models. Being widowed was also shown to have a strong relationship with having T2DM in both models. Men who were either not in the work force or not on the pension displayed an increased risk of diabetes (although this was not significant for pensioners when adjusted for age). Current smokers tended to be less likely to be diabetic (although this was not observed in the age-adjusted model), whereas past smokers had an increased likelihood of diabetes. Obese participants showed an increased probability of T2DM, when compared to participants in normal weight ranges. In addition, participants with T2DM were also found to have larger waist circumferences. Men in the cohort who reported a family history of diabetes, showed a robust association with current T2DM status. As well, when controlled for age, those with a family history of obesity were moderately more likely to have T2DM. Finally, those diagnosed as hypertensive, were also at increased risk of T2DM (Additional file ##SUPPL##1##2##).</p>", "<p>When all of the eligible effectors were entered in to the final model, there were numerous determinants of T2DM detected. There was a slight influence of age on diabetes observed in the cohort, with men aged above 55 tending towards an increased risk for the condition when compared with their younger counterparts. The overall trend of men with higher incomes showing a reduced possibility of T2DM neared significance. Widowed men were at greater risk of developing diabetes when compared with married/partnered men (RR = 1.91 (1.18, 3.08)). The waist circumference relationship observed initially was also conserved in the final model, with men with larger waists again showing an elevated risk of diabetes (RR = 1.02 (1.01, 1.04)). A family history of diabetes was again shown to strongly increase risk of having the condition (RR = 1.64 (1.21, 2.24)).</p>", "<title>Osteoarthritis &amp; Rheumatoid Arthritis</title>", "<p>The prevalence of the musculoskeletal conditions under consideration were 9.7% for osteoarthritis (n = 118) and 5.0% for rheumatoid arthritis (n = 60). In both cases, almost four out of five cases occurred in men over 55 years (77.6% for osteoarthritis and 76.7% for rheumatoid arthritis). There is a noted impact of age on the association with the selected exposure variables and these musculoskeletal conditions. When age was accounted for in the binomial models, there was a reduced risk for osteoarthritis in men born overseas and those with lower waist circumferences (Additional file ##SUPPL##1##2##).</p>", "<p>The predictive model of osteoarthritis (Additional file ##SUPPL##2##3##) confirms age as a determinant of this condition, with the eldest age group showing a significantly increased risk (RR = 2.98 (1.49, 5.93)) (and a strong trend in the 55–64 age group). Of note, being obese (whilst showing a weak association in the binomial models) had a strong association with osteoarthritis in the final model (RR = 3.78 (1.35, 10.58)) (Additional file ##SUPPL##2##3##).</p>", "<p>In the case of rheumatoid arthritis, none of the selected factors proved to be significant, although there was a slight tendency for such men to be unemployed (p = .028).</p>" ]

[ "<title>Discussion</title>", "<p>The prevalence of the chronic diseases examined in this study was generally similar to available estimates from national and local data sources. The higher cancer prevalence in comparison to age-matched men nationally was the noticeable exception, and this has previously been demonstrated in two other studies of the sampling area [##UREF##19##24##,##UREF##20##25##].</p>", "<p>There was a clear effect of age on the conditions examined (with the possible exception of asthma) as has been shown previously in both men and women [##UREF##21##26##, ####REF##12887292##27##, ##REF##10688541##28####10688541##28##], however data from this study suggest that a number of social and demographic factors also contribute significantly to disease risk for each of the conditions studied, over and above age.</p>", "<title>Angina</title>", "<p>The higher rate of mortality from cardiovascular disease is one of the major drivers of the life-expectancy gap between men and women. In this cohort, angina was reported by 6.5% of all participants, a similar prevalence to a previous study undertaken in the same region at approximately the same time (5.7% of participants aged 35+) [##REF##3409497##30##], but higher than that reported in the 2004–5 NHS (4.2% of males 35–80) [##UREF##0##1##]. The high prevalence of obesity, T2DM and other risk factors for cardiovascular disease in this cohort may account for the discrepancy.</p>", "<p>The effect of age was most noticeable in participants with angina, with men aged 65+ showing a markedly elevated risk of episodes of angina relative to younger men in accordance with observations of other population based studies [##REF##12543623##31##,##REF##18179397##32##].</p>", "<p>Both hypertensive and diabetic men in the cohort showed an increased age-adjusted risk for angina. Likewise, elevated cholesterol was identified as a predictor of ever having angina in this multiadjusted model. All are well established risk factors for angina in men [##UREF##0##1##,##UREF##2##3##]. We also confirmed the independent relationship between obesity and angina. Zdrackovic (2007) in a twin registry study has recently pointed to a stronger genetic influence on the relationship between obesity and angina than previously reported [##REF##16515433##33##]. Accordingly, a combination of family history of not only angina, but also obesity should prompt the assessment of and aggressive management of risk factors.</p>", "<title>Asthma</title>", "<p>The prevalence of men with asthma in Australia (9.0% of the total population) is amongst the highest in the developed world. In this cohort, 9.4% of men reported having being ever diagnosed with asthma, with over half in the youngest age group. Whilst this prevalence is slightly higher than that observed in comparable studies of the region [##UREF##22##29##,##REF##15992315##34##] and population surveillance data [##UREF##0##1##], it does provide limited support to recent observations of rising levels of asthma in ageing men in the face of overall decreases in other sub-groups [##REF##17157658##35##]. It is suggested that this is, at least in part, related to the increase in obesity that has been observed in such men [##UREF##2##3##,##REF##15867841##36##]. Asthmatic men in our study had higher waist circumferences in all level of analyses, independent of BMI. Such a finding is consistent with other epidemiological studies involving asthmatic men (see [##REF##7572960##37##] for summary).</p>", "<p>Whilst there have been numerous studies demonstrating a protective effect of marriage on developing asthma, to our knowledge this has been the first cohort study that has specifically shown an age-independent increase in asthma susceptibility for divorced or separated men. In an analysis of a group of middle aged British men, Ebrahim (1995) [##REF##17298453##38##] speculated that the observed increase in asthmatic symptoms amongst recently separated men was most likely a combination of an increase in detrimental behaviour (alcohol consumption, smoking and physical inactivity) and the removal of spousal support. This suggestion is supported by recent data indicating that separated men aged 55+ are least likely to adhere to a written asthma plan [##REF##7479626##39##].</p>", "<p>The absence of any significant effect of smoking behaviour on asthma prevalence in this study is not in accordance with the common view of smoking being linked to adult-onset asthma. It is possible that smokers with airway disease had been classified by their treating practitioners as having chronic obstructive airways disease, and that those with asthma had been successfully convinced not to smoke. Also, there are a relatively high proportion of current smokers in the cohort who could be termed heavy consumers (i.e. greater than 25/day [##REF##15516665##40##]). It has been suggested that such smokers, through a complex interaction of inherited and environmental factors may not display asthmatic-type symptoms [##UREF##23##41##].</p>", "<title>Cancer</title>", "<p>The rising proportion of elderly men with cancer is leading to an increased investigation into the conditions which promote its development. Numerous demographic and behavioural factors have been found to associate with various types of cancer. A high proportion of men in this cohort had been diagnosed with some form of cancer when compared with age-matched men from local (4.1% of men aged over 35 years) and national surveillance data (3.3% of Australian men over 35 years). Whilst interpretation of this disparity is limited by the comparatively low number of participants in the study, a specialized health atlas of the region has identified that four out of the six highest Standardised Incidence Ratios for cancer are found in this study's catchment area [##UREF##24##42##]. This is currently the focus of intense investigation by local health authorities [##UREF##25##43##]. Interestingly we observed that age-matched men from the highest SEIFA quintile were at an increased risk of ever having cancer. In surveillance studies, lower SEIFA quintiles have generally been shown to associate with increased morbidity and mortality [##REF##17050114##44##]. It has been argued that improvements in recent years in cancer screening and awareness may have created an artificially high impression of cancer prevalence amongst affluent American men [##REF##9507716##45##]. Accordingly those men in areas of high social advantage within the study area (and better access to health services) may have an increased rate of cancer detection by physician. In contrast, men not currently employed, who have previously been shown to have a low rate of health care utilization [##REF##16343685##46##,##REF##11978676##47##], had one of the lowest rates of cancer in this study. It is tempting to speculate that these men may have cancers diagnosed late and therefore be prone to an increased cancer-related mortality.</p>", "<title>Type II diabetes mellitus (T2DM)</title>", "<p>T2DM is a common condition of the older male. In this cohort, 15.6% of the men examined at clinic, had T2DM by either self-report or fasting glucose or HbA1c, a figure slightly higher than the prevalence reported by the AusDiab study (12.3% of men aged over 35 years [##REF##17623818##48##]). Our data was collected 3 years later than AusDiab and the higher prevalence in our study most likely relates to the very high prevalence of obesity in the cohort [##REF##17594505##19##].</p>", "<p>In all levels of the analyses there were strong associations with the obesity (BMI, waist circumference, family history) in an order consistent with many other studies (see [##REF##12242454##49##, ####REF##11551001##50##, ##REF##11723347##51####11723347##51##] for summary). One of the strongest associations observed with diabetes in this cohort, was the increased risk in widowed men. Following the loss of spousal support, widowed men (both middle aged and elderly) have been shown to exhibit poor self care, for example a reduction in physical activity and sub-optimal macronutrient intake [##REF##1740599##52##]. Currently, there are some limited multi-disciplinary programmes for widowed men in Australia (support groups, 'tool-shed' social meetings etc.), which in part seek to demonstrate appropriate management of diabetes and appear to be having a positive effect on presenting complications [##REF##17272799##53##].</p>", "<p>There is also considerable evidence that supports the inverse association between income and T2DM, particularly in elderly men [##UREF##26##54##,##REF##17166533##55##], as has been reported by others in both men and women in the same geographic region [##UREF##27##56##]. Whilst multifactorial in nature, a large portion of this effect appears to reside in the (perceived and real) expense of maintaining a well-balanced diet, and an over consumption of high fat/sugar foods at lower cost [##REF##18073361##57##]. The other factors found to associate with diabetes in this study (namely, smoking and hypertension) were broadly consistent with other studies (see [##REF##17684469##58##,##REF##18163497##59##] for summary).</p>", "<title>Osteoarthritis &amp; Rheumatoid Arthritis</title>", "<p>The prevalence of musculo-skeletal conditions (particularly osteoarthritis, rheumatoid arthritis and osteoporosis) has been increasing in both in Australia [##UREF##2##3##] and abroad [##REF##12096230##60##,##REF##12809109##61##]. The proportion of elderly men with such conditions is also increasing, and to a greater extent than in ageing females [##REF##18312205##62##]. In this study, the prevalence rates of ever-diagnosed osteoarthritis and rheumatoid arthritis for men in the sampling region was the same as that reported from national sources [##UREF##2##3##].</p>", "<p>As expected, the strongest determinant of both osteoarthritis and rheumatoid arthritis was an increasing age [##UREF##2##3##]. Given the projected increased prevalence of these conditions in an ageing population and the associated impact on disability and public health costs, there is an urgent need for research identifying earlier markers and preventative treatments for these conditions. There is also a large body of evidence that identifies obesity as a risk factor for developing osteoarthritis [##UREF##28##63##, ####REF##11033593##64##, ##REF##3358403##65####3358403##65##]. Moreover once osteoarthritis is established, its progression and consequent limitations are more pronounced in obese men [##REF##16899803##66##]. Whilst it was generally assumed that this effect is mediated through a decrease in physical activity [##REF##17266077##67##], our findings are consistent with recent studies showing a minimal or non-existent relationship between osteoarthritis and physical activity patterns [##REF##10760642##68##]. The mechanism by which obesity increases the risk of osteoarthritis is likely more than a simple mechanical effect [##UREF##29##69##]. The combined problem of an ageing and increasingly obese population and the morbidity and costs associated with osteoarthritis emphasise the importance of further study and primary prevention.</p>", "<p>In a study of this type there are several limitations of the design that may limit findings. Firstly, the use of self-report data (whilst common to most health surveys and epidemiological studies) likely underestimates the true prevalence of any conditions, an effect repeatedly observed in male respondents [##UREF##2##3##]. Obviously, the type of self-report data used (current vs. 'ever-diagnosed by doctor' conditions) necessitates caution when comparing rates of disease amongst different sources. The prevalence of any condition can often differ substantially by region (even between local and statistical divisions). Indeed data from the latest National Health Survey advises standard errors of up to 25% for some conditions (e.g. angina, cancer) [##UREF##0##1##]. Second, the use of point-prevalence data in trying to examine associations with such dynamic diseases is not ideal, although this is common in most epidemiological studies of this type. Third, whilst the study included a wider range of variables than most, the complex nature of the conditions examined meant that much of the variability remains unexplained. Whilst including too many predictors can dilute an analysis of this type, inclusion of additional data might have further clarified factors associated with disease risk (e.g. alcohol consumption, nutrient, and energy intake). Finally, the directionality of many of the observed relationships cannot be determined in cross-sectional studies of this sort.</p>" ]

[ "<title>Conclusions &amp; implications</title>", "<p>This analysis was designed to give an indication of the chronic disease burden in one of the few cohort studies specific to the ageing male worldwide, and the biological, behavioural and environmental settings that associate with these conditions. Whilst many proposals and strategies have been developed to address the problem of chronic disease and ageing populations, the unique challenges posed by elderly men still receives disproportionate attention.</p>", "<p>This study demonstrated that a high proportion of men are currently suffering from chronic disease, including most of the conditions recently identified as National Health Priorities in Australia. It is clear from this study and others like it, that these conditions are further exacerbated with age. Furthermore, the prevalence of conditions examined in this study were largely equivalent to available data for age-matched men from the region, providing further qualified support to the representativeness of the cohort to the local population.</p>", "<p>Obesity was associated with most of the diseases studied, and for some, having one or more obese parents added to the risk (angina, diabetes), even if those affected were not themselves obese, (at least at the time point examined). Many men had multiple, often related, conditions (angina, cancer, diabetes) and this multi-disease state is particularly prominent in elderly men [70], underscoring the importance of primary prevention. Apart from age and obesity a number of social and demographic factors were associated with chronic disease prevalence. Spousal support is demonstrably important; separated and widowed men have an increased prevalence of asthma and diabetes, respectively. Men from low-income households were at greater risk of diabetes. Participants not in the work force (most likely due to their disease) were shown to be more likely to have angina or cancer.</p>", "<p>The ongoing collection of data from this cohort, with a high retention rate to date [##REF##17594505##19##], will provide clearer information about cause and effect relationships. Since DNA has also been collected and stored, rapid advances in technology will permit an assessment of gene-environment interactions in the future.</p>", "<p>The challenges that the ageing male presents in the current global fight against chronic disease have important policy and public health implications. Men still occupy the majority of the workforce, and in the background of an ageing population, understanding the many facets of the development of disease and disability are vital for any functional and productive society.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>An increasing proportion of Australia's chronic disease burden is carried by the ageing male. The aim of this study was to determine the prevalence of asthma, cancer, diabetes, angina and musculoskeletal conditions and their relationship to behavioural and socio-demographic factors in a cohort of Australian men.</p>", "<title>Methods</title>", "<p>Self-reports of disease status were obtained from baseline clinic visits (August 2002 – July 2003 &amp; July 2004 – May 2005) from 1195 randomly selected men, aged 35–80 years and living in the north-west regions of Adelaide. Initially, relative risks were assessed by regression against selected variables for each outcome. Where age-independent associations were observed with the relevant chronic disease, independent variables were fitted to customized multiadjusted models.</p>", "<title>Results</title>", "<p>The prevalence of all conditions was moderately higher in comparison to national data for age-matched men. In particular, there was an unusually high rate of men with cancer. Multiadjusted analyses revealed age as a predictor of chronic conditions (type 2 diabetes mellitus, angina, cancer &amp; osteoarthritis). A number of socio-demographic factors, independent of age, were associated with chronic disease, including: low income status (diabetes), separation/divorce (asthma), unemployment (cancer), high waist circumference (diabetes), elevated cholesterol (angina) and a family history of obesity (angina).</p>", "<title>Conclusion</title>", "<p>Socio-demographic factors interact to determine disease status in this broadly representative group of Australian men. In addition to obesity and a positive personal and family history of disease, men who are socially disadvantaged (low income, unemployed, separated) should be specifically targeted by public health initiatives.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>GAW &amp; MTH conceived of the study. SAM participated in the design, management and coordination of the study and drafted the manuscript. AWT provided crucial initial infrastructural support and contributes to the management of the study. GAW acts as Chief Investigator and continues to manage the study. MTH participated in the study design and coordination. SMM performed statistical analyses. All of the authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2458/8/261/prepub\"/></p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>The authors would like to acknowledge the clinic and recruitment staff for their vital efforts. Particular thanks are extended to our participants and their families for their invaluable contributions.</p>", "<p>The study was initially supported by The University of Adelaide's Florey Foundation and is currently partially funded by the South Australian Premier's Science and Research Fund. FAMAS members include: Prof Gary Wittert, Prof Janet Hiller, Prof Villis Marshall, Prof Wayne Tilley, Dr Peter O'Loughlin, Dr Megan Warin, Dr Matthew Haren.</p>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Table 1. Risk of angina, asthma and cancer by personal, behavioural and socioeconomic factors (attached).</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p>Table 2. Risk of type 2 diabetes, osteoarthritis and rheumatoid arthritis by personal, behavioural and socioeconomic factors (attached).</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional file 3</title><p>Table 3. Multivariate model of personal, behavioural and socioeconomic predictors of selected chronic diseases (attached).</p></caption></supplementary-material>" ]

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[ "<media xlink:href=\"1471-2458-8-261-S1.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>", "<media xlink:href=\"1471-2458-8-261-S2.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>", "<media xlink:href=\"1471-2458-8-261-S3.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>" ]

{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>The control of vaccine preventable diseases is one of the major advances of public health [##REF##17074423##1##]. Although immunisation uptake is high for most routine immunisations in western countries, yet high risk groups, including children with underlying diseases, have often low immunisation coverage [##REF##17074423##1##]. Chronic diseases such as neurological and cardiovascular disorders are associated with high hospitalisation rates [##REF##15710789##2##,##REF##16480796##3##], and some immunisations including influenza and conjugate pneumococcal vaccines may prevent admission into hospital, medical visits, and other negative effects in these patients [##REF##17163386##4##,##REF##17148632##5##]. Despite mathematical models suggest that focusing immunisations on high risk groups may be suitable [##REF##17347643##6##,##REF##10806982##7##], parents of children at risk may underestimate incidence and severity of vaccine preventable diseases and may not be appropriately informed about safety and efficacy of available vaccines [##REF##10806982##7##, ####REF##17257463##8##, ##REF##16890330##9##, ##REF##16905036##10####16905036##10##]. Moreover immunisation delays often occur because of false contraindications linked to an underlying condition that, on the contrary, may represent an indication to immunisation [##REF##16905036##10##, ####REF##16953011##11##, ##REF##12519624##12####12519624##12##]. Immunisation rates are usually monitored by immunisation registries [##REF##16480494##13##]; in the U.S. vaccine delivery is shifting from the public sector to the private sector, with an emphasis on vaccination in the context of primary care and the medical home [##REF##15995043##14##]. Patients with chronic diseases may refer to private providers, that often do not submit data to a national or regional registry [##REF##16480494##13##,##REF##17984712##15##]. The reasons leading parents to choose a private practice for immunisation have not been well studied yet. This study aims, primarily, to describe the characteristics of two populations attending a private or a public immunisation service in the same metropolitan area. Secondarily this study has the objective to determine if the prevalence of patients with chronic diseases by type of service differs, and to study if the choice of different providers is associated with timeliness in immunisation.</p>" ]

[ "<title>Methods</title>", "<title>Background on local immunization policies</title>", "<p>In Italy immunisations against diphtheria, tetanus, pertussis, hepatitis B, poliomyelitis, Haemophilus influenzae type b, mumps-measles-rubella are universally offered to all infants [##REF##17206224##16##]. Children with underlying diseases are recommended to receive influenza, pneumococcal, meningococcal and varicella vaccines [##UREF##0##17##,##UREF##1##18##]. A national survey performed in 2003 showed that immunisation coverage within 24 months of age for influenza, pneumococcal, and varicella vaccines was less than 3% in the general population, and less than 10% in high risk groups [##UREF##2##19##,##UREF##3##20##]. Most immunisations are delivered in Italy by the public health service while private immunisation practices administer nearly 3% of all vaccines with wide differences among Regions [##UREF##3##20##].</p>", "<p>The national immunisation schedule includes: three doses of DTaP, Polio (IPV), HepB, Hib in the first year of life; a first dose of MMR between the 12^thand the 14^thmonth; a second dose of MMR at 5 years of age. Conjugate pneumococcal, conjugate meningococcal C, and varicella vaccines are recommended at national level for selected risk groups only.</p>", "<title>Setting</title>", "<p>We conducted the present study in a private paediatric research hospital with nearly 400 beds, and in a public immunisation service in the same area. The two facilities offer the same immunisations with the same schedule but with different charges for families (Table ##TAB##0##1##).</p>", "<title>Study design</title>", "<p>We performed a cross-sectional study on parents of children 2 – 36 months of age in a private hospital immunisation service attended by outpatients or to a public immunisation office serving the same metropolitan area of Rome, Italy, with approximately 53.000 inhabitants.</p>", "<p>One interviewer (EP) visited the two practices two days a week during office hours, and systematically performed a face to face interview to parents of vaccinees from January to July 2006 until reaching the desired sample size (Figure ##FIG##0##1##; Figure ##FIG##1##2##).</p>", "<p>A questionnaire including information on children immunisation history, the reasons for delay or incomplete immunisation, and the reasons for choosing a public or a private hospital service, was administered to parents before the child was immunised after obtaining informed consent.</p>", "<title>Definitions</title>", "<p>For influenza vaccine, coverage was calculated as the proportion of children older than 6 months who had received at least one dose in the past season. For conjugate pneumococcal and meningococcal C vaccines, coverage was the proportion of children older than three months who received at least one dose of vaccine. For varicella vaccine, coverage was the proportion of children older than 12 months who ever received at least one dose of vaccine.</p>", "<p>Delay in immunisations was defined for DTP, Polio, HBV and Hib as: a) first dose received later than 105 days of age; OR b) second dose received later than 70 days after the first dose; OR c) third dose received after 380 days of age. For MMR and varicella delay was defined as an immunisation after the 15^thmonth of age. These time intervals were calculated according to national recommendations for vaccine administration [##UREF##3##20##].</p>", "<title>Sample size</title>", "<p>The sample size was calculated to detect a difference in the prevalence of high risk children in the two settings. Assuming a prevalence rate of children with underlying diseases of 3% in the general population [##UREF##2##19##] and a risk ratio of 5, a total population of 200 children (100 per group) was considered sufficient to detect a difference in prevalence between those observed in the public and in the hospital practice with a power of 80% and a level of significance of 5%.</p>", "<title>Analysis</title>", "<p>Chi square or Fisher exact test for categorical variables and the Student's t test for continuous variables were used for assessing statistical significance at the univariate level. A logistic regression model was used to asses the role of different variables as determinants of timeliness in immunisation. Odds ratios and their 95% confidence intervals were used as measures of effect in the logistic regression model.</p>" ]

[ "<title>Results</title>", "<p>A total of 202 parents of children 2–36 months of age were interviewed, 104 (51.5%) in the public office, and 98 (48.5%) in the hospital practice. (Figure ##FIG##0##1a,1b##).</p>", "<p>The general features of parents interviewed and their children in the two groups are shown in Table ##TAB##1##2##.</p>", "<p>Although several characteristics of parents and children seen in the two practices were similar, foreign parents and graduated mothers were more represented in families who requested immunisation in the public service, whereas families interviewed in the hospital practice had more frequently firstborn female children to be vaccinated, who were breast fed for a longer period, who had a lower birth weight, and who were more frequently hospitalised in the previous period.</p>", "<p>The overall immunisation coverage for influenza vaccine was 1.9% (2 patients) in children in the public office and 1.0% (1 patient) in those immunised in the hospital practice (P = 0.52). Immunisation with conjugate pneumococcal vaccine was administered to 70 (67.3%) children in the public practice and 63 (64.0%) in the hospital office (P = 0.65), whereas 59 (56.7%) of them in public service and 49 (50.0%) in hospital office were immunised with conjugate meningococcal C vaccine (P = 0.33). Finally, none of the children in the public practice and 5 (5.10%) in the hospital office were immunised against varicella vaccine (P = 0.02)</p>", "<p>Nine (9.2%) out of the 98 patients immunised in the hospital had an underlying disease which was an indication for influenza, pneumococcal, and meningococcal C immunisation. Out of them only 1 child was immunised against influenza; 6 against pneumococcus, and 4 against meningococcus C at the time of the interview. Underlying diseases included: three cases of congenital heart disease, two cases of a neurological disease, and one case of a genetic syndrome. None of the children immunised in the public service had an underlying disease (P = 0.001).</p>", "<p>The Italian Ministry of Health also recommends influenza immunisation of households of patients belonging to risk groups [##UREF##3##20##]. Among the parents of these children only 2/9 mothers and 4/9 fathers received influenza immunisation.</p>", "<p>Although the number of children with an immunisation delay was substantial in both practices, they were nearly twice in the hospital practice than in the public service (DTP, polio, HBV, and Hib: 39.8% vs 22.1%; P = 0.005; MMR: 20.0% vs 12.5%; P = 0.15). Causes of delayed or missing immunisation reported from parents were mostly related to misinformation (58%), and child illness (15.6%).</p>", "<p>Determinants of delayed immunisation was studied through a logistic regression model (Table ##TAB##2##3##). The univariate analysis showed that a lower education of parents, a low birth weight, and immunisation in the hospital predicted an immunisation delay, but none of these variables were significant in the multivariate model. None of the variables included in the analysis were associated as well with delay of MMR immunisation, while children with a father from a foreign country were more likely to receive pneumococcal immunisation (OR: 3.18; 95% CI: 1.08 – 9.42).</p>", "<p>The most frequent reason for choosing the hospital service was that parents felt safe in a hospital environment (39%). On the other hand parents whose children were immunised in the public service felt that the office was easy to reach (20.2%) or were advised by the family paediatrician (19.2%).</p>", "<p>Even if a higher proportion of children with chronic conditions requiring immunisation in the hospital was expected, it should be noted that 38% of families interviewed in the hospital service had previously visited a public office for immunisation and 38% of their children were not immunised due to a false contraindication, that most of all was represented by the child's underlying disease. Another reason for attending the hospital practice was unavailability of some vaccines due to a temporary shortage of Meningococcal, Pneumococcal or Varicella vaccines, because of their different policies compared with universally recommended vaccines. Finally 8% of children immunised in the hospital had previous contacts with the same hospital.</p>" ]

[ "<title>Discussion and conclusion</title>", "<p>This study shows that children with underlying diseases, which often represent an indication to certain immunisations, were more likely to attend the hospital immunisation provider rather than the public office of their own district.</p>", "<p>Families of children with low birth weight preferred more often immunisation in the hospital and were more likely to be immunised against influenza. Parents who chose to immunise their children in the hospital have the perception of hospital as a safer environment; this finding underlines how lack of information or misinformation and the perception of an underlying disease as a contraindication, play a substantial role in the choice of health care providers and inappropriate consultation.</p>", "<p>Despite recommendations in place, coverage for immunisations indicated in high risk groups is low in our country [##UREF##1##18##,##UREF##2##19##]. Other studies performed in hospital settings and on high risk group children showed that immunisation coverage among these groups remains low and immunisations are often delayed. [##REF##16223649##21##, ####UREF##4##22##, ##REF##17214863##23##, ##UREF##5##24##, ##REF##17020728##25##, ##REF##16203946##26##, ##REF##7980737##27##, ##REF##2386382##28####2386382##28##].</p>", "<p>We found that families of children with immunisation delay attending the hospital practice had often previously attempted to vaccinate their children. This observation underlines the potential to improve immunisation timeliness through simple information and educational activities.</p>", "<p>Different approaches have been proposed for overcoming immunisation barriers in high risk groups. There is evidence that enhanced information focusing on vaccines benefits and how to manage their potential adverse effects, increased availability of vaccine offices, use of missed opportunities, and gratuity of immunisation are efficacious in increasing immunisation uptake [##REF##16953011##11##, ####REF##12519624##12##, ##REF##16480494##13####16480494##13##].</p>", "<p>We also observed that being immunised in a hospital setting was a predictor of delayed immunisation for routine vaccines. We speculate that this finding may be associated with misinformation and false contraindications, and it is in line with the frequent parents' perception of hospital as a safe environment.</p>", "<p>Regarding the determinants of appropriateness of immunisation we did not find significant associations with any of the variables included in the analysis. However the study may have not been adequately powered to address this issue. Nevertheless, we found a signal toward an association between an higher education of parents and timeliness of immunisation.</p>", "<p>Although the number of vaccinees may be small in a private hospital practice, this finding underlines how offering immunisations indicated in high risk groups in a hospital setting may result in a significant benefit. Hospitals are likely to attract high risk groups which often are immunised late.</p>", "<p>One advantage of this study was the inclusion of different immunisation settings serving the same population although these results are not necessarily generalizable to other settings. Our findings show that an integration between public and hospital settings, and an effort to improve communication on vaccines to parents, may significantly increase immunisation rates in high risk groups and in the general population and prevent the delays in immunisation.</p>" ]

[ "<title>Discussion and conclusion</title>", "<p>This study shows that children with underlying diseases, which often represent an indication to certain immunisations, were more likely to attend the hospital immunisation provider rather than the public office of their own district.</p>", "<p>Families of children with low birth weight preferred more often immunisation in the hospital and were more likely to be immunised against influenza. Parents who chose to immunise their children in the hospital have the perception of hospital as a safer environment; this finding underlines how lack of information or misinformation and the perception of an underlying disease as a contraindication, play a substantial role in the choice of health care providers and inappropriate consultation.</p>", "<p>Despite recommendations in place, coverage for immunisations indicated in high risk groups is low in our country [##UREF##1##18##,##UREF##2##19##]. Other studies performed in hospital settings and on high risk group children showed that immunisation coverage among these groups remains low and immunisations are often delayed. [##REF##16223649##21##, ####UREF##4##22##, ##REF##17214863##23##, ##UREF##5##24##, ##REF##17020728##25##, ##REF##16203946##26##, ##REF##7980737##27##, ##REF##2386382##28####2386382##28##].</p>", "<p>We found that families of children with immunisation delay attending the hospital practice had often previously attempted to vaccinate their children. This observation underlines the potential to improve immunisation timeliness through simple information and educational activities.</p>", "<p>Different approaches have been proposed for overcoming immunisation barriers in high risk groups. There is evidence that enhanced information focusing on vaccines benefits and how to manage their potential adverse effects, increased availability of vaccine offices, use of missed opportunities, and gratuity of immunisation are efficacious in increasing immunisation uptake [##REF##16953011##11##, ####REF##12519624##12##, ##REF##16480494##13####16480494##13##].</p>", "<p>We also observed that being immunised in a hospital setting was a predictor of delayed immunisation for routine vaccines. We speculate that this finding may be associated with misinformation and false contraindications, and it is in line with the frequent parents' perception of hospital as a safe environment.</p>", "<p>Regarding the determinants of appropriateness of immunisation we did not find significant associations with any of the variables included in the analysis. However the study may have not been adequately powered to address this issue. Nevertheless, we found a signal toward an association between an higher education of parents and timeliness of immunisation.</p>", "<p>Although the number of vaccinees may be small in a private hospital practice, this finding underlines how offering immunisations indicated in high risk groups in a hospital setting may result in a significant benefit. Hospitals are likely to attract high risk groups which often are immunised late.</p>", "<p>One advantage of this study was the inclusion of different immunisation settings serving the same population although these results are not necessarily generalizable to other settings. Our findings show that an integration between public and hospital settings, and an effort to improve communication on vaccines to parents, may significantly increase immunisation rates in high risk groups and in the general population and prevent the delays in immunisation.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Improving immunisation rates in risk groups is one of the main objectives in vaccination strategies. However, achieving high vaccination rates in children with chronic conditions is difficult. Different types of vaccine providers may differently attract high risk children.</p>", "<title>Aim</title>", "<p>To describe the characteristics of two populations of children who attended a private and a public immunisation provider in the same area. Secondarily, to determine if prevalence of patients with underlying diseases by type of provider differs and to study if the choice of different providers influences timeliness in immunisation.</p>", "<title>Methods</title>", "<p>We performed a cross-sectional study on parents of children 2 – 36 months of age who attended a private hospital immunisation service or a public immunisation office serving the same metropolitan area of Rome, Italy. Data on personal characteristics and immunisation history were collected through a face to face interview with parents of vaccinees, and compared by type of provider. Prevalence of underlying conditions was compared in the two populations. Timeliness in immunisation and its determinants were analysed through a logistic regression model.</p>", "<title>Results</title>", "<p>A total of 202 parents of children 2–36 months of age were interviewed; 104 were in the public office, and 98 in the hospital practice. Children immunised in the hospital were more frequently firstborn female children, breast fed for a longer period, with a lower birthweight, and more frequently with a previous hospitalisation. The prevalence of high risk children immunised in the hospital was 9.2 vs 0% in the public service (P = 0.001). Immunisation delay for due vaccines was higher in the hospital practice than in the public service (DTP, polio, HBV, and Hib: 39.8% vs 22.1%; P = 0.005). Anyway multivariate analyses did not reveal differences in timeliness between the public and private hospital settings.</p>", "<title>Conclusion</title>", "<p>Children with underlying diseases or a low birthweight were more frequently immunised in the hospital. This finding suggests that offering immunisations in a hospital setting may facilitate vaccination uptake in high risk groups. An integration between public and hospital practices and an effort to improve communication on vaccines to parents, may significantly increase immunisation rates in high risk groups and in the general population, and prevent immunisation delays.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>All Authors participated to the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final version of the manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-2458/8/278/prepub\"/></p>" ]

[ "<title>Acknowledgements</title>", "<p>We wish to thank the immunisation services' personnel for their precious co-operation in this study.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Co-operation rate of parents to the interview in the public immunization service.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Co-operation rate of parents to the interview in the Pediatric hospital.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Comparison of the immunisation services included in the study.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">Public immunisation service</td><td align=\"left\">Hospital provider</td></tr></thead><tbody><tr><td align=\"left\">Approximate average number of immunisations per year</td><td align=\"left\">50.000</td><td align=\"left\">3.000</td></tr><tr><td align=\"left\">DTaP, Polio, Hib, HepB</td><td align=\"left\">Free</td><td align=\"left\">Fully charged</td></tr><tr><td align=\"left\">Pneumococcal conjugate vaccine</td><td align=\"left\">Copayment</td><td align=\"left\">Fully charged</td></tr><tr><td align=\"left\">Meningococcal C conjugate vaccine</td><td align=\"left\">Copayment</td><td align=\"left\">Fully charged</td></tr><tr><td align=\"left\">Influenza vaccine</td><td align=\"left\">Copayment</td><td align=\"left\">Fully charged</td></tr><tr><td align=\"left\">Varicella vaccine</td><td align=\"left\">Copayment</td><td align=\"left\">Fully charged</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Characteristics of vaccinees and their families recorded by type of practice</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\"><bold>Public immunisation service (N = 104)</bold></td><td align=\"center\"><bold>Hospital provider (N = 98)</bold></td><td align=\"center\"><bold>Total (N = 202)</bold></td><td align=\"center\"><bold>P-value</bold></td></tr></thead><tbody><tr><td align=\"left\">Mother's mean age, years (range)</td><td align=\"center\">34.7 (19 – 47)</td><td align=\"center\">34.7 (24 – 45)</td><td align=\"center\">34.7 (19–47)</td><td align=\"center\">0.96</td></tr><tr><td align=\"left\">Mother from foreign country, n (%)</td><td align=\"center\">17 (16.3%)</td><td align=\"center\">7 (7.2%)</td><td align=\"center\">24 (11.9%)</td><td align=\"center\">0.04</td></tr><tr><td align=\"left\">Graduated mother, n (%)</td><td align=\"center\">59 (56.7%)</td><td align=\"center\">40 (41.2%)</td><td align=\"center\">99 (49.2%)</td><td align=\"center\">0.03</td></tr><tr><td align=\"left\">Working mother, n (%)</td><td align=\"center\">75 (72.1%)</td><td align=\"center\">67 (69.0%)</td><td align=\"center\">142 (70.3%)</td><td align=\"center\">0.04</td></tr><tr><td align=\"left\">Father's mean age, years (range)</td><td align=\"center\">37.3 (26 – 52)</td><td align=\"center\">37.8 (23 – 63)</td><td align=\"center\">37.6 (23–63)</td><td align=\"center\">0.48</td></tr><tr><td align=\"left\">Father from foreign country, n (%)</td><td align=\"center\">13 (12.5%)</td><td align=\"center\">5 (5.1%)</td><td align=\"center\">18 (8.9%)</td><td align=\"center\">0.06</td></tr><tr><td align=\"left\">Graduated father, n (%)</td><td align=\"center\">47 (45.2%)</td><td align=\"center\">37 (37.7%)</td><td align=\"center\">84 (41.6%)</td><td align=\"center\">0.30</td></tr><tr><td align=\"left\">Working father, n (%)</td><td align=\"center\">103 (99%)</td><td align=\"center\">96 (97.9%)</td><td align=\"center\">199 (98.5%)</td><td align=\"center\">0.50</td></tr><tr><td align=\"left\">Children age, months, mean (range)</td><td align=\"center\">9.8 (2–22)</td><td align=\"center\">11.8 (2–35)</td><td align=\"center\">10.8 (2–35)</td><td align=\"center\">0.02</td></tr><tr><td align=\"left\">Male children, n (%)</td><td align=\"center\">62 (59,6%)</td><td align=\"center\">40 (40.8%)</td><td align=\"center\">102 (50.5%)</td><td align=\"center\">0.008</td></tr><tr><td align=\"left\">Firstborn child, n (%)</td><td align=\"center\">56 (53.8%)</td><td align=\"center\">69 (70.4%)</td><td align=\"center\">125 (61.9%)</td><td align=\"center\">0.01</td></tr><tr><td align=\"left\">No. of households, mean (range)</td><td align=\"center\">3.5 (2 – 5)</td><td align=\"center\">3.4 (2–6)</td><td align=\"center\">3.5 (2–6)</td><td align=\"center\">0.50</td></tr><tr><td align=\"left\">Children with Previous hospitalisations (%)</td><td align=\"center\">15 (14.4%)</td><td align=\"center\">23 (23.7%)</td><td align=\"center\">38 (18.8%)</td><td align=\"center\">0.09</td></tr><tr><td align=\"left\">Birthweight, g, mean (range)</td><td align=\"center\">3270.0(1500–4400)</td><td align=\"center\">3065.0(1180 – 4500)</td><td align=\"center\">3170 (1180–4500)</td><td align=\"center\">0.01</td></tr><tr><td align=\"left\">Breast feeding duration, months, mean (range)</td><td align=\"center\">4.21 (0 – 15)</td><td align=\"center\">5.3 (0 – 14)</td><td align=\"center\">4.7 (0–15)</td><td align=\"center\">0.04</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Likelihood of on-time vaccination (multivariate analysis results are reported only for those variables who resulted significant at the univariate analysis)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>DTP, HBV, POLIO, Hib</bold></td></tr></thead><tbody><tr><td/><td align=\"right\"><bold>Univariate analysis, OR (95% CI)</bold></td><td align=\"right\"><bold>Multivariate analysis, OR (95% CI)</bold></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td align=\"left\">Mother's age &lt; 30 yrs</td><td align=\"right\">0.52 (0.22–1.24)</td><td/></tr><tr><td align=\"left\">Mother from foreign country</td><td align=\"right\">1.17 (0.43–3.13)</td><td/></tr><tr><td align=\"left\">Mother degree</td><td align=\"right\"><bold>0.46 (0.24–0.90)</bold></td><td align=\"right\">1.387 (0.662;2.903)</td></tr><tr><td align=\"left\">Working mother</td><td align=\"right\">0.88 (0.44–1.79)</td><td/></tr><tr><td align=\"left\">Father's age</td><td align=\"right\">0.29 (0.08–1.01)</td><td/></tr><tr><td align=\"left\">Father from foreign country</td><td align=\"right\">0.86 (0.25–2.75)</td><td/></tr><tr><td align=\"left\">Father degree</td><td align=\"right\"><bold>0.41 (0.20–0.82)</bold></td><td align=\"right\">2.102 (0.963;4.589)</td></tr><tr><td align=\"left\">Working father</td><td align=\"right\">0.22 (0.01–3.11)</td><td/></tr><tr><td align=\"left\">Child age &lt; 12 months</td><td align=\"right\">0.74 (0.40–1.34)</td><td/></tr><tr><td align=\"left\">Vaccinee's male gender</td><td align=\"right\">0.97 (0.51–1.85)</td><td/></tr><tr><td align=\"left\">Child with previous hospitalization</td><td align=\"right\"><bold>2.19 (1.00–4.81)</bold></td><td align=\"right\">1.757 (0.823;3.750)</td></tr><tr><td align=\"left\">Child birthweight &lt; 2500 gr</td><td align=\"right\"><bold>0.41 (0.14–1.13)</bold></td><td align=\"right\">1.000 (1.000;1.001)</td></tr><tr><td align=\"left\">Child breastfeeding duration &lt; 6 months</td><td align=\"right\">0.79 (0.38–1.65)</td><td/></tr><tr><td align=\"left\">Firstborn child</td><td align=\"right\">1.44 (0.73–2.85)</td><td/></tr><tr><td align=\"left\">Private immunisation office</td><td align=\"right\"><bold>2.32 (1.22–4.54)</bold></td><td/></tr></tbody></table></table-wrap>" ]

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[ "<graphic xlink:href=\"1471-2458-8-278-1\"/>", "<graphic xlink:href=\"1471-2458-8-278-2\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>First described by Klemperer and Rabin [##REF##12118790##1##], the solitary fibrous tumor of the pleura (SFTP) is a localized benign neoplasm arising from the submesothelial mesenchymal layer [##REF##17075563##2##] even if malignant forms have also been described [##REF##14720142##3##]. With about 800 cases reported in the world literature, this rare entity contrast with the primary diffuse pleural mesothelioma that have an incidence of 3000 new cases every year in the USA [##UREF##0##4##]. In over half of patients the tumor is asymptomatic, but if symptoms occur then chest pain, cough and dyspnea are the most common complaints. Complete en bloc surgical resection is the treatment of choice for these neoplasms offering a cure in all patients with benign form even if tumor recurrence may occur also in tumors with benign histological features [##UREF##0##4##,##REF##16788942##5##]. We describe an unreported case of SFTP, to our knowledge, manifesting with syncope episodes when coughing.</p>" ]

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[ "<title>Discussion</title>", "<p>It is well known that solitary fibrous tumors of the pleura are incidentally discovered during chest x-ray examination because these neoplasms often have a silent clinical course for several years [##UREF##0##4##,##REF##11156076##6##]. It has been described in all ages, but the peak of incidence is in the sixth and seventh decades of life [##REF##12118790##1##]. The larger the tumor, the more likely it is that there will be symptoms [##REF##17075563##2##,##UREF##0##4##]. Systemic symptoms such as weight loss, nocturnal sweating, chills, weakness, digital clubbing, hypertrophic osteoarthropathy, and hypoglycemia have also been reported [##REF##11156076##6##,##REF##2162571##7##]. Hypertrophic osteoarthropathy (Pierre Marie-Bamberg syndrome) [##REF##11156076##6##, ####REF##2162571##7##, ##UREF##1##8####1##8##], is related to the abnormal production of hyaluronic acid by tumor cells and affect up to 20% of patients. In less than 5% of cases, SFTP can secrete insulin-like growth factor II which causes refractory hypoglycemia [##REF##12118790##1##,##REF##2162571##7##]. Sometimes, large tumors might present an unusual onset, such as the case of Shaker and <italic>et al</italic>, [##UREF##1##8##] in which a woman with leg edema and dyspnea caused by a large SFTP compressing the right atrium and the inferior vena cava is described. In our case, the large tumor presented with episodes of situational syncope when coughing. Situational syncope is a neurally-mediated syncope related to a reflex response that, when triggered, determines vasodilatation and bradycardia. Neurally-mediated syncope is usually classified as vasovagal (common faint), or situational [##UREF##2##9##]. Suggestive for vasovagal syncope are a long history of syncope, a youthful age, a sudden and unpleasant sight, pain or sound, prolonged standing in hot and/or crowded places. It often associates with nausea and vomiting. Situational syncope is diagnosed if syncope occurs during or after urination, defecation, cough or swallowing [##UREF##2##9##]. In our case the syncope occurred immediately after coughing, without nausea and vomiting; in addition, the patient was old and reported a trauma. We, therefore, hypothesize that coughing, due to the stimulation of the phrenic nerve, resulted in a high intrathoracic pressure producing an exaggerated Valsalva responce that decreased venous return and, consequently, cardiac output. At this regard it should be noticed that the accidental phrenic nerve injury produces cough and dyspnea and this evenience is well documented during right atrial catheterization procedures [##REF##16781380##10##]. All these details ruled out the possibility of a common faint, and consequently a diagnosis of situational syncope when coughing was made. The negative results of the cardiovascular tests associated to the presence of a large thoracic mass convinced us to consider the cough syncope related to the stimulation of the phrenic nerve by the neoplasm. In fact, after surgical removal of the tumor, the patient is free from syncope episodes confirming the direct implication of the solitary fibrous tumor of the pleura in the neurologic manifestations.</p>" ]

[ "<title>Conclusion</title>", "<p>Generally SFTP is a localized, benign tumor which may have different clinical pictures. It is curable using a careful and complete resection, provided that the surgical margins are free from neoplastic cells. In our case its resection definitively resolved the episodes of situational syncope due, in our opinion, to the large thoracic mass compressing the phrenic nerve.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Solitary fibrous tumor of the pleura is a rarely encountered clinical entity which may have different clinical pictures. Although the majority of these neoplasms have a benign course, the malignant form has also been reported.</p>", "<title>Case presentation</title>", "<p>We herein describe a case of 72 year-old man with head, facial, and thoracic traumas caused by neurally-mediated situational syncope when coughing. The diagnostic work-up including chest x-ray, CT and PET, revealed a large solitary mass of the left hemithorax. Radical surgical resection of the mass was performed through a left lateral thoracotomy and completed with a wedge resection of the lingula. Hystological examination of the surgical specimen showed an encapsulated mass measuring 12 × 11.5 × 6 cm consistent with a solitary fibrous tumor of the pleura. It's surgical removal definitively resolved the neurologic manifestations. The patient had no postoperative complications. At two years follow-up the patient is free from recurrence and without clinical manifestations.</p>", "<title>Conclusion</title>", "<p>In our case its resection definitively resolved the episodes of situational syncope due, in our opinion, to the large thoracic mass compressing the phrenic nerve</p>" ]

[ "<title>Case presentation</title>", "<p>A 72 year-old man was admitted to the hospital for head injury, facial and left hemithorax contusions. The patient referred to had fainted after coughing; the same symptoms had occurred six and three months earlier. Diverticulosis of the colon was the only disease reported by the patient in his medical history. He denied smoking, drug or alcohol abuse. Physical examination showed dullness to percussion and decreased breath sound in the affected hemithorax. The neurological examination was negative. Blood pressure was 170/80 mmHg, heart rate was 90 beats/minute and rhythmic. Laboratory findings, arterial gas analysis, electrocardiogram, and brain computed tomography were negative.</p>", "<p>A chest x-ray revealed fractures of three left ribs plus a large medium-basal opacity on the left hemithorax (Figure ##FIG##0##1a##). Computed tomography (CT) of the thorax confirmed the presence of a well-delineated, homogeneous, solid mass of 11 × 8 cm, extending for about 10 cm on the vertical axis. The mass presented a mild enhancement after contrast injection and calcifications in the basal part. It was close to the chest wall, adjacent to the left pulmonary artery, pulmonary artery trunk, and left ventricle with no signs of infiltration (Figure ##FIG##0##1b##). Bronchoscopy showed an insignificant bleeding from the upper left bronchus. Positron emission tomography (PET) revealed a mild positivity of the lesion (Figure ##FIG##1##2##). Echocardiogram, Holter ECG monitoring, and carotid Doppler ultrasonography were negative. With suspected diagnosis of SFTP, the patient underwent surgery. Through a left lateral thoracotomy, the neoplasm was carefully isolated, and its origin from the visceral pleura of the pulmonary lingula segment became evident. The adhesions with the phrenic nerve were cut preserving the nerve integrity. The mass excision was performed with clear surgical margins and completed with a wedge resection of the lingula. The postoperative course was uneventful, a good re-expansion of the left lung was obtained, and the patient was discharged on the fifth postoperative day.</p>", "<p>Pathological examination showed a 12 × 11, 5 × 6 cm encapsulated tumor mass (Figure ##FIG##2##3a##), whitish in color, with whorled appearance and calcification on cut section (Fig. ##FIG##2##3b##). Microscopic examination showed fibroblast-like structures within the collagen (Figure ##FIG##3##4##). The diagnosis of benign SFTP was confirmed by mmunohistochemical analysis (CD34+, BCL-2+, SMA-, S100-).</p>", "<p>After two years of follow-up the patient is in good clinical condition without recurrence of disease or clinical symptoms.</p>", "<title>Consent</title>", "<p>Written and informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>LS conceived the idea, did supervision of manuscript preparation and proof reading initiated treatment, did surgical procedures and approved the final version of the paper. MN, AP, LR, DT proof reading, initiated treatment, did surgical procedures. UC, MDS wrote the manuscript and carried out literature review; MMC contributed to data management and preparing of the manuscript. All authors read and approved the final manuscript.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><italic>Left panel</italic>: chest x-ray showing a large opacity on the left side. <italic>Right panel</italic>: the CT scan of the chest showing a solid mass of 11 × 8 cm in the left hemi thorax, with vertical extension of 10 cm, mild enhancement after contrast medium infusion and some calcifications in the basal part (asterisk). The lesion is in close relation with chest wall, left pulmonary artery (LPA) and pulmonary trunk (PT), without signs of local infiltration.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Moderate activity of the mass on Positron Emission Tomography study.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><italic>Left panel</italic>: surgical specimen with detail of the wedge resection of the lingula. <italic>Right panel</italic>: solitary fibrous tumor of the pleura, whorled fibrous tissue is evident on the cut section.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Tumor consists of elongated cells that display a storiform pattern of growth and abundant stromal collagen (hematoxylin &amp; eosin stain; magnification 100 ×). At higher magnification, tumor cells appear of small size, spindle, with no cytologic atypia (insert; magnification 400 ×).</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1477-7819-6-86-1\"/>", "<graphic xlink:href=\"1477-7819-6-86-2\"/>", "<graphic xlink:href=\"1477-7819-6-86-3\"/>", "<graphic xlink:href=\"1477-7819-6-86-4\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>The mortality rate of people suffering from schizophrenia has been estimated to be twice as high as in the general population[##UREF##0##1##]. More than two thirds of this excess mortality is due to 'natural' causes[##REF##9519087##2##], with death due to cardiovascular complications being the leading cause of this excess mortality[##UREF##1##3##].</p>", "<p>The first reports of an increased risk of impaired glucose tolerance in people suffering from schizophrenia appeared in the literature several years before the first antipsychotic became available[##UREF##2##4##,##UREF##3##5##]. Soon after chlorpromazine was discovered reports suggesting an association between chlorpromazine and diabetes started appearing[##UREF##4##6##]. Since then many studies have been published firmly establishing a clear link between antipsychotics and diabetes mellitus, more with atypical than typical antipsychotics [##UREF##5##7##, ####UREF##6##8##, ##UREF##7##9##, ##REF##15630069##10####15630069##10##]. This led to a US Food and Drug Administration (FDA) recommendation in 2003 for including a warning about association with hyperglycaemia and diabetes on product labels for all atypical antipsychotics[##UREF##7##9##].</p>", "<p>While it is not entirely clear how antipsychotics are linked to increased risk of impaired glucose tolerance and diabetes, weight gain and obesity are major side effects of many antipsychotics [##REF##16319406##11##, ####REF##10553730##12##, ##REF##10868465##13####10868465##13##]. Obesity itself leads to hypertension, type II diabetes and coronary heart disease, many of the same problems that people with schizophrenia are already at an increased risk for[##REF##10553730##12##].</p>", "<p>We have not come across any research studying the association between antipsychotic use and weight gain in a Pakistani population. In this study, we have tried to find out if use of antipsychotics is associated with increase in weight and body mass index (BMI) in this population.</p>" ]

[ "<title>Methods</title>", "<p>The study was a case note review of all patients who had been prescribed antipsychotic medication in the psychiatry outpatient clinic of the Aga Khan University Hospital (AKUH) over a 4-year period. Patients were identified using the Psychiatric Assessment System (PAS), which records the basic demographic and clinical details including the medication prescribed, of patients presenting to the psychiatry clinics at the AKUH for the first time. All patients have their height recorded on the first visit and weight on every visit.</p>", "<p>We calculated mean weight and BMI (weight in kg/height in m²) at baseline, 3 months and 6 months. A World Health Organization (WHO) expert consultation has suggested that the BMI cut-off points for determining overweight and obesity for Asian populations may be lower than Caucasian populations[##REF##14726171##14##]. The consultation suggested the intervals of &lt; 18.5, 18.5 to 23, 23 to 27.5 and ≥ 27.5, representing the categories of being underweight, increasing but acceptable risk, increased risk, and higher risk, respectively. We have used the same cut-offs in this study.</p>", "<p>An increase in weight of 7% or more compared to the baseline is considered by licensing authorities as clinically significant weight gain[##REF##10548137##15##]. We calculated how many patients had achieved clinically significant weight gain at 3 months and 6 months.</p>", "<p>Statistical analyses were performed in SPSS v.15 (SPSS Inc., Chicago, IL, USA). We calculated means (with standard deviations) for quantitative variables and proportions (percentages) for categorical characteristics. We used a paired t test to determine if patients had achieved a statistically significant increase in weight and BMI from baseline. p Values &lt; 0.05 were considered significant.</p>" ]

[ "<title>Results</title>", "<p>We found a total of 145 patients who had been seen at least once in the psychiatry clinic of AKUH and had been prescribed an antipsychotic medication. All of these had had their weight recorded at baseline. A total of 81 patients had at least 1 further weight measurement at least 3 months after the baseline measurement. In all, 33 patients had their weight measured at all 3 time points; baseline, 3 months and 6 months. A total of 56 people had been weighed at baseline and 3 months, and 60 people at baseline and 6 months.</p>", "<p>The baseline sociodemographic and clinical characteristics of the sample are given in Table ##TAB##0##1##.</p>", "<p>The mean weight and BMI of the total sample at baseline, 3 months and 6 months are shown in Table ##TAB##1##2##. Among all patients for whom we could calculate BMI (n = 140) 50% (70/140) had a BMI in the overweight or higher range (&gt; 23) at baseline, 61% at 3 months and 63% at 6 months.</p>", "<p>Patients for whom we had weight readings at baseline and 3 months (n = 56) showed a mean weight gain of 1.88 kilograms (63.51 vs 65.4 kg). This difference was statistically significant (t = -3.16, p value = 0.003). Patients for whom we had weight readings at baseline and 6 months (n = 60) showed a mean weight gain of 3.29 kilograms (62.5 vs 65.79 kg). This difference was also statistically significant (t = -2.95, p value = 0.004).</p>", "<p>The difference in mean BMI at baseline and 3 months was 0.74 (24.27 and 25.02 respectively), which was statistically significant (p = 0.002). The difference in mean BMI between baseline and 6 months was 1.3 (23.84 and 25.18 respectively) and this increase was also statistically significant (p value = 0.002)</p>", "<p>In patients for whom we had at least 1 further weight measurement after baseline, 48% (39/81) showed a clinically significant weight gain. In all, 51% (19/37) of patients on risperidone, 71% (8/11) on olanzapine and 16% (1/6) on quetiapine achieved clinically significant weight gain. However, the numbers were too small to meaningfully assess differences in the propensity of different antipsychotics to cause clinically significant weight gain.</p>", "<p>We did a secondary analysis, dividing patients into groups by psychotic disorders, (schizophrenia, delusional disorder, drug-induced psychosis) and non-psychotic disorders (all other diagnoses) but the differences between the weights of these groups were non-significant at all time points (p value 0.671 at baseline, 0.238 at 3 months and 0.645 at 6 months).</p>", "<p>A total of 91 patients were taking other psychotropic(s) besides an antipsychotic medication; 34 of these were taking SSRIs, 7 TCAs, 17 anticholinergics, 25 mood stabilisers (out of these 13 were taking valproic acid), 12 benzodiazepines, and 8 zolpidem. In all, 12 patients were taking other antidepressants including Mirtazapine (3), venlafaxine (5), and Mianserin (4).</p>" ]

[ "<title>Discussion</title>", "<p>In this study we found that almost 50% of patients had a BMI in the overweight or higher range according to the WHO suggested cut-offs for Asian populations at the start of the study. On average patients gained about 2 kg and 3.5 kg in weight from baseline in 3 and 6 months, respectively. This correlated with a BMI increase of 0.74 in 3 months and 1.3 in 6 months. About 48% of patients for whom we had at least 1 more weight reading after 3 or 6 months achieved a clinically significant weight gain.</p>", "<p>In the study by Zipursky <italic>et al. </italic>[##REF##16319406##11##] patients receiving olanzapine or haloperidol had a mean weight gain of 15.4 kg and 7.5 kg respectively. Allison <italic>et al. </italic>[##REF##10553730##12##] in their systematic review reported a range of weight gain from 0.04 kg for ziprasidone to 4.45 kg for clozapine. Taylor and McAskill [##REF##10868465##13##] concluded that all atypical antipsychotics, with the exception of ziprasidone (aripiprazole had not been marketed in 2000), have been associated with weight increases, with clozapine having the highest risk. The weight gain in our study was closer to the Allison than the Zipursky study. The main reason for this difference could be that in the Zipursky study patients were not recruited if they had received prior antipsychotic treatment for more than 16 cumulative weeks.</p>", "<p>The overall prevalence of diabetes mellitus in Pakistan has been reported to be between 8.6% and 13.9%, depending on the province of residence [##REF##10646320##16##, ####REF##8750223##17##, ##UREF##8##18####8##18##]. This is far higher than the prevalence of diabetes of 1.2 to 6.3% reported from the US [##UREF##6##8##] or around 3% reported from the UK [##UREF##9##19##]. Any drug that causes weight gain is, therefore, likely to have even more serious consequences in terms of morbidity and mortality for the Pakistani population.</p>", "<p>One of the limitations of our study was that almost all the patients had already received one or more antipsychotics for variable lengths of time before they first presented to the clinic at the AKUH. That may explain whey the weight gain in our study was not as stark as the Zipursky study[##REF##16319406##11##]. Another limitation of the study is that there was no control group of patients who were not taking antipsychotic medications. This would have shed some light on how much of the weight gain might be attributable to suffering from a psychiatric illness and how much to taking of antipsychotic medications.</p>" ]

[ "<title>Conclusion</title>", "<p>Antipsychotics are associated with statistically significant weight gain in the Pakistani population. This may be even more hazardous for this population as the prevalence of diabetes mellitus is already higher than many other countries. It is important that while initiating an antipsychotic medication in this patient population, psychiatrists should counsel patients about the risk of weight gain associated with antipsychotic use, the increased risk of morbidity and mortality associated with weight gain, and the lifestyle changes such as changes in dietary habits and regular exercise that the patients can adopt to counter that risk.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>It has been known for a long time that use of antipsychotics, particularly atypical antipsychotics, is associated with weight gain and increase in risk of metabolic disturbances. In this study we have tried to find out if use of antipsychotics is associated with increase in weight and body mass index (BMI) in the Pakistani population.</p>", "<title>Methods</title>", "<p>We performed a case note review of all patients who had been prescribed antipsychotic medication at the psychiatry outpatient clinic of a tertiary care university hospital in Pakistan over a 4-year period.</p>", "<title>Results</title>", "<p>A total of 50% of patients had a BMI in the overweight or higher range at baseline. Patients showed a mean weight gain of 1.88 kg from baseline in 3 months and 3.29 kg in 6 months. Both of these values were statistically significant. The increase in mean BMI from baseline was 0.74 and 1.3 in 3 months and 6 months, respectively. In patients for whom we had at least one further weight measurement after baseline, 48% (39/81) showed a clinically significant weight gain.</p>", "<title>Conclusion</title>", "<p>Pakistani patients are just as likely to put on weight during antipsychotic treatment as patients from other countries. Considering that this population already has a much higher prevalence of diabetes mellitus compared to the Western countries, the consequences of increased weight may be even more serious in terms of increased morbidity and mortality.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>SA carried out the literature review, wrote the protocol, and wrote the initial draft of the paper. RK performed data extraction and was responsible for data entry into SPSS. SPI wrote the statistical part of the protocol/paper and carried out the statistical analyses. All authors were responsible for drafting the final form of the paper and approved the manuscript.</p>" ]

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[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Patient demographics and clinical characteristics at baseline</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Parameter</bold></td><td align=\"left\"><bold>Value</bold></td></tr></thead><tbody><tr><td align=\"left\">Age, years median (interquartile range)</td><td align=\"left\">31 (24–43)</td></tr><tr><td align=\"left\">Gender (n = 141):</td><td/></tr><tr><td align=\"left\">Male</td><td align=\"left\">79 (56%)</td></tr><tr><td align=\"left\">Female</td><td align=\"left\">62 (44%)</td></tr><tr><td align=\"left\">Marital status (n = 138):</td><td/></tr><tr><td align=\"left\">Single</td><td align=\"left\">75 (51%)</td></tr><tr><td align=\"left\">Married</td><td align=\"left\">52 (35.4%)</td></tr><tr><td align=\"left\">Widowed</td><td align=\"left\">7 (4.8%)</td></tr><tr><td align=\"left\">Divorced</td><td align=\"left\">3 (2%)</td></tr><tr><td align=\"left\">Separated</td><td align=\"left\">1 (0.7%)</td></tr><tr><td align=\"left\">Psychiatric diagnosis (n = 145):</td><td/></tr><tr><td align=\"left\">Schizophrenia</td><td align=\"left\">85 (57.8%)</td></tr><tr><td align=\"left\">Depression</td><td align=\"left\">21 14.3%</td></tr><tr><td align=\"left\">Bipolar disorder</td><td align=\"left\">16 (10.9%)</td></tr><tr><td align=\"left\">Delusional disorder</td><td align=\"left\">6 (4.1%)</td></tr><tr><td align=\"left\">Learning disability</td><td align=\"left\">5 (3.4%)</td></tr><tr><td align=\"left\">Dementia</td><td align=\"left\">3 (2%)</td></tr><tr><td align=\"left\">Substance misuse</td><td align=\"left\">3 (2%)</td></tr><tr><td align=\"left\">Obsessive/compulsive disorder (OCD)</td><td align=\"left\">2 (1.4%)</td></tr><tr><td align=\"left\">Anorexia nervosa</td><td align=\"left\">2 (1.4%)</td></tr><tr><td align=\"left\">Attention-deficit hyperactivity disorder (ADHD)</td><td align=\"left\">1 (0.7%)</td></tr><tr><td align=\"left\">Personality disorder</td><td align=\"left\">1 (0.7%)</td></tr><tr><td align=\"left\">Antipsychotic prescribed (n = 145):</td><td/></tr><tr><td align=\"left\">Risperidone</td><td align=\"left\">75 (51%)</td></tr><tr><td align=\"left\">Olanzapine</td><td align=\"left\">23 (15.6%)</td></tr><tr><td align=\"left\">Quetiapine</td><td align=\"left\">9 (6.1%)</td></tr><tr><td align=\"left\">Aripiprazole</td><td align=\"left\">3 (2%)</td></tr><tr><td align=\"left\">Clozapine</td><td align=\"left\">1 (0.7%)</td></tr><tr><td align=\"left\">Typical antipsychotics</td><td align=\"left\">34 (23.1%)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Mean (SD) weight and body mass index (BMI)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\"><bold>Baseline</bold></td><td align=\"left\"><bold>3 months</bold></td><td align=\"left\"><bold>6 months</bold></td></tr></thead><tbody><tr><td align=\"left\">Weight, kg</td><td align=\"left\">63.28 (16.99)</td><td align=\"left\">65.40 (18.01)</td><td align=\"left\">65.79 (15.79)</td></tr><tr><td align=\"left\">BMI, kg/m²</td><td align=\"left\">23.65 (5.45)</td><td align=\"left\">25.02 (5.48)</td><td align=\"left\">25.18 (4.93)</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>SD, standard deviation.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Bupropion HCl is known to cause seizures both when given at therapeutic doses or following accidental or intentional overdose in a dose-dependent manner [##REF##6406457##1##, ####REF##6406456##2##, ##REF##2500425##3##, ##REF##1744061##4##, ##REF##12526723##5##, ##REF##15261357##6##, ##UREF##0##7####0##7##]. It is also known that factors which include the excessive use of alcohol and sedatives, history of head trauma or prior seizure, and substance abuse, to mention a few, are associated with increased risk of bupropion-induced seizures [##UREF##0##7##]. In addition, postmarketing surveillance reports have indicated that there have been rare cases of adverse neuropsychiatric events or reduced alcohol tolerance in patients who are taking alcohol during treatment with bupropion [##UREF##0##7##]. Despite these latter reports, previous studies of the pharmacokinetic and/or pharmacodynamic interactions between alcohol and bupropion have revealed no significant pharmacodynamic interactions in animals [##REF##3923380##8##], and no pharmacokinetic interactions in healthy human volunteers [##REF##6432553##9##]. Furthermore, there are no studies specifically investigating the interaction between alcohol and bupropion-induced seizures in animals or man. Therefore, the objective of this study was to evaluate the effect of ethanol pretreatment on single-dose bupropion HCl-induced seizures in the Swiss albino mouse model.</p>" ]

[ "<title>Materials and methods</title>", "<p>The study protocol and any amendment(s) or procedures involving the care and use of animals were reviewed and approved by an appropriate ethics committee following internationally approved guidelines (Charles River Laboratories Preclinical Services Inc.'s (CRM) Institutional Animal Care and Use Committee; Charles River Laboratories, Wilmington, MA, USA). During the study, the animals were maintained in a facility fully accredited by the Standards Council of Canada (SCC) and the care and use of the animals was conducted in accordance with the guidelines of the Canadian Council on Animal Care (CCAC).</p>", "<title>Animals</title>", "<p>Experimentally naïve female Swiss Crl: CD1 (ICR) albino mice (<italic>Mus Musculus</italic>; Charles River Canada Inc., St. Constant, Quebec, Canada) of approximately 7 weeks of age, and weighing 17.3 to 28.6 g were housed individually in stainless steel wire mesh-bottomed cages equipped with an automatic watering valve in an environmentally controlled vivarium (temperature 22 ± 3°C; relative humidity 50 ± 20%) with a 12-h light/dark cycle. All animals were acclimated to their cages and to the light/dark cycle for 3 days before the initiation of treatment. In addition, all animals had free access <italic>ad libitum </italic>to a standard certified pelleted commercial laboratory diet (PMI Certified Rodent Diet 5002; PMI Nutrition International Inc., St Louis, MO, USA) and tap water except during designated procedures. Animals were randomly assigned to 8 treatment groups of 10 mice per group, using a computer-generated randomisation scheme, ensuring stratification by body weights. Four groups were pretreated with ethanol followed by treatment with increasing doses of bupropion HCl as follows: group 1, ethanol 2.5 g/kg + 0 mg/kg (vehicle); group 2, ethanol 2.5 g/kg + 100 mg/kg; group 3, ethanol 2.5 g/kg + 110 mg/kg; and group 4, ethanol 2.5 g/kg + 120 mg/kg. The other four groups were only treated with the same increasing doses of bupropion HCl as follows: group 5, 0 mg/kg (vehicle only); group 6, 100 mg/kg; group 7, 110 mg/kg; and group 8, 120 mg/kg. The doses of bupropion HCl 100 to 120 mg/kg selected for this study are higher than the low dose of 12.5 mg/kg used in a previous study [##REF##3923380##8##] because more recent studies have revealed that bupropion HCl at low doses of 15 to 30 mg/kg does not induce seizures but protects albino mice against seizures induced by maximal electroshock (anticonvulsant), and at high doses of 100 to 160 mg/kg is proconvulsant in the mice [##REF##15363958##10##]. Animals in poor health or at the extremes of the prespecified body weight range (18 to 30 g) were not assigned to treatment groups and unassigned animals were released from the study.</p>", "<title>Drugs</title>", "<p>Bupropion HCl was obtained from Biovail Corporation, Steinbach, Manitoba, Canada, in white powder form. The dose formulations of bupropion HCl were prepared on each day. The appropriate amount of bupropion HCl was weighed and dissolved in an appropriate amount of 0.9% NaCl and then vortexed until a solution was obtained. On each day of treatment, the single doses of bupropion HCl dose were administered by intraperitoneal (IP) injection in a dose volume of 10 ml/kg and dose concentrations of 0, 10, 11, and 12 mg/ml for the 0, 100, 110, and 120 mg/kg doses. The actual dose administered was based on the most recent body weight of each animal. In the applicable treatment groups (groups 1 to 4), each animal was pretreated with ethanol in a dose volume of 10 ml/kg 5 min prior to bupropion dosing. Ethanol was obtained in liquid form from Les Alcools de Commerce Inc., Montreal, Quebec, Canada. Ethanol 2.5 g/kg was administered as a dose volume of 10 ml/kg, and a dose concentration of 0.25 g/ml. Vehicle was 0.9% sodium chloride (NaCl) for injection USP and was obtained from Baxter Healthcare Corporation, Deerfield, IL, USA.</p>", "<title>Study procedure</title>", "<p>All animals were examined twice daily for mortality and signs of ill health or reaction to treatment, except on the days of arrival and necropsy when they were examined only once. After the acclimation period and randomisation, on the day prior to the initiation of treatment, all animals were weighed and the individual body weights were used for dose volume calculation. Treatment was then initiated and lasted for 4 consecutive days with equal numbers of animals from each group dosed on each day. On the days of treatment, approximately 5 min prior to bupropion HCl or vehicle dosing, animals in groups 1 to 4 were pretreated with a single dose of ethanol 2.5 g/kg IP in a dose volume of 10 ml/kg. These animals then received the assigned dose of bupropion HCl or vehicle IP. Animals in groups 5 to 8 were not pretreated with ethanol but received their assigned dose of bupropion HCl or vehicle by the IP route. Thereafter, the animals were placed in clear perspex observation boxes containing a foam base for padding and observed for the occurrence of seizures for 5 h, followed by a 5 min assessment at 24 h post dose. The presence or absence of seizures, the number of seizures, the onset, duration and intensity of seizures were all recorded. The intensity of each convulsion was graded using Charles River Laboratories, Inc.'s grading system of mild: head and tail slightly extended and little jerking; moderate: head and tail fully extended and some jerking; or severe: head and tail fully extended and strong jerking. In addition, the presence or absence of ataxic gait, paralysis, and catatonic episodes (without a grading of the intensity or number) were recorded over each 15 min observation period. Any animal that had a single episode of severe seizure lasting longer than 1 min or any animal displaying greater than 40 separate episodes of severe seizures over a 1-h period was sacrificed for humane reasons. At the end of the 5-h observation period, all animals were returned to their home cages, and as deemed necessary, additional bedding, food (on cage floor) and water bottles were provided if an animal was still showing adverse effects from the administration of study drugs.</p>", "<title>Assessment of convulsant activity</title>", "<p>The primary outcome variable was the percentage of mice that had seizures. This was the number of animals with seizures (mild, moderate or severe) divided by the total number of animals in each group multiplied by 100. In addition, the convulsive dose of bupropion HCl required to induce seizures in 50% of mice (CD₅₀), was calculated for the dose-response curves for bupropion HCl treatment alone and the ethanol/bupropion HCl treatment. The secondary outcome variables were the mean (SD) seizures per mouse in each group, and the duration of seizures.</p>", "<title>Data presentation and statistical analysis</title>", "<p>Data was summarised and presented in tables by treatment groups for the primary outcome variable, the percentage of convulsing mice, and the two secondary outcome variables, the mean (SD) seizures per mouse in each group, and the duration of seizures. The CD₅₀values were calculated using the PROBIT procedure in SAS (SAS Inc., Cary, NC, USA). The 95% confidence limits for CD₅₀were calculated according to the method of Litchfield and Wilcoxon [##REF##18152921##11##]. A total of 10 mice per group (total of 40 animals) were used to calculate the CD₅₀for the bupropion alone treatments, and 39 animals for the CD₅₀for the ethanol/bupropion HCl treatments. The number of seizures per mouse was analysed using analysis of variance (ANOVA) on the rank-transformed values, with presence of ethanol (yes/no), bupropion dose, and presence of ethanol-by-bupropion dose interaction as fixed effects in the model. p Values of ≤ 0.05 were considered statistically significant.</p>" ]

[ "<title>Results</title>", "<p>In all groups, except the group treated with vehicle only (group 5), a convulsive effect was observed following the administration of bupropion HCl and/or ethanol. The onset of convulsion was about 9 min following the administration of single doses of bupropion HCl, however, this was highly variable between animals in the same group and across the dose levels for the bupropion HCl alone and ethanol/bupropion HCl treatments. The intensity of the seizures observed following bupropion HCl alone treatment were only mild and moderate (Table ##TAB##0##1##). Following ethanol pretreatment, overall, there was an increase in the intensity of the bupropion HCl-induced seizures at all the doses. In the 100 mg/kg dose group (group 5), there were marked increases in the number of mild, moderate and severe seizures. In the 110 and 120 mg/kg dose groups, there was a redistribution of the intensity of the seizures resulting in reductions in the mild seizures but a fivefold and twofold increase, respectively, in the moderate seizures (Table ##TAB##0##1##).</p>", "<p>There were no deaths in the study. One animal treated with ethanol/bupropion HCl 110 mg/kg had excessive convulsions and was therefore euthanised for humane reasons. A variety of clinical signs were observed in the mice following the administration of bupropion HCl, some of which include paralysis, ataxic gait, catatonia, increased respiratory rate, twitching, tremors, increased activity, decreased activity, partially closed eyes, etc. Clinical signs were not dose dependent and pretreatment with ethanol had no effect on the signs observed.</p>", "<title>Percentage of convulsing mice</title>", "<p>Administration of single doses of IP bupropion HCl alone induced seizures in mice in a dose-dependent manner with the 120 mg/kg dose showing the largest effect. The percentage of convulsing mice were 0%, 20%, 30% and 60% in the 0 (vehicle only = 0.9% NaCl), 100, 110, and 120 mg/kg dose groups, respectively (Table ##TAB##1##2## and Figure ##FIG##0##1##). Pretreatment with ethanol produced a larger bupropion HCl-induced convulsive effect at all the doses including the ethanol + vehicle only group. There was a marked increase in the percentage of convulsing mice (70% of convulsing mice) at the ethanol/bupropion HCl 100 mg/kg dose, compared to bupropion HCl alone treatment, which was maintained at the ethanol/bupropion HCl 110 and 120 mg/kg doses, resulting in a flat dose-response curve (Table ##TAB##1##2## and Figure ##FIG##0##1##). Ethanol/vehicle (group 1) treatment induced a 10% incidence of seizures.</p>", "<p>The CD₅₀or convulsive dose₅₀, the convulsive doses of bupropion HCl required to induce seizures in 50% of mice, were 116.72 (CI: 107.95, 126.20) and 89.40 (CI: 64.92, 123.10) mg/kg for the dose-response curves for bupropion alone and ethanol/bupropion HCl treatments, respectively (Figure ##FIG##0##1##). The CD₅₀of 116.72 (CI: 107.95, 126.20) mg/kg for bupropion HCl alone treatment is similar to the value of 119.7 (CI: 104.1, 137.6) mg/kg reported previously for IP bupropion HCl in Swiss mice [##REF##15363958##10##].</p>", "<title>Mean convulsions per mouse</title>", "<p>The analysis of variance results showed a significant overall effect of ethanol pretreatment and bupropion dose on the number of bupropion HCl-induced seizures, and a borderline significant overall ethanol-bupropion interaction effect at the p ≤ 0.10 level (Table ##TAB##2##3##). Single-dose bupropion HCl alone treatment induced a dose-dependent increase in the mean (SD) seizures per mouse from 0 in the vehicle only-treated group (bupropion HCl 0 mg/kg) to 2.20 (4.49) seizures per mouse in the 110 mg/kg dose group, which was maintained at the 120 mg/kg dose group (mean (SD) convulsions per mouse = 2.10 (1.97)). Pretreatment with ethanol markedly and significantly increased the mean (SD) seizures per mouse compared to bupropion HCl alone treatment only in the 100 mg/kg dose group (ethanol/bupropion HCl = 10.90 (17.28); bupropion HCl alone = 0.20 (0.42); p = 0.0019). There were no statistically significant differences between the mean (SD) seizures per mouse obtained for ethanol/bupropion HCl versus bupropion alone treatments for the 0, 110 and 120 mg/kg dose groups (Table ##TAB##2##3##).</p>", "<title>Duration of convulsions</title>", "<p>Administration of single doses of bupropion HCl alone induced only short and medium duration seizures. The number of short seizures increased with dose to a maximum of 22 at the 110 mg/kg dose with a slight decrease to 18 at the 120 mg/kg dose (Table ##TAB##3##4##). In contrast, pretreatment with ethanol increased the total numbers of bupropion HCl-induced short and medium seizures, as well as caused long seizures. In addition, the number of short, medium and long seizures was markedly highest at the 100 mg/kg dose followed by a marked reduction at the 110 mg/kg dose and a further reduction at the 120 mg/kg dose only for the medium and long seizures (Table ##TAB##3##4##).</p>" ]

[ "<title>Discussion</title>", "<p>The pharmacokinetic and pharmacodynamic interactions of ethanol with antidepressant drugs are well known [##REF##1092511##12##, ####REF##7420073##13##, ##REF##6628520##14##, ##REF##6144139##15##, ##REF##1813903##16##, ##REF##9260032##17####9260032##17##]. Interactions between ethanol and psychotropic drugs could be additive, synergistic (potentiation) or antagonistic [##REF##6144139##15##]. Even though there are published reports of animal [##REF##3923380##8##] and human [##REF##6432553##9##,##REF##6436033##18##] studies investigating the pharmacokinetic and/or pharmacodynamic interactions between alcohol and bupropion, there are no published studies precisely evaluating the effects of alcohol on the convulsive liability of bupropion. This study was therefore designed to investigate the effect of ethanol pretreatment on single-dose bupropion HCl-induced seizures in the Swiss albino mice. The results of the primary outcome variable showed that bupropion HCl alone treatment in the dosage range 0 to 120 mg/kg was associated with a dose-dependent increase in the percentage of mice with bupropion HCl-induced seizures. This finding is consistent with previous reports that indicate bupropion induces seizures in a dose-dependent manner in animals [##REF##15363958##10##,##REF##15866510##19##] and humans [##REF##6406456##2##,##REF##2500425##3##,##UREF##0##7##]. Pretreatment with ethanol resulted in markedly increased percentage of mice with bupropion HCl-induced seizures at the 100 mg/kg dose, which was maintained at the 110 and 120 mg/kg doses. The latter results are consistent with a 3.5-, 2.3- and 1.2-fold increase in the percentage of convulsing mice at the 100, 110 and 120 mg/kg doses, respectively, following ethanol pretreatment. In addition, ethanol pretreatment resulted in a flat dose-response within the dosage range of 100 to 120 mg/kg studied. The CD₅₀for bupropion HCl alone treatment, a well known index of convulsive liability, of 116.72 (CI: 107.95, 126.20) mg/kg is similar to the value of 119.7 (CI: 104.1, 137.6) mg/kg reported previously for bupropion HCl in Swiss mice [##REF##15363958##10##], and confirms the validity of this animal model. Pretreatment with ethanol resulted in a 23% reduction in the CD₅₀value for bupropion HCl-induced seizures.</p>", "<p>The results of the secondary outcome variables were generally consistent with the results of the primary outcome variable. Bupropion HCl alone treatment induced a dose-dependent increase in the mean seizures per mouse up to the 110 mg/kg dose, which was maintained at the 120 mg/kg dose. Ethanol pretreatment resulted in a marked and statistically significant 54-fold increase in bupropion HCl-induced mean seizures per mouse only at the 100 mg/kg dose. There were no significant differences in bupropion HCl-induced mean seizures per mouse at the 110 and 120 mg/kg doses following ethanol pretreatment. With respect to the duration of seizures, bupropion HCl alone treatment only induced short and medium duration seizures, which when combined was dose dependent up to the 110 mg/kg dose. Ethanol pretreatment increased the duration of the seizures overall, resulting in more episodes of short, medium, and long duration bupropion HCl-induced seizures, but particularly in the 100 mg/kg dose group.</p>", "<p>The results of this study are in conflict with the results of previous studies that reported no pharmacodynamic interactions between alcohol and bupropion in mice [##REF##3923380##8##], and no pharmacokinetic interactions in normal healthy volunteers [##REF##6432553##9##]. The reason for the discrepant previous results may be because those studies of the pharmacokinetic and pharmacodynamic interactions between alcohol and bupropion in normal healthy volunteers [##REF##6432553##9##,##REF##6436033##18##] used a low dose of bupropion (100 mg, approximately 1.5 mg/kg) that is unlikely to be associated with the occurrence of seizures since bupropion-induced seizures are dose dependent. Similarly, a previous study [##REF##3923380##8##] investigating the interactive effect of combined treatment with alcohol and bupropion in adult albino mice utilised a low dose of bupropion (12.5 mg/kg IP) which is much lower than the convulsive doses of 100 to 160 mg/kg IP, with a CD₅₀of 119.7 (CI: 104.1, 137.6) mg/kg and CD₉₇of 156.7 mg/kg, that were subsequently reported for bupropion in mice by other investigators [##REF##15363958##10##]. In addition, lower doses of bupropion (15 to 30 and 5 to 10 mg/kg, respectively), which did not induce seizures, have been reported to protect against seizures evoked by maximal electroshock [##REF##15363958##10##] and nicotine [##REF##10991997##20##] in mice. However, one group has reported that the combination of bupropion with alcohol abolished the impairment in auditory vigilance and mental slowness observed following the administration of alcohol alone in normal healthy volunteers (a pharmacodynamic interaction) even though they used a low dose of bupropion (100 mg) and found no pharmacokinetic interaction [##REF##6436033##18##].</p>", "<p>The mechanism of bupropion HCl-induced seizures is unknown [##UREF##1##21##,##REF##7665537##22##]. Similarly, the mechanism for the synergistic interaction reported here between ethanol and bupropion HCl is also unknown. This interaction is unlikely to be solely due to pharmacokinetic reasons since a previous crossover study that investigated the interactions between alcohol and bupropion found no such interactions [##REF##6432553##9##]. This previous study, also in normal healthy human volunteers, examined the effect of administration of oral bupropion HCl 100 mg followed by the administration of ethanol found no changes in the pharmacokinetics of bupropion, and vice versa [##REF##6432553##9##].</p>", "<p>The observed interaction between ethanol and bupropion reported in the present study has potential clinical implications. It has been recognised that the seizure risk of bupropion is increased in subjects undergoing abrupt withdrawal from alcohol [##REF##2500425##3##,##REF##1744061##4##], hence, bupropion administration is contraindicated in such patients [##UREF##0##7##]. However, the more recent although rare postmarketing reports of adverse neuropsychiatric events or reduced alcohol tolerance in patients who are drinking alcohol during treatment with bupropion [##UREF##0##7##], suggests that there is an interaction between alcohol and bupropion following coadministration, consistent with the findings of this study. Consequently, patients should be cautioned to not consume alcohol with bupropion. Nonetheless, there is good evidence that many patients on bupropion, as well as other anti-depressants, continue to use alcohol [##UREF##2##23##].</p>", "<p>In conclusion, the results of this study demonstrate that ethanol pretreatment followed by single-dose IP bupropion HCl resulted in an increase in the number and percentage of convulsing mice, mean seizures per mouse, the intensity, and the duration of the seizures. Following ethanol pretreatment, the CD₅₀for bupropion HCl alone treatment was reduced from 116.7 to 89.0 mg/kg, representing a 23% reduction. The dose-related increase in the percentage of convulsing mice and mean seizures per mouse is consistent with previous reports that bupropion-induced seizures are dose dependent in animals and humans. The observed pharmacodynamic interaction between ethanol and bupropion-induced seizures in this study is novel and the mechanism is unknown. However, it has potential clinical implications for the prescribing of bupropion. It also implies that caution should be used when bupropion is prescribed to patients either using alcohol or at high risk of doing so.</p>" ]

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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Bupropion HCl is a widely used antidepressant that is known to cause seizures in a dose-dependent manner. Many patients taking antidepressants will consume alcohol, even when advised not to. Previous studies have not shown any interactions between bupropion HCl and alcohol. However, there have been no previous studies examining possible changes in seizure threshold induced by a combination of alcohol and bupropion HCl.</p>", "<title>Methods</title>", "<p>Experimentally naïve female Swiss albino mice (10 per group) received either single doses of bupropion HCl (ranging from 100 mg/kg to 120 mg/kg) or vehicle (0.9% NaCl) by intraperitoneal (IP) injection in a dose volume of 10 ml/kg, and single-dose ethanol alone (2.5 g/kg), or vehicle, 5 min prior to bupropion dosing. The presence or absence of seizures, the number of seizures, the onset, duration and the intensity of seizures were all recorded for 5 h following the administration of ethanol.</p>", "<title>Results</title>", "<p>The results show that administration of IP bupropion HCl alone induced seizures in mice in a dose-dependent manner, with the 120 mg/kg dose having the largest effect. The percentage of convulsing mice were 0%, 20%, 30% and 60% in the 0 (vehicle), 100, 110, and 120 mg/kg dose groups, respectively. Pretreatment with ethanol produced a larger bupropion HCl-induced convulsive effect at all the doses (70% each at 100, 110 and 120 mg/kg) and a 10% effect in the ethanol + vehicle only group. The convulsive dose of bupropion HCl required to induce seizures in 50% of mice (CD₅₀), was 116.72 mg/kg for bupropion HCl alone (CI: 107.95, 126.20) and 89.40 mg/kg for ethanol/bupropion HCl (CI: 64.92, 123.10).</p>", "<title>Conclusion</title>", "<p>These results show that in mice alcohol lowers the seizure threshold for bupropion-induced seizures. Clinical implications are firstly that there may be an increased risk of seizures in patients consuming alcohol, and secondly that formulations that can release bupropion more readily in alcohol may present additional risks to patients.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>The study was conceived by PHS and RW, was designed by LM and SF who were also involved in data acquisition, the first draft of the paper was by PHS, it was carried out in part by LM, and the statistical analysis was by RF. Funding for the conduct of this study and the manuscript preparation was provided by Biovail Laboratories International SRL.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Dose-response curves of the percentage of convulsing mice following the administration of bupropion HCl alone (closed circles) and the effect of ethanol pretreatment on bupropion HCl-induced seizures (open circles) in the Swiss albino mice</bold>. The 50% convulsing dose (CD₅₀) values, the convulsant doses of bupropion HCl required to induce seizures in 50% of mice were 116.72 (CI: 107.95, 126.20) and 89.40 (CI: 64.92, 123.10) mg/kg for the dose-response curves for bupropion alone and ET + bupropion HCl, respectively. Doses of bupropion HCl administered intraperitoneally (IP) were 0 (vehicle or ET + vehicle only), 100, 110, and 120 mg/kg. Ethanol pretreatment was with 2.5 g/kg IP 5 min prior to administration of bupropion HCl. Each data point is the percentage of convulsing mice in n = 10 mice. ET, ethanol + vehicle; S, vehicle (0.9% NaCl).</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Effect of ethanol pretreatment on bupropion HCI-induced convulsions: intensity of convulsions</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Dose (mg/kg), n =</bold><break/><bold> 10 per group</bold></td><td align=\"left\" colspan=\"6\"><bold>Intensity of convulsions</bold></td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"left\" colspan=\"2\"><bold>Mild</bold></td><td align=\"left\" colspan=\"2\"><bold>Moderate</bold></td><td align=\"left\" colspan=\"2\"><bold>Severe</bold></td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td></tr></thead><tbody><tr><td align=\"left\">0 (V or ET +V)</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">0</td></tr><tr><td align=\"left\">100</td><td align=\"left\">1</td><td align=\"left\">37</td><td align=\"left\">1</td><td align=\"left\">65</td><td align=\"left\">0</td><td align=\"left\">7</td></tr><tr><td align=\"left\">110</td><td align=\"left\">21</td><td align=\"left\">10</td><td align=\"left\">1</td><td align=\"left\">5</td><td align=\"left\">0</td><td align=\"left\">0</td></tr><tr><td align=\"left\">120</td><td align=\"left\">19</td><td align=\"left\">17</td><td align=\"left\">2</td><td align=\"left\">4</td><td align=\"left\">0</td><td align=\"left\">0</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Effect of ethanol pretreatment on bupropion HCI-induced convulsions: percentage of convulsing mice</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Dose (mg/kg), n = 10</bold><break/><bold> per group</bold></td><td align=\"left\" colspan=\"2\"><bold>No. of convulsing mice</bold></td><td align=\"left\" colspan=\"2\"><bold>Percentage of convulsing mice</bold></td></tr><tr><td/><td colspan=\"4\"><hr/></td></tr><tr><td/><td align=\"left\"><bold>Bupropion</bold><break/><bold> HCl</bold></td><td align=\"left\"><bold>ET + Bupropion</bold><break/><bold> HCl</bold></td><td align=\"left\"><bold>Bupropion</bold><break/><bold> HCl</bold></td><td align=\"left\"><bold>ET + Bupropion</bold><break/><bold> HCl</bold></td></tr></thead><tbody><tr><td align=\"left\">0 (vehicle or<break/> ET+vehicle)</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">0%</td><td align=\"left\">10%</td></tr><tr><td align=\"left\">100</td><td align=\"left\">2</td><td align=\"left\">7</td><td align=\"left\">20%</td><td align=\"left\">70%</td></tr><tr><td align=\"left\">110</td><td align=\"left\">3</td><td align=\"left\">7</td><td align=\"left\">30%</td><td align=\"left\">70%</td></tr><tr><td align=\"left\">120</td><td align=\"left\">6</td><td align=\"left\">7</td><td align=\"left\">60%</td><td align=\"left\">70%</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Effect of ethanol pretreatment on bupropion HCI-induced convulsions: mean standard deviation (SD) convulsions per mouse</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Dose (mg/kg), n =</bold><break/><bold> 10 per group</bold></td><td align=\"left\" colspan=\"2\"><bold>Total no. of convulsions</bold></td><td align=\"left\" colspan=\"2\"><bold>Mean (SD) convulsions per mouse</bold></td><td/></tr><tr><td/><td colspan=\"5\"><hr/></td></tr><tr><td/><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td><td align=\"left\"><bold>p Value</bold></td></tr></thead><tbody><tr><td align=\"left\">0 (V or ET +V)</td><td align=\"left\">0</td><td align=\"left\">2</td><td align=\"left\">0.00 (0.00)</td><td align=\"left\">0.20 (0.63)</td><td align=\"left\">0.1027*</td></tr><tr><td align=\"left\">100</td><td align=\"left\">2</td><td align=\"left\">109</td><td align=\"left\">0.20 (0.42)</td><td align=\"left\">10.90 (7.28)†</td><td/></tr><tr><td align=\"left\">110</td><td align=\"left\">22</td><td align=\"left\">15</td><td align=\"left\">2.20 (4.49)</td><td align=\"left\">1.50 (1.72)</td><td/></tr><tr><td align=\"left\">120</td><td align=\"left\">21</td><td align=\"left\">21</td><td align=\"left\">2.10 (1.97)</td><td align=\"left\">2.10 (3.35)</td><td/></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Effect of ethanol pretreatment on bupropion HCI-induced convulsions: duration of convulsions</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Dose (mg/kg),</bold><break/><bold>n = 10 per</bold><break/><bold> group</bold></td><td align=\"left\" colspan=\"6\"><bold>Duration of convulsions</bold></td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"left\" colspan=\"2\"><bold>No. of short convulsions</bold><break/><bold> (0 to 10 s)</bold></td><td align=\"left\" colspan=\"2\"><bold>No. of medium</bold><break/><bold>convulsions</bold><break/><bold> (11 to 30 s)</bold></td><td align=\"left\" colspan=\"2\"><bold>No. of long convulsions</bold><break/><bold> (≥ 31 s)</bold></td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td><td align=\"left\"><bold>BUP</bold></td><td align=\"left\"><bold>ET + BUP</bold></td></tr></thead><tbody><tr><td align=\"left\">0 (V or ET +V)</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">0</td></tr><tr><td align=\"left\">100</td><td align=\"left\">1</td><td align=\"left\">78</td><td align=\"left\">1</td><td align=\"left\">17</td><td align=\"left\">0</td><td align=\"left\">14</td></tr><tr><td align=\"left\">110</td><td align=\"left\">22</td><td align=\"left\">6</td><td align=\"left\">0</td><td align=\"left\">3</td><td align=\"left\">0</td><td align=\"left\">6</td></tr><tr><td align=\"left\">120</td><td align=\"left\">18</td><td align=\"left\">17</td><td align=\"left\">3</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">3</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.</p><p>BUP, bupropion HCl; ET, ethanol; V, vehicle or 0.9% sodium chloride (NaCl).</p></table-wrap-foot>", "<table-wrap-foot><p>n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.</p><p>ET, ethanol; vehicle, 0.9% sodium chloride (NaCl).</p></table-wrap-foot>", "<table-wrap-foot><p>n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.</p><p>*p Value for overall ethanol-bupropion interaction effect (ethanol effect, overall p = 0.0183; bupropion dose effect, overall p = 0.0007).</p><p>†p = 0.0019 for pairwise comparison with corresponding mean value for bupropion alone treatment.</p><p>BUP, bupropion HCl; ET, ethanol; SD, standard deviation; V, vehicle or 0.9% sodium chloride (NaCl).</p></table-wrap-foot>", "<table-wrap-foot><p>n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.</p><p>BUP, bupropion HCl; ET, ethanol; V, vehicle or 0.9% sodium chloride (NaCl).</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1744-859X-7-11-1\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>The peer reviewed literature on dairy cow mortality is relatively sparse. In a review on dairy cow mortality, Thomsen and Houe [##REF##17205832##1##] concluded that the number of published studies is surprisingly low, especially seen in relation to the large impact of dairy cow mortality on animal welfare and the farmer's economy.</p>", "<p>Mortality risk defined as unassisted death and euthanasia in Danish dairy cows has increased significantly since 1990. The mortality risk has increased from approximately 2% in 1990 to 5% in 2005. This increase is seen for all major dairy breeds and for all parities. There has been only a slight increase in mortality risk during the period 2002 to 2005 (from approximately 4.7% to 4.9%). Throughout the years the mortality risk has been approximately twice as high in older cows (parity 3 or older) as in younger cows [##REF##15154682##2##,##UREF##0##3##]. At first glance, this development seems very negative, as mortality risk in Danish dairy cows has more than doubled since 1990. The increased mortality can be caused by an increasing number of cows dying unassisted or by an increasing number of euthanized cows (or both). Cows dying unassisted probably often constitute an animal welfare problem, as cows dying unassisted in many cases will suffer from fear or pain before death. The situation concerning euthanasia is, however, more complex. An increase in the number of euthanized cows might be due to an increased number of seriously ill cows. This situation also has negative impacts on animal welfare. If, on the other hand, an increase in the number of euthanized cows is not a consequence of increased morbidity, but caused by an altered threshold for euthanasia of cows among farmers, it might have a positive impact on animal welfare. More seriously ill cows might be euthanized at an early stage and thus not put through a (perhaps long) period of suffering associated with disease and treatment attempts.</p>", "<p>The objectives of this study were to examine the proportion of dead Danish dairy cows that had been euthanized in 2002 and 2006, to examine the development over time in the threshold for euthanasia of cows based on interviews with farmers, to evaluate the farmers' perceptions regarding the background of this development and finally to analyse the welfare implications.</p>" ]

[ "<title>Methods</title>", "<p>In Denmark, farmers are required by law to report all deaths in cows to the Danish Cattle Database. Registration is based on the mandatory identification by ear tags and a central computerized tracking system. Consequently, a registration rate very close to 100% is achieved. Until now, these registrations have not distinguished between cows dying unassisted and cows being euthanized on the farm. They were all simply recorded as 'dead'. Until recently, it was therefore unknown how many cows died unassisted and how many were euthanized and whether there has been a change in the prevalence of euthanized cows over the years. Over the course of 7 weeks in 2002, and again in 2006, we randomly identified four cows on a daily basis that had been reported as 'dead' to the Danish Cattle Database. The random samples in each of the two years were independent. Dead cows included unassisted dead and euthanized cows, but not cows slaughtered. As all Danish dairy farmers are required by law to report dead cows to the Danish Cattle Database, the sample population for the study was all Danish dairy cows/herds. The 196 farmers who had reported the selected dead cows were asked to participate in a questionnaire survey that was part of a larger study on dairy cow mortality [##REF##15154682##2##] (data from the 2002 questionnaire survey has previously been presented in [##REF##15154682##2##]). The number of farmers participating in each questionnaire survey was determined based on a sample size calculation that is presented in [##REF##15154682##2##]. The sampling protocol guaranteed that an equal number of cows, which had died every day of the week, were sampled. Only cows of dairy breeds and currently in commercial milk-producing herds were included. Cows from herds where all cows were euthanized because of occurrence of bovine spongiform encephalopathy (BSE) were not included (until July 2008, a total of 14 Danish herds have been infected with BSE [##UREF##1##4##]). Cows from educational or experimental herds were also excluded from the study. Immediately following the daily sampling protocol, a letter of introduction was sent to the farmers. The letter explained the background and purpose of the study and guaranteed confidentiality. To minimize recall bias, farmers were contacted by telephone 2 to 7 days following the mailing of the letter. If the farmer was not reached by phone, we attempted calling each subsequent day up to a maximum of four days. The farmer was censored if we failed to make contact. If farmers were excluded from the study an additional dead cow was identified from the database and the owner contacted in the same manner as described above. The resulting data set contained 196 dead cows and the corresponding interview from the 196 owners of these animals in each of the two years sampled (in total 392 farmers interviews).</p>", "<p>The farmers were asked whether the cow died unassisted or was euthanized (closed question). Additionally, the farmers were asked their opinion regarding changes in their practice concerning euthanasia over the past 5 years using a closed question. The possible answers were: 1) I have lowered my threshold for euthanasia compared to 5 years ago, 2) my threshold is unchanged compared to 5 years ago, and 3) my threshold has increased compared to 5 years ago. If the farmer had started his/her operation less than 5 years ago the question was classified as 'not relevant'.</p>", "<p>In the 2006 questionnaire survey the new sample of farmers were interviewed in exactly the same manner as described above. Additionally, they were interviewed about the reasons behind possible changes in their practice concerning euthanasia using an open question. Trends in prices of live dairy cows and beef meat over time were compiled from public statistics and used in the discussion of possible changes in the practice concerning euthanasia.</p>" ]

[ "<title>Results and discussion</title>", "<p>The proportion of euthanized cows was approximately similar in the two surveys from 2002 and 2006 (Figure ##FIG##0##1##). Confidence intervals for the two years overlap and in fact the 'true' proportion of euthanized cows might be higher in 2006 than in 2002 and visa versa. In both surveys the farmers generally reported that their threshold for euthanasia had lowered compared to 5 years earlier (Figure ##FIG##1##2##). This was especially the case in the 2002 survey. Here 55% of the farmers reported a lower threshold and therefore reported euthanasia relatively more frequent (expressed as the percentage of cows in the herd euthanized per year) than 5 years earlier. Almost all the other farmers reported that they had the same threshold and therefore euthanized the same relative number of cows as 5 years earlier. In 2006, 54% reported a lower threshold and therefore performed euthanasia relatively more frequent than 5 years earlier, 33% reported unchanged threshold and performed euthanasia at the same level as previously and 10% reported higher threshold and euthanized relatively fewer cows than 5 years earlier. A total of 7 farmers participated in both the 2002 and the 2006 questionnaire survey. Recall bias regarding whether the cow died unassisted or was euthanized is unlikely due to the short time from the death of the cow to the interview. Recall bias regarding the changes in practice concerning euthanasia over time is more likely. Farmers might have difficulty remembering exactly how their policy regarding euthanasia was 5 years ago. However, our results regarding changes in euthanasia practice were relatively clear and recall bias most likely only affected our results to a minor degree. Additional evidence saying that the threshold for euthanasia has lowered during the years is not available at the moment. A number of technicalities prevent us from using information about health remarks from slaughterhouses for such an evaluation. Additional research is needed in this area.</p>", "<p>Interviews with the farmers in 2006 revealed a number of possible reasons (trends) behind the change in behaviour (Figure ##FIG##1##2##). Decreasing average profits per cow, decreasing value of the individual cow, increasing labour costs and increasing veterinary expenses during the last decade affected the farmer's decision-making concerning treatment versus euthanasia. Currently, the individual cow is not valuable to the farmer in the same way as 10 or 20 years ago [##UREF##2##5##,##UREF##3##6##]. Figure ##FIG##2##3## illustrates the development in meat prices and prices of live cows from 1990 to 2006. It can be seen that prices have decreased steadily from 1990 to 2001–2003. Hereafter prices have increased again by approximately 20%. Additionally, the costs of treating a seriously ill cow have increased steadily. Consequently, the farmer's interest in intensive treatment of seriously ill cows has decreased and euthanasia has become an attractive alternative to treatment attempts. The increasing prices of meat and live cows during the last few years might explain the higher proportion of farmers having increased their threshold for euthanasia in 2006 compared to 5 years earlier compared with the situation in 2002 and 5 years before that.</p>", "<p>Additionally, many farmers stated that changes in the practice concerning the veterinary inspection at the Danish slaughterhouses have affected their behaviour. A cow that was considered fit for transport a few years ago, is now often considered unfit for transport. If a farmer chooses to send such a cow for slaughter, the case may be considered as a violation of animal protection laws and consequently result in a fine to the farmer. To avoid this situation, many farmers therefore probably choose to euthanize cows whose fitness for transport is questionable. Some of the cows that are currently euthanized in the herds would probably have been sent to slaughter a few years ago. Besides a relatively new law against the slaughter of cows in the last tenth of pregnancy, these changes at the slaughterhouses are not caused by changes in any laws or regulations [##UREF##4##7##], but are most likely a result of a generally increased debate about and focus on the welfare of animals being transported for slaughter in Denmark in recent years [##UREF##5##8##]. This debate on animal welfare might have affected the awareness of veterinarians at the slaughterhouses and caused them to lower their threshold between what is acceptable and not when it comes to the transportation of animals.</p>", "<p>We have no evidence indicating whether the number of seriously ill cows has increased during the last decade and therefore we are not able to quantify to what extent an increasing prevalence of euthanized cows might also have been affected by an increasing number of seriously ill cows. The number of recorded disease treatments in Danish dairy cows has, however, not changed significantly during the last 10–15 years [##UREF##6##9##]. It should be noted that the number of recorded disease treatments is not necessarily a good indicator of the 'true' disease status in the population. There are a number of steps from a cow being sick to a treatment record. If the farmer does not observe that the cow is sick, no treatment is initialised and hence no treatment is recorded. If the farmer observers that the cow is sick, he/she might for different reasons decide not to treat the cow [##REF##12018446##10##]. Again, no treatment is recorded. And finally, if the farmer observes a sick cow and decides to treat her, the treatment might not be recorded correctly.</p>", "<p>As a consequence of the findings from this study, as of the end of 2007, farmers are required to report to the Danish Cattle Database whether a dead cow died unassisted or was euthanized thus allowing differentiation between unassisted dead and euthanized cows in the future and evaluation of the welfare implications of possible changes in the proportion of euthanized cows.</p>" ]

[ "<title>Results and discussion</title>", "<p>The proportion of euthanized cows was approximately similar in the two surveys from 2002 and 2006 (Figure ##FIG##0##1##). Confidence intervals for the two years overlap and in fact the 'true' proportion of euthanized cows might be higher in 2006 than in 2002 and visa versa. In both surveys the farmers generally reported that their threshold for euthanasia had lowered compared to 5 years earlier (Figure ##FIG##1##2##). This was especially the case in the 2002 survey. Here 55% of the farmers reported a lower threshold and therefore reported euthanasia relatively more frequent (expressed as the percentage of cows in the herd euthanized per year) than 5 years earlier. Almost all the other farmers reported that they had the same threshold and therefore euthanized the same relative number of cows as 5 years earlier. In 2006, 54% reported a lower threshold and therefore performed euthanasia relatively more frequent than 5 years earlier, 33% reported unchanged threshold and performed euthanasia at the same level as previously and 10% reported higher threshold and euthanized relatively fewer cows than 5 years earlier. A total of 7 farmers participated in both the 2002 and the 2006 questionnaire survey. Recall bias regarding whether the cow died unassisted or was euthanized is unlikely due to the short time from the death of the cow to the interview. Recall bias regarding the changes in practice concerning euthanasia over time is more likely. Farmers might have difficulty remembering exactly how their policy regarding euthanasia was 5 years ago. However, our results regarding changes in euthanasia practice were relatively clear and recall bias most likely only affected our results to a minor degree. Additional evidence saying that the threshold for euthanasia has lowered during the years is not available at the moment. A number of technicalities prevent us from using information about health remarks from slaughterhouses for such an evaluation. Additional research is needed in this area.</p>", "<p>Interviews with the farmers in 2006 revealed a number of possible reasons (trends) behind the change in behaviour (Figure ##FIG##1##2##). Decreasing average profits per cow, decreasing value of the individual cow, increasing labour costs and increasing veterinary expenses during the last decade affected the farmer's decision-making concerning treatment versus euthanasia. Currently, the individual cow is not valuable to the farmer in the same way as 10 or 20 years ago [##UREF##2##5##,##UREF##3##6##]. Figure ##FIG##2##3## illustrates the development in meat prices and prices of live cows from 1990 to 2006. It can be seen that prices have decreased steadily from 1990 to 2001–2003. Hereafter prices have increased again by approximately 20%. Additionally, the costs of treating a seriously ill cow have increased steadily. Consequently, the farmer's interest in intensive treatment of seriously ill cows has decreased and euthanasia has become an attractive alternative to treatment attempts. The increasing prices of meat and live cows during the last few years might explain the higher proportion of farmers having increased their threshold for euthanasia in 2006 compared to 5 years earlier compared with the situation in 2002 and 5 years before that.</p>", "<p>Additionally, many farmers stated that changes in the practice concerning the veterinary inspection at the Danish slaughterhouses have affected their behaviour. A cow that was considered fit for transport a few years ago, is now often considered unfit for transport. If a farmer chooses to send such a cow for slaughter, the case may be considered as a violation of animal protection laws and consequently result in a fine to the farmer. To avoid this situation, many farmers therefore probably choose to euthanize cows whose fitness for transport is questionable. Some of the cows that are currently euthanized in the herds would probably have been sent to slaughter a few years ago. Besides a relatively new law against the slaughter of cows in the last tenth of pregnancy, these changes at the slaughterhouses are not caused by changes in any laws or regulations [##UREF##4##7##], but are most likely a result of a generally increased debate about and focus on the welfare of animals being transported for slaughter in Denmark in recent years [##UREF##5##8##]. This debate on animal welfare might have affected the awareness of veterinarians at the slaughterhouses and caused them to lower their threshold between what is acceptable and not when it comes to the transportation of animals.</p>", "<p>We have no evidence indicating whether the number of seriously ill cows has increased during the last decade and therefore we are not able to quantify to what extent an increasing prevalence of euthanized cows might also have been affected by an increasing number of seriously ill cows. The number of recorded disease treatments in Danish dairy cows has, however, not changed significantly during the last 10–15 years [##UREF##6##9##]. It should be noted that the number of recorded disease treatments is not necessarily a good indicator of the 'true' disease status in the population. There are a number of steps from a cow being sick to a treatment record. If the farmer does not observe that the cow is sick, no treatment is initialised and hence no treatment is recorded. If the farmer observers that the cow is sick, he/she might for different reasons decide not to treat the cow [##REF##12018446##10##]. Again, no treatment is recorded. And finally, if the farmer observes a sick cow and decides to treat her, the treatment might not be recorded correctly.</p>", "<p>As a consequence of the findings from this study, as of the end of 2007, farmers are required to report to the Danish Cattle Database whether a dead cow died unassisted or was euthanized thus allowing differentiation between unassisted dead and euthanized cows in the future and evaluation of the welfare implications of possible changes in the proportion of euthanized cows.</p>" ]

[ "<title>Conclusion</title>", "<p>Other studies have shown that mortality risk in Danish dairy cows has increased significantly since 1990. Results from interviews with farmers, however, indicate that the threshold for euthanasia of cows among farmers has lowered. This situation might have a positive impact on animal welfare as more seriously ill cows are euthanized in the herds and not put through a period of suffering associated with disease and treatment or transport to a slaughterhouse in poor condition.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Mortality risk in Danish dairy cows has more than doubled since 1990 (from 2% in 1990 to 5% in 2005). Until now, registrations about dead cows in the Danish Cattle Database have not included information about whether the cow died unassisted or was euthanized.</p>", "<title>Methods</title>", "<p>We interviewed a random sample of 196 Danish dairy farmers that had reported a dead cow to the Danish Cattle Database in 2002 and 196 dairy farmers that had reported a dead cow in 2006. Our objectives were to evaluate the proportion of euthanized cows, changes in the behaviour of farmers regarding euthanasia of cows over the years and possible reasons for these changes.</p>", "<title>Results</title>", "<p>It seems that the threshold for euthanasia of cows among farmers has changed. Farmers generally reported a lower threshold for euthanasia compared to 5–10 years ago.</p>", "<title>Conclusion</title>", "<p>The threshold for euthanasia of cows has, according to the dairy farmers, become lower. This might have positive impacts on animal welfare as more seriously ill cows are euthanized in the herds and not put through a period of suffering associated with disease and treatment or transported to a slaughterhouse in poor condition.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>PTT participated in the planning of the study, conducted the study, analysed the results and drafted the manuscript. JTS participated in the planning of the study and helped drafting the manuscript. Both authors read and approved the final manuscript.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Proportion of a random sample of 196 dead cows that were euthanized or died unassisted in 2002 and 2006, respectively</bold>. 95% confidence intervals are indicated by vertical lines.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>The distribution of answers to a question regarding the farmers' practice concerning euthanasia of cows in 2002 and 2006, respectively, compared with 5 years earlier.</bold> 95% confidence intervals are indicated by vertical lines.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Indexed prices of beef meat and live cows sold for dairy purposes in Denmark from 1990 to 2006 (1990 = index 100).</bold> All prices are calculated in Danish kroner and adjusted for inflation. (Modified after [##UREF##7##11##, ####UREF##8##12##, ##UREF##9##13##, ##UREF##10##14##, ##UREF##11##15##, ##UREF##12##16####12##16##]).</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1751-0147-50-33-1\"/>", "<graphic xlink:href=\"1751-0147-50-33-2\"/>", "<graphic xlink:href=\"1751-0147-50-33-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>High chemical complexity of herbal medicines makes quality control through chemical analysis difficult. For example, a common Chinese medicinal herb <italic>Cortex Cinnamomi </italic>(<italic>Rougui</italic>) revealed over 90 volatile components in a gas chromatography-mass spectroscopy (GC-MS) experiment, only 60 of which were chemically identified [##REF##15019037##1##]. Little chemical information is known about herbal medicines [##UREF##0##2##]. Mok and Chau categorized authentication and quality control of herbal medicines into the 'component-based' and 'pattern-based' approaches [##UREF##1##3##]. For a refined classification, we refer to these two approaches in this article as the compound-oriented approach and pattern-oriented approach respectively. The compound-oriented approach includes the marker approach and the multi-compound approach. The marker approach takes into account herbal medicines with known components, while the multi-compound approach may include some unknown components whose chemical properties are known. The pattern-oriented approach, such as fingerprint analysis, evaluates all data acquired from analytical instrument. For example, fingerprints and bioactivities have recently been correlated to the quality control of herbal medicines [##REF##16213118##4##, ####UREF##2##5##, ##REF##16132135##6####16132135##6##]. Instead of measuring all elements within a biological system, systems biology aims to reveal intrinsic trends of the system [##REF##12931164##7##,##REF##15889489##8##]. Metabonomics focuses on the investigation of high-throughput data from metabolite profiling. The key advantage of metabonomics is to provide an integrative and systematic view of metabolism, which may also reveal the quality of herbal medicines [##REF##15656647##9##,##REF##16939360##10##].</p>", "<p>Different ingredients within a herbal medicine may have synergistic effects. These active ingredients must be identified and quantified for better understanding of the action mechanisms of herbal medicines. For the moment, the conventional marker approach is not successful as only a few chemical markers are available for herbal medicines in the American Herbal Pharmacopeia and Chinese Pharmacopoeia. For example, in the Chinese Pharmacopoeia (2005 edition) only one chemical marker chlorogenic acid (C₁₆H₁₈O₉) is recommended for the identification of <italic>Flos Lonicerae </italic>(<italic>Jinyinhua</italic>) (content ≥ 1.5%) and <italic>Flos Chrysanthemi </italic>(<italic>Juhua</italic>) (content ≥ 0.2%) [##UREF##3##11##]. These two herbs cannot be differentiated with the only chemical marker. Likewise, <italic>Fufang Danshen Diwan </italic>is a formulated herbal medicine that consists of three herbs but there is only one chemical marker salvianic acid A (C₉H₁₀O₅) recommended for this particular medicine in the Chinese Pharmacopoeia (2005 edition) [##UREF##3##11##].</p>", "<p>The pattern-oriented approach such as fingerprinting is more useful than compound-oriented approach in most cases. Chemical fingerprints are characteristic for herbal medicines, and are therefore useful in the quality control of herbal medicines [##REF##16472540##12##, ####UREF##4##13##, ##UREF##5##14##, ##UREF##6##15####6##15##]. The information-rich chemical fingerprints can be obtained from advanced instruments [##UREF##7##16##, ####UREF##8##17##, ##UREF##9##18##, ##UREF##10##19##, ##REF##15620519##20##, ##UREF##11##21####11##21##]. All detectable chemical components of a herbal medicine may be shown in fingerprints. Chemometrics helps analyze and interpret useful information from raw data, e.g. alignment of shifts of retention time in chromatography, data assessment and comparison, smoothing and filtering, deconvolution and resolution of overlapping peak clusters, in determination of the chromatographic and spectral profiles of pure components [##UREF##12##22##, ####REF##16174438##23##, ##REF##15032363##24##, ##UREF##13##25##, ##UREF##14##26##, ##UREF##15##27####15##27##]. This article summarizes recent advances in both compound-oriented and pattern-oriented approaches in terms of experimentation, instrumentation and data processing.</p>", "<title>Compound-oriented approach</title>", "<title>Marker approach</title>", "<p>According to the Chinese Pharmacopoeia (2005 edition), identification and quantification of chemical markers are crucial to the quality control of herbal medicines. A total of 525 quantitative assays of chemical markers were documented in the Chinese Pharmacopoeia (2005 edition) for assessment of herbal medicinal materials, plant lipids, herbal extracts and formulations [##UREF##3##11##]. Chemistry of these markers is known and their analytical procedures and reference standards are available for quality control. Chau <italic>et al</italic>. used near infrared spectroscopy (NIR) to quantitatively determine the content of berberine and total alkaloid in <italic>Cortex Phellodendri </italic>(<italic>Guanhuangbo</italic>) [##REF##17512816##28##]. The content of berberine determined by high-performance liquid chromatography-diode array detection (HPLC-DAD) was used as a critical parameter to confirm the accuracy of the data obtained from NIR according to a linear model of partial least squares (PLS) regression. In another study, high-performance liquid chromatography (HPLC) and HPLC-DAD were used to assess the quality consistency of a formulated Chinese medicine <italic>Qingfu Guanjie Shu </italic>(capsule) using four marker compounds, namely sinomenine, paeoniflorin, paeonol and curcumin. [##UREF##16##29##]. Lin <italic>et al</italic>. used liquid chromatography-tandem mass spectrometers (LC-MS/MS), solid phase extraction, and the marker glycyrrhetic acid to simultaneously validate <italic>Radix Glycyrrhizae </italic>(<italic>Gancao</italic>) and quantify the target compound in the samples [##UREF##17##30##]. Quantitative studies of markers and identification of active ingredients were carried out for the quality control of herbal medicines [##UREF##18##31##, ####REF##16515793##32##, ##REF##17029978##33##, ##UREF##19##34####19##34##]. Gas chromatography (GC), gas chromatography-mass spectroscopy (GC-MS), thin layer chromatography (TLC), thin layer chromatography-ultraviolet spectrophotometry (TLC-UV), capillary electrophoresis (CE) and capillary zone electrophoresis (CZE) were also proposed for the quality control of herbal medicines [##UREF##20##35##, ####UREF##21##36##, ##UREF##22##37##, ##UREF##23##38##, ##UREF##24##39####24##39##].</p>", "<title>Multi-compound approach</title>", "<p>Compared with the marker approach, the multi-compound approach uses multiple compounds with known chemical properties and does not necessarily require chemical markers. Chemometric deconvolution and resolution are major methods in this approach. In the Chinese Pharmacopoeia (2005 edition) [##UREF##3##11##], multiple compounds, instead of a single compound, are recommended for the quality control of herbal medicines. For example, total flavonol glycosides (i.e. quercetin, isorhamnetin and kaempferol) as well as total terpene lactones (i.e. bilobalide, and ginkgolides A, B and C) were used for the quality control of a ginkgo leaf product [##UREF##3##11##]. However, analyzing multiple compounds in a single chromatogram may not be easy. These chromatograms often contain overlapping peaks, which may not be resolved by changing chromatographic conditions. One possible solution is the use of chemical and/or instrumental methods that take advantage of spectra with very close retention times, e.g. mass spectra, ultraviolet spectra or other chemical properties containing variations large enough to resolve overlapping chromatographic profiles [##REF##15336357##40##, ####UREF##25##41##, ##REF##16168606##42##, ##UREF##26##43####26##43##].</p>", "<p>Chemometric resolution methods (CRM) were used extensively in the past decades to 'purify' chromatographic peak profiles of complex mixture systems such as herbal medicines [##UREF##4##13##]. The qualitative and quantitative chemical information obtained by CRM did help discover the active ingredients of herbal medicines and study the synergistic effects of the ingredients [##REF##15019037##1##].</p>", "<p>Previously, both iterative and non-iterative resolution methods were used to study the volatile and non-volatile components in herbal medicines [##REF##16539851##44##, ####REF##11206787##45##, ##REF##14601831##46####14601831##46##]. Many non-iterative resolution methods such as heuristic evolving latent projection (HELP), alternative moving window factor analysis (AMWFA), (subwindow factor analysis) SFA, evolving window orthogonal projection (EWOP) were useful in discovering more than ten components of herbal medicines [##REF##15137973##47##, ####UREF##27##48##, ##REF##16430907##49##, ##REF##15478995##50##, ##REF##11269523##51####11269523##51##]. Using GC-MS coupled with HELP, Li <italic>et al</italic>. identified 38 volatile chemical components of <italic>Radix Paeoniae Rubra </italic>(<italic>Chishao</italic>), which accounted for 95.21% of all detectable components [##UREF##28##52##]. In another study, 69 components of <italic>Radix Rehmanniae Preparata </italic>(<italic>Shudihuang</italic>) were separated, of which 59 were identified using standard spectra in the database of the National Institute of Standards and Technology (NIST). The 26 identified methyl esters accounted for about 94.29% of the total number of components [##UREF##29##53##]. Most of the iterative methods including (orthogonal projection approach) OPA and (iterative orthogonal projection resolution) IOP were applied to determine the chemical composition of herbal medicines [##REF##11206787##45##,##REF##14601831##46##]. With these chemometric methods, 65 volatile chemical constituents of <italic>Rhizoma et Radix Notopterygii </italic>(<italic>Qianghuo</italic>) were identified out of the 98 separated chemicals. Qi <italic>et al</italic>. resolved the overlapping chromatographic peaks in <italic>Resina Draconis </italic>(<italic>Xuejie</italic>) using HPLC-DAD. Therefore, using chemometric methods with hyphenated instruments was powerful in the analysis of herbal medicines [##UREF##30##54##]. Zeng <italic>et al</italic>. used PCA and generalized rank annihilation factor analysis (GRAFA) to process HPLC-DAD data sets obtained from <italic>Radix Salviae Miltiorrhizae </italic>(<italic>Danshen</italic>) and <italic>Radix Notoginseng </italic>(<italic>Sanqi</italic>) [##UREF##31##55##]. Ye <italic>et al</italic>. simultaneously analyzed seven major saponins of <italic>Danshen Diwan </italic>with HPLC-DAD and electron spray ionization-mass spectrometry (ESI-MS) [##UREF##32##56##].</p>", "<p>Chemometric methods including spectral correlative chromatography (SCC), multi-component spectral correlative chromatography (MSCC), AMWFA were proposed for comparing three-dimensional (3D) or two-dimensional (2D) chromatographic profiles and integrating presence or absence information [##REF##16430907##49##,##UREF##33##57##, ####REF##15612753##58##, ##UREF##34##59####34##59##]. SCC was used to compare pure or selective herbal medicine components [##UREF##34##59##]. Both MSCC and AMWFA were used to analyze complex herbal medicines. The main feature of MSCC is the construction of an orthogonal projection matrix using abstract spectra acquired from decomposition of original fingerprint data sets. For comparison, the pure and mixed spectra are projected to the matrix for presence or absence information of target components. AMWFA was used to extract pure chromatographic and spectral profiles of common components of related herbal medicines [##REF##16430907##49##,##REF##15612753##58##,##UREF##34##59##]. All these new chemometric algorithms were applied in the identification, quantification, comparison of chemical components and quality control of herbal medicines [##REF##14658022##60##, ####REF##15335044##61##, ##UREF##35##62##, ##UREF##36##63##, ##REF##17606017##64##, ##UREF##37##65##, ##UREF##38##66####38##66##].</p>", "<title>Pattern-oriented approach</title>", "<title>Single pattern approach</title>", "<title>Fingerprint analysis</title>", "<p>The pattern-oriented approach analyzes fingerprints obtained from one-, two- or higher dimensional chromatographic and/or spectral instruments. Single pattern approach focuses on one type of pattern (e.g. chemical fingerprints of chromatograms and spectra) for the quality control of herbal medicines. Significant progress was made in 2D chromatographic profiles obtained from HPLC, CE, GC as well as 3D ones from hyphenated instruments [##UREF##9##18##,##UREF##10##19##,##UREF##39##67##, ####REF##16999970##68##, ##UREF##40##69##, ##REF##17161980##70####17161980##70##]. Yan <italic>et al</italic>. combined 3D fingerprints from HPLC-DAD instruments with principal component analysis (PCA) to monitor <italic>Qingkailing </italic>(a proprietary Chinese medicine injection formulation) produced by various manufacturers [##REF##16196137##71##]. High-speed counter-current chromatography (HSCCC) and high-performance liquid chromatography-coulometric electrode array detector (HPLC-CEAD) were used to obtain the fingerprint of <italic>Radix Salviae Miltiorrhizae </italic>(<italic>Danshen</italic>) collected from various localities [##REF##14753780##72##, ####REF##15584232##73##, ##UREF##41##74####41##74##]. Binary chromatographic fingerprints from HPLC-DAD and GC-MS were used to analyze the aporphinoid and quinolizidine alkaloids of <italic>Caulophyllum robustum </italic>(<italic>Leiyemudan</italic>). Similarity index and the cluster analysis method were used to analyze the quality of ten batches of samples of <italic>Caulophyllum robustum </italic>[##UREF##42##75##]. Fan <italic>et al</italic>. developed multiple chromatographic fingerprinting including two HPLC fingerprints for the quality assessment of <italic>Danshen Diwan </italic>[##UREF##43##76##]. Thin layer chromatography scan (TLCS) provided unique fingerprints which differentiated passiflora and other herbs grown under various conditions [##UREF##11##21##,##UREF##44##77##]. Fourier transform infrared spectroscopy (FT-IR), NIR and two-dimensional correlation infrared spectroscopy (2D-IR) spectroscopy were used to construct spectral fingerprints of complex herbal medicines [##REF##15620519##20##,##REF##17512816##28##,##UREF##45##78##,##UREF##46##79##]. New fingerprinting techniques have recently emerged, such as oscillating fingerprints [##UREF##47##80##], electrochemistry fingerprints [##UREF##48##81##,##UREF##49##82##], X-ray diffraction (XRD) second derivative fingerprints [##UREF##50##83##] and high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MSn) [##UREF##51##84##].</p>", "<title>Chemometric methods for fingerprint analysis</title>", "<p>It is important to extract useful information from chromatographic/spectral fingerprints which contain chemical information of all detectable components of herbal medicines. Chau <italic>et al</italic>. developed a software package Computer Aided Similarity Evaluation (CASE) for data processing under Matlab [##UREF##52##85##]. Gong <italic>et al</italic>. developed a series of chemometric methods to extract information from fingerprints [##REF##15032363##24##,##REF##12885076##86##,##REF##17723488##87##]. Xu <italic>et al</italic>. used target peak alignment (TPA) to correct the retention time shifts and multiplicative scattering correction (MSC) for response correction [##REF##16962124##88##]. In addition, wavelet technique was used for data pre-processing of fingerprints [##UREF##53##89##]. Comparison, assessment and discrimination analysis are key steps to process the 'standardized' fingerprints of herbal medicines. Chemometric methods are used to compare data among herbs with hundreds or even thousands of chemical components. This information can be further used to identify the origins of herbs, or to authenticate herbs. For example, research has made significant progress since the introduction of the similarity index and pattern recognition analysis [##UREF##54##90##, ####UREF##55##91##, ##UREF##56##92##, ##UREF##57##93##, ##REF##15909545##94####15909545##94##]. Information fusion and determination of relative entropy critical value were proposed for the similarity analysis of fingerprints of herbal medicines [##UREF##54##90##, ####UREF##55##91##, ##UREF##56##92##, ##UREF##57##93####57##93##]. PCA, orthogonal projection technique (OP), 2D-IR worked well on data discrimination analysis of fingerprints [##REF##15909545##94##, ####REF##16622674##95##, ##REF##17386812##96##, ##UREF##58##97####58##97##]. Data deconvolution methods helped obtain chromatographic and spectral profiles of individual pure components [##REF##15137973##47##, ####UREF##27##48##, ##REF##16430907##49##, ##REF##15478995##50##, ##REF##11269523##51##, ##UREF##28##52##, ##UREF##29##53####29##53##]. Furthermore, chemical fingerprint databases of herbal medicines were developed according to chemometric methods [##UREF##59##98##,##UREF##60##99##].</p>", "<title>Multi-pattern approach</title>", "<p>The multi-pattern approach assesses the quality of herbal medicines according to multiple patterns. For example, both chromatographic fingerprints and biological activity profiles are used for the quality control of herbal medicines. This approach targets the discovery of bioactive ingredients, assessment of medical effects, correlation between chemical fingerprints and pharmacological indices, and quality control [##REF##16132135##100##, ####REF##16496683##101##, ##REF##16213118##102##, ##UREF##61##103##, ##REF##17276058##104##, ##UREF##62##105####62##105##]. Some studies used chromatographic fingerprints from liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectroscopy (LC-DAD-APCI-MS) followed by data processing. Wang <italic>et al</italic>. found 32 potential bioactive components of <italic>Radix Angelicae Sinensis </italic>(<italic>Danggui</italic>) in rabbit plasma and over ten new compounds [##REF##16132135##100##]. Using a <italic>Herba Houttuyniae </italic>(<italic>Yuxingcao</italic>) injection, Lu <italic>et al</italic>. evaluated its anti-inflammatory effects and established the correlation between the chemical components of the injection and the bioactivities of the active ingredients [##REF##16496683##101##,##REF##16213118##102##]. Wang <italic>et al</italic>. devised a method which combined metabolic profiling and liquid chromatography-diode array detection-mass spectroscopy (LC/DAD-MS) with chemometrics to study <italic>Danggui Buxue Tang</italic>, a Chinese medicine formulation [##UREF##61##103##]. Aided by PCA, Yu <italic>et al</italic>. discovered major bioactive components of <italic>Aquilegia oxysepala </italic>(<italic>Jianeloudoucai</italic>) [##REF##17276058##104##]. Evidently, the multi-pattern approach provides a reliable means for the quality control of herbal medicines.</p>" ]

[ "<title>Chemometric methods for fingerprint analysis</title>", "<p>It is important to extract useful information from chromatographic/spectral fingerprints which contain chemical information of all detectable components of herbal medicines. Chau <italic>et al</italic>. developed a software package Computer Aided Similarity Evaluation (CASE) for data processing under Matlab [##UREF##52##85##]. Gong <italic>et al</italic>. developed a series of chemometric methods to extract information from fingerprints [##REF##15032363##24##,##REF##12885076##86##,##REF##17723488##87##]. Xu <italic>et al</italic>. used target peak alignment (TPA) to correct the retention time shifts and multiplicative scattering correction (MSC) for response correction [##REF##16962124##88##]. In addition, wavelet technique was used for data pre-processing of fingerprints [##UREF##53##89##]. Comparison, assessment and discrimination analysis are key steps to process the 'standardized' fingerprints of herbal medicines. Chemometric methods are used to compare data among herbs with hundreds or even thousands of chemical components. This information can be further used to identify the origins of herbs, or to authenticate herbs. For example, research has made significant progress since the introduction of the similarity index and pattern recognition analysis [##UREF##54##90##, ####UREF##55##91##, ##UREF##56##92##, ##UREF##57##93##, ##REF##15909545##94####15909545##94##]. Information fusion and determination of relative entropy critical value were proposed for the similarity analysis of fingerprints of herbal medicines [##UREF##54##90##, ####UREF##55##91##, ##UREF##56##92##, ##UREF##57##93####57##93##]. PCA, orthogonal projection technique (OP), 2D-IR worked well on data discrimination analysis of fingerprints [##REF##15909545##94##, ####REF##16622674##95##, ##REF##17386812##96##, ##UREF##58##97####58##97##]. Data deconvolution methods helped obtain chromatographic and spectral profiles of individual pure components [##REF##15137973##47##, ####UREF##27##48##, ##REF##16430907##49##, ##REF##15478995##50##, ##REF##11269523##51##, ##UREF##28##52##, ##UREF##29##53####29##53##]. Furthermore, chemical fingerprint databases of herbal medicines were developed according to chemometric methods [##UREF##59##98##,##UREF##60##99##].</p>" ]

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[ "<title>Conclusion</title>", "<p>The compound-oriented and pattern-oriented approaches to the quality control of herbal medicines have been significantly improved in terms of analytical instruments, biological screening methods and chemometrics. Among all the advanced techniques, multi-pattern approach will have a great potential for further development.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>The current approaches to the quality control of herbal medicines are either compound-oriented or pattern-oriented, the former targeting specific components with some known chemical properties and the latter targeting all detectable components. The marker approach uses specific chemical compounds with known molecular structures, while the multi-compound approach uses both chemical compounds with known structures and those with partial chemical information e.g. retention times, mass spectra and ultraviolet spectra. Apart from chromatographic techniques, new techniques such as oscillating and electrochemistry fingerprints have been developed for quality control. Chemometric resolution methods are widely used for component deconvolution and data comparison. Pattern recognition techniques are used for authentication of herbal medicines.</p>" ]

[ "<title>Abbreviations</title>", "<p>2D-IR: two-dimensional correlation infrared spectroscopy; AMWFA: alternative moving window factor analysis; CE: capillary electrophoresis; CRM: chemometric resolution methods; CZE: capillary zone electrophoresis; DAD: diode array detection; ESI-MS: electron spray ionization-mass spectrometry; EWOP: evolving window orthogonal projection; FSMWEFA: fixed-size moving window evolving factor analysis; FT-IR: Fourier transform infrared spectroscopy; GC: gas chromatography; GC-MS: gas chromatography-mass spectroscopy; GRAFA: generalized rank annihilation factor analysis; HELP: heuristic evolving latent projection; HPLC: High-performance liquid chromatography; HPLC-CEAD: high-performance liquid chromatography-coulometric electrode array detector; HPLC-DAD: high-performance liquid chromatography-diode array detection; HPLC-ESI-MSn: high-performance liquid chromatography electrospray ionization tandem mass spectrometry; HSCCC: high-speed counter-current chromatography; IOP: iterative orthogonal projection resolution; LC-DAD-APCI-MS: liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectroscopy; LC/DAD-MS: liquid chromatography-diode array detection-mass spectroscopy; LC-MS: liquid chromatography-mass spectroscopy; LC-MS/MS: liquid chromatography-tandem mass spectrometers; MS: mass spectroscopy, MSCC: multi-component spectral correlative chromatography; NIR: near infrared spectroscopy, OP: orthogonal projection technique; OPA: orthogonal projection approach, PCA: principle component analysis; PLS: partial least squares; SCC: spectral correlative chromatography; SFA: subwindow factor analysis, TLC: thin layer chromatography; TLC-UV: thin layer chromatography-ultraviolet spectrophotometry; XRD: X-ray diffraction.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>ZZ and FC conceived the classification of approaches to quality control of herbal medicines. ZZ drafted the manuscript. FC supervised the project and revised the manuscript. HC and CYC provided references and helped revise the manuscript. TL and SW advised on the manuscript. DM, COC and YL were responsible for some studies in this work. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>This work was financially supported by the University Grants Committee of the Hong Kong SAR CMRFD project (A0E/B-10/01), Innovation and Technology Committee of the Hong Kong SAR ITF-TCFS project (GHP/037/05), Taskforce for Development of the Hong Kong Polytechnic University BB8H and BB6R projects, National Nature Foundation Committee of China (20475066 and 10771217) and International Cooperative Project for Traditional Chinese Medicine of the Ministry of Science and Technology of China (2006DFA41090 and 2007DFA40680).</p>" ]

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[ "<title>Background</title>", "<p>Obesity has been increasing in every state in the nation [##REF##10546690##1##] and has reached an all time high in children [##REF##18505949##2##]. Due to the adverse health affects of obesity the total cost for medical care and disability associated with obesity was estimated to be 99.2 billion dollars in 1995 [##REF##8615344##3##]. Currently there are only two pharmaceutical drugs used for the treatment of obesity and these drugs have not been demonstrated to result in weight loss beyond 5 years [##REF##15315867##4##]. Much more successful results have been obtained by performing bariatric surgery, however there are significant risks associated with these procedures. The isolation of a small molecule that can activate lipolysis could have therapeutic potential for the treatment of obesity. By extension there is a great need to identify which genes regulate fat metabolism to identify potential mechanisms of targeted molecular medicine.</p>", "<p>Several monogenic obesity genes have been cloned in mice including leptin, leptin receptor, carboxypeptidase, melanocortin-4 receptor, and the orexigenic agouti protein (for a review see [##REF##15703762##5##]). Mutations in melanocortin-4 receptor have been observed more than any other monogenic obesity disease at 1–6% of the surveyed obese population. A variety of other genes have been shown to be associated with the obesity phenotype including: UCP1-3, PPARγ, and several adrenergic receptors. A proline to alanine at position 12 of PPARγ has clearly been shown to have effects on lipid metabolism. The number of genes expected to be involved in obesity remains to be determined.</p>", "<p>The zebrafish model organism <italic>Danio rerio </italic>is currently the simplest model organism complete with the full complement of vertebrate organs that can be used in forward genetic screens. ENU mutagenesis screens have been performed to isolate mutant zebrafish with defects in their temperature maintenance in an attempt to identify genes potentially involved in regulating lipid metabolism [##REF##10841731##6##]. This was based in part on the observation that mice missing the leptin protein or receptor have problems regulating their temperature. However, to date no one has completed a forward chemical mutagenesis screen to result in the identification of genes regulating fat metabolism using zebrafish.</p>", "<p>The yolk sac is maternally derived and represents the sole source of energy for the embryo and larva during early zebrafish development. Significantly, the yolk sac is a quantifiably finite source of energy that is largely consumed during the first week of larval development. These characteristics give the yolk sac distinct advantages for assaying changes in organismal lipid metabolism.</p>" ]

[ "<title>Methods</title>", "<title>Materials</title>", "<p>Zebrafish were obtained from the Zebrafish International Resource Center. All small molecules were obtained from Sigma chemicals.</p>", "<title>Maintenance of Zebrafish</title>", "<p>Adult and embryonic zebrafish were maintained according to protocols described in The Zebrafish Book [##UREF##0##7##].</p>", "<title>Measurement of Total Fatty Acids and Cholesterol</title>", "<p>Pools of 30 to 50 healthy 3 dpf larvae were selected for daily incubations with fresh E3 media containing small molecules until 7 dpf before they were clarified by centrifugation at 11,000 rcf for 3 min. Supernatant was removed and larvae were frozen for chemical analysis. The pool of larvae was subjected to saponification, conversion to methyl esters, and extraction with hexane. A known mass of heptadecanoic acid was added to the larvae pool before saponification. The methyl esters were quantified by gas chromatography and the mass was calculated from the ratio of the area of methyl heptadecanoate to those of other methyl esters. The conditions for the gas chromatograph have been described previously [##REF##15236180##8##]. For cholesterol measurements at least 30 zebrafish were treated with 1 mM of nicotinic acid or nicotinamide from 3 to 10 dpf before extraction and quantitation of total cholesterol [##REF##16407385##9##].</p>", "<title>Transcript Quantitation by Q-RT-PCR</title>", "<p>Twenty 6 dpf zebrafish larvae were exposed to molecules for 11 hr and flash frozen with liquid nitrogen. RNA was quickly isolated using Qiagen RNeasy mammalian tissue protocol, incorporating Qiashredder. RNA levels were quantified using Nanodrop spectrophotometer and cDNA was synthesized using equivalent amounts of RNA using Superscript II Reverse Transcriptase (Invitrogen). cDNA was quantitated in triplicate by real-time PCR. Primers pairs used were for GPR109a (also known as HM74a, TTTCGACGCTCCCATTCTGGATGA and AGGACGAACTCGCTGAACAGAACA for 60 bp) CD36, AGATGGTTCCTCTTTCCACCCGTT and ACAGGCAGCAAGTACCGATACACA for 144 bp), FABP4 (TGAGCAGGGCGTCATCACTATGAA and TTGTGGTCTTTCCTTCCCAGGTCT for 176 bp), and these were normalized to PP1A, AGAATTTCAGGCAGTTGTGCACGG and TGTGGTTTGTGAAGTCACCTCCCT Qiagen Hot Start DNA Polymerase was used for PCR: 15 m 95°C, 45 × (25 s 94°C, 25 s 60°C, 25 s 68°C), and hold 10°C. All reaction single product sizes were first verified by visualization on agarose gels and melting curve analysis. Fluorescein was included in the reaction to provide a stable fluorescence baseline. Data was collected with the Bio-Rad iCycler iQ system using SYBR green to detect amplicon production. Analysis was performed by the comparative Ct method so both treated and untreated samples were normalized to housekeeping gene product peptidyl-prolyl isomerase A (PPIA) as an internal control.</p>", "<title>Hypoxic stress to Zebrafish larvae</title>", "<p>Multi-well 48 well plates with 0.5 mL of zebrafish embryo (E3; [##UREF##0##7##]) were preconditioned to anoxic conditions by placement into a Bio-Bag type A environmental chamber (Becton Dickinson) as described previously [##REF##11404478##10##]. Using a plate containing at least 20 of 6 dpf zebrafish, the water was removed and replaced with preconditioned hypoxic E3 before initiating a second Bio-Bag type A environmental chamber reaction. Zebrafish were visually monitored for lack of response to shaking stimuli. After approximately 2 hours, zebrafish were harvested by rapidly removing solution and flash freezing in tubes with liquid nitrogen for Q-RT-PCR analysis.</p>", "<title>Small Molecule Treatments with Nile Red and Fluorescence Microscopy</title>", "<p>Embryos were raised in embryo 3 media (E3: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 10E-5 methylene blue) supplemented with 200 μM PTU [##UREF##0##7##]. Starting at 3 days post-fertilization (dpf), 30 to 50 healthy larvae were placed into 48 well plates containing E3 with 10 mM Tris pH 7.4 and 10 ng/mL nile red at a density of 10 larvae per well. Nile red was dissolved in 50,000 × stock solution at 500 μg/mL in acetone. In order to quantitatively prepare the nile red incubation solution 10 μL of stock solution was first carefully pipetted into 50 mL E3 media to prepare a 10× solution. This was diluted to a final working concentration of 10 ng/mL. Incubating larvae at higher levels of nile red produced a bright signal throughout the larvae. Incubation times ranged from 6 hours to overnight to allow for the nile red to equilibrate and penetrate into the depths of the yolk sac and central nervous system of 7 dpf larvae as monitored by fluorescent microscopy. Incubations were performed without shaking at 28°C. Media was replaced daily until 7 dpf by transferring larvae to plates containing solutions pre-heated to 28°C using baskets. To first determine the working concentration, groups of 3 dpf zebrafish were exposed to a ten-fold serial dilution of small molecules and allowed to develop. Ten-fold sub-lethal dose of molecules was chosen as the working concentrations for experiments. Molecules were refreshed on a daily basis starting at 3 dpf. Media was supplemented with 100 μM nicotinic acid, 100 μM nicotinamide, 50 μM resveratrol (10 mM stock dissolved in DMSO), or 50 μM resveratrol with 100 μM norepinephrine. At 7 dpf larvae were examined by fluorescence microscopy. Nile red was visualized on a Zeiss Axioplan microscope using a Texas Red filter. Camera exposure time was established using untreated larva for signal normalization. Larvae to be compared were placed in a single depression slide so that the media and depth were consistent.</p>", "<title>Morpholino Injection</title>", "<p>At least 30 embryos were injected with 2 nL of 0.3 mM (0.6 pmol) anti-sense morpholino per protocols for each of three separate experiments as described previously [##REF##15464585##11##]. Morpholinos were designed to target the zebrafish ortholog to a G-protein coupled receptor the loss of function of which has previously been shown to result in a decrease in fat in <italic>C. elegans </italic>screen [##REF##17176039##12##]. The <italic>C. elegans </italic>GenePair corresponds to locus tag H09F14.1. The zebrafish ortholog corresponds to mRNA [accession XM_001340648], which is GPR142a. A morpholino with sequence CGGGCGTCGTGCCATTGTGCCAGTC was used to target the GPR142a mRNA. As a negative control, three unrelated morpholinos were injected targeting the ortholog to B3 (TATACGAAAATGAGCGACCGTGTTG) and heat shock protein. From 3 to 7 dpf zebrafish larvae were incubated with daily refreshing of nile red before relative quantification by fluorescence microscopy.</p>" ]

[ "<title>Results</title>", "<title>Nile Red Can Penetrate the Deep Tissues of 7 dpf Zebrafish Larvae to Produce a Specific Fluorescent Signal Restricted to Lipid Rich Tissues Without Exerting Toxic Effects</title>", "<p>Early development of the zebrafish proceeds quickly to insure greater chance for survival in nature. By just 3 dpf the protruding mouth has been formed. Thus oral delivery of small molecules can be taken into consideration by this stage of development. At 3 dpf zebrafish siblings generally have equivalent amounts of lipid content, thus providing a good baseline for quantifying subsequent changes in lipid metabolism. Volume measurements reveal that over 50% of the volume of the yolk sac is depleted for development from 4 to 7 dpf (Fig ##FIG##0##1A##. [##REF##12529643##13##]). For these reasons we believe this developmental time window is particularly ideal for providing a potentially sensitive read out of fat metabolic activity identify molecular activities regulating vertebrate animal fat metabolism.</p>", "<p>To establish the staining protocol, a 10 fold series of concentrations of nile red were tested starting from the established concentration used previously in <italic>C. elegans </italic>genetic screens [##REF##17176039##12##]. A concentration of 10 ng/mL final nile red concentration was ideal for obtaining a signal significantly greater than background yet not present throughout the entire embryo. The bulk of signal was restricted to the lipid rich yolk sac (Fig. ##FIG##0##1B##). No overt nile red associated toxicity or effects on viability were observed in zebrafish. Groups of 6 dpf zebrafish were incubated with nile red for times ranging from 30 min to overnight. We determined that at least 8 hours were needed to detect signal in the deeper regions throughout the embryo.</p>", "<title>Pharmacological Targeting of PPARγ, SIRT-1, beta-adrenergic receptors, and GPR109a is Generally Evolutionarily Conserved from 7 dpf Zebrafish Larvae to Man</title>", "<p>To be of greatest clinical relevance the zebrafish animal model should be responsive to known mammalian pharmacology regulating fat metabolism where both increases or decreases in fat content should be detectable under the assay conditions. To examine zebrafish pharmacology and assay sensitivity, we targeted PPARγ or the hormone responsive lipase (Fig. ##FIG##1##2##). Then total fat, total cholesterol and gene expression of markers of differentiation were quantified.</p>", "<p>Insulin is the most potent known physiological inhibitor of lipolysis, while nicotinic acid is a potent pharmacological inhibitor of lipolysis [##REF##15183629##14##]. Treatment of zebrafish larvae from 3 to 7 dpf with 100 μM nicotinic acid but not nicotinamide resulted in detectable increases in total fat, whereas treatment with 100 μM resveratrol caused significant decreases in detectable fat (Fig. ##FIG##2##3A##). This decrease in fat was even further decreased when resveratrol was supplemented with 20 μM norepinephrine consistent with previous results performed using murine adipocytes [##REF##15175761##15##]. Treatment with 1 mM nicotinic acid from 3 to 10 dpf also caused a significant 24% decrease in total cholesterol from 42 ng cholesterol per mg protein to 32 ng/mg (Fig. ##FIG##2##3B##). Nicotinamide did not significantly alter the total cholesterol (46 ng/mg). These results are consistent with the long known ability of nicotinic acid but not nicotinamide to decrease total cholesterol in the clinic.</p>", "<p>Even though nicotinic acid has been used in the clinic for over 50 years to treat high cholesterol, it was only recently that the nicotinic acid G-protein coupled receptor was identified [##REF##12563315##16##], thus elucidating a critical link in the mechanism of action for one of the most effective drugs known to prevent cardiac mortality [##REF##3782631##17##]. This receptor has also been identified as a gene up-regulated in response to hypoxia or interferon gamma treatment in macrophages [##REF##16386710##18##]. The closely related molecule nicotinamide, does not bind to the high affinity nicotinic acid receptor with any appreciable affinity, but like nicotinic acid it does provide NAD (Fig. ##FIG##1##2##). Thus nicotinamide is a negative control for nicotinic acid signal transduction through the high affinity nicotinic acid G-PCR GPR109a. Nicotinic acid treatment to activate GPR109a caused the total fat content of zebrafish larvae to increase, while nicotinamide did not exert any dramatic change in total fat content (Fig. ##FIG##2##3A##). Thus, nicotinic acid responsive anti-lipolytic activity is present in 6 dpf zebrafish similar to mammals.</p>", "<p>Since the high affinity nicotinic acid G-protein coupled receptor is of such great clinical significance we examined the transcriptional regulation of GPR109a/HM74a after nicotinic acid treatment and hypoxic stress by quantitative RT-PCR. Hypoxic stress applied to 6 dpf zebrafish larvae caused induction of GPR109a transcription (Fig. ##FIG##2##3B##) consistent with previous observations linking hypoxia mediated up-regulation GPR109a in monocytes [##REF##16386710##18##]. Treatment with nicotinic acid by itself caused an increase in GPR109a transcription that was synergistically enhanced by hypoxia treatment, thus suggesting a positive feedback loop (Fig. ##FIG##2##3C##).</p>", "<p>To determine whether zebrafish PPARγ is responsive to classical PPARγ activators, we exposed 6 dpf zebrafish to 20 nM 15d-PGJ₂overnight. The prostaglandin 15d-PGJ₂is the most potent known endogenous activator of PPARγ. Adipocyte fatty acid-binding protein (FABP4) and CD36 are classic PPARγ target genes in mammals [##REF##11089532##19##]. Treatment of zebrafish larvae with 15d-PGJ₂overnight caused a greater than 10 fold increase in the amount of FABP4 and CD36 transcripts in zebrafish similar to what has been observed in mammals (Fig. ##FIG##2##3D##). Nicotinic acid activation of GPR109a leads to localized endogenous 15d-PGJ₂biosynthesis at professional antigen presenting cells (Langerhans dendritic cells and macrophages, of which there are many in 6 dpf zebrafish larvae), which similarly activates PPARγ [##REF##16386710##18##] leading to increased transcription of FABP4 (also known as aP2) and CD36 target genes in mammals (Fig. ##FIG##1##2##). Treatment of zebrafish with nicotinic acid resulted in increased gene expression of FABP4 and CD36, while treatment with the related NAD precursor nicotinamide did not cause any appreciable change. Together these results are consistent with a high degree of evolutionary conservation in zebrafish related to mammals for PPARγ (15d-PGJ₂and nicotinic acid), SIRT-1 (resveratrol), beta-adrenergic receptor (norepinephrine, isoproterenol), or GPR109a (nicotinic acid but not nicotinamide) pharmacology as related to fat metabolism (Fig. ##FIG##1##2##).</p>", "<title>Small Molecules Regulating Fat Metabolism are Visually Detectable in Live Zebrafish larvae</title>", "<p>To determine whether we could visually detect both increases and decreases in this whole animal assay of fat metabolism we incubated zebrafish larvae in nile red containing embryo media and captured fluorescence microscopy images at 7 dpf. Exposure time used for capturing all images were normalized to that initially obtained by automatic exposure of untreated stained solvent only zebrafish larvae. For determining whether we could detect pharmacological decreases in fat we used activators of the beta-adrenergic receptor (isoproterenol or noradrenaline) or SIRT-1 (nicotinamide adenine dinucleotide, resveratrol, or potentially Vaticanol B). To determine whether we could visually detect increases in fat we used specific activators of PPARγ (the thiazolidinedione drug troglitazone or 15d-PGJ₂, Fig. ##FIG##1##2##). Resveratrol treatment resulted in highly reproducibly detectable decreases in fat. Conversely, treatment with just 20 nM of the highly stable PPARγ activator troglitazone resulted in visually detectable increases in fat (Fig. ##FIG##3##4## and ##FIG##4##5##). Treatment with the SIRT-1 inhibitor sirtinol was less consistent in results due to poor solubility combined with randomness of larval movement activity. Nonetheless, some fish did exhibit increased detectable fat.</p>", "<p>Several tetramers of resveratrol derived from acetone extracts of tree bark have garnered increased attention owing to their ability to inhibit the map kinase pathway and thus suppress cancer cell line growth. Vaticanol B in particular was distinctively more active than resveratrol in its ability to inhibit LPS-mediated production of nitric oxide, TNFα, and PGE₂[##REF##17475668##20##]. However, it is not known how this compound affects fat metabolism. Thus we examined fat metabolism using the zebrafish larvae nile red fluorescence assay. Treatment of zebrafish with Vaticanol B revealed a fat reducing activity that was approximately equivalent in potency to that of resveratrol itself (Fig. ##FIG##3##4A##). Targeting of the beta-adrenergic receptor with norepinephrine or the more chemically stable molecule isoproterenol also resulted in reproducibly detectable decreases in fat content (Fig. ##FIG##3##4B##). All examined pharmacological data support the notion that signal transduction regulating fat and cholesterol metabolism through beta-adrenergic receptors, SIRT-1, and the high affinity nicotinic acid G-protein coupled receptor GPR109a are highly evolutionarily conserved from fish to man. Significantly, whole zebrafish nile red fluorescence microscopy also enables examination of overt pharmacological toxicity.</p>", "<title>Anti-sense knockdown of zebrafish ortholog to G-protein coupled receptor previously identified in C. elegans nile red fat metabolism screen, reveals that GPR142 may be a good target of future obesity drug development</title>", "<p>Genetic screens have previously been used to identify 305 out of 16,757 screened genes the loss of function of which results in decreased fat content in <italic>C. elegans </italic>[##REF##17176039##12##]. From this screen we selected two G-protein coupled receptors to examine by anti-sense knockdown in zebrafish given their drug accessibility. We limited our choices to G-PCRs since they are the most commercially successful class of pharmaceutical drug targets. One G-PCR was chosen because the <italic>C. elegans </italic>loss of function results in increased fat content (<italic>C. elegans </italic>H09F14.1) while the other leads to decreases in fat (<italic>C. elegans </italic>F56B6.5). Knockdown of the zebrafish ortholog to <italic>C. elegans </italic>F56B6.5 resulted in developmental defects thus suggesting that complete antagonism of this GPCR may be toxic. Knockdown of H09F14.1 however, did cause detectable decreases in fat (Fig. ##FIG##5##6##). The zebrafish gene corresponds to GPR142a. Thus GPR142a is a candidate drug target for development of anti-obesity therapeutics.</p>" ]

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[ "<title>Conclusion</title>", "<p>The zebrafish yolk sac is a finite maternally derived energy source that predominately dwindles in size during the first week of development (Fig. ##FIG##0##1##). In this study we determine that the rate by which the zebrafish yolk sac diminishes is predictably affected by small molecules known to regulate mammalian fat metabolism via PPARγ adipogenic and hormone sensitive lipolytic pathways (Fig. ##FIG##1##2## and ##FIG##2##3##). Both direct (15d-PGJ₂) and indirect (nicotinic acid) PPARγ agonists caused increased FABP4 and CD36 gene expression in 7 dpf zebrafish larvae (Figure ##FIG##2##3D##). Conversely, beta-adrenergic agonist exposure (norepinephrine or isoproterenol) led to decreases in detectable fat in zebrafish larvae (Fig. ##FIG##2##3A## and ##FIG##4##5B##).</p>", "<p>Our examination of nicotinic acid pharmacology in zebrafish larvae also supports a high degree of evolutionary conservation for GPR109a signaling. Nicotinic acid functions as a potent inhibitor of adipocyte lipolysis by signaling through the high affinity nicotinic acid G-protein coupled receptor GPR109a. This causes Gi-coupled decreases in cyclic AMP Protein kinase A, which inhibits hormone sensitive lipase (Fig. ##FIG##1##2##[##REF##15183629##14##,##REF##12563315##16##]). Accordingly, zebrafish larvae exposed to nicotinic acid from 3 to 7 dpf have resultant increases in total fat (Fig. ##FIG##2##3A##). Zebrafish exposed to nicotinic acid from 3 to 10 dpf dramatically decreased (24% less, Fig. ##FIG##2##3B##) levels of total cholesterol roughly similar to that observed for patients treated for dyslipidemia with pharmacological niacin administration, which typically results in a decrease in total cholesterol of 4–16% [##REF##17705685##21##]. This result supports that this window of zebrafish development is particularly sensitive to effectors of cholesterol metabolism and that zebrafish may be useful for the development of potential drugs to treat dyslipidemia that work through GPR109a.</p>", "<p>While changes in fat content are visually quantifiable under these <italic>in situ </italic>whole zebrafish larvae conditions, this assay is limited by drug delivery. Our experiments using the only OTC anti-obesity drug Orlistat (also known as Alli or Xenical) failed to detect changes in zebrafish larvae fat content due to water insolubility (unpublished observations). For testing molecules with poor solubility in zebrafish it is necessary to inject them. This makes the zebrafish assay not as high throughput, but it does work and an examination of the logP of octanol:water partitioning can generally predict whether injection will be necessary [##REF##12642353##22##].</p>", "<p>Of all the molecules tested here, we see the most consistently dramatic effects in reducing nile red detectable fat after treatment with 100 μM resveratrol (Fig. ##FIG##4##5##). Resveratrol has been shown to increase mitochondrial biogenesis and to shift the health of mice fed high calorie diets to that of mice fed standard diets, significantly increasing their lifespan and motor function [##REF##17086191##23##]. Resveratrol can also function as a direct inhibitor of fatty acid synthase [##REF##16611078##24##]. Thus resveratrol is being considered for the clinical treatment of obesity. Given that fat reduction is known to extend lifespan in mice [##REF##12543978##25##], these studies provided a potential mechanistic explanation for resveratrol-mediated increases in lifespan observed in every model organism tested to date.</p>", "<p>Newly discovered pathways are now being targeted for therapeutic clinical obesity drug development. Knockdown of zebrafish GPR142a causes a significant decrease in detectable fat without causing overt developmental defects (Fig. ##FIG##5##6##), thus supporting the notion that this may be a good target for the development of anti-obesity drugs (Fig. ##FIG##5##6##). Little is known regarding GPR142. The first published report of GPR142 based purely on genomic sequence mentioned its presence in zebrafish. GPR142 is expressed throughout the brain as well as spleen, liver, kidney, and testes [##REF##16378626##26##]. No examination of adipose tissue expression has been reported to date. GPR142 is an orphan G-PCR – neither endogenous nor xenobiotic ligands are known. Given the high level of GPR142 expression in so many tissues and its drug accessibility as receptor, this would appear to be an ideal anti-obesity drug target. More GPR142 research is needed to determine whether it is expressed in adipose tissue and what the effect of GPR142 antagonism may have on fat content in mammals along with possible toxicity.</p>", "<p>While it was commonly believed that hormone sensitive lipase is the rate-limiting enzyme in lipolysis, the recent discovery of triglyceride lipases revealed a more complex picture [##REF##15550674##27##]. Hormone sensitive lipase turns out to be the major lipase for catecholamine and natriuretic peptide-stimulated lipolysis, whereas adipose triglyceride lipase mediates basal triglyceride lipolysis [##REF##16644234##28##]. What more this basic mechanism of lipolysis involving triglyceride lipase followed by hormone sensitive lipase and lastly monoglyceride lipase is conserved from yeast to man but is poorly understood [##REF##16267052##29##].</p>", "<p>Our experiments reveal for the first time that the direct addition of NAD causes a decrease in total fat content (Fig. ##FIG##4##5A##). These results are consistent with observations of the CD38 knockout mouse, which leads to a five-fold increase in NAD levels that results in prevention of high fat diet induced obesity through increased metabolic activity [##REF##17585054##30##]. Along these lines, drugs targeting CD38 inhibition or NAD supplementation are only just recently being considered in obesity research. In one of the few examples examining the direct application NAD to an animal, intranasal administration of NAD was determined to confer great protection from transient focal ischemia in rats. This was quantified by infarct size measured in the brain hemisphere respectively connected with the given nostril where the negative control was the untreated opposite hemisphere [##REF##17127275##31##]. The whole zebrafish nile red fluorescence fat metabolism assay can help narrow the list of potential future drug targets for treating clinical obesity.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The alarming rise in the obesity epidemic and growing concern for the pathologic consequences of the metabolic syndrome warrant great need for development of obesity-related pharmacotherapeutics. The search for such therapeutics is severely limited by the slow throughput of animal models of obesity. Amenable to placement into a 96 well plate, zebrafish larvae have emerged as one of the highest throughput vertebrate model organisms for performing small molecule screens. A method for visually identifying non-toxic molecular effectors of fat metabolism using a live transparent vertebrate was developed. Given that increased levels of nicotinamide adenine dinucleotide (NAD) via deletion of CD38 have been shown to prevent high fat diet induced obesity in mice in a SIRT-1 dependent fashion we explored the possibility of directly applying NAD to zebrafish.</p>", "<title>Methods</title>", "<p>Zebrafish larvae were incubated with daily refreshing of nile red containing media starting from a developmental stage of equivalent fat content among siblings (3 days post-fertilization, dpf) and continuing with daily refreshing until 7 dpf.</p>", "<title>Results</title>", "<p>PPAR activators, beta-adrenergic agonists, SIRT-1 activators, and nicotinic acid treatment all caused predicted changes in fat, cholesterol, and gene expression consistent with a high degree of evolutionary conservation of fat metabolism signal transduction extending from man to zebrafish larvae. All changes in fat content were visually quantifiable in a relative fashion using live zebrafish larvae nile red fluorescence microscopy. Resveratrol treatment caused the greatest and most consistent loss of fat content. The resveratrol tetramer Vaticanol B caused loss of fat equivalent in potency to resveratrol alone. Significantly, the direct administration of NAD decreased fat content in zebrafish. Results from knockdown of a zebrafish G-PCR ortholog previously determined to decrease fat content in <italic>C. elegans </italic>support that future GPR142 antagonists may be effective non-toxic anti-obesity therapeutics.</p>", "<title>Conclusion</title>", "<p>Owing to the apparently high level of evolutionary conservation of signal transduction pathways regulating lipid metabolism, the zebrafish can be useful for identifying non-toxic small molecules or pharmacological target gene products for developing molecular therapeutics for treating clinical obesity. Our results support the promising potential in applying NAD or resveratrol where the underlying target protein likely involves Sirtuin family member proteins. Furthermore data supports future studies focused on determining whether there is a high concentration window for resveratrol that is effective and non-toxic in high fat obesity murine models.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>WTP developed the original conceptual framework for the study, performed most of the experiments, and prepared the manuscript. KSJ performed initial experiments developing the zebrafish-based assay. LAW measured cholesterol. RJJ measured total phospholipids and helped prepare the manuscript. APA and HLR performed quantitative real-time PCR. All authors read and approved the final version.</p>" ]

[ "<title>Acknowledgements</title>", "<p>This work was supported by a grant from the Biomedical Research and Technology Transfer 02-002 awarded to WTP. We are most grateful to the assistance of the following particular individuals. Without their help it would not have possible to execute these experiments. This list includes Drs. Christine Beattie, Dr. James D. Jontes, and Smith Malireddy at Ohio State University and Brenda Grizzle.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>A large portion of the yolk sac is absorbed during this sensitive developmental time window used to detect small molecule-mediated changes in fat metabolism (A).</bold> Extended incubation at 5 ng/mL nile red provides ideal signal to noise ratio for detection of anatomically localized fat stores (B). Overnight incubation was even better with the signal in the gall bladder often disappearing (shown in Fig. 4).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Pathways regulating PPARγ-mediated adipocyte differentiation are shown on the left, while G-PCR targeted pathways working via cAMP-mediated signal transduction to regulate lipolysis are shown on the right</bold>. The pharmacologically targeted G-PCRs involved in lipolysis are GPR109a/HM74a or beta-adrenergic receptors. Nicotinic acid similarly affects both processes, inhibiting lipolysis and promoting adipocyte differentiation through PPARγ activation. However, nicotinic acid is also a precursor to NAD, which exerts the opposite effect (left). Resveratrol can both directly inhibit fatty acid synthase and cause activation of SIRT-1 activity, both of which are known to decrease fat content in adipocytes. Vaticanol B is an only just recently characterized tropical tree bark-derived resveratrol tetramer determined to have much greater anti-inflammatory activity than resveratrol. Thus, we predict that Vaticanol B may be similarly involved in controlling fat metabolism.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Zebrafish respond to mammalian regulators of fat metabolism in an evolutionarily conserved manner</bold>. Similar to pharmacological application of niacin in the clinic, nicotinic acid inhibits zebrafish lipolysis (A), decreases total cholesterol (B), and increases expression of the most potent quantitative scavenger of oxidized lipids, CD36 and the classic marker of adipocyte differentiation, FABP4 (D). Zebrafish larvae were exposed to nicotinic acid (NA), nicotinamide (NAM), resveratrol (R), or resveratrol with norepinephrine (NE) from 3–7 dpf before extraction of total phospholipids for quantitation by gas chromatography (A) or quantitation of transcript levels (C and D). For cholesterol measurements zebrafish were treated with 1 mM concentrations of NA or NAM from 3–10 dpf (B).</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Pharmacology causing decreases or increases in fat metabolism is visually detectable in a quantitative fashion by using live zebrafish nile red fluorescence microscopy.</bold> Zebrafish were incubated with daily refreshing of fish water containing nile red with or without 100 μM resveratrol (R) or 10 nM troglitazone (TZD). Exposure time for all fluorescent images was set to that of untreated zebrafish (left). Bright field images are provided to show lack of overt toxicity.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Conservation of pharmacological pathways and consistency amongst groups of animals are shown</bold>. NAD or resveratrol is known to activate SIRT-1. Conversely, sirtinol is an inhibitor of SIRT-1. Vaticanol B (Vat B) is a resveratrol tetramer natural product predicted to activate SIRT-1 as well (A). Isoproterenol or norepinephrine activation of beta-adrenergic receptors causes a decrease in detectable fat. Conversely, activation of PPARγ promotes adipocyte diferentation as predicted for treated zebrafish larvae. This slows the normal reduction in fat stores seen during this window of development.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Antagonists of GPR142 are candidate future obesity drugs</bold>. Anti-sense knockdown of G-protein coupled receptor gene products the loss of function previously determined to cause decrease in fat content [##REF##17176039##12##]. Incubation with the stable beta-adrenergic agonist isoproterenol is shown as a positive control causing increased lipolysis for comparison.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1743-7075-5-23-1\"/>", "<graphic xlink:href=\"1743-7075-5-23-2\"/>", "<graphic xlink:href=\"1743-7075-5-23-3\"/>", "<graphic xlink:href=\"1743-7075-5-23-4\"/>", "<graphic xlink:href=\"1743-7075-5-23-5\"/>", "<graphic xlink:href=\"1743-7075-5-23-6\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Free extracellular DNA has been retrieved in many environments, both aquatic and terrestrial [##REF##17961479##1##]. Naked DNA is actively excreted by growing cells, depending on biotic and abiotic factors [##REF##12676729##2##, ####UREF##0##3##, ##REF##7968924##4####7968924##4##], or is passively released in the environment by decaying cells. In hypersaline systems the water availability is reduced with the consequence of inferring water stress to cells. The osmotic stress in particular affects cell turgor and membrane integrity, leading to death by osmotic lysis of cells not adapted to hypersaline conditions [##UREF##1##5##], and in turn to the release of the cellular content, including nucleic acids.</p>", "<p>Free dissolved DNA is used by microorganisms as source of N, P and C [##REF##18156329##6##,##REF##16195451##7##], and in the deep-sea environment has been hypothesised to constitute a key trophic resource, substantially contributing to P cycling [##REF##16195451##7##]. Other than a nutrient source, dissolved DNA may have a genetic function as a source of genes acquirable by natural transformation. In terrestrial and aquatic environments several bacterial strains have been discovered to be naturally competent, <italic>i.e. </italic>to have the capacity to acquire exogenous naked DNA [##REF##7968924##4##]. The acquisition of new genetic traits by horizontal gene transfer can constitute an evolutionary strategy for the selection of natural microbial communities [##REF##10830951##8##]. In harsh and stressful conditions, in particular, gene exchange and rearrangements are estimated to increase in order to promote genome plasticity, increasing DNA repairing rates and evolutionary adaptation mechanisms [##REF##3396864##9##,##REF##1906416##10##].</p>", "<p>In natural ecosystems the majority of extracellular DNA is converted in deoxyribose, inorganic orthophosphate, purines and pyrimidines by the enzymatic hydrolytic action of nucleases, present in most of the microbial habitats [##UREF##2##11##,##REF##16348813##12##]. Besides the biological degradation, DNA is a chemically unstable molecule that decays spontaneously mainly through hydrolysis and oxidation [##REF##8469282##13##]. Several physical and chemical factors can moreover compromise the integrity of naked DNA molecules once they are released by cells. Once free in the environment, DNA fragments are no longer preserved by cellular DNA repair mechanisms and, even if not severely degraded, they accumulate environmentally inflicted damages. Despite all these factors, extracellular DNA has been demonstrated to be preserved in soil, sediments, freshwater and seawater for different time periods [##REF##17961479##1##], and even geologically ancient DNA has been retrieved from fossil materials [##UREF##3##14##,##REF##12024211##15##]. Dell'Anno and Danovaro [##REF##16195451##7##] estimated that the deep-sea sediments constitute the largest reservoir of extracellular DNA in the Earth's oceans, with 0.50 ± 0,22 Gt of extracellular DNA contained in the first 10 cm of sediments, and its residence time, resulted by the balance of release and degradation, is 9.5 years. In particular, in the sediments underlying the deep anoxic hypersaline lake l'Atalante Danovaro et al. [##REF##15816935##16##] retrieved the highest concentration of extracellular DNA reported in a natural environment.</p>", "<p>In hypersaline environments salt has been shown to have a stabilising effect on nucleic acids, protecting biological macromolecules against heat degradation [##REF##12382120##17##,##REF##8202372##18##]. On the other side, the reduced water activity of a salty environment has consequences on DNA conformation. The reduction of the hydration of the DNA molecules decreases the stabilisation of the structure that in high water activity environments is conferred by weakly bound water molecules [##REF##3785407##19##].</p>", "<p>The aim of this work is to study the persistence of extracellular DNA and its potential for gene exchange in extreme environments characterised by hypersaline and anoxic conditions. The deep hypersaline anoxic lakes of the Eastern Mediterranean Sea are unique deep-sea habitats originated from the dissolution of buried salt deposits emerging at the topography due to the strong faulting activity of the area. They are characterised by a salinity above 30%, absence of light, elevated pressure, variable pH values and ionic compositions. The sharp density difference between brines and normal sea water acts as a barrier, avoiding oxygen exchange, therefore the brines become oxygen-free and rich in hydrogen sulphide. Despite these harsh conditions, the brines that fill the lakes contain highly adapted active microbial communities [##REF##15637281##20##]. The brines are separated from the upper seawater by a steep interface layer with salinity values ranging from seawater to the brine physio-chemical parameters. This layer is an enrichment phase for complex microbial networks rich in taxonomical and functional biodiversity that are stratified along the depth and salinity profile [##REF##16525471##21##].</p>" ]

[ "<title>Methods</title>", "<title>Brines and bacterial strains</title>", "<p>Brines used in this study were recovered in 2003 from the deep submarine lakes Urania, Bannock, Discovery, L'Atalante located in the Eastern Mediterranean Sea, during a cruise of the R/V Urania [##REF##15637281##20##]. Brines were sterilised by filtration on 0.22 μm pore size filters immediately after recovery, and then filtered a second time before the survival/preservation experiments.</p>", "<p>Strains used in the work were isolated from the seawater/brine interface of each basin [##REF##16525471##21##] and maintained in Plate Count Broth (PCB) (Difco, Milan, Italy) supplemented with 5% NaCl (PCB5%). The strains have been identified by sequencing of the PCR-amplified 16S rRNA gene and comparison of the sequence with public databases [##REF##16525471##21##]. The list of the strains and the isolation media are reported in table ##TAB##0##1##.</p>", "<title>Survival of bacterial cells in the brines</title>", "<p>Cells were harvested by centrifugation from 10 ml of overnight cultures incubated at 28°C in the medium PCB5%. Cells were washed with sterile NaCl solution (5%), and then resuspended in 1 ml of the same solution. One hundred μl of the bacterial suspension were inoculated in 10 ml of filter sterilised brine and incubated at 15°C, the <italic>in situ </italic>temperature of the hypersaline anoxic lakes. At defined time intervals, up to 140 days (figure ##FIG##0##1, A1, B1##), 100 μl of the incubated suspension was collected, immediately subjected to serial dilutions in NaCl solution (5%) and plate counted on agarised PCB5% in triplicate. At the same time intervals (figure ##FIG##0##1, A2, B2##) the optical density at 600 nm wavelength (OD₆₀₀) of an aliquot of the suspension was analysed in a spectrophotometer (Beckman, DU640).</p>", "<title>Preservation of plasmid DNA in the brines</title>", "<p>To simulate the release of DNA from the decaying cells, known amounts of purified plasmid pZR80(gfp) were incubated in the filtered sterile brines of the four basins or, as a control, in deep-sea water. The incubations have been carried out simulating the <italic>in situ </italic>conditions, at 15°C in the dark and in the absence of oxygen. At selected time intervals triplicate aliquots of the plasmid-containing solution were collected and the relative abundance of the different plasmid conformations was analysed by gel electrophoresis. Plasmid pZR80(gfp), used in this study, was constructed by the insertion of a gfp-cassette coding for a Green fluorescent protein in the plasmid pZR80-2 [##UREF##8##28##]. Briefly, the 1.1-kb gfp-cassette was amplified by standard PCR by using p<italic>PnptII::gfp </italic>plasmid as template [##REF##11916719##29##] and primers PnptII1F-SphI (5'-ATTATT<underline>GCATGC</underline>AACCGGAATTGCCAGCT-3') and TendR-SphI (5'-ATTATT<underline>GCATGC</underline>CCAATTCCTGGCAGTTTATG-3'), both containing a SphI restriction site (underlined) with 5' overhang. The SphI digested PCR product was ligated in the SphI linearized pZR80-2 plasmid and used to transform competent <italic>E. coli </italic>cells.</p>", "<p>Plasmid pZR80(gfp) was extracted from an overnight culture of the strain <italic>E. coli </italic>(pZR80(gfp)) with the QuiaPrep Mini Kit (Quiagen, Milan, Italy), quantified determining the optical density at 260 nm wavelength in a spectrophotometer (Beckman DU640). 212 μl of the plasmid preparation was inoculated to 10 ml of anoxic sterile filtered brines (final concentration 2 μg ml^-1), and incubated in the dark at 15°C. At defined time intervals (figure ##FIG##2##3##) up to 32 days, a 100 μl aliquot of the plasmid containing brines were collected. All the inoculation and collection operations were conducted in an anoxic glove box. The recovered plasmid was desalted by dialysis against sterile milliQ water on a floating 0.1 μm pore size filter (25 mm diameter, Millipore, Milan, Italy) and stored at -20°C. At the end of the time course experiment 10 μl of each desalted aliquots were visualized by agarose gel electrophoresis in 0.5× TBE buffer stained with ethidium bromide. The relative abundance of the different plasmid conformations were estimated from the brightness of the plasmid bands in the electrophoresis gel picture using the software QuantityOne (Bio-Rad, Milan, Italy).</p>", "<title>Transformability of DNA after incubation into the brines</title>", "<p>Desalted plasmid preparations recovered after incubation in the brines were used as donor DNA for the transformation of naturally competent cells of <italic>Acinetobacter baylii </italic>strain BD413, as described by Rizzi et al. [##REF##18165369##30##]. Briefly, bacterial cells of <italic>A. baylii </italic>BD413 were recovered by centrifugation of 50 ml of an overnight culture in LB medium (Amersham Biotech, Milano, Italy), washed in sterile saline solution (NaCl 9 g l^-1), resuspended in 10 ml of saline solution containing 15% (V/V) glycerol, divided in 100 μl aliquots and stored at -80°C. Cell concentration in each aliquot was 3.4 ± 0.8 × 10⁸cfu/ml. Sixteen μl of donor plasmid DNA were mixed with a thawed aliquot of naturally competent <italic>A. baylii </italic>cells, placed on a sterile mixed cellulose esters 0.22 μm pore size filter (47 mm diameter, Millipore, Milan, Italy), and positioned on the surface of an LB agar plate. After overnight incubation, the cells were recovered from the filter by resuspension in 5 ml of saline solution, serially diluted and plated on Luria Bertani (LB) (Amersham Biotech, Milan Italy) agar to count the total number of cells present on the filter, and LB agar supplemented with 100 μg/ml kanamycin to selectively count the transformant cells that acquired the plasmid. Transformation frequency was calculated as the number of Km^Rtransformants over the total number of cells. Results are an average of triplicate experiments. Randomly selected colonies were checked for the expression of the <italic>gfp </italic>gene contained in the pZR80(gfp) plasmid, coding for a Green fluorescent protein, by epifluorescence microscopy (Zeiss Axioplan).</p>", "<title>Live/dead staining</title>", "<p>Cells from 200 μl of overnight cultures in medium PCB5% were collected by centrifugation (10 minutes 10000 <italic>g</italic>) and resuspended in 50 μl of sterile NaCl solution (5%). Five hundred μl of brine was added to the suspension. After 10 minutes of incubation 10 μl of the suspension was diluted with 100 μl of sterile water, and added with 3 μl of SybrGreen and 3 μl of propidium iodide solutions following the manufacturers instructions (Live/Dead staining kit, Molecular Probes). After 20 minutes of incubation at room temperature in the dark, the suspension was filtered on black polycarbonate filters (Millipore, Milan, Italy) and observed in epifluorescence (Zeiss Axioplan).</p>" ]

[ "<title>Results and discussion</title>", "<title>Survival in the brines of bacteria isolated from the seawater-brine interface</title>", "<p>The interface between deep sea-water and the hypersaline anoxic brines is a thin layer of few meters over the brines that hosts high bacterial density and diversity [##REF##16525471##21##]. Bacteria sinking through the interface encounter conditions of increasing salinity and, as a consequence, increasing osmotic stress. Seven strains have been selected, based on the following criteria: i) isolation from the seawater-brine interface of the four hypersaline lakes, L'Atalante, Bannock, Discovery, Urania, that have brines with very different chemical composition [##REF##15637281##20##] and ii) belonging to different taxonomic groups, <italic>i.e. </italic>gamma-proteobacteria and high G+C sporeforming bacteria of the family <italic>Bacillaceae </italic>(table ##TAB##0##1##). The seven strains have been isolated from the less saline layers of the hypersaline basins, <italic>i.e. </italic>the seawater-brine interface, and do not exhibit halophilic features, being able to grow in absence of salt. They are moderately halotolerant, since all of them tolerate up to 10–12% of NaCl in the growth medium (table ##TAB##0##1##). Simulating a sink in the lower hypersaline layers of the lakes, the cells of the strains isolated from the different lakes were grown on medium with intermediate salinity (5%) and then incubated in the corresponding hypersaline brines. Figure ##FIG##0##1(A1, B1)## shows the dramatic decrease in cell viability over time of contact with the brines. All the strains are subjected to a strong stress immediately after the exposure to the brines, since the number of the viable cells decreased by 5–11 orders of magnitude in the first minute. The stress is osmotic and leads to cell lysis rather than just a loss of viability, as demonstrated by the parallel decrease in optical density of the suspension (figure ##FIG##0##1, A2, B2##). The majority of the cells that were not completely lysed, showed nevertheless damaged membranes after 10 minutes of incubation in the brines, as demonstrated by their staining with propidium iodide (figure ##FIG##1##2##).</p>", "<p>The rate of viability loss seems to depend on the type of brine rather than type of microorganism. In the Discovery brine both the strains tested, <italic>Bacillus firmus </italic>11D and <italic>Alteromonas macleodii </italic>5D, did not show any remnant living cell after only 2 and 24 hours of incubation respectively. When compared with the other 3 brines, the Discovery one is indeed characterised by the most extreme composition, with a concentration of MgCl₂that reaches 5 M [##REF##15637281##20##], and has been shown to be highly hostile toward life [##REF##17298378##22##]. In the other brines all the strains exhibited longer survival times, demonstrating viable cells up to 30–93 days, depending on the strains. <italic>Halobacillus trueperi </italic>11A was the most resistant of all the strains tested, showing the complete disappearance of viable cells after 144 days of incubation in L'Atalante brines. Further experiments of cross inoculations would confirm whether the various degrees of lethal effect of the brines depend only by their different composition, or could be related to the peculiar physiological features of the microbes colonising each lake.</p>", "<p>Based on these results it is possible to hypothesise that, as a consequence of the osmotic stress encountered during sinking through the depth profile of the basins, cells not adapted to hypersalinity decay releasing their cellular content, including nucleic acids, during osmotic lysis. The following part of the work aims to understand the fate of naked DNA once released from the decaying cells.</p>", "<title>Survival in the brines of plasmid DNA and transformation potential</title>", "<p>Figure ##FIG##2##3A## indicates the gel electrophoresis pictures showing the fate of the plasmid DNA during a 32 day-long incubation, whereas Figure ##FIG##2##3B## shows the relative quantification of the main plasmid bands as a mean of different experiments. In all the incubation experiments the DNA proved to be highly preserved, and the total plasmid quantity did not show remarkable degradation for the first 15 days of incubation (p &lt; 0.01 in L'Atalante, Urania, Discovery brines). This result confirms previous findings by DeFlaun and Paul [##REF##24196018##23##] who performed short term experiments of 36 hours incubation using sterile seawater. In non sterile conditions extracellular DNA is degraded in few hours both in seawater and in freshwater [##REF##24196018##23##,##UREF##4##24##], due to enzymatic DNA degradation [##REF##17961479##1##]. In this work the preservation of dissolved DNA has been studied in 0.22 μm pore size filtered systems, in order to selectively investigate the effects of the four anoxic brines with different ionic composition without the interference of cells that are retained on the filter. For the first 3 days of incubation brines or seawater did not show any apparent effect on DNA. After this period in the L'Atalante brine the supercoiled conformation of the plasmid (CCC form) decreased up to 70 ± 15%. The Discovery brine, which was the most aggressive toward living cells, induced a decrease up to 43 ± 25% of the CCC form between 18 and 32 days of incubation. The exposure to Urania and Bannock brines as well as the seawater did not severely affect the total quantity of DNA over the 32 days of the experiment, but mainly affected the conformation of the plasmid molecule. Except for incubation in L'Atalante brines, the CCC form decrease was minor, between 0 and 29 ± 16%, while the other forms increased in variable percentages when compared with the respective bands at the beginning of the experiment (figure ##FIG##2##3##). The incubation of pZR80(gfp) plasmid in seawater or Urania brine lead to the appearance after 8 days of a DNA band with higher electrophoretic mobility than the supercoiled form, that could be attributed to the linear plasmid. The results showed that seawater or hypersaline brines in anoxic conditions in the absence of cells and suspended material retained on 0.22 μm filters had a partial, in L'Atalante and Discovery brines, or negligible, in the other cases, degradative effect on the overall DNA molecules. This could be due to a nicking effect on the plasmid DNA molecules, leading to the opening of the supercoiled form toward more relaxed conformations. These high values of DNA preservation confirm previous findings in sediments underlying the brines of the L'Atalante lake, which reported high number of spores [##UREF##5##25##], exceptionally high concentrations of extracellular DNA [##REF##15816935##16##], and in general a high level of organic matter preservation [##UREF##6##26##].</p>", "<p>To investigate the biological effect of the degradation or changes in conformation induced by brines or seawater, we tested the efficiency of the rescued plasmids in transformation. Plasmid pZR80(gfp) recovered after incubation in seawater and brines was used to transform naturally competent cells of <italic>A. baylii </italic>BD413, and the efficiency of transformation was calculated. To exclude that differences in transformation efficiency were due to differences in quantity of the donor DNA rather than in its quality, we estimated the minimum quantity of donor DNA that did not affect the transformation efficiency. The results demonstrated that applying between 20 and 50 ng of donor DNA per transformation assay did not significantly alter the transformation frequency (p &lt; 0.03), giving an average of 1.9 ± 0.4 × 10^-3transformants/total cells (figure ##FIG##3##4##). Based on these data, the transformation efficiency of pZR80(gfp) incubated in seawater or brines was calculated using equal concentrations of 25 ng of donor DNA per assay. The results (figure ##FIG##4##5##) showed that the incubation in all the four brines or in seawater did not affect the biological activity of extracellular dissolved plasmid DNA. pZR80(gfp) maintained similar values of transformation frequency for 32 days, with an average value of 5.6 ± 3.1 10^-4transformants/total cells. Transformation frequency was on average lower than that calculated using pure plasmid extracts, probably due to incomplete desalting of plasmid preparations recovered from seawater and brines. Brines of the Urania basin that showed negligible degrading effects on dissolved DNA (figure ##FIG##2##3##), induced nevertheless a significant (p = 0.037) increase in transformation frequency after 22 days of incubation from 4.9 ± 0.3 × 10^-4to 1.2 ± 0.2 × 10^-3. This result could be due to a kind of effect at molecular level induced by the brines on the dissolved DNA, that was not visible by agarose gel electrophoresis and should be further explored. The dependence of the frequency of transformation upon the topological form of the plasmid has been described [##UREF##7##27##].</p>", "<p>The integrity of the genetic information acquired by horizontal gene transfer was confirmed by evaluating the expression of the <italic>gfp </italic>gene which codifies for a green fluorescent protein that conferred fluorescent phenotype to the transformants (<italic>data not shown</italic>).</p>" ]

[ "<title>Results and discussion</title>", "<title>Survival in the brines of bacteria isolated from the seawater-brine interface</title>", "<p>The interface between deep sea-water and the hypersaline anoxic brines is a thin layer of few meters over the brines that hosts high bacterial density and diversity [##REF##16525471##21##]. Bacteria sinking through the interface encounter conditions of increasing salinity and, as a consequence, increasing osmotic stress. Seven strains have been selected, based on the following criteria: i) isolation from the seawater-brine interface of the four hypersaline lakes, L'Atalante, Bannock, Discovery, Urania, that have brines with very different chemical composition [##REF##15637281##20##] and ii) belonging to different taxonomic groups, <italic>i.e. </italic>gamma-proteobacteria and high G+C sporeforming bacteria of the family <italic>Bacillaceae </italic>(table ##TAB##0##1##). The seven strains have been isolated from the less saline layers of the hypersaline basins, <italic>i.e. </italic>the seawater-brine interface, and do not exhibit halophilic features, being able to grow in absence of salt. They are moderately halotolerant, since all of them tolerate up to 10–12% of NaCl in the growth medium (table ##TAB##0##1##). Simulating a sink in the lower hypersaline layers of the lakes, the cells of the strains isolated from the different lakes were grown on medium with intermediate salinity (5%) and then incubated in the corresponding hypersaline brines. Figure ##FIG##0##1(A1, B1)## shows the dramatic decrease in cell viability over time of contact with the brines. All the strains are subjected to a strong stress immediately after the exposure to the brines, since the number of the viable cells decreased by 5–11 orders of magnitude in the first minute. The stress is osmotic and leads to cell lysis rather than just a loss of viability, as demonstrated by the parallel decrease in optical density of the suspension (figure ##FIG##0##1, A2, B2##). The majority of the cells that were not completely lysed, showed nevertheless damaged membranes after 10 minutes of incubation in the brines, as demonstrated by their staining with propidium iodide (figure ##FIG##1##2##).</p>", "<p>The rate of viability loss seems to depend on the type of brine rather than type of microorganism. In the Discovery brine both the strains tested, <italic>Bacillus firmus </italic>11D and <italic>Alteromonas macleodii </italic>5D, did not show any remnant living cell after only 2 and 24 hours of incubation respectively. When compared with the other 3 brines, the Discovery one is indeed characterised by the most extreme composition, with a concentration of MgCl₂that reaches 5 M [##REF##15637281##20##], and has been shown to be highly hostile toward life [##REF##17298378##22##]. In the other brines all the strains exhibited longer survival times, demonstrating viable cells up to 30–93 days, depending on the strains. <italic>Halobacillus trueperi </italic>11A was the most resistant of all the strains tested, showing the complete disappearance of viable cells after 144 days of incubation in L'Atalante brines. Further experiments of cross inoculations would confirm whether the various degrees of lethal effect of the brines depend only by their different composition, or could be related to the peculiar physiological features of the microbes colonising each lake.</p>", "<p>Based on these results it is possible to hypothesise that, as a consequence of the osmotic stress encountered during sinking through the depth profile of the basins, cells not adapted to hypersalinity decay releasing their cellular content, including nucleic acids, during osmotic lysis. The following part of the work aims to understand the fate of naked DNA once released from the decaying cells.</p>", "<title>Survival in the brines of plasmid DNA and transformation potential</title>", "<p>Figure ##FIG##2##3A## indicates the gel electrophoresis pictures showing the fate of the plasmid DNA during a 32 day-long incubation, whereas Figure ##FIG##2##3B## shows the relative quantification of the main plasmid bands as a mean of different experiments. In all the incubation experiments the DNA proved to be highly preserved, and the total plasmid quantity did not show remarkable degradation for the first 15 days of incubation (p &lt; 0.01 in L'Atalante, Urania, Discovery brines). This result confirms previous findings by DeFlaun and Paul [##REF##24196018##23##] who performed short term experiments of 36 hours incubation using sterile seawater. In non sterile conditions extracellular DNA is degraded in few hours both in seawater and in freshwater [##REF##24196018##23##,##UREF##4##24##], due to enzymatic DNA degradation [##REF##17961479##1##]. In this work the preservation of dissolved DNA has been studied in 0.22 μm pore size filtered systems, in order to selectively investigate the effects of the four anoxic brines with different ionic composition without the interference of cells that are retained on the filter. For the first 3 days of incubation brines or seawater did not show any apparent effect on DNA. After this period in the L'Atalante brine the supercoiled conformation of the plasmid (CCC form) decreased up to 70 ± 15%. The Discovery brine, which was the most aggressive toward living cells, induced a decrease up to 43 ± 25% of the CCC form between 18 and 32 days of incubation. The exposure to Urania and Bannock brines as well as the seawater did not severely affect the total quantity of DNA over the 32 days of the experiment, but mainly affected the conformation of the plasmid molecule. Except for incubation in L'Atalante brines, the CCC form decrease was minor, between 0 and 29 ± 16%, while the other forms increased in variable percentages when compared with the respective bands at the beginning of the experiment (figure ##FIG##2##3##). The incubation of pZR80(gfp) plasmid in seawater or Urania brine lead to the appearance after 8 days of a DNA band with higher electrophoretic mobility than the supercoiled form, that could be attributed to the linear plasmid. The results showed that seawater or hypersaline brines in anoxic conditions in the absence of cells and suspended material retained on 0.22 μm filters had a partial, in L'Atalante and Discovery brines, or negligible, in the other cases, degradative effect on the overall DNA molecules. This could be due to a nicking effect on the plasmid DNA molecules, leading to the opening of the supercoiled form toward more relaxed conformations. These high values of DNA preservation confirm previous findings in sediments underlying the brines of the L'Atalante lake, which reported high number of spores [##UREF##5##25##], exceptionally high concentrations of extracellular DNA [##REF##15816935##16##], and in general a high level of organic matter preservation [##UREF##6##26##].</p>", "<p>To investigate the biological effect of the degradation or changes in conformation induced by brines or seawater, we tested the efficiency of the rescued plasmids in transformation. Plasmid pZR80(gfp) recovered after incubation in seawater and brines was used to transform naturally competent cells of <italic>A. baylii </italic>BD413, and the efficiency of transformation was calculated. To exclude that differences in transformation efficiency were due to differences in quantity of the donor DNA rather than in its quality, we estimated the minimum quantity of donor DNA that did not affect the transformation efficiency. The results demonstrated that applying between 20 and 50 ng of donor DNA per transformation assay did not significantly alter the transformation frequency (p &lt; 0.03), giving an average of 1.9 ± 0.4 × 10^-3transformants/total cells (figure ##FIG##3##4##). Based on these data, the transformation efficiency of pZR80(gfp) incubated in seawater or brines was calculated using equal concentrations of 25 ng of donor DNA per assay. The results (figure ##FIG##4##5##) showed that the incubation in all the four brines or in seawater did not affect the biological activity of extracellular dissolved plasmid DNA. pZR80(gfp) maintained similar values of transformation frequency for 32 days, with an average value of 5.6 ± 3.1 10^-4transformants/total cells. Transformation frequency was on average lower than that calculated using pure plasmid extracts, probably due to incomplete desalting of plasmid preparations recovered from seawater and brines. Brines of the Urania basin that showed negligible degrading effects on dissolved DNA (figure ##FIG##2##3##), induced nevertheless a significant (p = 0.037) increase in transformation frequency after 22 days of incubation from 4.9 ± 0.3 × 10^-4to 1.2 ± 0.2 × 10^-3. This result could be due to a kind of effect at molecular level induced by the brines on the dissolved DNA, that was not visible by agarose gel electrophoresis and should be further explored. The dependence of the frequency of transformation upon the topological form of the plasmid has been described [##UREF##7##27##].</p>", "<p>The integrity of the genetic information acquired by horizontal gene transfer was confirmed by evaluating the expression of the <italic>gfp </italic>gene which codifies for a green fluorescent protein that conferred fluorescent phenotype to the transformants (<italic>data not shown</italic>).</p>" ]

[ "<title>Conclusion</title>", "<p>At the interface between deep seawater and brine in the hypersaline anoxic lakes of the Eastern Mediterranean Sea bacterial communities are sharply stratified according to the increasing levels of salinity. The process of particulate sinking throughout the depth profile along the interface could expose non-adapted bacteria to higher salinity, with consequent osmotic stress that induces the lysis of the cells thereby releasing nucleic acids. With this work we demonstrated that this process is likely to occur in brines with different chemical composition, the released DNA is preserved biologically active in these extreme environments as in seawater, and the preserved DNA retains its transforming potential. Even though the role 0.22 μm filterable agents like DNAses, viruses or ultramicrobacteria should be further evaluated to estimate biological degradation of extracellular DNA, and the presence of naturally competent bacteria should be demonstrated, this work gives a first insight on the potential role of dissolved extracellular DNA in hypersaline environments to constitute a reservoir of genetic information that can be acquired by horizontal gene transfer mediated by natural transformation.</p>" ]

[ "<title>Background</title>", "<p>Extracellular dissolved DNA has been demonstrated to be present in many terrestrial and aquatic environments, actively secreted, or released by decaying cells. Free DNA has the genetic potential to be acquired by living competent cells by horizontal gene transfer mediated by natural transformation. The aim of this work is to study the persistence of extracellular DNA and its biological transforming activity in extreme environments like the deep hypersaline anoxic lakes of the Mediterranean Sea. The brine lakes are separated from the upper seawater by a steep chemocline inhabited by stratified prokaryotic networks, where cells sinking through the depth profile encounter increasing salinity values and osmotic stress.</p>", "<title>Results</title>", "<p>Seven strains belonging to different taxonomic groups isolated from the seawater-brine interface of four hypersaline lakes were grown at medium salinity and then incubated in the brines. The osmotic stress induced the death of all the inoculated cells in variable time periods, between 2 hours and 144 days, depending on the type of brine rather than the taxonomic group of the strains, <italic>i.e. Bacillaceae </italic>or gamma-proteobacteria. The Discovery lake confirmed to be the most aggressive environment toward living cells. In all the brines and in deep seawater dissolved plasmid DNA was substantially preserved for a period of 32 days in axenic conditions. L'Atalante and Bannock brines induced a decrease of the supercoiled form up to 70 and 40% respectively; in the other brines only minor changes in plasmid conformation were observed. Plasmid DNA after incubation in the brines maintained the capacity to transform naturally competent cells of <italic>Acinetobacter baylii </italic>strain BD413.</p>", "<title>Conclusion</title>", "<p>Free dissolved DNA is likely to be released by the lysis of cells induced by osmotic stress in the deep hypersaline anoxic lakes. Naked DNA was demonstrated to be preserved and biologically active in these extreme environments, and hence could constitute a genetic reservoir of traits acquirable by horizontal gene transfer.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>SB designed and coordinated the work and wrote the manuscript. EC carried out the incubation and transformation experiments. FM carried out extracellular DNA quantifications. IT participated in microscopy analyses. CC was responsible of sample collection and at-sea operations. DD participated in conceiving and designing the work and revised the manuscript. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>We thank the captain and the crew of the R/V Urania for the expert assistance at sea. This work has been possible in the framework of the MIDDLE project, thanks to the support from the European Science Foundation (ESF, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.esf.org\">http://www.esf.org</ext-link>) under the EUROCORES Programme, Ecosystem Functioning and Biodiversity in the Deep Sea (EuroDEEP, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.esf.org/eurodeep\">http://www.esf.org/eurodeep</ext-link>), through contract No. ERAS-CT-2003-980409 of the European Commission, DG Research, FP6. Partial support came from the European Community FP5 project BIODEEP, contract EVK3-2000-22057.</p>", "<p>We also thank anonymous reviewers for the helpful suggestions for manuscript improvement.</p>" ]

[ "<fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p><bold>Survival of bacterial cells in the brines.</bold> Figure 1A1, 1B1: time series plate count quantification of different strains incubated in the brine of isolation, <italic>i.e. </italic>L'Atalante, Bannock, Urania (A1) and Discovery (B1). Note that the x-axis scale in B1 is in minutes, while in A1 is in days. Figure 1A2, 1B2: time series measurement of the optical density (OD₆₀₀) of the strains incubated in the brines of L'Atalante, Bannock, Urania (A2) and Discovery (B2). Error bars are within the range of 0.3–71% of each value.</p></caption></fig>", "<fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p><bold>Epifluorescence microscopy visualization of the strains <italic>B. licheniformis </italic>12B (A) and <italic>H. meridiana </italic>18B (B) after 10 minutes of incubation in the Bannock brines, stained with Propidium iodide (red) and SybrGreen (green). </bold>A1, B1: visualisation at Propidium iodide excitation/emission wavelength (494/617 nm) of the cells with damaged cell wall. A2, B2: visualisation at SybrGreen I excitation/emission wavelength (494/519 nm) of the totality of the cells.</p></caption></fig>", "<fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p><bold>Stability of plasmid DNA in the brines and seawater.</bold> A: gel electrophoresis of pZR80(gfp) incubated for increasing periods of time in brines and seawater. B: relative quantification of the different plasmid conformations, OC (open circular), linear, CCC (covalently closed circular).</p></caption></fig>", "<fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Frequency of transformation of naturally competent cells of <italic>A. baylii </italic>BD413 with different quantities of plasmid pZR80(gfp).</p></caption></fig>", "<fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Frequency of transformation of naturally competent cells of <italic>A. baylii </italic>BD413 with plasmid pZR80(gfp) incubated for different time periods in brines and seawater.</p></caption></fig>" ]

[ "<table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Strains used in the work, taxonomic identification and salinity tolerance</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"center\">Strain name</th><th align=\"center\">lake of isolation¹⁾</th><th align=\"center\">Medium of isolation</th><th align=\"center\">closest relative</th><th align=\"center\">homology %²⁾</th><th align=\"center\" colspan=\"2\">NaCl tolerance (%)</th></tr></thead><tbody><tr><td/><td/><td/><td/><td/><td align=\"center\">min</td><td align=\"center\">max</td></tr><tr><td colspan=\"7\"><hr/></td></tr><tr><td align=\"center\">6A</td><td align=\"center\">L'Atalante</td><td align=\"center\">DSMZ-246³⁾</td><td align=\"center\"><italic>Alteromonas marina</italic></td><td align=\"center\">98</td><td align=\"center\">0</td><td align=\"center\">10</td></tr><tr><td align=\"center\">11A</td><td align=\"center\">L'Atalante</td><td align=\"center\">DSMZ-372</td><td align=\"center\"><italic>Halobacillus trueperi</italic></td><td align=\"center\">100</td><td align=\"center\">0</td><td align=\"center\">10</td></tr><tr><td align=\"center\">12B</td><td align=\"center\">Bannock</td><td align=\"center\">Marine Broth (Difco 2216)³⁾</td><td align=\"center\"><italic>Bacillus licheniformis</italic></td><td align=\"center\">98</td><td align=\"center\">0</td><td align=\"center\">12</td></tr><tr><td align=\"center\">18B</td><td align=\"center\">Bannock</td><td align=\"center\">DSMZ-246³⁾</td><td align=\"center\"><italic>Halomonas meridiana</italic></td><td align=\"center\">99</td><td align=\"center\">0</td><td align=\"center\">10</td></tr><tr><td align=\"center\">5D</td><td align=\"center\">Discovery</td><td align=\"center\">DSMZ-246³⁾</td><td align=\"center\"><italic>Alteromonas macleodii</italic></td><td align=\"center\">99</td><td align=\"center\">0</td><td align=\"center\">10</td></tr><tr><td align=\"center\">11D</td><td align=\"center\">Discovery</td><td align=\"center\">DSMZ-246</td><td align=\"center\"><italic>Bacillus firmus</italic></td><td align=\"center\">99</td><td align=\"center\">0</td><td align=\"center\">10</td></tr><tr><td align=\"center\">13U</td><td align=\"center\">Urania</td><td align=\"center\">Marine Broth (Difco 2216)</td><td align=\"center\"><italic>Halomonas meridiana</italic></td><td align=\"center\">99</td><td align=\"center\">0</td><td align=\"center\">10</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>¹⁾this is also the brine in which the strain has been incubated for the survival experiments</p><p>²⁾the strains have been identified based on the percentage homology on the 16S rRNA sequence with the sequences contained in public databases</p><p>³⁾medium diluted 1:10 with sterile seawater</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1746-1448-4-10-1\"/>", "<graphic xlink:href=\"1746-1448-4-10-2\"/>", "<graphic xlink:href=\"1746-1448-4-10-3\"/>", "<graphic xlink:href=\"1746-1448-4-10-4\"/>", "<graphic xlink:href=\"1746-1448-4-10-5\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Tyrosine phosphorylation plays an important role in several signaling pathways regulating cell growth, differentiation, cell cycle, apoptosis and neuronal functions [##REF##15817824##1##,##REF##14718383##2##]. The phosphorylation/dephosphorylation balance is controlled by protein tyrosine kinases and phosphatases. PTPs can be distinguished into four classes: 1) classical PTPs that can be subdivided into transmembrane, receptor-like enzymes, and the intracellular, nonreceptor PTPs, 2) dual-specificity PTPs (Ser and Tyr phosphatases), 3) low molecular weight PTP and 4) the Asp-based PTPs (Tyr/Ser phosphatase activity) [##REF##15186772##3##].</p>", "<p>Classical PTPs have been reported to play a key role in neural functions, from development to cognitive function. For example, RPTPs such as PTPδ, PTPσ, LAR, and especially PTPRO, are important players in axonal growth and guidance during development [##REF##15829633##4##]. Studies on PTPσ-KO (RPTP) mice have shown involvement of this PTP in the regulation of the developing hypothalamo-pituitary axis [##REF##10080192##5##,##REF##10080191##6##] and in the development of the CNS architecture [##REF##12237861##7##]. PTPBL-KO (non receptor like PTP-NRPTP) mice display impaired motor nerve repair in a model of sciatic nerve crush lesion [##REF##15226483##8##] and PTPMEG (NRPTP) interacts with key intracellular players leading to the stimulation of the channel activity of NMDA receptors [##REF##10748123##9##].</p>", "<p>In the present study we focused our attention on a NRPTP, PTPH1, and on its possible role on neural functions. Indeed PTPH1 has been shown to be expressed in the CNS [##REF##7644504##10##] but little is currently known on its potential impact on CNS functions. PTPH1 (also called PTPN3) belongs to a sub-family of non receptor cytosolic PTPs characterized by the presence of a FERM domain (band 4.1, ezrin, radixin, moesin) at its N-terminus, responsible for the interaction with transmembrane proteins and/or phospholipids in the cell membrane [##REF##7544351##11##, ####REF##9341175##12##, ##REF##7983158##13####7983158##13##]. In addition PTPH1 has a PDZ domain in the central part responsible for the interaction with other proteins, whereas the single catalytic domain is located at the C-terminus.</p>", "<p>PTPH1 activity has been involved in a variety of cellular functions including TCR-signaling [##REF##10940933##14##, ####REF##10820377##15##, ##REF##14672952##16####14672952##16##], cell cycle regulation [##REF##7544351##11##,##REF##14672952##16##,##REF##10364224##17##], endoplasmic reticulum assembly [##REF##11087817##18##], cardiac sodium channel modulation [##REF##16930557##19##] and TNFα converting enzyme inhibition [##REF##12207026##20##].</p>", "<p>Recently, our group has demonstrated that PTPH1 dephosphorylates GHR <italic>in vitro </italic>and in cellular assays [##REF##12907755##21##] and results in an increase of body weight in the functional PTPH1-knockout (KO) mice via modulation of IGF1 secretion [##REF##17921143##22##] thus demonstrating its <italic>in vivo </italic>relevance.</p>", "<p>PTPH1 has been shown in the rat to be highly expressed in thalamic nuclei as well as various cortical areas [##REF##7644504##10##]. However, no information is currently available on its impact on CNS functions. To address this question we have further characterized our PTPH1-KO mice line through behavioral and anatomical approaches. PTPH1 expression and localization was evaluated by LacZ staining in the brain and a behavioral test battery evaluated PTPH1 loss on CNS functions such as locomotor activity (open field), anxiety-like behavior (open field and elevated plus maze), motor ability, coordination and learning (accelerating rotarod), spatial working memory (Y maze) and nociceptive sensitivity (hot plate).</p>" ]

[ "<title>Methods</title>", "<title>Animals</title>", "<p>PTPH1-KO and wild type littermates (F2 generation, 87.5% C57Bl/6 – 12.5% 129S6SvEv) aged 3–4 months were used for behavioral phenotyping. Mice were individually caged and maintained in a 12:12 hours light: dark cycle (lights on at 7 am) at 21 ± 1°C with food and water available <italic>ad libitum</italic>. Protection of animals used in the experiment was in accordance with Directive 86/609/EEC, enforced by the Italian D.L. No. 116 of January 27, 1992. Physical facilities and equipment for accommodation and care of animals were in accordance with the provisions of EEC Council Directive 86/609. Tail snips from mice were collected and genotyped as previously reported [##REF##17921143##22##].</p>", "<title>PTPH1 KO design</title>", "<p>PTPH1-KO mice were generated using the Velocigene technology [##REF##12730667##23##], as described in details elsewhere [##REF##17921143##22##]. Briefly a mouse BAC containing the PTPH1 gene was modified: an in-frame LacZ reporter sequence and a neomycin-selectable marker replaced exons 15 to 22 encoding for the PDZ and the catalytic domain of PTPH1. BAC electroporation into embryonic stem cells was performed. F1 heterozygous mice were bred to generate F2 PTPH1-KO mice. Line breeding and animal care were performed in Charles River Italy and France.</p>", "<title>LacZ staining procedure and immunohistochemistry</title>", "<p>PTPH1-KO and WT mice, 12 months old, n = 2, male and females, were sacrificed by ip overdose of thiopental (5%), perfused with paraformaldehyde 4%, then washed in PBS and incubated overnight at 37°C in the solution containing the substrate for beta-galactosidase (beta-gal, encoded by the LacZ cassette) coupled to a NBT salt. The organs and the tissues in the sections display a green/blue staining where PTPH1 gene is normally expressed. After rinsing into PBS, organs were postfixed in PFA 4% for 1 hour, then incubated in 50% glycerol overnight at 4°C and finally maintained in 70% glycerol at room temperature. LacZ staining was observed through a low magnification microscope and described by an operator blind to the genotypes.</p>", "<p>LacZ staining was also performed on CNS sections. Mice (n = 3, 12 months old) were sacrificed by ip injection of an overdose of thiopental (5%), perfused with PBS and PFA 4%. Brains were removed and postfixed overnight at 4°C in PFA 4%, then placed overnight at 4°C in 15% and finally in 30% sucrose buffer. The brains were then included in O.C.T. (Tissue-Tek) and sections were cut on slides with a cryostat at 20 μm thickness. The slides were incubated in LacZ staining solution (see above) overnight at 37°C, washed thrice in PBS (5 min each) and either counterstained with H&amp;E (Merck KGaA) [##REF##12730667##23##] or co-expressed with NeuN immunostaining. Briefly, sections were incubated for 3 hours at room temperature in blocking solution (Vectastain Kit), washed in TBS (Tris-buffered saline), incubated overnight at 4°C with a solution containing the primary antibody mouse anti-mouse Neuronal Nuclei (Chemicon MAB377, 1/1000). The staining was revealed by ABC kit secondary antibody (mouse Vectastain Kit), and DAB (Sigma). After dehydration, sections were transferred onto coverslips. LacZ staining and co-expression with NeuN-immunoreactivity (NeuN-ir) was observed by microscopy and described by an operator blind to the genotypes.</p>", "<title>Semiquantitative RT-PCR for beta galactosidase gene</title>", "<p>Semiquantitative RT-PCR for PTPH1 and beta-gal gene expression was performed in different brain areas of PTPH1-KO and WT mice in order to confirm the presence of beta-gal expression in the KO tissues, replacing PTPH1 PDZ and catalytic domain. Brains from KO and WT mice (n = 5, 6 months old) were freshly removed and rinsed in HBSS. Hippocampus, cerebellum, cortex, striatum, midbrain and olfactory bulbs were dissected. Total RNA was extracted using Trizol Reagent (Invitrogen) and cleaned-up by RNAeasy columns from Qiagen. 5 μg of total RNA were used to perform the RT-PCR reaction (SuperScript II RT kit, Invitrogen). The primer sequences for LacZ amplification were the following: LacZ – forward 5'-GAT GTA CGT GCC CTG GAA CT/reverse 5'-GGT CCC ACA CTT CAG CAT TT. In order to load equally the reaction mixes, a 300 bp fragment of Histone 2A was amplified as a house keeping gene with the following primers: H2Az forward – 5' CGT ATT CAT CGA CAC CTG AAA; H2Az reverse – 5' CTG TTG TCC TTT CTT CCC GAT.</p>", "<title>Behavioral phenotyping test battery</title>", "<p>Neurological functions of PTPH1-WT and KO mice (males and females, 11 weeks-old, n = 10 per gender per genotype) were assessed through a behavioral test battery.</p>", "<p>The sequence of the test battery was chosen from the least invasive to the most ones. The schedule of the testing sessions included one week of recovery from one test to the next, as reported in Table ##TAB##0##1##.</p>", "<title>Open field</title>", "<p>After one hour of adaptation in the testing room, each mouse was placed in an open field chamber (50 cm²wide with white floor and walls) (ViewPoint Life Sci. Inc.) to test locomotor activity and anxiety-like behaviors. Locomotion was recorded for one hour by a video camera and analyzed automatically by VideoTRACK^®software (ViewPoint Life Sci. Inc.). Locomotor activity was evaluated by calculating the total path length traveled, whereas the relative time spent in the center was taken as indicative of anxiety-like behavior [##REF##9826725##24##]. The tests were performed in two sessions with equivalent group representation.</p>", "<title>Elevated plus maze</title>", "<p>After one hour of adaptation in the testing room, anxiety-like behavior was tested for each mouse by EPM within one session. The apparatus consists of four arms (29.5 cm long and 5 cm wide each). Two arms are open whereas the 2 others are limited by 2 black walls (20 cm high). The number of entries of each mouse in the open and closed arms was recorded by a video camera during a period of 5 minutes and analyzed by the SMART Video-Tracking Software (ViewPoint Life Sci. Inc.). The total number of entries into the arms is an index of locomotion, whereas the percentage of time spent and percentage of entries in the closed arms is an index of anxiety-like behaviors [##REF##3110839##25##].</p>", "<title>Accelerated rotarod</title>", "<p>Motor ability, coordination and learning were evaluated by using an Accelerated Rotarod apparatus for mice (Cat. # 7650 by Jones and Roberts, distr. by Basile Instr., Italy). The apparatus was placed within the animal colony room and was cleaned after each trial. Mice were tested for their abilities to maintain a balance on a rotating bar, which accelerated from 4 to 40 rpm/min in a 5 min trial. Latency to fall off was measured within one session and all mice underwent four trials (one every 30 min) [##REF##4384609##26##, ####REF##11752130##27##, ##REF##11875043##28####11875043##28##]. The differences at the rotarod performances in WT and KO were assessed by a single set of trials [##REF##11752130##27##,##REF##11875043##28##]. This set-up allows a major focus on the early phases of motor learning, involving a strong activation of prefrontal cortex and of the associative areas of basal ganglia and cerebellum [##REF##12015240##29##,##REF##12742261##30##].</p>", "<title>Y-maze alternation</title>", "<p>After one hour of adaptation in the testing room, mice were tested on a Y maze apparatus (40 cm long/8 cm wide arms with transparent walls) to investigate spatial working memory [##REF##10547924##31##]. The number and the sequence of the arm entries for each mouse were recorded during 5 minutes. The locomotion index was calculated as the overall number of arm entries, whereas the working memory index was calculated as following: number of exact alternations (entries into three different arms consecutively)/possible alternations (i.e. the number of arms entered minus 2) × 100.</p>", "<title>Hot plate</title>", "<p>Thermal sensitivity was assessed by a hot plate apparatus for mice (Cat. # 7280 by Biol. Research Apparatus, distr. by Basile Instr., Italy) and lasted a maximum of 45 seconds, time at which damages could occur [##REF##9150302##32##]. The apparatus was placed in the animal colony room and all the mice were tested within one session. Animals were placed on a surface heated at 52.5°C and the latency (seconds) to shake or lick the paw was recorded by the operator.</p>", "<title>Statistics</title>", "<p>Statistical comparisons were performed by unpaired two-tailed T-test (p &lt; 0.05) and two-way ANOVA (p &lt; 0.05) followed by post-hoc test as necessary. In the accelerated rotarod, two-way ANOVA with repeated measures followed by T-test was used. Results are expressed as mean ± SEM.</p>" ]

[ "<title>Results</title>", "<title>LacZ staining in whole mount</title>", "<p>In PTPH1-KO adult animals, LacZ staining in the brain was observed in the cerebellum, hippocampus and in the thalamic nuclei. In addition a strong staining was observed in the cerebral cortex, tenia tecta and septum (Figures ##FIG##0##1a, 1b##).</p>", "<title>LacZ staining in sections and RT-PCR results</title>", "<p>No gross cytoarchitectural brain differences were observed by simple visual observation at the microscope in the cortex, hippocampus and thalamus in PTPH1-KO mice compared to WT littermates.</p>", "<p>LacZ staining was performed on frozen brain sections to confirm and to describe the expression of PTPH1 at the brain structural level (Table ##TAB##1##2##).</p>", "<p>In cortical regions, LacZ was expressed in the external pyramidal (III) and internal granular layer (IVA) of the cerebral cortex (Figures ##FIG##1##2a, 2b##), in the retrosplenial cortex (Figures. ##FIG##2##3a, 3b 3c##, ##FIG##3##4a## and ##FIG##3##4b##) and indusium griseum (Figures ##FIG##2##3a, 3b##). In the cerebellum, in spite of a strong staining in the whole mount (Figure ##FIG##1##2##), only a faint LacZ signal was observed in sections (Figures ##FIG##4##5a, 5b##) in particular in the granule cells, close to the nuclei. The RT-PCR on cortical and cerebellar extracts confirmed the presence of LacZ expression in these brain areas (Figure ##FIG##5##6##).</p>", "<p>In subcortical regions, LacZ was detected in the anterior ventral, mediodorsal, ventrolateral, anteromedial and central lateral thalamic nuclear groups (Figure ##FIG##6##7a##). In more caudal thalamic areas, LacZ was again detectable in the posterior thalamic nuclear group (Po), and to a lesser extent in posteromedial, in posterolateral and in reticular thalamic nuclei and also in the dorsal lateral geniculate nuclei (Figures ##FIG##6##7b, 7c, 7d## and ##FIG##6##7e##). In the tenia tecta, LacZ staining visible in the whole mount preparation was confirmed (Figures ##FIG##1##2##, ##FIG##7##8a, 8b##). The RT-PCR on substriatal regions including the thalamus, the midbrain and the pontine areas confirmed the presence of LacZ expression in some of these brain areas (Figure ##FIG##5##6##). To exclude any potential impact of LacZ blood signal contamination in brain areas, RT-PCR on 5 to 20 μl of whole blood was carried out and did not reveal any significant signal [Additional files ##SUPPL##0##2##, ##SUPPL##1##3##].</p>", "<p>In the hippocampus, LacZ expression was observed in the cytoplasm of a few pyramidal cells and through the fibers of the oriens and radiatum layer in a rostral caudal spread (Figure ##FIG##8##9a##). In rostral sections, LacZ was expressed in the septohippocampal nuclei (Figure ##FIG##7##8a##). In more caudal sections LacZ was present in the CA1 and CA3, and in a lesser extent in the CA2 (Figures ##FIG##8##9b, 9c## and ##FIG##8##9d##). In the CA3 LacZ was strongly expressed in the oriens and pyramidal cell layer (Figure ##FIG##8##9d##), but its intensity was reduced in the radiatum and oriens compared to CA1 (Figure ##FIG##8##9b##). No staining was detected in the lacunosum-molecular layer in CA1, CA2 and CA3 (Figures ##FIG##8##9b, 9c## and ##FIG##8##9d##). The dentate gyrus showed a strong positive LacZ signal in the molecular layer, but not in the hilus (Figure ##FIG##8##9e##).</p>", "<title>Behavioral phenotyping</title>", "<p>As previously demonstrated, PTPH1-KO mice were healthy, reproduced normally and did not show any phenotypic traits distinguishing them from their WT littermates by simple visual observations [##REF##17921143##22##,##REF##17339465##33##]. An increased body weight has been detected in PTPH1-KO mice compared to WT littermates, more pronounced in male mice and probably due to an enhanced GHR sensitivity, that leads to increased IGF-1 mRNA and protein expression in liver and plasma, respectively [##REF##17921143##22##].</p>", "<p>In EPM, open field test and hot plate tests (anxiety-related behavior and thermal pain sensitivity), PTPH1-KO male and female mice did not show any significant differences in comparison with their WT littermates (data not shown).</p>", "<p>In the accelerated rotarod and Y-maze test, significant differences were observed between PTPH1-KOs and WTs based on gender and genotype factors. In the accelerating rotarod test PTPH1-WT mice did not show any gender differences (P_2WAY= 0.5824 (WT gender vs WT activity); P_AUC= 0.3218 (PTPH1-WT male vs female)) (Figure ##FIG##9##10a##). PTPH1-KO male mice displayed an overall significant better performance compared to their matched female littermates (P_2WAY= 0.007 (KO gender vs KO activity) (Figure ##FIG##9##10b##). Post-hoc T-test analyses showed that the difference was significant at the second trial of the test (P₀= 0.109; P₃₀= 0.015; P₆₀= 0.067; P₉₀= 0.835), and the area under the curve for PTPH1-KO male mice was significantly higher (by 50%) compared to the matched values of the female littermates (P = 0.0194) (Figure ##FIG##9##10c##).</p>", "<p>Considering this gender effect, the follow up analysis was carried out in males or females assessing genotype effects on activity. PTPH1-KO female mice performed significantly worse compared to their matched WT littermates, starting from the second trial and onwards (P₀= 0.171; P₃₀= 0.002; P₆₀= 0.028; P₉₀= 0.025) (Figure ##FIG##9##10d##). No significant differences were observed in PTPH1-KO male mice compared to their matched WT littermates (P₀= 0.92; P₃₀= 0.363; P₆₀= 0.222; P₉₀= 0.135) (Figure ##FIG##9##10e##).</p>", "<p>In the Y-maze test, no differences were detected between PTPH1-KO and WT female mice either in working memory (P_female= 0.972) or in locomotion indices (P_female= 0.73; Figures ##FIG##10##11a, 11b##). On the other hand, PTPH1 KO male mice displayed a significantly higher working memory index (percentage of exact alteration; P_male= 0.041) but similar locomotion activity (total arm entries) (P_male= 0.348) compared to their matched WT littermates (Figures ##FIG##10##11a, 11b##).</p>" ]

[ "<title>Discussion</title>", "<p>PTPs are key factors in multiple signaling pathways, leading to modulated functional activities in various cell types [##REF##15900534##34##,##REF##17057753##35##]. Among all PTP forms, PTPH1 has been shown <italic>in vitro </italic>to modulate cardiac sodium channel Na_v1.5 [##REF##16930557##19##], that it is also known to be expressed in the axons of cerebral cortex, cerebellum, thalamus and brain stem [##REF##12499865##36##]. Moreover, PTPH1 contains a domain with high sequence homology with the members of the band 4.1 superfamily protein, FERM. This domain mediates the linkage of actin filaments to the plasma membrane [##REF##8167019##37##], and therefore may be involved in cytoskeleton-membrane interactions, crucial for axon functionality. To further understand the potential role of PTPH1 in neural functions <italic>in vivo</italic>, we first investigated its expression pattern in embryonic and adult PTPH1-KO mice CNS by LacZ staining, and second its role in CNS functions by behavioral phenotype characterization.</p>", "<p>In rat embryonic stage Es19, PTPH1 expression through FISH analyses has already been shown in the dorsal thalamic nuclei, which give rise to the thalamo-cortical connections in adulthood [##REF##7644504##10##]. Thus, it has been suggested to play a role in the maintenance of these connections in adults. We replicated these data in PTPH1-KO mice at Es14 and Es16 embryonic stages. PTPH1 is expressed in the hypothalamic area and but also in the dorsal root ganglia of the spinal cord, excluding the spinal cord itself [Additional file ##SUPPL##2##1##] [##REF##9648846##38##]. Moreover, at postnatal P1, PTPH1 expression is also present in peripheral organs such as muscles and intestines as in the adults [##REF##17921143##22##]. On the other hand, the CNS expression at P1 appears weaker than in the adults suggesting a pattern of PTPH1 expression corresponding to specific developmental stages of the CNS as well as peripheral organs (data not shown). These changes in expression may play a role in various developmental functions that need to be further understood.</p>", "<p>In PTPH1-KO adults, LacZ is expressed in different CNS areas such as cerebral and retrosplenial cortices (Figures ##FIG##0##1##, ##FIG##1##2##, ##FIG##2##3## and ##FIG##3##4##), hippocampus (Figure ##FIG##8##9##), thalamus (preferentially ventral thalamus) (Figure ##FIG##6##7##), cerebellum (Figure ##FIG##4##5##) and in the region of the tenia tecta (Figures ##FIG##0##1##, ##FIG##7##8##). This data confirms previously observed expression patterns in the rat brain by Sahin et al. [##REF##7644504##10##] and extends the observation to other brain regions. We, furthermore, demonstrate that PTPH1 is expressed within the cytoplasm and close to the cell membrane of neurons in most of the brain area investigated (Figures ##FIG##3##4a, 4b##). It is known that the FERM domain is indeed necessary for PTPH1 localization close to the plasma membrane in Jurkat T cells [##REF##10940933##14##] and it could be responsible for the punctate expression pattern of PTPH1 in the cytosol of the neurons (Figure ##FIG##3##4b##) [##REF##12062040##39##]. This supports the concept that PTPH1 may be involved in cytoskeleton-membrane interaction within extended neuronal population in the CNS, potentially playing a role in various neuronal functions.</p>", "<p>Indeed the neural expression of PTPH1 in CA1, CA3 and DG of the hippocampus (Figures ##FIG##8##9a, 9b, 9c, 9d## and ##FIG##8##9e##), in the retrosplenial cortex (Figures ##FIG##2##3##, ##FIG##3##4##) and in a series of thalamic nuclei (Figures ##FIG##6##7a, 7b, 7c, 7d## and ##FIG##6##7e##) suggests an involvement of PTPH1 in the modulation of the memory circuit. Both hippocampus and retrosplenial cortex are key regions in the spatial working memory functions [##REF##11356886##40##, ####REF##16426767##41##, ##REF##15325783##42##, ##REF##17699661##43##, ##REF##10467569##44##, ##REF##16708393##45##, ##REF##16585789##46####16585789##46##]. Moreover, several thalamic nuclei have also been shown to be important in the memory process [##REF##16540567##47##,##REF##11484055##48##]. For example, a strong loss of dorsomedial and ventral posterior thalamic neurons is associated with severe cognitive and memory disabilities in patients affected by traumatic brain injury [##REF##15456707##49##]. Lesions in the lateral thalamus may lead to important working memory defects in rodents [##REF##15298780##50##]. The anterior thalamic nuclei project via the retrosplenial cortices to the hippocampus [##REF##11549742##51##,##REF##7593752##52##], thus underlying the importance of both these circuits and of PTPH1 in the mnemonic process.</p>", "<p>Another interesting PTPH1-positive area is the indusium griseum (Figures ##FIG##2##3a, 3b##) whose role in the adult brain is not clear. It is thought to be part of the limbic system, receiving afferents from the entorhinal and pyriform cortex and projecting to the septohippocampal nuclei, olfactory tubercle (presumably the tenia tecta) and the medial frontal cortex [##REF##7722001##53##,##REF##15168114##54##]. The expression of PTPH1 in these specific regions suggests a potential role in the processing/integration of memory and sensory information to the SHi and likely the cortex.</p>", "<p>Indeed PTPH1 expression is also detectable in the pyramidal neurons in layer III and IVA of the cerebral cortex of the mouse (Figures ##FIG##1##2a, 2b##), in agreement with Sahin's findings in the rat brain. The middle layers (III and IV) of the cerebral cortex are key sites for thalamic inputs [##REF##15772374##55##,##REF##11312303##56##] especially for VPM and VPL, primary thalamic nuclei for somato-sensory information integration [##REF##11576674##57##]. Furthermore a strong cortico-cortical communication has been assessed between these two layers [##REF##12466210##58##], thus suggesting a role for PTPH1 as key regulator in the transmission of the thalamo-cortical and cortico-cortical information.</p>", "<p>The cerebellar cortex is also positive for PTPH1 expression, in particular in the cytoplasm of granule cells (Figure ##FIG##4##5b##). The cerebellum is known to be the main structure for motor learning functions. In particular, the cerebellar cortex seems to be involved in the early learning phases of motor activities [##REF##6502205##59##,##REF##15217344##60##] that include also a strong activation of other areas such as prefrontal cortex and basal ganglia [##REF##12015240##29##,##REF##12742261##30##]. PTPH1 expression in the granule cells seems to indicate a potential involvement in the processing of afferent information to the purkinje cells, since it is known that afferents fibers to the cerebellar cortex will project in part through the granule cell layer.</p>", "<p>PTPH1 expression pattern observed in our analysis points out a potential involvement of this phosphatase in numerous CNS processing functions such as locomotion, sensorial integration, learning and memory. In this study, the behavioral phenotyping of the PTPH1-KO mice allowed us to test these hypotheses <italic>in vivo</italic>. Indeed, as already demonstrated by our group [##REF##17921143##22##] and also by others [##REF##17339465##33##], PTPH1-KO mice are healthy and do not display any phenotype, distinguishing them from their matched WT littermates, detectable by simple visual observation. Therefore PTPH1-WT and KO mice underwent a battery composed by five behavioral tests, from the least to the most invasive (Table ##TAB##0##1##), with the tolerable limitation of the handling bias.</p>", "<p>Behavioral testing revealing locomotor dysfunctions, such as open-field, EPM and Y-maze did not highlight differences between the two genotypes (Figure ##FIG##10##11b##), suggesting that PTPH1 does not play a critical role in the integration of locomotor information.</p>", "<p>Anxiety-like behaviors measured by open-field (as path in the center) and EPM (as time spent in the open arms), exploiting rodents natural aversion to open space, did not show any differences between the two genotypes (data not shown), leading to the conclusion that PTPH1 may not be involved in the integration of thalamo-limbic information, key paths for anxiety behavior processing. Similar conclusions can be drawn from the lack of difference between the genotypes regarding integration of nociceptive information, based on hot plate test.</p>", "<p>In the behavioral test, that partly depends on working memory performances (Y maze), PTPH1-KO male mice showed a slightly better short-term memory than their WT littermates (Figure ##FIG##10##11a##). Thus, PTPH1 may be involved in the integration of memory information. This was further strengthened by results obtained with a test assessing learning and coordination, the rotarod. Contrary to other behaviors where little differences have been observed, learning and coordination capacities in PTPH1-KO female mice are significantly impaired (Figures ##FIG##9##10b, 10c##). The low rotarod performance on the early trials, compensated by the last trial, is suggestive of a delay in learning acquisition (Figures ##FIG##9##10b, 10d##).</p>", "<p>As reported in Pilecka et al., our PTPH1-KO mice express the non-catalytic part of PTPH1 in frame with the enzymatically active part of LacZ gene. LacZ is widely used as a reporter for promoter activity in KO mice and all those mice express a modified protein, whose full function is not known. So far it was never reported a function of LacZ alone in cognition and we consider quite unlikely that this is the case in our mice. Thus, it is very likely that the behavioral phenotype we detect in our mice is linked to the deletion of the catalytic domain of PTPH1.</p>", "<p>The impairment in learning and coordination of PTPH1-KO female mice may be resulting from the involvement of PTPH1 in the GH signaling pathway [##REF##12907755##21##]. Indeed our group has already shown that PTPH1-KO mice display higher GHR response <italic>in vivo </italic>and consequently a higher expression of its down-stream effector hormone, the IGF1 in liver and plasma [##REF##17921143##22##]. GHR is highly expressed in most areas of the CNS, in particular in the choroid plexus, hippocampus, putamen, thalamus and hypothalamus. Similarly IGF1 and IGF1-receptors are localized predominantly in hippocampus, but also in amygdala, cerebellum and cortex [##REF##14585082##61##]. Although IGF1 is considered a neuroprotective hormone, it can be produced in the CNS, it is primarily synthesized in the liver and can cross the blood-brain barrier [##REF##16432628##62##, ####REF##7664680##63##, ##REF##10751445##64##, ##REF##12239123##65####12239123##65##]. The GH-IGF1 axis is also known to influence cognitive functions due to several neuroprotective effects on the hippocampus [##REF##15833590##66##]. Furthermore it has been recently pointed out that old conditional liver-IGF1-KO mice display impaired spatial learning and memory [##REF##16731792##67##]. The presence of PTPH1 in key CNS regions, as well as the consequent deregulation of the GH-IGF1 axis in KO mice, strengthens the concept that the PTPH1 network (CNS and downstream peripheral effectors) may be involved in cognitive functions.</p>", "<p>The behavioral tests assessing working memory and specifically learning revealed not only a genotype effect but also a gender effect, as mentioned above. Sex hormones are known to modulate the somatotropic system [##REF##12845224##68##,##REF##9329378##69##]. In humans, testosterone has an important effect on GH axis, in part by its aromatization to estradiol. Administration of estrogens, or aromatized androgen, modulates GH axis neuroregulation [##REF##9329378##69##,##REF##9861545##70##]. In particular, chronic E2 administration has been shown to reduce GH-induced IGF1 increased expression in liver and plasma via a negative feedback mechanism, while acute E2 administration leads to the expected GH-induced IGF1 release [##REF##3335211##71##]. Furthermore, it has been reported that estrogens play not only regulatory functions on neuroendocrine systems but can also have stimulatory or inhibitory impacts on the inter-connectivity of the hippocampal structure depending on the gender [##REF##16574776##72##, ####REF##11487652##73##, ##REF##14750972##74##, ##REF##11256441##75####11256441##75##], meaning that the same stimulus can have opposite effects in male <italic>vs </italic>female mice. Thus, the cognitive behavioral differences observed in our KO mice are underlying the potential impact of the PTPH1 network on neuroendocrine regulation as well as on cellular architecture within specific brain regions.</p>" ]

[ "<title>Conclusion</title>", "<p>In conclusion, we have demonstrated that PTPH1 is expressed in neural populations present in adult brain areas mainly involved in locomotor and cognitive functions. The behavioral assessments have allowed us to reveal PTPH1 functionality especially within cognitive domains. Better understanding the interplay between various phosphatases regulating CNS functions, which now includes PTPH1, will be key in the future to unravel some of the complexity of CNS signaling pathways necessary for information processing.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions.</p>", "<title>Methods</title>", "<p>We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions.</p>", "<title>Results</title>", "<p>In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females.</p>", "<title>Conclusion</title>", "<p>These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.</p>" ]

[ "<title>List of abbreviations</title>", "<p>PTPH1: protein tyrosine phosphatase H1; KO: knock-out; WT: wild type; CNS: central nervous system; PTKs: protein tyrosine kinases; PTPs: protein tyrosine phosphatases; RPTPs: receptor-like protein tyrosine phosphatases; NRTPTs: nonreceptor PTPs; FERM: 4.1, Ezrin, Radixin, Moesin; TACE: TNFα converting enzyme;GH: growth hormone; GHR: growth hormone receptor; IGF1: insulin-like growth factor 1; BAC: bacterial artificial chromosome; ip: intraperitoneal; PBS: phosphate buffered saline; PFA: paraformaldehyde; NBT: nitrobluetetrazolium; beta-gal/LacZ: beta-galactosidase; NeuN: Neuronal Nuclei; HBSS: Hank's balanced salts solution; H2A: Histone 2A; EPM: Elevated plus maze; AUC: area under the curve; Es: embryonic stage; E2: estradiol; CA: Cornu Ammonis; DG: Dentate Gyrus; MDL: mediodorsal lateral thalamic nuclei; AV: anteroventral thalamic nuclei; VM: ventromedial thalamic nuclei; VPM: ventral posteromedial thalamic nuclei; VL: ventrolateral thalamic nuclei; VPL: ventral posterolateral thalamic nuclei; LD: laterodorsal thalamic nuclei; Po: posterior thalamic nuclei; Rt: reticular thalamic nuclei; VPPC: ventral posteromedial parvicel thalamic nuclei; DLG: lateral geniculate nucleus; PF: parafascicular thalamic nuclei; nu: nuclei; SHi: septohippocampal muclei; Tt: tenia tecta; Ig: indusium griseum.</p>", "<title>Competing interests</title>", "<p>The present work is part of CP’s PhD program at the University of Eastern Piedmont, in close collaboration with MerckSerono International S.A.. MCM, PT, VM, BG, PFZ are employed by MerckSerono International S.A., which is involved in the discovery and the commercialization of therapeutics for the prevention and treatment of human diseases.</p>", "<title>Authors' contributions</title>", "<p>The study was devised by CP and MCM and carried out by CP. PT was responsible for the genotyping of all the adult animals that have been used in this study. MA performed the LacZ staining experiment on adult mice. VM and BG have been deeply involved in the first editing of the manuscript and all the authors contributed to modifications in subsequent drafts. PFZ has been involved in critically revising the manuscript and has given the final approval of the version to be published. All the authors read and approved the final version of the manuscript.</p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>This work was fully supported by MerckSerono International S.A.. We would like to thank Rob Hooft van Huijsduijnen, Andrea Graziani and Linda Chaabane for their scientific support and for kindly reviewing the manuscript. A special thank to Sonia Carboni and Gabriele Dati for their helpful comments and advices on immunohistochemistry and to Niels Adams for his support on LacZ staining data. </p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>PTPH1-KO adult mouse brain. <bold><italic>a</italic></bold>: whole brain, dorsal view, staining in cerebellum (Cb) and cortex (Co); <bold><italic>b</italic></bold>: LacZ staining on brain, sagittal view: detection in the tenia tecta (Tt), cortex (Co), thalamus (Th), hippocampus (H), retrosplenial cortex (Rc), septum (S) and in the granule cell layer of cerebellum.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold><italic>a</italic></bold>: PTPH1-KO cerebral cortex (10×, scale bar: 220 μm). <bold><italic>b</italic></bold>: positive cytoplasmatic and perinuclear LacZ staining (blue dots) in the external pyramidal (III) and internal granular layer (IVA) (63×, scale bar: 10 μm).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>PTPH1-KO cerebral cortex. <bold><italic>a</italic></bold>: LacZ detection in retrosplenial cortex (Rc) and indusium griseum (ig) staining (4×, scale bar: 80 μm). <bold><italic>b</italic></bold>: detail of the Rc and ig (40×, scale bar: 20 μm); <bold><italic>c</italic></bold>: positive cytoplasmatic staining of the neurons of Rc (63× scale bar: 10 μm); the interneural LacZ signals are due to the presence of trans-sectioned axons and dendrites.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>PTPH1-KO cerebral cortex. <bold><italic>a</italic></bold>: colocalization of NeuN-ir and LacZ staining signal in the Rc (100×, scale bar: 4.5 μm).; <bold><italic>b</italic></bold>: detail of the cytoplasmatic signal of LacZ in neurons.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p>PTPH1-KO cerebellar cortex. <bold><italic>a</italic></bold>: faint LacZ staining in the granule cell layer (20×, scale bar: 16.5 μm); <bold><italic>b</italic></bold>: perinuclear staining in the granule cell layer of the cerebellum (63×; scale bar: 5 μm).</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p>RT-PCR for beta-galactosidase expression in brain extracts. Beta-gal mRNA is expressed in PTPH1-KO cerebellum, cortex, hippocampus and substriatal regions (midbrain, thalamic nuclei, pontine region); no beta-gal expression detected in WT brain extracts (first lane of each block); histone H2A gene was used as positive control (second lane).</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p>PTPH1-KO thalamus [##UREF##0##76##]. <bold><italic>a</italic></bold>: LacZ expression detected in several thalamic nuclei (4×; scale bar: 165 μm): mediodorsal (MD), central lateral (CL), anteroventral (AV), anteromedial (AM), ventromedial (VM) ventral posteromedial (VPM) and ventrolateral (VL) thalamic nuclear groups. MHb: medial habendular nuclei. <bold><italic>b</italic></bold>: LacZ expression detected in the ventral posteromedial thalamic nuclei (VPM) and it is present also in ventrolateral (VL), ventromedial (VM), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt) thalamic nuclei (2.5×, scale bar: 130 μm). <bold><italic>c</italic></bold>: LacZ is expressed in the dorsal lateral geniculate nucleus (DLG) and in the lateroposteral thalamic nuclear group. In this caudal section LacZ staining is more intense in the posterior nucleus, but present also in VPM, VPL and VPPC (ventral posteromedial parvicel) thalamic nuclei (2.5×, scale bar: 13 μm). <bold><italic>d</italic></bold>: Detail of beta-gal expression in neural cell body of VPL and VPM at 40× (scale bar: 20 μm) and <bold><italic>e</italic></bold>: at 63× (scale bar: 10 μm).</p></caption></fig>", "<fig position=\"float\" id=\"F8\"><label>Figure 8</label><caption><p>PTPH1-KO adult mouse brain. <bold><italic>a</italic></bold>: beta-gal expression detected in the dorsal tenia tecta (Dtt) and in the septohippocampal nuclei (SHi) (4×; scale bar: 165 μm). <bold><italic>b</italic></bold>: Detail of cytoplasmatic LacZ staining in the Dtt and SHi (10×; scale bar: 70 μm).</p></caption></fig>", "<fig position=\"float\" id=\"F9\"><label>Figure 9</label><caption><p>PTPH1-KO hippocampus [##UREF##0##76##]. <bold><italic>a</italic></bold>: Hippocampus at 4×; <bold><italic>b</italic></bold>: CA1 area of hippocampus shows very intense LacZ staining in both oriens and radiatum layers and to a less extent in the pyramidal cell layer (20×; scale bar: 90 μm). <bold><italic>c</italic></bold>: CA2 area of hippocampus displays LacZ-positive staining in the pyramidal cell layer. <bold><italic>d</italic></bold>: CA3 area shows an intense beta-gal expression in the oriens and pyramidal cell layer, and in a less extent in the radiatum (20×). <bold><italic>e</italic></bold>: The dentate gyrus (DG) displays a strong LacZ staining in the molecular layer and not in the hilus (20×) (scale bar: 20 μm). pyr: pyramidal cell layer; oriens: oriens layer; rad: radiatum layer; mol: molecular layer: gr: granule cell layer; lac/mol: lacunosum-molecular layer.</p></caption></fig>", "<fig position=\"float\" id=\"F10\"><label>Figure 10</label><caption><p>Rotarod test on PTPH1-WT and KO mice (n = 10) males and females. <bold><italic>a</italic></bold>: WT males and WT females do not display any significant different performance at the rod (P_2WAY= 0.5824) <bold><italic>b</italic></bold>: KO males and KO females display a significant different performance (P_2WAY= 0.007) (post-hoc T-test: P₀= 0.109; P₃₀= 0.015; P₆₀= 0.067; P₉₀= 0.835). <bold><italic>c</italic></bold>: 50% difference in the area under the curve represented in figure 10a (unpaired T-test, P=0.0194). <bold><italic>d</italic></bold>: Female KO mice display a worse performance at the rod compared to WT females (P₀= 0.171; P₃₀= 0.002; P₆₀= 0.028; P₉₀= 0.025) <bold><italic>e</italic></bold>: No significant difference in the performance on the rod between male KO and WT mice. <bold><italic>a, b, d, e</italic></bold>: All the data were analyzed by Two-way Anova followed by T-test; *: p &lt; 0.05; **: p &lt; 0.01.</p></caption></fig>", "<fig position=\"float\" id=\"F11\"><label>Figure 11</label><caption><p>Y-maze behavioral test on PTPH1-WT and KO mice (n = 10) males and females. <bold><italic>a</italic></bold>: Male KO mice display higher working memory index compared to WT male littermates (P_male= 0.041); no differences recorded in the female mice. <bold><italic>b</italic></bold>: No significant differences recorded in the locomotion index, represented by the total arm entries between PTPH1-WT and KO males and females. T-test, *: p &lt; 0.05.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Schedule of the behavioral test battery.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Age (wks)</bold></td><td align=\"center\">8</td><td align=\"center\">9</td><td align=\"center\">10</td><td align=\"center\">11</td><td align=\"center\">12</td><td align=\"center\">13</td><td align=\"center\">14</td><td align=\"center\">15</td></tr></thead><tbody><tr><td align=\"left\"><bold>10M+10F</bold></td><td align=\"center\">arrival</td><td align=\"center\">quarantine</td><td align=\"center\">adaptation</td><td align=\"center\"><bold>Open Field</bold></td><td align=\"center\"><bold>EPM</bold></td><td align=\"center\"><bold>Rotarod</bold></td><td align=\"center\"><bold>Y-maze</bold></td><td align=\"center\"><bold>Hot plate</bold></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Qualitative estimation of LacZ staining intensity in the different brain areas.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Brain Area</bold></td><td align=\"left\"><bold>Intensity of LacZ staining</bold></td><td align=\"left\"><bold>Brain Area</bold></td><td align=\"left\"><bold>Intensity of LacZ staining</bold></td></tr></thead><tbody><tr><td align=\"left\">Cerebral cortex</td><td align=\"left\">+</td><td align=\"left\">Dorsal Tenia Tecta</td><td align=\"left\">++</td></tr><tr><td align=\"left\">Retrosplenial cortex</td><td align=\"left\">++</td><td align=\"left\">Septohippocampal nu</td><td align=\"left\">+</td></tr><tr><td align=\"left\">CA1 oriens layer</td><td align=\"left\">+++</td><td align=\"left\">VPL</td><td align=\"left\">+</td></tr><tr><td align=\"left\">CA1 radiatum layer</td><td align=\"left\">+++</td><td align=\"left\">MDL</td><td align=\"left\">++</td></tr><tr><td align=\"left\">CA1 pyramidal cell layer</td><td align=\"left\">+</td><td align=\"left\">AV</td><td align=\"left\">++</td></tr><tr><td align=\"left\">CA2 oriens layer</td><td align=\"left\">-</td><td align=\"left\">VPM</td><td align=\"left\">++</td></tr><tr><td align=\"left\">CA2 radiatum layer</td><td align=\"left\">-</td><td align=\"left\">VL</td><td align=\"left\">+</td></tr><tr><td align=\"left\">CA2 pyramidal layer</td><td align=\"left\">+</td><td align=\"left\">VM</td><td align=\"left\">++</td></tr><tr><td align=\"left\">CA3 oriens layer</td><td align=\"left\">+</td><td align=\"left\">Po</td><td align=\"left\">++</td></tr><tr><td align=\"left\">CA3 radiatum layer</td><td align=\"left\">+/-</td><td align=\"left\">LD</td><td align=\"left\">+</td></tr><tr><td align=\"left\">CA3 pyramidal layer</td><td align=\"left\">+</td><td align=\"left\">Rt</td><td align=\"left\">+</td></tr><tr><td align=\"left\">DG granular cell layer</td><td align=\"left\">-</td><td align=\"left\">DLG</td><td align=\"left\">++</td></tr><tr><td align=\"left\">DG molecular layer</td><td align=\"left\">+++</td><td align=\"left\">VPPC</td><td align=\"left\">++</td></tr><tr><td align=\"left\">DG hilus</td><td align=\"left\">-</td><td align=\"left\">PF</td><td align=\"left\">-</td></tr><tr><td align=\"left\">Fascicola cinereum</td><td align=\"left\">++</td><td align=\"left\">cerebellum</td><td align=\"left\">+</td></tr><tr><td align=\"left\">Indisium griseum</td><td align=\"left\">+</td><td/><td/></tr></tbody></table></table-wrap>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p>Semiquantitative RT-PCR for beta galactosidase gene in blood samples. <bold><italic>a</italic></bold>: white and red blood cells count in PTPH1-WT and KO mice; no major differences were found in the hematological composition in WT and KO mice. <bold><italic>b</italic></bold>: Beta-gal mRNA signal was present in PTPH1-KO hippocampus and cortex and not in the WTs; histone H2A gene was used as positive control. <bold><italic>c</italic></bold>: RT-PCR for beta-gal/H2A on 4 increasing amounts of whole blood (WB): 5, 10, 15 and 20 μl. No signal for beta-gal or H2A was detectable using 5 and 10 μl of WB, due to the low amount of total RNA; a faint signal for H2A was detectable on 15 and 20 μl of WB and a faint band for beta-gal was present only in KO mice, representing the maximum blood contamination in the whole mouse brain. Thus, blood contamination is minimum and it cannot interfere with the main source of signal.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional file 3</title><p>Additional methods. This document provides the methods and the references that have been used to perform the experiments represented in Additional file ##SUPPL##0##2##</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>LacZ staining on PTPH1-WT and KO embryos. PTPH1-WT embryos do not show any staining either at embryological stage 14 (Es14) or at Es16. PTPH1-KO embryos display a positive LacZ staining in the hypothalamic area and but also in the dorsal root ganglia of the spinal cord, excluding the spinal cord itself.</p></caption></supplementary-material>" ]

[ "<table-wrap-foot><p>-: absent; +/-: faint; +: present; ++: intense; +++: very intense staining. Cornu Ammonis (CA), Dentate Gyrus (DG), mediodorsal lateral (MDL), anteroventral (AV), ventromedial (VM) ventral posteromedial (VPM), ventrolateral (VL), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt), ventral posteromedial parvicel (VPPC) thalamic nuclei, lateral geniculate nucleus (DLG), parafascicular thalamic nuclei (PF), nuclei (nu).</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>The field of human genetics is currently experiencing an explosion of genetic data, as genotyping technology becomes more inexpensive and accessible. This creates an analytical challenge for genetic epidemiologists. This challenge is exaggerated in the case of complex diseases since the phenotype is likely the result of many genetic and environmental factors[##REF##14614241##1##,##REF##15522460##2##]. The limitations of traditional parametric statistical tools in searching for such interactions motivate the development of novel computational methods[##REF##15522460##2##, ####REF##17924838##3##, ##REF##12108579##4####12108579##4##].</p>", "<p>Recently, our group proposed a Grammatical Evolution Neural Network (GENN), a machine-learning approach designed to detect gene-gene interactions in the presence or absence of marginal main effects[##UREF##0##5##,##REF##18265411##6##]. GENN performs both variable selection and statistical modelling without the computational burden of exhaustively searching all possible variable combinations. GENN uses an evolutionary computation algorithm (grammatical evolution) to build neural networks (NN). NN analogize the parallel processing of the human brain, and are used as non-linear statistical data modeling tools to model complex relationships between inputs and outputs or to find patterns in data[##REF##3683570##7##].</p>", "<p>GENN has been shown to have high power to detect interactions in a range of empirical studies with both real and simulated data[##UREF##0##5##,##REF##18265411##6##,##UREF##1##8##,##UREF##2##9##]. The performance of GENN has been compared to other NN strategies and found to have significantly improved performance in large datasets[##REF##18265411##6##]. GENN has also been shown to efficiently scale to large datasets[##REF##18265411##6##].</p>", "<p>In the current study, we assess the robustness of GENN to several common types of noise. Data originally simulated for Ritchie et al 2003[##REF##12548676##10##] was used to examine the impact of genotyping error (GE), missing data (MS), phenocopy (PC), and genetic heterogeneity (GH) in six different gene-gene interaction models. Additionally, we examine the impact of all possible combinations of these types of noise to detect potentially synergistic effects. Also, we compare the performance of GENN to that of another method designed to detect interactions – Multifactor Dimensionality Reduction (MDR)[##REF##16296945##11##].</p>" ]

[]

[ "<title>Results</title>", "<p>The results of all analyses were first considered under a very strict definition of power. Initially, power was defined as the proportion of times across dataset replicates the method found all functional loci, with no false positives. For the majority of models (those without GH), a two-locus model was simulated, so the correct two loci, with no false positives must be chosen as the best final model under this definition of power. For those models with GH, two different two-locus models that both independently confer disease risk were simulated. Therefore, under this strict power definition, all four functional loci must be included in the final best model, with no false positives.</p>", "<p>The results of the MDR analyses under this definition of power, as found in[##REF##12548676##10##] are shown in Table ##TAB##0##1##. GENN results using the same definition of power are shown in Table ##TAB##1##2##. Similar trends are seen for both methods. MS and GE have a very minimal effect on the power. PC decreases the power of both methods, though MDR slightly outperforms GENN in the presence of this type of noise. GH decreases power the most for both methods, which is unsurprising with this strict power definition. Even without the analytical challenge presented by two competing models, it is more difficult to detect a four-locus model than a two locus model. Additionally, PC and GH in the current simulations may also result in greater decreases in power because they account for an overall greater percentage of error in the datasets as compared to MS and GE (50% compared to 5%).</p>", "<p>No synergistic effect is seen between PC and GE or MS for either method. However, different synergistic effects are seen with other combinations of error in comparing the results of the two methods. The MDR results show a major synergistic effect of PC and GH, as any combination of PC and GH, alone or with other types of error, causes the greatest decrease in power. This is unsurprising, because those datasets with only one single type of error that had the greatest drop in power were PC and GH as mentioned above. One would expect to find similar results when these two types of error are combined. The GENN results, however, do not demonstrate this effect. When PC and GH are present, the power is comparable to GH alone. However, there does seem to be a synergistic effect of GH and MS for GENN that is not seen with MDR under this strict definition of power. This effect is lessened for other definitions of power.</p>", "<p>It is important to note that for most combinations of error, the power of GENN is generally comparable to that of MDR under this original definition of power. The evolutionary computation approach of GENN does not require an exhaustive exploration of all possible combinations of variables, as MDR does. This is an important advantage in terms of computation time; however, this advantage must not come at the cost of power.</p>", "<p>Recognizing the extremely stringent nature of the original definition of power, especially for the models with GH, the results were re-examined. While it would be ideal for a method to identify all important signals within a dataset, it is important to know what signals a method is able to detect. To gain a more complete understanding of the performance of GENN on models with GH, several other definitions of power were considered. Again, these results are generally compared to those of MDR[##REF##17654613##15##].</p>", "<p>First, the power of each method to detect the primary genetic model (not including any loci from the second model or any false positive loci) was considered. The MDR and GENN results under this definition of power are shown in Tables ##TAB##2##3## and ##TAB##3##4## respectively. As these results demonstrate, the power increases greatly to detect one of the two underlying disease models than to find all four functionally loci. Also, the power of GENN is comparable, if not a little higher than that of MDR under this definition.</p>", "<p>Next, the power of each method to detect either underlying genetic model was examined. The MDR and GENN results are shown in Tables ##TAB##4##5## and ##TAB##5##6## respectively. Again, the power increases for both methods as the definition is expanded. It is important to note that the power of GENN is generally higher than that of MDR in these results. The power of GENN under this definition is actually comparable to its power to detect these genetic models in the complete absence of error, shown in Table ##TAB##1##2##.</p>", "<p>Finally, the power of each method to detect only correct loci was examined. Under this definition of power, any combination of the four total functional loci simulated as the best model is considered important. The results are shown in Tables ##TAB##6##7## and ##TAB##7##8##. Note this is a different definition than was used in[##REF##17654613##15##]. In [##REF##17654613##15##], the power to detect any correct loci, regardless of false-positive loci in the model was examined. Currently, only models with no false positive loci are considered. For both methods, the power is highest for models with genetic heterogeneity under this definition of power. This is expected since this final definition is the least strict, and it is encouraging that this increase is so large compared to the original very stringent definition. The comparison between the MDR and GENN results demonstrate a substantial difference between the power results of the two methods. GENN has substantially higher power to detect only correct loci than MDR.</p>" ]

[]

[ "<title>Conclusion</title>", "<p>The results of the current study demonstrate that GENN is relatively robust to common types of noise. GENN has excellent power in the presence of GE and/or MS, but is more impacted by PC and GH. Strikingly, when the performance is compared to that of MDR, GENN has higher power to detect only true positive loci. GENN's advantage over MDR in the presence of GH may be due to the search process used (an evolutionary strategy instead of an exhaustive search), and/or specific operators (i.e. Boolean operators[##REF##4460277##16##]) used in the grammar.</p>", "<p>These results are encouraging, but it will be important to assess the performance of GENN to detect even more complex models, particularly involving GH and PC. Theoretical and empirical studies should focus on improving the overall performance, as well as evaluating GENN's relative strengths and weaknesses compared to other computational methods in the field.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>With the advent of increasingly efficient means to obtain genetic information, a great insurgence of data has resulted, leading to the need for methods for analyzing this data beyond that of traditional parametric statistical approaches. Recently we introduced Grammatical Evolution Neural Network (GENN), a machine-learning approach to detect gene-gene or gene-environment interactions, also known as epistasis, in high dimensional genetic epidemiological data. GENN has been shown to be highly successful in a range of simulated data, but the impact of error common to real data is unknown. In the current study, we examine the power of GENN to detect interesting interactions in the presence of noise due to genotyping error, missing data, phenocopy, and genetic heterogeneity. Additionally, we compare the performance of GENN to that of another computational method – Multifactor Dimensionality Reduction (MDR).</p>", "<title>Findings</title>", "<p>GENN is extremely robust to missing data and genotyping error. Phenocopy in a dataset reduces the power of both GENN and MDR. GENN is reasonably robust to genetic heterogeneity and find that in some cases GENN has substantially higher power than MDR to detect functional loci in the presence of genetic heterogeneity.</p>", "<title>Conclusion</title>", "<p>GENN is a promising method to detect gene-gene interaction, even in the presence of common types of error found in real data.</p>" ]

[ "<title>Findings</title>", "<title>Grammatical Evolution Neural Networks (GENN)</title>", "<p>GENN methodology and software have been previously described[##UREF##0##5##,##REF##18265411##6##]. The steps of GENN are shown in Figure ##FIG##0##1##. Grammatical Evolution is a variation on genetic programming that uses a Backus-Naur Form grammar to create a computer program using a genetic algorithm[##UREF##3##12##]. A genetic algorithm is an array of bits that encodes definitions in the grammar (a set of rules that is used to construct computer programs – NN in this case). Then the program is executed and fitness is recorded. The genetic algorithm evolves chromosomes until an optimal solution is found, using balanced classification error as the fitness function (lower error represents higher fitness). GENN automatically selects the inputs from a pool of variables, optimizes synaptic weights, and evolves the architecture of the network, automatically selecting the appropriate network architecture for a dataset.</p>", "<p>In the case of missing data the algorithm does not include that observation in the calculation of classification error. Only the particular missing instance is ignored, not all data for an entire individual or entire locus. Configuration parameters used in the current analyses were: 10 demes, migration every 25 generations, population size of 200 per deme, maximum of 200 generations, crossover rate of 0.9, tournament selection, standard two-point crossover, selection and a reproduction rate of 0.1[##UREF##1##8##].</p>", "<title>Multifactor Dimensionality Reduction (MDR)</title>", "<p>The steps of MDR, and details of the MDR analyses presented here have been previously described[##REF##16296945##11##]. Briefly, in the first step, the data is divided into a training set and an independent testing set for cross validation. Second, a set of <italic>n </italic>genetic and/or environmental factors are selected. These factors and their multi-factor classes are divided in <italic>n</italic>-dimensional space. Then the ratio of cases to controls is calculated within each multifactor class. Each multifactor cell class is then labelled \"high risk\" or \"low risk\" based on the ratio calculated, reducing the <italic>n</italic>-dimensional space to one dimension with two levels. The collection of these multifactor classes composes the MDR model for a particular combination of factors. For each possible model size (one-locus, two-locus, etc.) a single MDR model is chosen that has the lowest number of misclassified individuals. In order to evaluate the predictive ability of the model, prediction error is calculated using 10-fold cross-validation. The result is a set of models, one for each model size considered. From these models, a final model is chosen based on minimization of prediction error and maximization of cross validation consistency (number of times a particular set of factors is identified across the cross validation subsets).</p>", "<title>Data Simulations</title>", "<p>Datasets were originally generated and described for Ritchie et al, 2003[##REF##12548676##10##]. Briefly, case-control data were generated under six different two-locus epistatic models, where the functional variables are single-nucleotide polymorphisms (SNPs). Data were generated using penetrance functions (shown in Figure ##FIG##1##2##), where a risk of disease is specified for each genotype combination, using software described in[##UREF##4##13##]. Each dataset is comprised of 200 cases and 200 controls, with a total of 10 biallelic SNPs each.</p>", "<p>Each model was simulated in the presence and absence of common sources of noise, including: 5% genotyping error (GE), 5% missing data (MS), 50% phenocopy (PC), and 50% genetic heterogeneity (GH). For each model, 100 datasets were generated in the absence of any type of noise, 100 datasets were generated for each type of noise, and 100 datasets were generated for each 2, 3, or 4-way combination of the sources of noise. 96 sets of 100 datasets were generated in total.</p>", "<p>As previously described[##REF##12548676##10##], GE was simulated using a directed-error model[##REF##12598690##14##] so that 5% of genotypes were biased towards one allele. MS were simulated by randomly removing 5% of genotype information. PC was simulated so that 50% of cases actually had genotypes that correspond to low-risk profiles according to the penetrance function of the corresponding epistasis model. This simulation corresponds to case status that is due to a random environmental effect. 50% GH was simulated by using a second penetrance function to define half of the cases, so that two different two-locus models predicted disease risk.</p>", "<title>Abbreviations</title>", "<p>GENN: Grammatical Evolution Neural Networks; MDR: Multifactor Dimensionality Reduction; GE: Genotyping error; PC: Phenocopy; MS: Missing data; GH: Genetic heterogeneity.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>AAM and MDR contributed to the design of the study. AAM, ACD, and TJF contributed to the data analysis. All four authors contributed to the manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The work for this project was supported by National Institutes of Health grants HL65962, GM62758, and AG20135.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>An overview of the GENN method</bold>. First, a set of parameters must be initialized in the configuration file. These parameters specify details for the evolutionary processes. Second, the data are divided into 10 equal parts for 10-fold cross-validation. Third, training begins by generating an initial population of random solutions using sensible initialization, which guarantees functional NNs in the initial population. Fourth, each newly generated NN is evaluated on the data in the training set and its fitness recorded. Fifth, a selection technique that is specified by the user is used to select the best solutions for crossover and reproduction in the evolutionary process. The cycle begins with the new generation, which is equal in size to the original population. This cycle continues until either a classification error of zero is found or a limit on the number of generations is reached. After each generation, an optimal solution is identified. At the end of GENN evolution, the overall best solution is selected as the optimal NN. Sixth, this best GENN model is tested on the 1/10 of the data left out to estimate the prediction error of the model. Steps two through six are performed ten times with the same parameters settings, each time using a different 9/10 of the data for training and 1/10 of the data for testing. At the end of a GENN analysis, 10 models are generated – one best model from each cross-validation interval. A final model is chosen based on maximization of the cross-validation consistency of variables/loci across the ten models.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Penetrance Functions used to simulate epistasis models.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Power of MDR (from Ritchie et al. 2003) to detect correct functional epistatic loci. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%)</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">None</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">99</td><td align=\"center\">99</td><td align=\"center\">82</td><td align=\"center\">84</td></tr><tr><td align=\"center\">GE</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">97</td><td align=\"center\">80</td><td align=\"center\">92</td></tr><tr><td align=\"center\">GH</td><td align=\"center\">3</td><td align=\"center\">41</td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">4</td></tr><tr><td align=\"center\">PC</td><td align=\"center\">90</td><td align=\"center\">99</td><td align=\"center\">45</td><td align=\"center\">32</td><td align=\"center\">30</td><td align=\"center\">32</td></tr><tr><td align=\"center\">MS</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">99</td><td align=\"center\">97</td><td align=\"center\">82</td><td align=\"center\">87</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">4</td><td align=\"center\">41</td><td align=\"center\">2</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">6</td></tr><tr><td align=\"center\">GE+PC</td><td align=\"center\">94</td><td align=\"center\">99</td><td align=\"center\">41</td><td align=\"center\">48</td><td align=\"center\">28</td><td align=\"center\">33</td></tr><tr><td align=\"center\">GE+MS</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">98</td><td align=\"center\">98</td><td align=\"center\">74</td><td align=\"center\">84</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">5</td><td align=\"center\">38</td><td align=\"center\">0</td><td align=\"center\">2</td><td align=\"center\">4</td><td align=\"center\">6</td></tr><tr><td align=\"center\">PC+MS</td><td align=\"center\">96</td><td align=\"center\">99</td><td align=\"center\">42</td><td align=\"center\">43</td><td align=\"center\">14</td><td align=\"center\">16</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">6</td><td align=\"center\">34</td><td align=\"center\">2</td><td align=\"center\">1</td><td align=\"center\">3</td><td align=\"center\">7</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td></tr><tr><td align=\"center\">GE+PC+MS</td><td align=\"center\">94</td><td align=\"center\">100</td><td align=\"center\">48</td><td align=\"center\">42</td><td align=\"center\">18</td><td align=\"center\">16</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">0</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Power of GENN to detect correct functional epistatic loci. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%)</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">None</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">96</td><td align=\"center\">91</td><td align=\"center\">69</td><td align=\"center\">72</td></tr><tr><td align=\"center\">GE</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">96</td><td align=\"center\">85</td><td align=\"center\">58</td><td align=\"center\">68</td></tr><tr><td align=\"center\">GH</td><td align=\"center\">7</td><td align=\"center\">4</td><td align=\"center\">15</td><td align=\"center\">16</td><td align=\"center\">14</td><td align=\"center\">16</td></tr><tr><td align=\"center\">PC</td><td align=\"center\">88</td><td align=\"center\">92</td><td align=\"center\">21</td><td align=\"center\">12</td><td align=\"center\">17</td><td align=\"center\">21</td></tr><tr><td align=\"center\">MS</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">99</td><td align=\"center\">82</td><td align=\"center\">42</td><td align=\"center\">74</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">3</td><td align=\"center\">6</td><td align=\"center\">14</td><td align=\"center\">16</td><td align=\"center\">11</td><td align=\"center\">9</td></tr><tr><td align=\"center\">GE+PC</td><td align=\"center\">92</td><td align=\"center\">95</td><td align=\"center\">19</td><td align=\"center\">11</td><td align=\"center\">12</td><td align=\"center\">16</td></tr><tr><td align=\"center\">GE+MS</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">93</td><td align=\"center\">75</td><td align=\"center\">48</td><td align=\"center\">58</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">9</td><td align=\"center\">9</td><td align=\"center\">13</td><td align=\"center\">15</td><td align=\"center\">10</td><td align=\"center\">11</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">1</td></tr><tr><td align=\"center\">PC+MS</td><td align=\"center\">65</td><td align=\"center\">85</td><td align=\"center\">18</td><td align=\"center\">13</td><td align=\"center\">7</td><td align=\"center\">9</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">3</td><td align=\"center\">9</td><td align=\"center\">2</td><td align=\"center\">2</td><td align=\"center\">7</td><td align=\"center\">3</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">5</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td></tr><tr><td align=\"center\">GE+PC+MS</td><td align=\"center\">62</td><td align=\"center\">81</td><td align=\"center\">14</td><td align=\"center\">9</td><td align=\"center\">9</td><td align=\"center\">9</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">0</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Power of MDR (from Ritchie et al. 2007) to detect primary genetic model in data with genetic heterogeneity. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%) to Detect Primary Model (5,10)</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">GH</td><td align=\"center\">30</td><td align=\"center\">18</td><td align=\"center\">19</td><td align=\"center\">25</td><td align=\"center\">8</td><td align=\"center\">8</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">39</td><td align=\"center\">18</td><td align=\"center\">19</td><td align=\"center\">25</td><td align=\"center\">8</td><td align=\"center\">8</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">11</td><td align=\"center\">18</td><td align=\"center\">5</td><td align=\"center\">3</td><td align=\"center\">4</td><td align=\"center\">2</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">28</td><td align=\"center\">23</td><td align=\"center\">19</td><td align=\"center\">19</td><td align=\"center\">9</td><td align=\"center\">13</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">10</td><td align=\"center\">18</td><td align=\"center\">8</td><td align=\"center\">4</td><td align=\"center\">3</td><td align=\"center\">3</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">29</td><td align=\"center\">22</td><td align=\"center\">21</td><td align=\"center\">20</td><td align=\"center\">7</td><td align=\"center\">4</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">12</td><td align=\"center\">22</td><td align=\"center\">5</td><td align=\"center\">5</td><td align=\"center\">2</td><td align=\"center\">4</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">16</td><td align=\"center\">17</td><td align=\"center\">4</td><td align=\"center\">3</td><td align=\"center\">1</td><td align=\"center\">3</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Power of GENN to detect primary genetic model in data with genetic heterogeneity. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%) to Detect Primary Model (5,10)</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">GH</td><td align=\"center\">34</td><td align=\"center\">49</td><td align=\"center\">20</td><td align=\"center\">13</td><td align=\"center\">8</td><td align=\"center\">11</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">48</td><td align=\"center\">49</td><td align=\"center\">20</td><td align=\"center\">13</td><td align=\"center\">8</td><td align=\"center\">10</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">10</td><td align=\"center\">11</td><td align=\"center\">3</td><td align=\"center\">0</td><td align=\"center\">4</td><td align=\"center\">3</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">31</td><td align=\"center\">35</td><td align=\"center\">12</td><td align=\"center\">11</td><td align=\"center\">9</td><td align=\"center\">9</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">11</td><td align=\"center\">8</td><td align=\"center\">3</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">2</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">43</td><td align=\"center\">29</td><td align=\"center\">13</td><td align=\"center\">7</td><td align=\"center\">8</td><td align=\"center\">5</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">3</td><td align=\"center\">8</td><td align=\"center\">3</td><td align=\"center\">1</td><td align=\"center\">5</td><td align=\"center\">3</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">4</td><td align=\"center\">8</td><td align=\"center\">1</td><td align=\"center\">3</td><td align=\"center\">0</td><td align=\"center\">2</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Power of MDR (from Ritchie et al. 2007) to detect either genetic model in data with genetic heterogeneity. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%) to Detect Either Model (5,10 or 3,4)</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">GH</td><td align=\"center\">70</td><td align=\"center\">34</td><td align=\"center\">42</td><td align=\"center\">41</td><td align=\"center\">20</td><td align=\"center\">19</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">69</td><td align=\"center\">34</td><td align=\"center\">42</td><td align=\"center\">41</td><td align=\"center\">20</td><td align=\"center\">19</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">24</td><td align=\"center\">35</td><td align=\"center\">9</td><td align=\"center\">8</td><td align=\"center\">7</td><td align=\"center\">5</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">65</td><td align=\"center\">40</td><td align=\"center\">42</td><td align=\"center\">31</td><td align=\"center\">18</td><td align=\"center\">22</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">27</td><td align=\"center\">35</td><td align=\"center\">10</td><td align=\"center\">8</td><td align=\"center\">7</td><td align=\"center\">6</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">64</td><td align=\"center\">44</td><td align=\"center\">42</td><td align=\"center\">41</td><td align=\"center\">16</td><td align=\"center\">11</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">23</td><td align=\"center\">38</td><td align=\"center\">9</td><td align=\"center\">10</td><td align=\"center\">4</td><td align=\"center\">6</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">31</td><td align=\"center\">36</td><td align=\"center\">9</td><td align=\"center\">7</td><td align=\"center\">4</td><td align=\"center\">3</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T6\"><label>Table 6</label><caption><p>Power of GENN to detect either genetic model in data with genetic heterogeneity. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%) to Detect Either Model (5,10 or 3,4)</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">GH</td><td align=\"center\">79</td><td align=\"center\">89</td><td align=\"center\">38</td><td align=\"center\">24</td><td align=\"center\">16</td><td align=\"center\">19</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">92</td><td align=\"center\">90</td><td align=\"center\">41</td><td align=\"center\">24</td><td align=\"center\">16</td><td align=\"center\">22</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">22</td><td align=\"center\">22</td><td align=\"center\">4</td><td align=\"center\">5</td><td align=\"center\">6</td><td align=\"center\">4</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">65</td><td align=\"center\">62</td><td align=\"center\">26</td><td align=\"center\">19</td><td align=\"center\">17</td><td align=\"center\">15</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">23</td><td align=\"center\">23</td><td align=\"center\">4</td><td align=\"center\">3</td><td align=\"center\">2</td><td align=\"center\">4</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">83</td><td align=\"center\">61</td><td align=\"center\">23</td><td align=\"center\">15</td><td align=\"center\">17</td><td align=\"center\">10</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">13</td><td align=\"center\">17</td><td align=\"center\">3</td><td align=\"center\">2</td><td align=\"center\">7</td><td align=\"center\">4</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">7</td><td align=\"center\">16</td><td align=\"center\">2</td><td align=\"center\">4</td><td align=\"center\">1</td><td align=\"center\">3</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T7\"><label>Table 7</label><caption><p>Power of MDR to detect only correct loci (from either/both genetic models) in data with genetic heterogeneity. GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%) to Detect Only Correct Loci</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">GH</td><td align=\"center\">71</td><td align=\"center\">36</td><td align=\"center\">46</td><td align=\"center\">45</td><td align=\"center\">26</td><td align=\"center\">21</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">71</td><td align=\"center\">36</td><td align=\"center\">46</td><td align=\"center\">45</td><td align=\"center\">26</td><td align=\"center\">21</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">29</td><td align=\"center\">39</td><td align=\"center\">11</td><td align=\"center\">8</td><td align=\"center\">10</td><td align=\"center\">7</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">68</td><td align=\"center\">41</td><td align=\"center\">48</td><td align=\"center\">36</td><td align=\"center\">21</td><td align=\"center\">29</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">30</td><td align=\"center\">39</td><td align=\"center\">13</td><td align=\"center\">9</td><td align=\"center\">9</td><td align=\"center\">10</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">65</td><td align=\"center\">46</td><td align=\"center\">45</td><td align=\"center\">45</td><td align=\"center\">20</td><td align=\"center\">16</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">27</td><td align=\"center\">42</td><td align=\"center\">10</td><td align=\"center\">17</td><td align=\"center\">10</td><td align=\"center\">11</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">34</td><td align=\"center\">39</td><td align=\"center\">11</td><td align=\"center\">15</td><td align=\"center\">8</td><td align=\"center\">10</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T8\"><label>Table 8</label><caption><p>Power of GENN to detect only correct loci (from either/both genetic models) in data with genetic heterogeneity.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Source of Noise</td><td align=\"center\" colspan=\"6\">Power (%) to Detect Only Correct Loci</td></tr><tr><td/><td colspan=\"6\"><hr/></td></tr><tr><td/><td align=\"center\">Model 1</td><td align=\"center\">Model 2</td><td align=\"center\">Model 3</td><td align=\"center\">Model 4</td><td align=\"center\">Model 5</td><td align=\"center\">Model 6</td></tr></thead><tbody><tr><td align=\"center\">GH</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">83</td><td align=\"center\">71</td><td align=\"center\">57</td><td align=\"center\">68</td></tr><tr><td align=\"center\">GE+GH</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">83</td><td align=\"center\">73</td><td align=\"center\">58</td><td align=\"center\">49</td></tr><tr><td align=\"center\">GH+PC</td><td align=\"center\">59</td><td align=\"center\">57</td><td align=\"center\">42</td><td align=\"center\">49</td><td align=\"center\">35</td><td align=\"center\">40</td></tr><tr><td align=\"center\">GH+MS</td><td align=\"center\">99</td><td align=\"center\">99</td><td align=\"center\">77</td><td align=\"center\">61</td><td align=\"center\">46</td><td align=\"center\">61</td></tr><tr><td align=\"center\">GE+GH+PC</td><td align=\"center\">63</td><td align=\"center\">81</td><td align=\"center\">39</td><td align=\"center\">40</td><td align=\"center\">23</td><td align=\"center\">32</td></tr><tr><td align=\"center\">GE+GH+MS</td><td align=\"center\">100</td><td align=\"center\">100</td><td align=\"center\">79</td><td align=\"center\">72</td><td align=\"center\">58</td><td align=\"center\">53</td></tr><tr><td align=\"center\">GH+PC+MS</td><td align=\"center\">57</td><td align=\"center\">68</td><td align=\"center\">41</td><td align=\"center\">50</td><td align=\"center\">31</td><td align=\"center\">31</td></tr><tr><td align=\"center\">GE+GH+PC+MS</td><td align=\"center\">50</td><td align=\"center\">74</td><td align=\"center\">37</td><td align=\"center\">42</td><td align=\"center\">31</td><td align=\"center\">34</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></table-wrap-foot>", "<table-wrap-foot><p>GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></table-wrap-foot>", "<table-wrap-foot><p>GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></table-wrap-foot>", "<table-wrap-foot><p>GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></table-wrap-foot>", "<table-wrap-foot><p>GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></table-wrap-foot>", "<table-wrap-foot><p>GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></table-wrap-foot>", "<table-wrap-foot><p> GE = 5% Genotyping Error; GH = 50% Genetic Heterogeneity; PC = 50% Phenocopy; MS = 5% Missing Data</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1756-0500-1-65-1\"/>", "<graphic xlink:href=\"1756-0500-1-65-2\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>In spite of the high reported rates of medical errors in various health care systems [##REF##11230064##1##, ####REF##1824793##2##, ##REF##7791255##3##, ##REF##15561651##4##, ##REF##15668306##5####15668306##5##], most studies of medical errors focus on either analysing incidents reported or assessing health care professionals' views [##REF##15335131##6##, ####REF##12486987##7##, ##REF##1987460##8##, ##REF##7791256##9####7791256##9##]. Furthermore, given the importance of using health care consumers' opinions and attitudes [##REF##12614708##10##, ####REF##16436486##11##, ##REF##15668304##12####15668304##12##], few studies have assessed attitudes of health care users with regard to medical errors [##REF##16879943##13##, ####REF##16037100##14##, ##REF##12078376##15##, ##UREF##0##16##, ##REF##17341749##17####17341749##17##]. Some of these studies have found out that consumers have increased expectations as well as an awareness of their rights and responsibilities [##REF##12928472##18##,##UREF##1##19##]. The importance of assessing consumers' views is demonstrated by the significant positive associations between satisfaction and improved compliance and continuity of care which ultimately leads to better outcomes, reduced rates of disease complications and the side effects of medications [##REF##16770368##20##,##REF##16749337##21##].</p>", "<p>Knowledge about medical errors by health care consumers should help to strengthen health care provision and improve clinical practice [##REF##12477944##22##]. Such knowledge could re-enforce the level of trust in health care systems in general and of professionals in particular. This is especially important given the publicity the media allocate to medical errors [##UREF##2##23##] as well as how the media play in modifying patients' health seeking behaviour [##REF##16333935##24##,##REF##16034948##25##].</p>", "<p>Furthermore, having data from health care recipients facilitates proper community education programs about medical errors that enable patients differentiate between side effects, normal course of a disease and adverse events resulting from medical errors. This is particularly applicable to elderly and illiterate patients who, for example, may not appreciate the difference between a medical error and the side effect of a medication. In addition, these programs may help providers educate patients about the role the individual and the system in the development of an error, thus reducing blames on doctors as a main source of errors. In addition, such programs would also help improve reporting of medical errors by consumers [##REF##15375095##26##]. Ultimately, consumers can have an active role in the quality of their own health care delivery, as partners rather than as passive users.</p>", "<p>Oman is a developing country located on the south-eastern tip of the Arabian Peninsula with a population of 2.24 million based on the 1993 census [##UREF##3##27##]. The Gross Domestic Product per capita income (GDP) was estimated to be 11,466 U.S Dollars in the year 2005 [##UREF##4##28##]. The health services in Oman are funded by the government and provided free for all Omanis and non-Omanis working in the government sector. The standards of health services are equivalent to the industrialized nations. In the year 2005, the crude death was 2.53 per 1000 population, the infant mortality rate was 10.28, the under-five mortality rate was 11.05 per 1000 live-birth and the life expectancy at birth was 74.28 years [##UREF##3##27##]. However, despite such improvements many Omanis travel to other countries seeking health care. This might reflect a trend that deserves an exploration of its causes such as lack of trust on safety of care delivered.</p>", "<p>Despite the benefits of exploring health care consumers' attitudes to medical errors, not much is known from developing health care systems. The objectives of this study were to assess health care consumers' perceptions of medical errors and to further examine factors affecting such perceptions. This study will be a starting point for further research in the field of patient's safety in Oman.</p>" ]

[ "<title>Methods</title>", "<p>The study was conducted in the North Al-Batinah region of Oman, from 15–26 January 2005. Two villages were selected because of close proximity to the College of Medicine and Health Sciences, Sultan Qaboos University (SQU), Muscat, Oman. All houses (250) in these two villages were included in the study. However, only 212 interviews took place (85% of the total) because some were unoccupied (families had moved away).</p>", "<p>Data were collected using face-to-face interviews with the paternal head of the family. When the father was not at home, the eldest member (either male or female) over 18 years of age was interviewed. Interviews were carried out by third and fourth year medical students as part of their Village Health Care course in the College of Medicine and Health Sciences. These students had been trained in a 3-day course on community surveys and face-to-face interviews. To assure data quality, all student interviews were supervised by Family and Community Medicine doctors.</p>", "<p>The questionnaire was developed after literature review, discussion with colleagues and pilot testing (by the medical students in their own village communities). The questionnaire was composed of three sections. Section one assessed demographic and other data (including age, sex, education, marital status, family income, usual source of health care, frequency of health care facility usage, history of chronic illnesses, and of any regular doctor appointments). Section two assessed participant's perceived knowledge about medical error definition (\"Do you know what is meant by medical error?\"). To follow up the participant's understanding, those who answered \"Yes\" were asked for at least one definition. Answers were then reviewed and coded into five categories: the prescription of wrong medications, the wrong diagnosis, a doctor's technical incompetence, technical incompetence of other staff and other examples such as staff attitude. These answers were then compared to our study definition. Subsequently, selection of answers falling under that definition was made. Section three had questions on related issues such as experience of medical errors and of its consequences. Answers to questions in section three vary between \"Yes/No\" format to selection of answers from a list of options. The study protocol was approved by the Medical Research and Ethics Committee of the College of Medicine and Health Sciences, SQU.</p>", "<title>Statistical Analysis</title>", "<p>For categorical variables, frequencies and percentages were recorded. Differences between groups were compared using Pearson's χ²or Fisher's exact tests (for cells that have less than 5 responses). For continuous variables, means and standard deviations (± SD) were calculated. Differences between groups were analysed using Student's t-tests. The distribution of medical errors follows a Poisson distribution or one of its variants. One of the rarely met assumptions of a Poisson model is that the mean must equal the variance. When the <italic>conditional </italic>variance is greater than the mean, an over-dispersed model may occur producing incorrect variance estimates that are biased downwards. When this occurs, a negative binomial model, which does not constrain the <italic>conditional </italic>variance to equal the mean, is preferred over a Poisson Model [##REF##7501743##29##,##UREF##5##30##]. Since there was significant over-dispersion, as denoted by the likelihood ratio test (p &lt; 0.001), the association between the perceived knowledge on medical errors definition and age was analysed using the negative binomial model.</p>", "<p>The associations between knowledge and various predictors were analysed using univariate and multivariate logistic regressions. The dependent outcome variable was the perceived knowledge of the meaning of medical error. Covariates included age, gender, educational level, marital status and family income.</p>", "<p>The multivariate logistic model was examined extensively to evaluate overall model fit and any assumptions. The overall fit was assessed using the Hosmer &amp; Lemeshow goodness-of-fit statistic [##REF##7055134##31##] and the area under the Receiver Operating Curve (ROC) [##REF##7063747##32##]. The Hosmer &amp; Lemeshow statistic analyses the actual versus the predicted responses; theoretically, the observed and expected counts should be close. Based on the χ²distribution, a Hosmer &amp; Lemeshow statistic with a <italic>p</italic>-value greater than 0.05 is considered a good fit. The ROC curve is a graph of the sensitivity against one minus specificity as the threshold cut-off is varied, and also calculates the area under the curve. The ROC curve provides a measure of the model's discriminatory power. A model with perfect prediction has an ROC of 1.0; an area of 0.5 provides no better discrimination than chance. Models with area under the ROC curve of greater than 0.7 are preferred. A <italic>priori </italic>two-tailed level of significance was set at the 0.05 level. Statistical analyses were performed using STATA version 10.0 software.</p>" ]

[ "<title>Results</title>", "<p>About half of the participants were male (53%; n = 112). The overall mean age was 34 ± 13 years with an age range from 15 to 94 years, literacy was 83% (n = 177) and 70% (n = 148) were married. Ninety three per cent (n = 197) stated that they had visited health care facilities (primary or secondary care) over 5 times a year, which included visits for curative/preventive services (e.g. vaccination). The average number of visits per person per year was 10.2 compared to the Ministry of Health (MoH) figures (an average of 4.4 per person per year in 2005) [##UREF##3##27##]. The discrepancy between rates is because the current study counted accompanying someone as a visit, compared to MoH statistics which count only visits for individual health care services. Forty six percent (n = 97) reported a history of chronic illness such as diabetes mellitus or recurrent low back pain. In 2005 non-communicable diseases represented 54.5% and 39.8% of outpatient and inpatient morbidity respectively [##UREF##3##27##]. Fifty eight percent (n = 124) stated that they saw their regular doctor on most visits.</p>", "<p>Questioned about understanding of \"what is meant by medical error\", 78% (n-165) responded \"Yes\". Of these, 34% to referred to wrong medication, 26.5% to wrong diagnosis, 13% to wrong operations and 4% to wrong injections. Interestingly, around 23% of the definitions given were referring to causes of medical errors rather than exact definition. These were related to professionals and patients such as lack of doctor's experience and not following doctor's advice (Table ##TAB##0##1##).</p>", "<p>Associations between perceived knowledge of medical error and various predictors were evaluated using both univariate and multivariate logistic regression models. The overall multivariate logistic regression model was statistically significant (LR χ²(7) = 35.61; p &lt; 0.001) and it accounted for 15.9% of the variance in perceived knowledge of medical errors definition (Pseudo R²= 15.88). The model fits reasonably well. The Pearson's χ²goodness-of-fit statistic, using 10 near equal-size groups as suggested by Hosmer &amp; Lemeshow, was 1.66 and the <italic>p</italic>-value was 0.990. The ROC curve was 0.76. The model correctly classified 81% of the cases.</p>", "<p>Age was negatively correlated with perceived medical error knowledge. This was significant in both univariate and multivariate regression models (Table ##TAB##1##2##). The older participants were less likely to be knowledgeable about medical errors. Specifically, after controlling for other variables, each year increase in age was associated with a 4% reduction in participant's perceived knowledge of medical error definition (CI: 1% to 7%; p = 0.045). There was a trend in both the univariate (OR 0.33; CI: 0.14 to 0.79; <italic>p </italic>= 0.012) and multivariate (OR 0.45; CI: 0.15 to 1.31; <italic>p </italic>= 0.144]) regression models for married participants to be less knowledgeable than their unmarried counterparts; this did not attain statistical significance in the multivariate logistic model (Table ##TAB##1##2##). There was a positive relationship between family income and perceived knowledge of medical error definition; the higher the family income, the more knowledge on its definition was seen. This trend was seen in both the univariate and multivariate regression models (Table ##TAB##1##2##).</p>", "<p>Using exploratory univariate statistics, other variables such as source of healthcare, frequency of healthcare use, history of chronic illness, and seeing one doctor regularly were found to have no significant effect on awareness of medical errors definition. These variables were excluded from the final logistic models (Table ##TAB##2##3##). Of those who believed they knew what was meant by medical error (n = 165), 49% (n = 80) had had experience of medical error in the preceding year, either by themselves or a family member. The outcomes included severe pain (44%; n = 36), substantial loss of time at work/school or other activities (19%; n = 15), disability (15%; n = 12) and death (9%; n = 7). The nature of the experience was diagnostic errors for 32 participants (40%); 21 (26%) and 22 (28%) of the participants stated that errors were due to surgical and medication errors, respectively. Ninety-five participants (58%) of those who believed they knew what a medical error was, felt that medical errors occurred often in the community, compared to 7 participants (4%) who felt that errors never happened.</p>", "<p>With regard to the causes of medical error for those who believed they knew its meaning (n = 165), 48.5% of the participants (n = 80) felt that uncaring health care professionals was the main cause (Table ##TAB##3##4##). Lack of training of health care professionals was identified as the next most frequent cause (46%; n = 76). Forty two percent of the participants (n = 70) appreciated that work overload was one of the causes of medical errors. A further 39% (n = 64) of the participants considered lack of time spent with the patient as the cause. When asked to list other causes of medical errors, none of the participants listed patients' factors although they listed some factors when asked to define medical error. It was also found that younger participants were more able to list one or more possible causes of medical errors compared to older participants. Specifically, each one year increase in age was associated with a 2% reduction in the likelihood of listing one or more possible cause of medical error (Incident Rate Ratio of 0.98: CI 0.97 to 0.99; p &lt; 0.001).</p>" ]

[ "<title>Discussion</title>", "<p>To our knowledge this is the first study in Oman to assess health care consumers' perceptions about medical errors. This study shows that the majority of participants believed they knew what is meant by the term 'medical error'. In Oman, the issue of medical errors is currently publicly discussed through newspapers, television and radio programs, actively encouraged by the Shura Council (State Consultative Council) and the Ministry of Health. This could explain increased community members awareness about medical errors. This finding is in line with what was found by Gallagher et al, that all participants were aware of the topic of errors in medicine [##REF##12597752##33##]. However, Blendon et al found that 68% of the public were unaware of the meaning of medical errors [##REF##12477944##22##]. The majority of participants being young and literate in the present study could explain our findings. Interestingly, other authors distinguished medical errors resulting from failures of a planned action (i.e. errors of execution) to those due to the wrong management plan (i.e. errors of planning) [##UREF##6##34##]. Participant statements not falling under the above definition were not considered as definitions of errors (Table ##TAB##0##1##). For example, statements such as given wrong medication, making wrong diagnosis or performing wrong surgery were considered as definitions of errors, whereas statements such as lack of doctor's experience and technical incompetence were considered as causes of errors.</p>", "<p>However, in the present study it was found that 23% of the definitions refer to causes of medical errors rather than the exact definition. Such a finding is similar to what was found by Van Vorst and colleagues in their study which showed that at least 41% of the 180 reported mistakes received were not judged to be medical mistakes when coded with a taxonomy designed to specifically describe medical errors [##REF##17341749##17##].</p>", "<p>This finding reflects the need to communicate with the community about the definition of medical error and its causes. Furthermore, it reflects the role health care professionals, mainly physicians, play in educating patients about investigations done and their results, diagnosis, medication/s they are taking and their side-effects. This may help community members to differentiate between an error and a side-effect of medication or a complication resulting from the normal course of a disease. Ultimately, this would improve patients' reporting of such outcomes, thus enable health care providers take needful actions.</p>", "<p>It is of interest to note that multivariate logistic regression showed an association between perceived knowledge of the meaning of medical error and age. Such associations could have two explanations. On the one hand, younger patients might be more likely to ask health care professionals for an explanation of events compared with their older counterparts. In addition, younger patients might have more knowledge on issues such as health care safety, thus empowering them to raise questions about their own care. However, our results could be linked to the findings of patient satisfaction studies which show that elderly patients are more satisfied with their health care provision than younger or more educated patients, regardless of the quality of care provided [##REF##11848270##35##].</p>", "<p>Forty-nine percent of the participants in our study stated that they had an experience with a medical error, either themselves or with one of their family members. This rate is similar to that reported by Blendon et al, in which 42% of the participants or their family members experienced a medical error [##REF##12477944##22##]. In contrast, a community survey about medical errors carried out by the European Commission showed that 23% of the patients or their family member had encountered a medical error [##UREF##7##36##]. Another study found that 22% of the patients stated that they or family members had experienced medical errors of some kind [##UREF##8##37##]. Furthermore, a large percentage of participants in the current study felt that errors were common. These rates reflects concerns among health care consumers that deserves consideration by health care systems such as the need to explore these experiences more and link the results with those of clinical audits [##UREF##8##37##,##REF##15335132##38##]. This will help providers identify strengths and weaknesses of their health care systems and plan for improving patient safety. Vigilance in these areas will ultimately help health care systems gain the trust of the communities they serve.</p>", "<p>Participants listed work overload (for health care professionals) and lack of time physicians spend with their patients as very important causes of medical errors. This finding is similar to that of a study which showed that physicians' stress, fatigue, overwork and inadequate time with their patients to be at the top of the causes of medical errors [##REF##12477944##22##]. These factors affect the doctor-patient and doctor-health professionals communication, and the educational role doctors ought to play. This reflects the need to look at these factors and reduce the communication gap among health care professionals and patients as it has been shown that gap in communication was a common cause of medical errors [##UREF##9##39##].</p>", "<p>Despite our participants' perceived knowledge of the meaning of medical error and estimates of the prevalence in the community, none of them listed patient factors as causes. This may reflect the lacking of medical knowledge from patients' side to describe medical errors, particularly related to its causes [##REF##17341749##17##]. Furthermore, health care consumers may not be aware of their own role in health care delivery or they may have assumed that only the health care system was being studied. This ultimately affects satisfaction with the quality of care delivered, because blame is directed to health care providers and institutions, thus affecting trust. These observations further re-enforce the passive role patients assume in their own health care, forgetting that patients can be experts in their own care and can thus play a major role in reducing medical errors such as adverse drug reactions [##REF##12805126##40##,##REF##7819893##41##]. This might then indirectly affect the health care system; patients might not follow physicians' recommendations, ultimately leading to a vicious cycle in which all drug side-effects or all disease complications may be assumed to be medical errors. Such findings reflect an essential need to educate the community members about the role individual patients and the system play in the development of medical errors. This would help in reducing the pressure on health care professionals either from the public or the media when it comes to medical errors.</p>", "<p>Lack of patient education about these as well as other causes of medical errors could be due to the defensive nature of many health care systems when medical errors are discussed [##REF##10720336##42##,##UREF##10##43##]. However, this can be set against the high preferences of patients towards disclosure of errors and the provision of more information about the underlying disease shown by some studies [##REF##12597752##33##,##UREF##11##44##]. Although one study showed that many people interviewed thought that patients were often at least partially responsible for errors in their health care, the public were less likely (than physicians) to attribute errors to patient factors [##REF##12477944##22##]. Participants in the current study were not explicitly asked about their role in medical errors and were left to comment in answers to open questions.</p>", "<p>This study has limitations. The first is that there was no independent verification that someone in the family suffered a medical error. However, health care consumers are now more oriented towards modern medicine with regard to their rights and responsibilities. Secondly, reliability and validity tests were not performed on the questionnaire, however, one could argue that the questionnaire was in fact not a proper survey tool, and hence would not require these tests to be conducted. Thirdly, the convenience sampling of the population in the two villages located close to the College of Medicine and Health Sciences, Sultan Qaboos University (SQU) could have affected the generalizability of our sample cohort. A larger study comprising the different areas of Oman is warranted to corroborate these findings.</p>" ]

[ "<title>Conclusion</title>", "<p>This study shows a majority of respondents believe they know what is meant by the term 'medical error'. Younger people are more likely to believe this than older people. Therefore, given the high rate of chronic illness and increased use of health care facilities by elderly people, more health education programmes should be directed to the older community members. These programmes should aim to increase awareness about the possible medical errors that might occur in health care delivery. Ultimately, this will help to differentiate between unfortunate drug side-effects and medical errors. Furthermore, a large percentage of the definitions given were referring to the causes of medical errors rather than exact definition. In addition, no participant raised patient factors as contributory causes to medical errors. There needs to be further education to increase patients' awareness about the meaning of a medical error and its causes and of patients' own active roles in the health care delivery. Finally, community surveys about medical errors should be supported by clinical audits in order to show the exact prevalence of medical errors in the system.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Errors have been the concern of providers and consumers of health care services. However, consumers' perception of medical errors in developing countries is rarely explored. The aim of this study is to assess community members' perceptions about medical errors and to analyse the factors affecting this perception in one Middle East country, Oman.</p>", "<title>Methods</title>", "<p>Face to face interviews were conducted with heads of 212 households in two villages in North Al-Batinah region of Oman selected because of close proximity to the Sultan Qaboos University (SQU), Muscat, Oman. Participants' perceived knowledge about medical errors was assessed. Responses were coded and categorised. Analyses were performed using Pearson's χ², Fisher's exact tests, and multivariate logistic regression model wherever appropriate.</p>", "<title>Results</title>", "<p>Seventy-eight percent (n = 165) of participants believed they knew what was meant by medical errors. Of these, 34% and 26.5% related medical errors to wrong medications or diagnoses, respectively. Understanding of medical errors was correlated inversely with age and positively with family income. Multivariate logistic regression revealed that a one-year increase in age was associated with a 4% reduction in perceived knowledge of medical errors (CI: 1% to 7%; p = 0.045). The study found that 49% of those who believed they knew the meaning of medical errors had experienced such errors. The most common consequence of the errors was severe pain (45%). Of the 165 informed participants, 49% felt that an uncaring health care professional was the main cause of medical errors. Younger participants were able to list more possible causes of medical errors than were older subjects (Incident Rate Ratio of 0.98; p &lt; 0.001).</p>", "<title>Conclusion</title>", "<p>The majority of participants believed they knew the meaning of medical errors. Younger participants were more likely to be aware of such errors and could list one or more causes.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>ASA–M, MAA–S, MHA–A and MK participated in the design of the study, data collection and in drafting the manuscript. ISA–Z, AMA–W and SR performed the statistical analysis, and interpretation of data. ASA–M, MHA–A and ISA–Z revised the manuscript. All authors have read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1472-6939/9/13/prepub\"/></p>" ]

[ "<title>Acknowledgements</title>", "<p>The authors would like to express their sincere thanks to the College of Medicine and Health Sciences and to the Department of Family Medicine and Public Health, Sultan Qaboos University. Also, we would like to thank residents and medical students who helped in data collection. Furthermore, thanks also go to Prof. John Alexander Raeburn, Dr. Rodger Martin, Dr. Brenda Leese and to Mr. Kassim Al-Riyami who assisted with the preparation of this manuscript.</p>" ]

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[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Participants' perceived definitions of medical errors</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Serial No.</bold></td><td align=\"left\"><bold>Definition categories</bold></td><td align=\"left\"><bold>Definitions given by participants</bold></td><td align=\"center\"><bold>Number (%)</bold></td></tr></thead><tbody><tr><td align=\"left\">1</td><td align=\"left\">Wrong prescription/dispensing of medication</td><td align=\"left\">Wrong medication</td><td align=\"center\">93 (34)</td></tr><tr><td align=\"left\">2</td><td align=\"left\">Wrong diagnosis</td><td align=\"left\">Wrong diagnosis</td><td align=\"center\">73 (26.5)</td></tr><tr><td align=\"left\">3</td><td align=\"left\">Doctors' technical in-competence</td><td align=\"left\">Wrong surgery</td><td align=\"center\">36 (13)</td></tr><tr><td/><td/><td align=\"left\">Technical incompetence*</td><td align=\"center\">10 (3.6)</td></tr><tr><td/><td/><td align=\"left\">Lack of doctor's experience*</td><td align=\"center\">7 (3)</td></tr><tr><td align=\"left\">4</td><td align=\"left\">Other staff technical in-competence (nurses and pharmacist)</td><td align=\"left\">Giving wrong injection</td><td align=\"center\">11 (4)</td></tr><tr><td/><td/><td align=\"left\">Pharmacist incompetence*</td><td align=\"center\">4 (1)</td></tr><tr><td align=\"left\">5</td><td align=\"left\">Others</td><td align=\"left\">Errors by doctors</td><td align=\"center\">5 (2)</td></tr><tr><td/><td/><td align=\"left\">Forgotten surgical items</td><td align=\"center\">2 (0.7)</td></tr><tr><td/><td/><td align=\"left\">Wrong vaccination</td><td align=\"center\">2 (0.7)</td></tr><tr><td/><td/><td align=\"left\">IV canula left in site for 25 days</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Error in first aid</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Wrong BP reading</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Wrong procedure</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Doctor ignorance*</td><td align=\"center\">14 (5)</td></tr><tr><td/><td/><td align=\"left\">Poor staff attitude*</td><td align=\"center\">4 (1)</td></tr><tr><td/><td/><td align=\"left\">Not updating patients*</td><td align=\"center\">2 (0.7)</td></tr><tr><td/><td/><td align=\"left\">Faulty equipment*</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Doctors overload*</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Slowness in giving care*</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Nurses ignorance*</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Wrong information by the patient+</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Not following doctors advise+</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Intake of un-prescribed medicine+</td><td align=\"center\">1 (0.4)</td></tr><tr><td/><td/><td align=\"left\">Intake of herbal medicine+</td><td align=\"center\">1 (0.4)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Total</bold></td><td/><td/><td align=\"center\"><bold>275</bold></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Univariate and multivariate logistic regression models (N = 212).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Independent Variable</bold></td><td align=\"center\"><bold>N</bold></td><td align=\"center\" colspan=\"2\"><bold>Univariate</bold></td><td align=\"center\" colspan=\"2\"><bold>Multivariate</bold></td></tr><tr><td/><td/><td colspan=\"2\"><hr/></td><td colspan=\"2\"><hr/></td></tr><tr><td/><td/><td align=\"center\"><bold>Odds Ratio [95% CI]</bold></td><td align=\"center\"><bold>p-value</bold></td><td align=\"center\"><bold>Odds Ratio [95% CI]</bold></td><td align=\"center\"><bold>p-value</bold></td></tr></thead><tbody><tr><td align=\"left\">Age</td><td align=\"center\">212</td><td align=\"center\">0.94 [0.91–0.96]</td><td align=\"center\">&lt;0.001</td><td align=\"center\">0.96 [0.93–0.99]</td><td align=\"center\">0.045</td></tr><tr><td align=\"left\">Male gender</td><td align=\"center\">112</td><td align=\"center\">0.63 [0.32–1.22]</td><td align=\"center\">0.169</td><td align=\"center\">0.82 [0.36–1.85]</td><td align=\"center\">0.629</td></tr><tr><td align=\"left\">Educational level</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> Illiterate</td><td align=\"center\">35</td><td align=\"center\">Ref</td><td/><td/><td/></tr><tr><td align=\"left\"> Preparatory</td><td align=\"center\">147</td><td align=\"center\">4.20 [1.92–9.19]</td><td align=\"center\">&lt;0.001</td><td align=\"center\">1.70 [0.61–4.77]</td><td align=\"center\">0.314</td></tr><tr><td align=\"left\"> Secondary &amp; above</td><td align=\"center\">30</td><td align=\"center\">8.50 [2.17–33.3]</td><td align=\"center\">0.002</td><td align=\"center\">1.58 [0.28–8.88]</td><td align=\"center\">0.603</td></tr><tr><td align=\"left\">Married</td><td align=\"center\">148</td><td align=\"center\">0.33 [0.14–0.79]</td><td align=\"center\">0.012</td><td align=\"center\">0.45 [0.15–1.31]</td><td align=\"center\">0.144</td></tr><tr><td align=\"left\">Family income</td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"> &lt;200</td><td align=\"center\">66</td><td align=\"center\">Ref</td><td/><td/><td/></tr><tr><td align=\"left\"> 200–500</td><td align=\"center\">102</td><td align=\"center\">2.19 [1.08–4.43]</td><td align=\"center\">0.029</td><td align=\"center\">1.99 [0.87–4.55]</td><td align=\"center\">0.104</td></tr><tr><td align=\"left\"> &gt;500</td><td align=\"center\">44</td><td align=\"center\">5.35 [1.70–16.8]</td><td align=\"center\">0.004</td><td align=\"center\">4.73 [1.37–16.4]</td><td align=\"center\">0.014</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Socio-demographic and educational variables of the study participants stratified by perceived knowledge of medication error definition (N = 212).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Characteristic</bold></td><td align=\"center\" colspan=\"3\"><bold>Knowledge of Medication Errors</bold></td></tr><tr><td/><td colspan=\"3\"><hr/></td></tr><tr><td/><td align=\"center\"><bold>No </bold>(n = 47)</td><td align=\"center\"><bold>Yes </bold>(n = 165)</td><td align=\"center\"><bold>p-value</bold></td></tr></thead><tbody><tr><td align=\"left\">Age, mean ± SD, in years</td><td align=\"center\">43 ± 17</td><td align=\"center\">31 ± 11</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\">Age category, n (%)</td><td/><td/><td/></tr><tr><td align=\"left\"> 15–24 years</td><td align=\"center\">5 (11%)</td><td align=\"center\">47 (28%)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\"> 25–34 years</td><td align=\"center\">10 (21%)</td><td align=\"center\">67 (41%)</td><td/></tr><tr><td align=\"left\"> 35–44 years</td><td align=\"center\">11 (23%)</td><td align=\"center\">27 (16%)</td><td/></tr><tr><td align=\"left\"> &gt;44 years</td><td align=\"center\">21 (45%)</td><td align=\"center\">24 (15%)</td><td/></tr><tr><td align=\"left\">Gender, n (%)</td><td/><td/><td/></tr><tr><td align=\"left\"> Female</td><td align=\"center\">18 (38%)</td><td align=\"center\">82 (50%)</td><td align=\"center\">0.167</td></tr><tr><td align=\"left\"> Male</td><td align=\"center\">29 (62%)</td><td align=\"center\">83 (50%)</td><td/></tr><tr><td align=\"left\">Educational Level, n (%)</td><td/><td/><td/></tr><tr><td align=\"left\"> Illiterate</td><td align=\"center\">17 (36%)</td><td align=\"center\">18 (11%)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\"> Reads &amp; writes/preparatory</td><td align=\"center\">27 (57%)</td><td align=\"center\">120 (72%)</td><td/></tr><tr><td align=\"left\"> Secondary and above</td><td align=\"center\">3 (6%)</td><td align=\"center\">27 (16%)</td><td/></tr><tr><td align=\"left\">Marital Status, married, n (%)</td><td align=\"center\">40 (85%)</td><td align=\"center\">108 (65%)</td><td align=\"center\">0.010</td></tr><tr><td align=\"left\">Family Income, n (%), in OR</td><td/><td/><td/></tr><tr><td align=\"left\"> &lt;200</td><td align=\"center\">23 (49%)</td><td align=\"center\">43 (26%)</td><td align=\"center\">0.004</td></tr><tr><td align=\"left\"> 200 – 500</td><td align=\"center\">20 (43%</td><td align=\"center\">82 (50%)</td><td/></tr><tr><td align=\"left\"> &gt;500</td><td align=\"center\">4 (9%)</td><td align=\"center\">40 (24%)</td><td/></tr><tr><td align=\"left\">Usual Source of Healthcare, n (%)</td><td/><td/><td/></tr><tr><td align=\"left\"> Local Health Center</td><td align=\"center\">33 (70%)</td><td align=\"center\">108 (65%)</td><td align=\"center\">0.272</td></tr><tr><td align=\"left\"> Local Hospital</td><td align=\"center\">6 (13%)</td><td align=\"center\">20 (12%)</td><td/></tr><tr><td align=\"left\"> Private Hospital</td><td align=\"center\">7 (15%)</td><td align=\"center\">37 (22%)</td><td/></tr><tr><td align=\"left\"> Others (e.g. Traditional Healer)</td><td align=\"center\">1 (2%)</td><td align=\"center\">0 (0%)</td><td/></tr><tr><td align=\"left\">Frequency of Healthcare Use, n (%)</td><td/><td/><td/></tr><tr><td align=\"left\"> 1–5</td><td align=\"center\">2 (4.3%)</td><td align=\"center\">13 (7.9%)</td><td align=\"center\">0.787</td></tr><tr><td align=\"left\"> 6–10</td><td align=\"center\">21 (45%)</td><td align=\"center\">72 (44%)</td><td/></tr><tr><td align=\"left\"> &gt;10</td><td align=\"center\">24 (51%)</td><td align=\"center\">80 (48%)</td><td/></tr><tr><td align=\"left\">History of Chronic Disease, n (%)</td><td align=\"center\">22 (47%)</td><td align=\"center\">75 (45%)</td><td align=\"center\">0.869</td></tr><tr><td align=\"left\">Seeing a Doctor Regularly, n (%)</td><td align=\"center\">26 (55%)</td><td align=\"center\">98 (59%)</td><td align=\"center\">0.617</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Participant responses to a list of causes of medical errors</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Cause</bold></td><td align=\"center\" colspan=\"2\"><bold>Number (%)</bold></td></tr><tr><td/><td colspan=\"2\"><hr/></td></tr><tr><td/><td align=\"center\"><bold>Yes</bold></td><td align=\"center\"><bold>No</bold></td></tr></thead><tbody><tr><td align=\"left\">Uncaring health care professional</td><td align=\"center\">80 (48.5)</td><td align=\"center\">85 (51.5)</td></tr><tr><td align=\"left\">Lack of training</td><td align=\"center\">76 (46)</td><td align=\"center\">89 (54)</td></tr><tr><td align=\"left\">Work overload</td><td align=\"center\">70 (42)</td><td align=\"center\">95 (58)</td></tr><tr><td align=\"left\">Lack of time spend with the patient</td><td align=\"center\">64 (39)</td><td align=\"center\">101 (61)</td></tr><tr><td align=\"left\">Shortage of doctors</td><td align=\"center\">57 (34.5)</td><td align=\"center\">108 (65.5)</td></tr><tr><td align=\"left\">Poor handwriting</td><td align=\"center\">37 (22)</td><td align=\"center\">128 (78)</td></tr><tr><td align=\"left\">Poor supervision</td><td align=\"center\">33 (20)</td><td align=\"center\">132 (80)</td></tr><tr><td align=\"left\">Complexity of medical care</td><td align=\"center\">26 (16)</td><td align=\"center\">139 (84)</td></tr><tr><td align=\"left\">Shortage of paramedical</td><td align=\"center\">24 (14.5)</td><td align=\"center\">141 (85.5)</td></tr><tr><td align=\"left\">Shortage of nurses</td><td align=\"center\">21 (13)</td><td align=\"center\">144 (87)</td></tr><tr><td align=\"left\">Lack of computerized medical record</td><td align=\"center\">11 (7)</td><td align=\"center\">154 (93)</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>Percents are out of total number (275). Please notice that some participants gave more than one definition.</p><p>* Causes of medical errors</p><p>+ Patient-related factor</p></table-wrap-foot>", "<table-wrap-foot><p>N = Number of participants in each category; CI = Confidence Interval; the variables were entered into the multivariate logistic regression model simultaneously; p-values were generated using both univariate and multivariate logistic regression models</p></table-wrap-foot>", "<table-wrap-foot><p>SD = Standard deviation; Percents are column percents; OR = Omani Rials; Differences between groups were analyzed using <italic>Student's </italic>t-test, <italic>Pearson's </italic>χ²test, and <italic>Fisher's Exact </italic>test whenever appropriate.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Posterior locked shoulder dislocation is an uncommon injury (2–4% of all shoulder dislocations) which may be misdiagnosed and overlooked in up to 60% of cases [##REF##17522876##1##]. The spectrum of associated injuries varies from the isolated impaction fracture of the anteromedial aspect of the humeral head (\"reverse Hill-Sachs lesion\") to more complex fracture types of the proximal humerus (less than 1%) and shoulder girdle [##REF##17522876##1##,##REF##17606784##2##]. The unrecognised dislocation-fracture pattern can jeopardise the joint mobility and the vascularity of the humeral head predisposing to chronic instability, osteonecrosis and osteoarthritis [##REF##17522876##1##].</p>", "<p>We present a case of a neglected four-part posterior fracture-dislocation of the proximal humerus in a young woman. The vascularity and integrity of the humeral head were at high risk due to a large reverse Hill-Sachs lesion (50% of the articular surface) and severely displaced tuberosities fractures. Open reduction and internal fixation of the humeral neck and greater tuberosity fractures in combination with grafting and transfer of the lesser tuberosity to the humeral defect led to joint stability, viability of the humeral head and favourable functional outcome.</p>" ]

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[ "<title>Discussion</title>", "<p>The rarity of incidence of posterior-fracture dislocation, the potential for delay in diagnosis and the lack of evidence-based management strategies make this specific injury type challenging to treat. Recently, Robinson <italic>et al. </italic>[##REF##17606784##2##] divided posterior-fracture dislocations into three subtypes according to the extent of fracture lines and the involvement of tuberosities. In Type I, a Neer Two-Part anatomic fracture is present without associated tuberosity fractures. In Type II, there is an additional fracture of the lesser tuberosity and in rare Type III both tuberosities are involved. The authors found the latter fracture type in 17 cases and noticed that in all of the cases, the greater and lesser tuberosities were held together giving the characteristic \"shield\" fragment which was first described by Edelson <italic>et al. </italic>[##REF##15125131##4##]. Even if internal comminution exists and more fracture lines are apparent (\"shattered shield\" configuration), the intact periosteal sleeve averts secondary displacement. In the present case, the tuberosities were substantially displaced outlining a Neer Four-Part fracture of the proximal humerus. This finding illustrates the variability of the fracture pattern and the complexity of the underlying mechanism of injury.</p>", "<p>Apart from the severity of injury and fracture deformity, the final prognosis is further affected by the extent of the underlying glenoid or reverse Hill-Sachs lesion [##REF##7068692##5##,##REF##15741636##6##]. As extensive erosion of the posterior margin of the glenoid fossa is rarely encountered even in long-standing dislocations [##REF##3805075##3##], the focus is concentrated on treatment of the anteromedial defect of the humeral head. Transfer of the subscapularis or lesser tuberosity, rotational osteotomy of the humerus and allograft or autograft reconstruction have been advocated for the treatment of medium (25–40% of articular surface) or large (more than 40%) defects in cases where the articular cartilage has been impressed but not destroyed [##REF##15741636##6##,##REF##15125117##7##]. Hemiarthroplasty has been suggested in patients with an impression fracture involving more than 50% of the articular surface or when the humeral head is very soft and not viable [##REF##15125117##7##]. However, in young patients, all efforts should be made to retain the humeral head and restore its shape, roundness and normal anatomy. Similar to our case, good results have been reported after reconstruction of defects equal to or greater than 40% of the articular surface using allograft or lesser tuberosity transfer [##REF##15756620##8##,##REF##8613444##9##]. Regardless of the selected treatment option, elevation of the cartilage with the adjacent bone from the impressed area and subsequent subchondral support should be carried out [##REF##17522876##1##].</p>", "<p>The transfer of lesser tuberosity instead of subscapularis alone was first introduced by Hawkins <italic>et al. </italic>[##REF##3805075##3##]. The osteotomised or fractured bone fragment offers better filling of the defect and more secure reinsertion of the tendon [##REF##15756620##8##]. Finkelstein <italic>et al. </italic>[##REF##7623169##10##] reported that full flexion, abduction, and external rotation were achieved at 3 months in seven acutely treated shoulders with a 20% to 45% humeral head defect. The authors stated that the technique allowed earlier joint mobilisation because of the increased confidence in the immediate stability of the repaired shoulder. Checchia <italic>et al. </italic>[##REF##9524341##11##] noted similar results but emphasised the importance of the time interval between injury and diagnosis. Specifically, posterior fracture-dislocations which were treated within 2 years of the injury had good shoulder function in comparison with neglected and misdiagnosed cases. However, Aparicio <italic>et al. </italic>[##REF##10664437##12##] found radiographic signs of glenohumeral arthritis in six out of seven cases. The mild dislocation arthropathy was attributed to the loss of the concavity-compression effect and alteration of joint biomechanics after lesser tuberosity transfer in a non-anatomic position.</p>", "<p>Although avascular necrosis of the humeral head is unpredictable and may occur in any posterior fracture-dislocation pattern, neglected injuries and fracture of the anatomic neck substantially increase the above incidence [##REF##15148610##13##]. Accurate reduction and stable internal fixation – even if performed late – enhance the probability of successful revascularisation of the humeral head and avoid the development of avascular necrosis [##REF##10360700##14##]. Head reperfusion seems to occur by the intact posteromedial vessels or alternatively by \"creeping substitution\" in cases with severe disruption of the arterial flow and soft tissue attachments [##REF##15741636##6##]. In the presented case, the impaction of demineralised bone matrix might contribute to the viability of humeral head due to its osteoconductive and osteoinductive properties [##REF##11886919##15##]. Even though it does not offer structural support, it is well suited for filling bone defects and cavities and it can be revascularised quickly. We believe that transposition of lesser tuberosity combined with allograft impaction can effectively address large humeral defects and decrease the potential of subchondral collapse or avascular necrosis.</p>" ]

[ "<title>Conclusion</title>", "<p>Posterior shoulder fracture-dislocation continues to be a \"diagnostic trap\" for the unaware physician despite the advances in imaging techniques and the continuous flow of information about the risk of missed diagnosis. In neglected injuries, open reduction of the humeral head, stable fixation of all of the associated fractures and filling of the anterolateral defect with graft and/or transfer of lesser tuberosity may lead to optimum result and good functional recovery.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Posterior shoulder fracture-dislocation is a rare emergency condition with poor prognosis when there is a delay in diagnosis and presence of associated injuries.</p>", "<title>Case presentation</title>", "<p>We present a case of a neglected four-part fracture-dislocation of the proximal humerus in a 34-year-old Greek woman. Except from the substantially displaced and comminuted tuberosity fractures, an anterolateral defect of approximately 50% of the articular surface was apparent. Open reduction of the humeral head was followed by reconstruction of the proximal humerus with allograft impaction, transfer of lesser tuberosity to the humeral defect and anatomic fixation of the greater tuberosity and humeral neck fractures. At two and a half years postoperatively, the humeral head was revascularised and properly articulated with the glenoid fossa.</p>", "<title>Conclusion</title>", "<p>The presented case underlines the variability of injury pattern, the potential of missed diagnosis and the need for preserving the humeral head in young patients regardless of the amount of articular surface defect and disruption of soft tissue attachments.</p>" ]

[ "<title>Case presentation</title>", "<p>A 34-year-old right-hand dominant Greek woman, presented at the Upper Limb Clinic of the Hospital complaining of persisting pain and stiffness in her right shoulder. The symptoms began 3 months earlier after a fall on her outstretched hand from a height of approximately 3 metres. The patient reported that the initial clinical assessment in the local emergency department and the anteroposterior radiograph of the right shoulder did not reveal any significant abnormality and a diagnosis of shoulder sprain and contusion was established. Pain medication was prescribed and a sling was applied for 10 days. After that time, the patient was re-examined and physical therapy with active and passive shoulder and upper limb exercises was commenced. As there was no improvement in pain and shoulder mobility, she was finally referred to our clinic for a second opinion and further evaluation.</p>", "<p>On physical examination, her shoulder looked flattened anteriorly and both acromion and coracoid processes appeared to be prominent at the anterior part of the shoulder. There was an internal rotation deformity of 30° and any effort to passively or actively move the glenohumeral joint was extremely painful. Forward elevation of 40°, no external rotation and inability to completely supinate the forearm were also identified. The patient did not have any neuromuscular deficit and her medical history was unremarkable in terms of previous injuries in the shoulder region or other medical comorbidities. The anteroposterior radiograph of the right shoulder illustrated the marked internal rotation of the proximal humerus and the typical \"lightbulb sign\". The greater and lesser tuberosities were fractured and displaced from each other and from the humeral head. A further undisplaced fracture line at the anatomic neck of the proximal humerus was also evident (Figure ##FIG##0##1A##). Because of the inherent patient difficulty to abduct the arm, an axillary view was not performed. The transthoracic lateral roentgenogram showed posterior extrusion of the humeral head from the glenoid fossa (Figure ##FIG##0##1B##). Furthermore, the computed tomography (CT) scan clearly delineated the locked posterior shoulder dislocation with the large anteromedial head defect (50% of the articular surface) and the comminuted fractures of both tuberosities (Figure ##FIG##0##1C##).</p>", "<p>According to these findings, open reduction and reconstruction of the proximal humerus was considered necessary. Under general anaesthesia, the patient was placed in a beach chair position and the glenohumeral joint was assessed via a deltopectoral approach. The axillary nerve was palpated to ascertain its position but it was not mobilised. The long head of the biceps was still intact and both tuberosities were localised and circumferentially released from the newly formed granulation tissue and immature callus. As the capsule was torn and detached along with the lesser tuberosity, mobilisation of the bone fragment in a \"trap-door\" manner allowed easy access and visualisation of the glenohumeral joint. The humeral head was found to be dislocated posteriorly, the posterior labrum was pulled out from the glenoid and a layer of fibrous tissue covered the glenoid cavity (Figure ##FIG##1##2A##). After meticulous removal of the scar tissue, the glenoid articular cartilage looked to be in good condition and the humeral head was reduced using long Darrach retractors in combination with extra-articular pressure. However, the joint was unstable even with a few degrees of internal rotation. Using three Panalok RC (Mitek Products, Ethicon) absorbable anchors with number-2 polyester braided sutures, the posterior capsule and labrum were repaired to the posterior glenoid rim. The large reverse Hill-Sachs lesion was addressed with transfer of the fractured lesser tuberosity and its attached subscapularis muscle to the anteromedial defect according to McLaughlin's technique modified by Hawkins <italic>et al. </italic>[##REF##3805075##3##]. Aiming to restore the sphericity of the humeral head and enhance the healing process, the bone bed of the defect was augmented with demineralised bone matrix allograft (Grafton^®DBM Putty, Osteotech, Eatontown, NJ) and stable fixation of the lesser tuberosity was achieved with two partially threaded 4.0 mm titanium screws (Figure ##FIG##1##2B##). The greater tuberosity and anatomic neck fractures were subsequently stabilised using three screws of the same type. Repair of the rotator interval was the last step performed and routine closure of the wound over a drain was achieved.</p>", "<p>Postoperatively, the extremity was placed in a sling with the shoulder in neutral rotation and slight abduction. At 4 weeks, passive shoulder and pendulum exercises were initiated and the patient was advised to use the sling for another 4 weeks. At 8 weeks, a more aggressive physical therapy with active assisted range-of-motion and strengthening exercises was instituted as plane X-rays showed maintenance of joint congruency and early signs of bone healing. Despite the instructions for examination at regular intervals, the patient did not return for follow-up until two and a half years postoperatively. She reported that her shoulder was totally painless without any limitations during daily activities. She could actively elevate and abduct her arm 150° and 120°, respectively. In internal rotation, she reached the L2 vertebra and external rotation was 40°. Plane radiographs (Figure ##FIG##2##3A##) and CT scan (Figure ##FIG##2##3B##) confirmed a good clinical result and absence of devascularisation or instability of the humeral head.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>BEC prepared and submitted the article. PP collected and analysed the data while CGD critically revised the manuscript. Each author read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Posterior shoulder fracture-dislocation</bold>. A) Anteroposterior radiograph of the right shoulder showing the internally rotated humerus and the characteristic \"lightbulb sign\" of its proximal part. Both tuberosities have been detached from their anatomic position. B) Transthoracic lateral radiograph of the right shoulder demonstrates the posterior dislocation of the humeral head. C) Axial computed tomography (CT) scan of the right shoulder. A locked posterior fracture-dislocation is recognised. The anteromedial defect is close to 50% of the articular surface. Fracture comminution of both tuberosities and low bone density of the humeral head are also visible.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Intraoperative photographs of the right shoulder</bold>. A) Mobilisation of the fractured lesser tuberosity revealed the posterior dislocation of the humeral head and the \"empty\" glenoid fossa. B) Appearance of the right shoulder after open reduction and stabilisation of the lesser tuberosity to the anteromedial defect with two 4.0 mm titanium screws.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Postoperative radiological evaluation</bold>. A) Anteroposterior radiograph of the right shoulder at two and a half years postoperatively. The fractures have been nicely healed and the humeral head shows no signs of avascular necrosis or post-traumatic arthritis. B) At the same time, an axial computed tomography (CT) scan of the right shoulder demonstrates the well-centred humeral head over the glenoid fossa.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1752-1947-2-260-1\"/>", "<graphic xlink:href=\"1752-1947-2-260-2\"/>", "<graphic xlink:href=\"1752-1947-2-260-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Proximal humeral fractures are a common injury with an incidence of approximately 5% of all fractures, with the majority being secondary to blunt trauma in an elderly population [##UREF##0##1##]. Despite the close proximity of the axillary artery and the surgical neck of humerus, injury to this artery is a rare complication of proximal humeral fractures. It is, however, associated with significant risks to both function and viability of the affected upper limb.</p>", "<p>Upper limb ischaemia secondary to such a cause requires prompt intervention to restore blood flow and subsequently treat the primary cause. Earlier reports have documented success in similar settings, using modified equipment not necessarily designed for use as an intravascular shunt [##REF##1432831##2##,##REF##12045642##3##].</p>", "<p>We present a case of delayed presentation of axillary artery pseudoaneurysm following proximal humeral fracture and discuss the use of a Javid™ carotid shunt (Bard carotid shunt, 17F tapered to 10F; Bard^®Javid™ Carotid Shunts, Bard Ltd., Forest House, Brighton Rd., Crawley, West Sussex, UK) in maintaining vascular perfusion during open reduction and internal fixation of the fracture.</p>" ]

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[ "<title>Discussion</title>", "<p>Despite the fact that a significant proportion of fractures of the surgical neck of the humerus are displaced, axillary artery injuries secondary to these fractures are rare [##UREF##0##1##,##REF##9308598##4##, ####REF##3694729##5##, ##REF##12379386##6##, ##REF##16283573##7####16283573##7##]. The majority affect the third part of the artery, due to its position of relative immobility, being tethered by the subscapular and thoracromial arteries [##UREF##0##1##,##REF##7427051##8##]. Most of these injuries lead to thrombosis of the axillary artery and acute lower limb ischaemia [##REF##9308598##4##,##REF##3694729##5##,##REF##4008042##9##]. Pseudoaneurysm formation of the axillary artery is rare following blunt and penetrating trauma to the shoulder, often presenting late as a pulsatile mass rather than acute limb ischaemia [##UREF##0##1##,##REF##12379386##6##,##REF##16283573##7##,##REF##9602783##10##].</p>", "<p>Endovascular treatment with a covered stent graft has been reported previously and is the treatment of choice in patients with pseudoaneurysm of the axillary artery without upper limb ischaemia [##REF##16283573##7##,##REF##15599485##11##]. Due to the presence of propagating thrombus and displaced fracture requiring open reduction and internal fixation, endovascular treatment was not an option in this patient. Following proximal and distal arterial control and thrombectomy, the limb was revascularised temporarily using a Javid™ shunt, which allowed safe internal fixation of the fracture before bypass grafting. The insertion of the Javid™ shunt served to confirm the viability of the limb and adequacy of distal thrombo-embolectomy. The use of temporary shunting of peripheral vasculature in order to maintain distal vascular perfusion is rarely employed in civilian surgical practice [##REF##1432831##2##,##REF##12045642##3##], however, it has been gaining popularity in the management of military trauma [##REF##17033547##12##, ####REF##15874928##13##, ##REF##17382222##14####17382222##14##]. Recent reports from Belfast, whereupon the use of intraluminal shunts has been advocated for the early restoration of blood flow following complex lower limb vascular injuries, have shown significant benefits in averting the incidence of fasciotomy, contractures, ischaemic nerve palsy and amputations [##REF##16618547##15##]. This Belfast approach of early shunting allows for a disciplined surgical approach with adequate time for wound debridement, safe fracture fixation and optimal vascular reconstruction. Reports from Operation Iraqi Freedom suggest that vascular shunts can be used safely to bypass complex vascular injuries encountered in forward surgical units, in order to allow transfer of injured patients for definitive vascular assessment and reconstruction [##REF##17033547##12##,##REF##17382222##14##]. The use of vascular shunts in these circumstances was associated with very low limb amputation rates [##REF##17382222##14##], even in patients in whom the shunt had thrombosed in transit [##REF##17033547##12##].</p>", "<p>The Javid™ shunt has the advantage over other types of non-vascular shunt employed [##REF##1432831##2##,##REF##12045642##3##], in that it is specifically designed for use as a carotid artery shunt. It is manufactured out of soft, kink free material, which is tapered towards the ends which are bulbous in nature. This allows the shunt to be clamped in place around the artery, thereby providing stability whilst surgery continues. It was felt that the Javid™ shunt was superior to the Pruitt-Inahara^®carotid shunt (an H-shaped carotid shunt, held in place using inflatable balloons) for this patient due to its ease of use, lack of extra lumens (which would easily be caught and cause the shunt to be dislodged), ability to interconnect two shunts and its specially designed clamps to hold the shunt in situ during extensive and vigorous mobilisation of the fractured bone during reduction and fixation. Although these shunt clamps may cause more damage to the arterial lumen than the balloon of the Pruitt-Inahara^®shunt, this damaged segment of the injured artery would in turn be ligated and bypassed.</p>" ]

[ "<title>Conclusion</title>", "<p>This case highlights the usefulness of a Javid™ shunt, over other forms of shunt, in prompt restoration of blood flow to effect limb salvage. It can be considered as a temporary measure whilst awaiting definitive revascularisation which can be performed following fracture fixation.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Axillary artery injury is a rare but severe complication of fractures of the surgical neck of the humerus.</p>", "<title>Case presentation</title>", "<p>We present a case of axillary artery pseudoaneurysm secondary to such a fracture, in a 82-year-old white woman, presenting 10 weeks after the initial injury, successfully treated with subclavian to brachial reversed vein bypass together with simultaneous open reduction and internal fixation of the fracture. We discuss the use of a Javid™ shunt during combined upper limb revascularisation and open reduction and internal fixation of the fractured humerus.</p>", "<title>Conclusion</title>", "<p>This case highlights the usefulness of a Javid™ shunt, over other forms of vascular shunts, in prompt restoration of blood flow to effect limb salvage. It can be considered as a temporary measure whilst awaiting definitive revascularisation which can be performed following fracture fixation.</p>" ]

[ "<title>Case presentation</title>", "<p>An 82-year-old, white woman with a history of alcohol abuse, presented to the accident and emergency department with a 4-hour history of an acutely ischaemic right upper limb with motor and sensory deficit. A hard tender, pulsatile mass was palpable in the right subclavian area with significant bruising; there was a palpable right subclavian pulse with no pulses distal to this. X-ray revealed a fracture of the surgical neck of the right humerus with the humeral head abducted and externally rotated, while the humeral shaft was displaced medially (Fig. ##FIG##0##1##).</p>", "<p>Ten weeks previously, she had presented with a fracture of the surgical neck of the right humerus following a fall whilst under the influence of alcohol. On that occasion, sensory and motor function of the limb had been recorded to be fully intact by the medical staff in Accident and Emergency and there had been a full complement of pulses. Given she had no neuro-vascular deficit in the affected limb, the vascular surgeons were not involved initially. Under guidance of the orthopaedic surgeons, she had been treated conservatively with a collar and cuff due to her age and history of current alcohol abuse. She was to have been followed up fortnightly in the orthopaedic fracture clinic – but failed to attend after her second visit. She had no neuro-vascular deficit on follow-up. She denied any further falls or trauma to the right upper limb.</p>", "<p>The acute nature of the current presentation together with neurological compromise prompted classification as category-II acute limb ischaemia (Society for Vascular Surgery/International Society for Cardiovascular Surgery classification) [##REF##9308598##4##] and urgent angiography was performed with a view to revascularisation. This revealed a pseudoaneurysm of the third part of the right axillary artery with complete occlusion of the right brachial artery distal to this (Fig. ##FIG##1##2##).</p>", "<p>Operative treatment was undertaken with initial exposure and control of the subclavian artery above the clavicle (Fig. ##FIG##2##3A##). Simultaneous exposure of the brachial artery in the antecubital fossa was performed and a size 3 Fogarty embolectomy catheter passed distally down the brachial artery. Both radial and ulnar arteries were found to contain thrombus which was cleared with good back flow. The proximal brachial and distal subclavian arteries were ligated in continuity. Two interconnected Javid™ shunts were inserted to carry blood flow from the subclavian to the brachial artery in order to maintain perfusion (Fig. ##FIG##2##3B##) during open reduction and internal fixation of the fractured humerus, after which a subclavian to brachial bypass was performed using reversed long saphenous vein. The fracture was temporarily stabilised using external splints to immobilize the limb whilst securing vascular continuity.</p>", "<p>Postoperatively, the patient had strong radial and ulnar pulses with complete resolution of her motor and sensory dysfunction within 72 hours. Her postoperative course was uncomplicated and she was discharged on the 10th postoperative day. Early postoperative duplex scan performed at 6 weeks revealed satisfactory function of the vein graft.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>SAS was first assistant (subclavian exposure), carried out the literature review and constructed the manuscript. RM was first assistant (brachial exposure), photographer, carried out the literature review and drafted and editing the manuscript. AH was primary surgeon (brachial exposure), constructed the idea behind the case report, was senior editor of the manuscript (critical revisions) and gave final approval. GDG was primary surgeon (subclavian exposure), constructed the idea behind the case report, was senior editor of the manuscript (critical revisions) and gave final approval.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained retrospectively from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>" ]

[ "<title>Acknowledgements</title>", "<p>Written on behalf of the East of Scotland Vascular Network.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Anteroposterior view of right shoulder 10 weeks after the primary injury, revealing malalignment of fracture ends and attempts at formation of primary callus (arrow).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Catheter angiogram depicting pseudoaneurysm formation of third part of axillary artery with complete occlusion of the distal right brachial artery.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Supraclavicular exposure of the subclavian artery</bold>. (A) The phrenic nerve is retracted before the division of the scalenus anterior muscle. (B) The subclavian artery is exposed and ligated distally, with blood flow to the right arm being maintained with the aid of a Javid shunt during open reduction and internal fixation of the fracture.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1752-1947-2-259-1\"/>", "<graphic xlink:href=\"1752-1947-2-259-2\"/>", "<graphic xlink:href=\"1752-1947-2-259-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Myocardial abscess (MA) is a suppurative infection of the myocardium, endocardium, native or prosthetic valves, perivalvular structures or the cardiac conduction system. It is a potentially life-threatening disease, where early recognition and institution of appropriate medical and surgical therapy is vital for patient survival. The overall mortality rate associated with <italic>Staphylococcus aureus </italic>endocarditis is 42%. If treated with appropriate antibiotics and surgery, the mortality rate falls to 25%. The presence of an intracardiac abscess results in a 13.7-fold increase in mortality. In the past, most cases of MA were found during autopsy; however, detection of MA can now be achieved antemortem, using noninvasive diagnostic modalities including transthoracic echocardiography (TTE), transoesophageal echocardiography (TOE), radionuclide scintigraphy, computed tomography (CT) scan and cardiac magnetic resonance imaging (CMRI).</p>" ]

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[ "<title>Discussion</title>", "<p>MA has been reported in about 20% of patients with IE [##REF##696668##1##]. They are usually adjacent to the area of valve infection and represent a direct extension of the infection [##REF##1277418##2##]. Rarely, embolization of septic material results in a metastatic myocardial abscess remote from the main focus [##REF##11525359##3##, ####REF##9639010##4##, ##REF##11515915##5##, ##REF##3389387##6####3389387##6##], as was the case here. The normal appearance of left-sided valves and concurrence of abscesses in extracardiac organs lead us to the conclusion that the left-sided MA had occurred as the result of embolization from the right-sided endocarditis.</p>", "<p>Antemortem diagnosis of MA remains a challenge and a high index of clinical suspicion is required. TTE has a sensitivity of 23% and specificity of 98.6% in diagnosing MA [##REF##8789176##7##]. At present, TOE is considered the investigation of choice but a recent prospective study of 115 patients revealed that it has only 48% sensitivity in diagnosing MA [##REF##17967599##8##]. CMRI is a noninvasive imaging modality with high temporal and spatial resolution. To the best of our knowledge, no studies have compared the diagnostic value of TOE and CMRI in such cases. However, there are a few case reports and studies which suggest good diagnostic yield with CMRI in diagnosing annular abscess [##REF##12638334##9##], sub-valvular abscess [##REF##11449169##10##] and pseudo-aneurysm [##REF##2028858##11##] in the setting of complicated IE. There may also be a complementary role for radionuclide imaging in diagnosing MA, where it can reveal a focal area of increased uptake in the myocardium suggesting the location of an abscess. It has low sensitivity but can be helpful in cases of prosthetic valve endocarditis where echocardiography may show too much scatter. Different radioisotopes including gallium-67, technetium-99 and indium-111 have been used in clinical practice with variable success [##REF##9735977##12##,##REF##10230285##13##].</p>", "<p>Patients with this lethal disease can be saved by aggressive antibiotic treatment and prompt surgical intervention [##REF##11515915##5##]. This can be best achieved by multidisciplinary care involving cardiologists, microbiologists, cardiac radiologists and cardiothoracic surgeons. Urgent surgery is recommended in most cases of MA since the perioperative risk and chances of rupture increase with the delay to surgery. However, the decision to perform emergency (same day) or urgent (1–2 days) surgery has to be made in individual cases depending on the clinical status of the patient, size of the abscess and thickness of the abscess wall. CMRI can provide useful morphological evaluation to help make this decision [##REF##11449169##10##].</p>" ]

[ "<title>Conclusion</title>", "<p>In conclusion, MA is a life-threatening illness. A high index of clinical suspicion is required to make a prompt diagnosis. Final diagnosis may need multimodality imaging. Many of these patients may present to district hospitals where appropriate imaging and surgical facilities may not be available, and an urgent transfer to a specialist cardiothoracic centre is imperative. An early diagnosis, aggressive medical therapy, multidisciplinary care and timely surgical intervention may save the patient's life in this otherwise fatal condition.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Myocardial abscess is a rare and potentially fatal condition. Metastatic myocardial abscess in the setting of infective endocarditis has been infrequently reported in the medical literature. To the best of the authors' knowledge no case of myocardial abscess affecting the free wall of the left ventricle secondary to infective endocarditis of a right-sided heart valve has been reported previously.</p>", "<title>Case presentation</title>", "<p>We report a case of tricuspid valve endocarditis caused by <italic>Staphylococcus aureus </italic>and resulting in a myocardial abscess on the posterior wall of the left ventricle, far from the active valvular infection. We also briefly discuss the role of different investigation modalities including cardiac magnetic resonance imaging in diagnosing myocardial abscess.</p>", "<title>Conclusion</title>", "<p>Myocardial abscess is a life-threatening illness. A high index of clinical suspicion is required to make a prompt diagnosis. Final diagnosis may need multi-modality imaging. An early diagnosis, aggressive medical therapy, multidisciplinary care and timely surgical intervention may save life in this otherwise fatal condition.</p>" ]

[ "<title>Case presentation</title>", "<p>A 28-year-old intravenous drug user was admitted in a district general hospital with a 2-week history of fever, malaise and myalgia. He had no past medical history of note. On examination he was pyrexial but haemodynamically stable. His cardiovascular examination revealed signs of tricuspid regurgitation. His respiratory, abdominal and neurological examination was normal. Clinically, the diagnosis of infective endocarditis (IE) was suspected. Three sets of blood cultures were drawn and empirical intravenous antibiotic treatment commenced.</p>", "<p>His blood tests showed leukocytosis with predominant neutrophilia and mild normochormic, normocytic anaemia. His electrocardiogram revealed non-specific ST-changes but no conduction abnormality. His chest X-ray was unremarkable. TTE confirmed vegetations on the tricuspid valve with severe regurgitation. All other valves were normal. Blood cultures grew <italic>S. aureus </italic>and vigorous antibiotic treatment was continued appropriately. However, the patient's condition continued to deteriorate with spiking fever and raised inflammatory markers. He was referred to the regional cardiothoracic centre for evaluation of valve surgery in view of uncontrolled infection.</p>", "<p>On arrival at the cardiothoracic centre, the patient was acutely unwell with a temperature of 38.5°C, pulse of 120 beats per minute, blood pressure of 100/70 and respiratory rate of 26 breaths per minute. He had signs of severe tricuspid regurgitation and right heart failure. His repeat chest X-ray showed multiple cavitating lesions depicting metastatic pulmonary abscesses. There was also evidence of splenic abscesses on his abdominal ultrasound scan. Repeat TTE confirmed vegetations on the tricuspid valve with severe regurgitation but additionally it showed a small echo-free space in the wall of the left ventricle, raising suspicion of an MA (Figure ##FIG##0##1##). TOE was planned to evaluate this further but the patient was unable to tolerate it. Urgent CMRI was obtained, which revealed a 4.5 cm diameter left ventricular posterior wall abscess contained by only a 2 mm thin layer of myocardium (Figure ##FIG##1##2## and Additional file ##SUPPL##0##1##). Urgent surgical intervention was planned but, unfortunately, the patient had a cardiac arrest prior to surgery and could not be resuscitated.</p>", "<title>Abbreviations</title>", "<p>CMRI: cardiac magnetic resonance imaging; IE: infective endocarditis; MA: myocardial abscess; TOE: transoesophageal echocardiography; TTE: transthoracic echocardiography.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>JI collected data, performed the literature search and drafted the manuscript. IA was involved in the literature search and manuscript review. WB supervised this patient's care and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient's next of kin for publication of this case report and the accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>We are grateful to Professor Mohan Sivananthan, Leeds General Infirmary, for providing expert opinion on MR images and Dr Fizah Shafiq, University of Brunel, for proofreading the manuscript.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Transthoracic echo – Short axis view showing abscess cavity.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Cardiac magnetic resonance imaging</bold>. A 4.5 cm diameter left ventricular posterior wall abscess contained by only a 2 mm thin layer of myocardium.</p></caption></fig>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Cardiac magnetic resonance imaging of our patient showing morphological features of myocardial abscess.</p></caption></supplementary-material>" ]

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[ "<graphic xlink:href=\"1752-1947-2-258-1\"/>", "<graphic xlink:href=\"1752-1947-2-258-2\"/>" ]

[ "<media xlink:href=\"1752-1947-2-258-S1.mpg\" mimetype=\"video\" mime-subtype=\"mpeg\"><caption><p>Click here for file</p></caption></media>" ]

{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Dermal sinus tracts are rare congenital lesions located in the midline characterized by a cutaneous pit or dimple. They are defined as developmental anomalies in which the end result can be abnormal communication between the dermis and intracranial structures. They incorporate a tract of cutaneous ectoderm from the dorsal midline skin that extends for a variable distance into the underlying mesenchymal tissue and in many instances penetrates the dura to end within the thecal sac adjacent to, or continuous with the neural tube [##REF##12949296##1##]. Sinuses may be asymptomatic or present clinically with varying degrees of drainage from their cutaneous openings, recurrent bouts of septic or aseptic meningitis, or mass effect on the cerebrospinal fluid (CSF) pathways and consequent hydrocephalus [##REF##17328285##2##].</p>", "<p>These lesions are almost always solitary and co-existence of double dermal sinuses has not been reported previously. We report a girl with asymptomatic double dermal sinuses.</p>" ]

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[ "<title>Discussion</title>", "<p>Congenital dermal sinus is a rare entity (1/2500), and consists of a tract lined by stratified squamous epithelium [##UREF##0##3##,##UREF##1##4##]. It is often detected at birth, and usually by the end of the first year. Typically, the pediatrician notes a midline dimple or cutaneous defect, containing one or more hairs [##UREF##2##5##].</p>", "<p>The most widely accepted theory regarding the embryogenesis of dermal sinus tracts and related anomalies proposed that they arise through faulty separation of the neuroectoderm from the overlying cutaneous ectoderm at the time of dysjunction between the third and eighth week of gestation [##REF##12949296##1##,##UREF##2##5##,##REF##11593240##6##].</p>", "<p>They have been reported all along the midline neuroaxis, from the nasion and occipital area down to the lumbar and sacral regions [##UREF##0##3##], most frequently in the lumbar and lumbosacral region (75%) and only 1% of all tracts along the spine are cervical [##REF##11593240##6##,##UREF##3##7##].</p>", "<p>Cranial sinuses are less frequent than their counterparts in the spinal region in which 85% are located near the external protuberance of the occipital bone, 11% at the nasion and 5% at the posterior parietal area [##REF##17328285##2##]. The sinus tract may end in subcutaneous tissue or extend any distance inward to its ultimate embryological terminus, which is the conus medullaris for lesions in the lumbosacral region or the central canal of the spinal cord for tracts at the thoracic or cervical level [##UREF##0##3##].</p>", "<p>Approximately one-half of the tracts terminate in a dermoid (83%) or epidermoid (13%) cyst or a teratoma (4%). The slow growth rate of these tumors often masks their presentation for years, although there are some patients with acute neurological deterioration [##UREF##0##3##,##REF##12854760##8##].</p>", "<p>A wide spectrum of clinical manifestations can occur ranging from asymptomatic dermal sinus to serious complications. There is no apparent timeframe for an asymptomatic lesion to later become symptomatic [##UREF##4##9##]. This process is believed to be benign until an episode of meningitis [##UREF##2##5##]. Meningitis resulting from dermal sinus tracts may occur at any age and is seen in infants and elderly patients. Patients may present with concomitant infections of the dermal sinus tract and underlying inclusion cysts [##REF##12854760##8##].</p>", "<p>MRI has become the reference study technique because of its ability to accurately depict the extent of the sinus tract and associated lesions [##REF##12854760##8##].</p>", "<p>Therapy is almost always surgical. The goal is obliteration of the tract with elimination of the communication between the skin and the neural structures. The earlier the lesion is detected and corrected, the less likely any long-term morbidity [##UREF##2##5##].</p>", "<p>The process of dysjunction occurs after closure of the neural tube at a time between the third and eighth week of gestation, whereas it develops during the third to fifth week of intrauterine life in the cranium. At the same time, the cutaneous portion of the neuroectoderm separates and fuses in the midline to form the overlying integument. Disorders of this process may lead to midline dermal anomalies such as dermal sinus tracts and inclusion cysts [##UREF##2##5##,##REF##11593240##6##,##REF##12854760##8##].</p>" ]

[ "<title>Conclusion</title>", "<p>We described a rare case of double dermal sinus. To our knowledge, there is no report of double or multiple dermal sinuses in the literature. Regarding the similar range of gestational age for dysjunction in the spinal and cranial neural tube, the occurrence of double dermal sinuses in one person, of which one is cranial and the other cervical, suggests that there is an underlying cause which affects separation of the neuroectoderm at an early gestational age during the third to eighth week of gestation.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Dermal sinus tracts are rare congenital lesions located in the midline characterized by a cutaneous pit or dimple. They occur all along the midline neuroaxis, from the nasion and occipital area down to the lumbar and sacral regions, most frequently in the lumbar and lumbosacral region.</p>", "<title>Case presentation</title>", "<p>Here we report a 5-year-old girl who presented with occasional headache. There were two dimples, one on the dorsal aspect of her head and another on her neck.</p>", "<title>Conclusion</title>", "<p>Dermal sinuses are almost always singular and the co-existence of double dermal sinuses has not been reported previously.</p>" ]

[ "<title>Case presentation</title>", "<p>This 5-year-old girl presented with occasional headache. She was the first child of nonconsanguineous parents without significant past medical history. On physical examination, the child was totally normal neurologically and generally. There were two dimples on the dorsal aspect of her head and neck. A fine dimple was noted at the midline occipital area above the inion, surrounded by a small smooth hairless area, harboring a few thick black hairs at the ostium without any discharge (Fig. ##FIG##0##1a##). The other dimple was at the midcervical area with a large mouth and hemangiomatous skin discoloration around the dimple (Fig. ##FIG##0##1b##). Brain magnetic resonance imaging (MRI) was performed, which was normal without bone defect and intracranial sinus or tract. Cervical MRI showed the sinus at the level of the C3–C4 vertebra with a tract ending before the spinal canal (Fig. ##FIG##1##2##). She had not experienced any previous infection and there was no intradural extension for both lesions, therefore further investigation or procedure was not done.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>All authors have contributed to the study and manuscript preparation.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient's next of kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>A: Photograph (posterior view) of the child's head showing a small opening in the midline occipital area <italic>(arrow) </italic>above the occipital protuberance and B: midcervical area.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Sagittal T1-weighted MR image showing the opening of the dermal sinus at the level of C3–C4 and the extension of the tract outside the spinal canal.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1752-1947-2-281-1\"/>", "<graphic xlink:href=\"1752-1947-2-281-2\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Fibroepithelial polyps (FEPs) are the most common benign lesions of the ureter. Most occur in the ureter and renal pelvis in adult patients, while a few occur in the posterior urethra or bladder, generally in children [##REF##11847584##1##]. However, FEPs of the ureter accompanied by calculi are rare. We review our experiences with three patients having FEP associated with calculi in the distal ureter to define this entity more clearly and its outcome following treatment.</p>" ]

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[ "<title>Conclusion</title>", "<p>Ureteral FEP was first reported in 1932 [##UREF##0##2##], and since then, there have been approximately 236 scientific papers on fibroepithelial polyps. Fibroepithelial polyps are rare, benign, mesodermal tumors of the urinary tract that are histologically composed of fibrous stroma covered with a transitional urothelium. They are considered the most common benign lesions of the ureter among other benign lesions such as leiomyomas, lymphangiomas, and neurofibromas. They are often smoothly marginated and cylindrical, sessile, or even frond like [##REF##11847584##1##]. Because of their histologic organization, FEPs are classified as benign hamartomas; however, malignant degeneration and cystic transformation have also been reported [##REF##14624899##3##,##REF##7754889##4##].</p>", "<p>Fibroepithelial polyps commonly present in adults in the third to fifth decades with a male-to-female ratio of 3:2. In adults, most FEPs occur in the ureter; 62% of these polyps are located in the upper ureter or uretero-pelvic junction, 15% are in the renal pelvis, and a small percentage is in the bladder or posterior urethra [##REF##11847584##1##]. Fibroepithelial polyps of the lower urinary tract usually occur in the posterior urethra, most often in children. They usually appear as solitary polyps; however, rare cases of multiple and bilateral appearances have been reported [##REF##14624899##3##,##REF##15661062##5##].</p>", "<p>Although the etiology of FEPs is unclear, they are thought to be either congenital slow-growing lesions or lesions that develop as a result of chronic urothelial irritants, such as infection, inflammation, calculi, or obstruction. The most significant signs and symptoms of the polyps are hematuria and flank pain [##REF##11847584##1##]. The pain is characteristically intermittent and colicky due to partial obstruction. Urinary frequency, dysuria, and pyuria are other less common findings [##REF##11847584##1##].</p>", "<p>On IVU or retrograde urograms, FEPs appear to be long, smooth ureteral filling defects; their position may change between images; and they are associated with varying degrees of hydronephrosis [##REF##11555017##6##]. It is important to distinguish FEPs from upper urinary tract carcinomas because management and prognosis can be significantly different. Debruyne and associates reported that unnecessary nephroureterectomies were performed in 42 of 112 patients (37%) with FEP because of an uncertain pre-operative diagnosis [##REF##7414778##7##].</p>", "<p>In the past, management of FEP was excision of the polyp and reanastomosis with an open procedure. Recently, with the advent of ureteroscopes, minimally invasive endoscopic treatment has become more popular. Usually, the polyps are grasped with forceps for traction and resected over the root area through the ureteroscope, and the base is fulgurated to prevent recurrence. The holmium:YAG laser is another modality for endoscopic resection. Carey and Bird successfully ablated multiple polyps in one ureter by using the holmium laser, and removed each polyp from the ureteral wall with grasping forceps. Also, ureteral stones are removed concurrently with a basket and a ureteral access sheath is used to facilitate the multiple passes of the ureteroscope and the removal of the polyps and stones from the proximal ureter [##REF##7941202##8##]. Percutaneous antegrade excision should be available for treating polyps in the renal pelvis and the upper ureter [##REF##14624899##3##]. Laparoscopic surgery might be preferred over open surgery when the polyps are too large to be fully cleaned by endoscopic surgery.</p>", "<p>Although close follow-up was recommended in the literature because of the risk of recurrence, the duration and frequency of follow-up are not clear. Although some studies have suggested cytological evaluation of urine in the postoperative follow-up, we do not agree with this because of the benign nature of FEP. Some studies have suggested control ureteroscopy associated with IVU in the follow-up [##REF##7941202##8##]. We think that yearly IVU should be helpful in the follow-up after the initial IVU 2 to 3 months after the endoscopic resection of FEP. However, the follow-up period might be changed depending on the clinical progression, signs and symptoms of the disease. Interestingly, although fibroepithelial polyps were solitary in children, in the cases presented here they were multiple, located in the distal part of the ureter and associated with an adjacent ureteral calculus; however, this scenario has rarely been reported in the literature. There have only been three prior case reports of ureteral FEP with adjacent urolithiasis in adults [[##REF##7941202##8##,##REF##9413761##9##], 10], and long-term or repeated inflammation of ureteral tissue by urinary crystals, calculi, stents, and infections has been implicated.</p>", "<p>Since chronic irritation has been reported to be an etiological factor in fibroepithelial polyps, calculi in the ureter may be responsible for the formation of FEP; however, the opposite might also be true. In the cases presented here, urinary retention and chronic irritation due to the obstruction formed by the ureteral stones might have been responsible for the ureteral polyps, but the accepted theory in the literature is stone formation due to urinary retention caused by FEP. In both situations, endoscopic management of FEP and calculi is safe, effective, and minimally invasive. We reviewed our experience in cases of FEP associated with calculi in the distal ureter and attempted to define this entity and its outcome more clearly after treatment. When a calculus and an accompanying polypoid mass are detected in the ureter, FEP, which is histologically benign in nature, should be kept in mind in differential diagnosis and both polyps and calculi should be managed at the same time, if possible.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Fibroepithelial polyps of the ureter are benign tumors arising from the mesodermal tissue in the ureteral wall. Their etiology remains unknown. Hematuria and obstructive urinary symptoms are the most common findings. The treatment of choice is endoscopic resection, and the prognosis for patients with these lesions is excellent.</p>", "<title>Case presentation</title>", "<p>We present three cases of fibroepithelial polyps associated with calculi in the distal part of the ureter. The patients were all women, aged 20, 45 and 52 years. Two patients were suffering from flank pain and dysuria while one patient was asymptomatic at the time of diagnosis. The patients were fully treated with endoscopic resection. To the best of our knowledge, this is the fourth report of adult ureteral fibroepithelial polyps associated with ureteral calculi in the English literature. The etiology, clinical features, diagnosis, and management of fibroepithelial polyps are discussed in this report.</p>", "<title>Conclusion</title>", "<p>Whenever polypoid lesions are detected especially at the distal part of the ureter, benign fibroepithelial polyps should be kept in mind for differential diagnosis. Additionally, although rarely seen, the co-existence of ureteral calculi with fibroepithelial polyps should be borne in mind.</p>" ]

[ "<title>Case presentation</title>", "<title>Case 1</title>", "<p>A 20-year-old woman presented to our clinic with intermittent right flank pain and dysuria. She had undergone an extracorporeal shockwave lithotripsy for a kidney stone 5 months before the current admission. Urine analysis showed mild microscopic hematuria. Intravenous urography demonstrated a 12-mm calculus in the right distal ureter, focal ureteral dilatation in the proximal part of the ureter, smoothly marginated tubular filling defects, and mild hydronephrosis. Also, there was another irregular filling defect at the level of the right ureteral orifice (Fig. ##FIG##0##1##). On cystoscopic examination of her bladder, there were two polypoid masses, grayish-white in color, about 2 cm long and 0.5 cm wide, prolapsing into the bladder from the right ureteral orifice with a thin stalk (Fig. ##FIG##1##2##). A urine sample was taken for immediate cytology and the polyps were grasped with forceps for traction and resected over the root area through the ureteroscope and manipulated for frozen biopsy. The results of the urine cytology were negative, and the analysis of the frozen section demonstrated a benign FEP. Then the ureteroscope was inserted into the right ureter through the guide wire and the calculus was fragmented by pneumatic lithotripsy and fragments were extracted with a basket. About 1 cm proximally to the calculi, multiple millimetric polypoid structures were exposed and all of them were resected as described above. The bases of the polyps were coagulated with an electrode to stop bleeding and prevent recurrences. A ureteral stent was placed to provide urine drainage and to avoid probable obstruction caused by edema, then removed 24 hours later.</p>", "<p>The final pathology report showed FEP. Histologically, the polyps had a core of loose fibrovascular stroma covered by a layer of normal transitional uroepithelia, associated with edema, congestion, and mononuclear cell infiltration. No significant cellular atypia or cytologic abnormality was observed.</p>", "<title>Case 2</title>", "<p>A 45-year-old woman presented to our clinic with left flank pain, dysuria, and hematuria. <italic>Escherichia coli </italic>was detected in a urine culture and treated with levofloxacin (500 mg PO q.d.) for 2 weeks. An intravenous urogram (IVU) showed two 0.6 cm calculi in the left distal ureter, a tubular filling defect proximal to the calculi, and moderate hydronephrosis.</p>", "<p>A left ureteroscopy showed a polypoid mass, grayish in color, about 1 cm long and 0.3 cm wide, with a thin stalk that projected into the lumen in the distal ureter. A urine sample was obtained for cytology, and then the polyp was grasped with forceps for traction and resected over the root area through the ureteroscope and manipulated for frozen biopsy. Urine cytology was negative and frozen biopsy results were in favor of benign FEP. The base of the polyp was coagulated by an electrode to stop bleeding and prevent recurrences. Then, pneumatic lithotripsy was performed, the resultant fragments were extracted, and double J stents were inserted for 4 weeks. The final pathology report revealed a FEP (Fig. ##FIG##2##3##).</p>", "<title>Case 3</title>", "<p>A 52-year-old woman presented to our clinic with an asymptomatic left distal ureteral stone diagnosed incidentally. IVU showed a calculus 12 mm long and located in her left distal ureter without any ipsilateral urinary drainage, indicating no renal dysfunction and right renal calculi. On cystoscopic examination, 2 polyps were detected, 5 mm in diameter and prolapsing into the bladder from the left ureteral orifices. The polyps were resected in the same way as described above and the calculus was managed with ureteroscopic lithotripsy. The final pathology report revealed a FEP.</p>", "<p>IVU was performed in all three patients 2 months after the operation; no pathological finding was detected except focal dilation in the distal ureter in Case 1 and residual dilatation in the ureter and the kidney in Cases 2 and 3.</p>", "<p>All patients subsequently underwent annual radiographic follow-ups with IVU, and no recurrences were detected.</p>", "<title>Abbreviations</title>", "<p>FEP: Fibroepithelial polyp; IVU: Intravenous urography.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>TT reviewed the literature, conceived and drafted the manuscript. BK and TC examined the patient, helped to record the data and prepare the manuscript. All of the authors read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patients for publication of this case series and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>" ]

[ "<title>Acknowledgements</title>", "<p>Sezgin Guvel is thanked for his help in editing and reviewing the manuscript. The authors declare that no funding has been received for the preparation of the manuscript.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Intravenous urographic demonstration of fibroepithelial polyps in distal ureter with filling defects</bold>.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Cystoscopic visualization of fibroepithelial polyps prolapsing into the bladder from the right ureteral orifice</bold>.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Fibroepithelial polyp of ureter that has a loose fibrovascular stroma (A) (hematoxylin and eosin, ×40) and covered by transitional epithelium (B) (hematoxylin and eosin, ×200).</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1752-1947-2-280-1\"/>", "<graphic xlink:href=\"1752-1947-2-280-2\"/>", "<graphic xlink:href=\"1752-1947-2-280-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Chronic lymphocytic leukemia of the B-cell-lineage (B-CLL) is the most common form of adult leukemia and predominantly a disease of older individuals. Due to the strong heterogeneity in the clinical course of B-CLL with survival ranging from months to decades, treatment regimens are strongly based upon clinical staging. Although recent data show that cytogenetic profiling of tumor cells and flow-cytometry characterization of certain surface and intracytoplasmic proteins have strong predictive value, treatment regimens are still strongly influenced by clinical parameters such as clinical presentation, laboratory values or lymphocyte doubling time [##REF##16304392##1##].</p>", "<p>CLL is generally treated at the onset of symptomatic disease, and initial treatment includes alkylator therapy (chlorambucil or cyclophosphamide) or purine nucleoside analogs, such as fludarabine alone or in combination with cyclophosphamide. Rituximab is a humanized murine monoclonal antibody directed against the B-cell surface protein CD20 and is active against most B-lineage lymphoid malignancies, including CLL. Initially, the use of rituximab in CLL was considered unsafe because of severe toxic reactions from tumor cell lysis in patients with very elevated blood cell counts [##REF##10498591##2##]. Here we describe a patient with B-CLL, who did not respond to prior chlorambucil and fludarabine chemotherapy developing a marked marrow toxicity to fludarabine, but was successfully treated with rituximab.</p>" ]

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[ "<title>Discussion</title>", "<p>In this report, we describe the remarkable clinical response of a patient with rapidly progressive B-CLL and poor response to standard chemotherapy and with successful treatment with rituximab monotherapy. Because, so far, there is no clinical evidence favoring early chemotherapeutic treatment of B-CLL, therapy is not usually considered until evidence of disease progression is observed. With the patient presented here, there was no indication for treatment at first presentation, where we diagnosed B-CLL stage Rai 0/Binet A. According to current standards, treatment was delayed until a fast lymphocyte doubling time and rapid progression of tumor mass were observed. The general prognosis of this patient was unclear, as genotyping revealed a deletion in the V_Hgene locus. Translocations in this chromosomal region are frequently found in patients suffering from multiple myeloma and are associated with a poor prognosis. The meaning of del 14q32 mutations in B-CLL is not yet clear [##REF##15763978##3##].</p>", "<p>Although fludarabine as monotherapy or in combination with cyclophosphamide appears to be a highly effective regimen in CLL, many patients are still treated with chlorambucil as a first line therapy.</p>", "<p>The complete lack of response to chlorambucil in our patient suggests an aggressive course of the disease, which was indeed documented by the rapid progression and deterioration of all hematological parameters.</p>", "<p>In this setting, fludarabine is usually a valid therapeutic option. Although the incidence of autoimmune hemolysis or thrombocytopenia is well recognized, this therapy was accompanied by severe marrow toxicity, with virtual disappearance of normal leucocyte precursors and megakaryocytes on a bone marrow aspirate, as well as by a lack of efficacy. Although pulmonary infections are still a dangerous consequence of the secondary humoral immune deficiency in CLL, the frequency and severity of the infections in our patient were certainly due to a combined humoral and cellular immune defect.</p>", "<p>The severity of the chemotherapy-induced marrow aplasia was surprising, and could be due to both a toxic effect, a lack of effect on CLL infiltration, or both. A further possibility would include a severe autoimmune reaction against all three lineages of hematopoiesis, including early precursors (i.e., amegakaryocytic thrombocytopenia).</p>", "<p>In our opinion, further chemotherapy was particularly dangerous in this patient with extremely reduced general conditions, an active pulmonary infection and functional pancytopenia. Therapy with alemtuzumab was considered, but preference was given to rituximab because of the less severe compromise of the cellular immune system by rituximab, and because of the possibility that autoimmune phenomena might play a role in the functional marrow aplasia.</p>", "<p>With rituximab monotherapy, response rates of 51% and 25% have been described as first-line treatment [##REF##12721250##4##] and in patients with several pretreatments [##REF##11520778##5##], respectively. The main drawback of rituximab monotherapy observed so far is the limited response to the induction therapy in pretreated patients [##REF##11520778##5##]. Hainsworth and coworkers did report that patients with small lymphocytic lymphoma (SLL) and CLL who had shown an initial response or stable disease after rituximab induction therapy could be successfully retreated at 6-month intervals [##REF##12721250##4##], but additional follow-up is required to fully assess the impact of this treatment strategy. Recently, we reported the efficacy and feasibility of a response-adjusted rituximab maintenance therapy in 12 patients with pretreated B-CLL [##UREF##0##6##].</p>", "<p>It is important to underline that the response to rituximab became apparent when the dosage of the drug was increased to a much higher level, according to a publication by the Keating group [##REF##11304768##7##] with dosages up to 2.25 g/m²in CLL patients. Both the weak expression of CD20 and the extremely elevated tumor burden of CLL patients (and certainly of the patient in this case) might explain the need for the higher dosage.</p>" ]

[ "<title>Conclusion</title>", "<p>Besides demonstrating the excellent response to rituximab in CLL, this case further suggests that maintenance therapy appears useful and feasible in CLL patients, which is in accordance with a recent report from our institution [##UREF##0##6##].</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Treatment of chronic lymphocytic leukemia of the B-cell-lineage is strongly based upon clinical staging because of the heterogeneous clinical course of this disease.</p>", "<title>Case presentation</title>", "<p>We describe a 62-year-old patient with newly diagnosed chronic lymphocytic leukemia of the B-cell-lineage who did not respond to several chemotherapy regimens including chlorambucil, fludarabine and cyclophosphamide, developing a marked neutropenia and thrombocytopenia with life-threatening infections. Further chemotherapy appeared not feasible because of bone marrow toxicity. The patient was treated with 600 mg/m²rituximab weekly followed by eight courses of biweekly therapy and then by long-term maintenance therapy, achieving almost complete remission of the symptoms and disease control.</p>", "<title>Conclusion</title>", "<p>After resistance to standard chemotherapy with chlorambucil and fludarabine, a patient with chronic lymphocytic leukemia of the B-cell-lineage was successfully treated with rituximab.</p>" ]

[ "<title>Case presentation</title>", "<p>A 62-year-old Caucasian woman (61 kg/160 cm) was first diagnosed with B-CLL stage Binet A/Rai 0 in July 2002. She showed marked leukocytosis with a white blood cell (WBC) count of 43.35 × 10⁹/l (82% lymphocytosis), accompanied by slight anemia (hemoglobin 11.1 g/dl) and a platelet count of 129 × 10⁹/l. Flow cytometric immunophenotyping (Fig. ##FIG##0##1##) of peripheral blood identified a CD5/CD19/CD20/CD23-positive clonal B lymphocyte population with kappa light chain expression and sIgM expression. CD10/CD103/CD38/CD43/CD22 and FMC7 were all negative. A chromosomal fluorescence in situ hybridization (FISH) analysis revealed del 14q32.</p>", "<p>Upon this first presentation, the patient's general conditions were good, there was no peripheral lymphadenopathy or hepatosplenomegaly detectable, hepatic and routine laboratory parameters were in the normal range, plasma proteins as detected by electrophoresis and immunofixation were without pathological findings. As a consequence, a watch and wait approach was chosen.</p>", "<p>Upon reevaluation in December 2002, a lymphocyte doubling time of less than 6 months was found, the patient had developed splenomegaly and cervical as well as axillary lymphadenopathy. The patient received five courses of chlorambucil until May 2003, but did not show significant regression of tumor lesions. Leukocytosis (230 × 10⁹/l) and nonhemolytic anemia (8.3 g/dl Hb) further deteriorated, accompanied by a 10% weight loss. Platelet counts remained normal at this time (174 × 10⁹/l). Administration of packed red cell transfusions was necessary. During the course of this first-line therapy, the patient developed a purulent bronchitis in March 2003 and shortly later, pneumonic pulmonary infiltrations.</p>", "<p>Fludarabine monotherapy was started in June 2003 at a dosage of 25 mg/m²day for 4 days. After the first cycle, the patient developed fever, a secondary antibody deficiency and a Coombs-positive severe anemia without detectable hemolysis. In addition, protracted thrombocytopenia developed. The patient became transfusion dependent for erythrocytes and platelets (Fig. ##FIG##1##2##).</p>", "<p>Upon immunoglobulin substitution, the therapy with chlorambucil was reassumed. Until September 2003, the patient received three courses of chlorambucil (0.18 mg/kg for 10 days). Under this regimen, the CLL progressed to Binet stage C with subtotal bone marrow infiltration by mature lymphocytes with almost no residual hematopoietic activity (absolute granulocyte count 140/μl). Leukocyte count was high (210 × 10⁹/l). Bone marrow aspiration showed subtotal bone marrow infiltration with displacement of normal hematopoiesis. At this time, a fludarabine/cyclophosphamide (FC) combination chemotherapy was started. After the first cycle, the thrombocytopenia did not improve, and the patient experienced a retinal bleeding. There was no response toward CLL-related symptoms. As a further complication indeed, the patient developed a severe life-threatening abscessing pneumonia that was empirically treated with different broad-spectrum antibiotics including linezolid and additional amphotericin B. Cultures from sputum, bronchoscopy and blood were repeatedly negative.</p>", "<p>Considering the remaining therapeutic options and the risks of any further aggressive chemotherapy in a severely pancytopenic patient, rituximab monotherapy was started with a weekly dose of 375 mg/m². After a hospitalization of 35 days, the patient could be dismissed with improved clinical condition, still transfusion-dependent.</p>", "<p>A few weeks later, pneumonia reactivation was diagnosed, accompanied by cholecystitis requiring hospitalization and new antibiotics treatment. Leukocyte count at this time was 20,000/μl with agranulocytosis (neutrophils 30/μl). Broad-spectrum antibiotics were given, and rituximab therapy was given at an increased dosage of 600 mg/m²weekly × 5, then 600 mg/m²biweekly × 8 without any other cytotoxic drugs.</p>", "<p>During this therapy of intensified rituximab, the patient became transfusion-free. Twelve weeks after the first dose of 600 mg/m²rituximab, anemia abated to a hemoglobin-level of 9.5 g/dl, platelet count progressively rose to 136 × 10⁹/l and leukocyte count decreased to 5.08 × 10⁹/l. Upon normalization of platelets in April 2004, the rituximab dose was reduced to 375 mg/m²in a 4-week schedule while maintaining the immunoglobulin substitution. This regimen was sustained 20 times until November 2005. During this therapy, the hematopoiesis recovered completely, with the hemoglobin level reaching 11.9 g/dl, a WBC count of 4.38 × 10⁹/l, and a platelet count of 1.38 × 10⁹/l. Both the lymphadenopathy as well as the splenomegaly regressed completely. Over the entire term of maintenance therapy, treatment of pulmonary infections by oral antibiotics became necessary three times but could be performed at the outpatient level.</p>", "<p>Due to the good condition of the patient, rituximab was tapered, with two administrations of 375 mg/m²in January and in April 2006. Since WBCs started to rise again 7 months after the last administration, rituximab maintenance therapy was reassumed in November 2006 at a dosage of 375 mg/m²every 3 months. The general condition of the patient remained good. In March 2007, hemoglobin was stable at 12.9 g/dl, leukocyte count was 11 × 10⁹/l (with 58% CD19+CD20+CD23+CD5+CD10-lymphocytes), and platelet count was 122 × 10⁹/l. With the exception of a minimal right-axillary lymphadenopathy, there were no pathological physical signs, and the Karnofsky Performance Scale score was rated 100%. Immunophenotyping of peripheral blood lymphocytes showed unchanged phenotype of leukemic cells that retained CD20 positivity. During the whole therapy, rituximab was tolerated well; a slow infusion rate was necessary on one or two occasions.</p>", "<title>Competing interests</title>", "<p>Isrid Sturm, Joachim Oertel, Jörg Westermann and Antonio Pezzutto declare that they have no competing interests. Stephan Oertel is now employed by Roche company which is vendor of rituximab.</p>", "<title>Authors' contributions</title>", "<p>IS, JO, SO, JW and AP were all involved in the diagnosis and treatment of the patient.</p>", "<title>Authors' note</title>", "<p>In the meantime, after 4 years of disease control, the patient presented with progressive disease (abdominal bulk), and because CD20 expression was still present, rituximab treatment was intensified (375 mg/m2 weekly).</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Immunophenotype of chronic lymphocytic leukemia of the B-cell-lineage at primary diagnosis in 2002: 61.7% of gated lymphocytes show CD20 expression.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Course of hemoglobin, leucocyte and thrombocyte numbers under therapy with chlorambucil, fludarabine, cyclophosphamide and rituximab 375 and 600 mg/m².</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1752-1947-2-275-1\"/>", "<graphic xlink:href=\"1752-1947-2-275-2\"/>" ]

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[ "<title>Background</title>", "<p>Structural chromosomal abnormalities are estimated to occur in around 0.5% of newborn infants, using moderate level of resolution in conventional cytogenetic analysis [##REF##1613759##1##]. Complex chromosomal rearrangements (CCRs) are defined as structural chromosomal rearrangements with at least three breakpoints and exchange of genetic material between two or more chromosomes. It is therefore not surprising to see CCR rarely in constitutional karyotypes. Moreover, some CCRs cannot be interpreted with standard cytogenetic methods at all [##REF##7449183##2##]. Complex chromosomal rearrangements are extremely rare but are often associated with mental retardation, congenital abnormalities, recurrent abortions and infertility [##REF##16160854##3##]. More than 130 constitutional CCRs have been documented so far [##REF##17100206##4##]. 12 of these were related with fertile men including the case we present [##REF##15004458##5##]. Providing genetic counseling for CCRs is very important and this can be offered before or after pregnancy as well as at the time of prenatal diagnosis [##REF##9187678##6##].</p>", "<p>Since the introduction of fluorescence in situ hybridization (FISH) techniques using whole chromosome painting probes [##REF##3458254##7##] in human cytogenetics, progress has been achieved concerning the ability to characterize chromosomal subregions by molecular cytogenetic methods. Recently, high resolution MCB technique was developed [##REF##10393418##8##] making it possible to identify different chromosome region specific areas at band and subband levels.</p>", "<p>Here we report a fertile male with mental retardation carrying balanced complex chromosomal rearrangements, involving chromosomes 1, 4 and 2. We also provide advice for genetic counseling of the fertile CCR carrier by discussing the possible mechanisms underlying the origin of CCR.</p>" ]

[ "<title>Materials and methods</title>", "<title>Clinical case report</title>", "<p>A couple was referred to us following three pregnancy losses out of four pregnancies. The mother was 26 years old and had one living child from her third pregnancy. The first one was lost at the first trimester, the second one was aborted at the third trimester due to fetal abnormality, the third one was finally born 4 years ago as a healthy male child, and the fourth one was lost at the first trimester again. The father was 33 years old, he had mental deficit since birth while his spermiogram was normal. There were no functional motor deficits apart from the severe mental retardation he suffered necessitating continuous support. The examination of their male child did not reveal any clinical evidence about any abnormality. The parents did not give consent to further evaluate their living child with chromosomal analysis.</p>", "<title>Banding cytogenetics</title>", "<p>Cytogenetic investigations from the couple were performed on peripheral blood samples using a high resolution technique after cell culture synchronization and BrdU incorporation [##UREF##1##23##].</p>", "<title>Molecular cytogenetics</title>", "<p>High resolution multicolor banding (MCB) based on microdissection derived region-specific libraries for chromosomes 1, 2 and 4 was carried out to further delineate the nature of chromosomal rearrangements as described before [##REF##11891523##22##]. Each of the 20 metaphase spreads were analyzed by using a fluorescence microscope (Axioplan 2 mot, Zeiss) equipped with appropriate filter sets to discriminate between a maximum of five fluorochromes and the counterstain DAPI (Diaminophenylindol). Image capturing and processing were carried out using an ISIS mFISH imaging system (MetaSystems, Altlussheim, Germany) for the evaluation of MCB.</p>" ]

[ "<title>Results</title>", "<p>Banding cytogenetic revealed a normal karyotype for the wife and a complex rearranged one for the spouse. A CCR involving chromosomes 1, 2 and 4 was detected and his karyotype was characterized as 46, XY, t (1; 4; 2) (p21~31; q31.3; q31) (see Fig ##FIG##0##1##). After performing FISH by using MCB (see Fig ##FIG##1##2##), the breakpoints were localized to 1p31.1, 2q24.3 and 4q31.3.</p>" ]

[ "<title>Discussion</title>", "<p>As existing difficulty of precise definition of CCR using standard cytogenetic methods [##REF##7449183##2##], detection of CCR in the chromosomes of a patient causes anxiety for patient and clinician, especially when it is balanced that can lead to genetic imbalance [##REF##15635069##9##]. Because precise identification of all the chromosomes involved in a CCR is the prerequisite to every appropriate genetic counseling. By combining high resolution techniques of chromosome banding with FISH we have an essential tool to determine whether a complex abnormal karyotype is apparent or not, this is especially important for prenatal diagnosis [##REF##9395262##10##].</p>", "<p>These rearrangements are usually ascertained by routine chromosome analysis of a child with mental retardation and congenital abnormalities [##REF##9637422##11##], recurrent abortions in female [##REF##3570293##12##], and infertility in man [##REF##14507821##13##]. Although most carriers of balanced translocations are phenotypically normal, in a small proportion (~6%) of these phenotypic abnormalities are reported [##REF##15635069##9##,##REF##1928105##14##]. Madan et al [1997] analyzed 60 cases with balanced CCRs [##REF##9187678##6##]. They found that for female CCR carriers the risk of abortions and abnormal livebirths is 52.6% and for male carriers the risk of abortion and abnormal livebirths is 60%, with a combined frequency of 53.7%. While liveborn infants possessing normal chromosomes have incidences of 31.6%, liveborn infants carrying balanced chromosomes have incidences of 50% [##REF##9187678##6##]. If chromosomal rearrangement is detected in a phenotypically normal individual, then this rearrangement is generally assumed to be truly balanced. These often represent familial cases. If, however, a chromosomal rearrangement is detected in a phenotypically abnormal individual; then usually a submicroscopic imbalance or other genetic defects exist. This situation often represents de novo cases. The incidence of live born infants with unbalanced chromosomes and variable degrees of phenotypic abnormalities is 18.4% [##REF##9187678##6##,##REF##17515301##15##]. An abnormal phenotype with apparently balanced rearrangements may be the result of chromosomal breakage disrupting a gene leading to abnormal gene expression or the presence of a submicroscopic deletion or duplication [##REF##15635069##9##]. The change of location and/or orientation of translocated genes can also influence the activity of regulatory sequences co-operating with the breakpoint flanking translocated genes [##REF##15017330##16##]. Recently Goumy et al [2006] described a boy with mild developmental delay and psychotic disorder. He had balanced complex rearrangements but no molecular abnormalities were detected by using FISH with whole chromosome painting (WCP), comparative genomic hybridization (CGH) and array-CGH [##REF##17100206##4##]. The results of array CGH belong to De Gregori et al. [2007] showed that 16 of 18 patients had imbalances while all cases had been interpreted as balanced by conventional cytogenetics. 11 of 16 CCRs associated with deletion. The phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions. There was no association between the severity of the pathology and the number of deletions or their sizes [##REF##17766364##17##].</p>", "<p>Moreover, the exact cytogenetic mechanisms underlying the origin of CCRs are unclear. A major catastrophe within the gamete (spermatogenesis) appears a vague yet plausible patognomonic mechanism for CCRs [##REF##17515301##15##]. Simple three-way translocations are predicted to form hexavalents at meiosis. By focusing solely on symmetric segregation (3:3), up to 20 possible gametic combinations could be devised among which only two were balanced. The number of unbalanced gametes increased significantly together with the possibility of asymmetric segregation and recombination during meiosis [##REF##15004458##5##]. Lespinasse et al. [2003] analyzed the localization of 90 chromosome breakpoints in 24 CCRs delineating random involvements of specific chromosomes in CCRs. However, they observed a non-random distribution of specific breakpoints at 1q25, 4q13, 6q27, 7p14, 9q12, 11p11, 12q21, 13q31 and 18q21 [##REF##14507821##13##]. Recently De Gregori et al. [2007] screened 59 balanced translocations including CCRs by using array comparative genome hybridization and 18 of these were found to be de novo balanced complex translocations. At the 22 breakpoints identified using a specific customized array, they could not find any specific DNA sequences. Thus, they were unable to determine the mechanisms underlying the concurrent breakage of several chromosomes with losses of parts of the broken portions and their random assortment. Considering that all the men fathering children with unbalanced translocation or CCRs were fertile, they came up with the following hypothesis: during spermatogenesis some cells escape the mechanism responsible for correct crossing-over; these undergo chaotic breaks resulting in the reunion of several chromosomes and thereby exposing the broken portions to exonuclease degradation [##REF##17766364##17##].</p>", "<p>The couple we present in here has only one living child out of four pregnancies. Their male child does not have any clinical abnormalities or any developmental delay. But we do not have any objective findings to confirm whether this child bears any chromosomal abnormalities. Even if the detected CCR looks balanced with MCB, the carrier of this CCR has severe mental deficiency. Several studies reported apparently balanced chromosomal rearrangements to be associated with significant risks of mental retardation and malformation [##REF##9637422##11##,##REF##15017330##16##]. Based on a review of apparently balanced translocations, Warburton [1982] concluded that the presence of a de novo apparently balanced translocation is associated with an increased risk of mental retardation with an odds ratio of 6.0–7.0 [##UREF##0##18##]. Moreover, the vast majority of male carriers show reduced fertility [##REF##7143391##19##,##REF##3068990##20##]. Disturbances in spermatogenesis as well as pre- and post implantation losses are discussed as reasons for this phenomenon [##REF##9187678##6##]. Zahed et al. [1998] suggested that the scarcity of the number of transmitting males with CCRs is usually attributed to either a lower risk of producing abnormal progeny therefore, a lower probability of ascertainment, or to infertility attributed to problems in chromosome pairing at spermatogenesis [##REF##9738865##21##]. There are only few reports on fertile male CCR carriers referred for cytogenetic evaluation due to spontaneous abortions of spouses or due to abnormal offsprings, which were well reviewed by Grasshoff et al. [2003] as presented here [##REF##15004458##5##].</p>", "<p>Identification of submicroscopic aberrations (below 3 Mb) and more detailed molecular profiling of the rearrangements require precise mapping of the breakpoints with other methods such as florescence in situ hybridization (FISH) with locus-specific probes or array CGH [##REF##15635069##9##]. Since the establishment of FISH technique in human cytogenetic, much progress has been achieved concerning the ability to characterize chromosomal subregions by molecular cytogenetic methods. Recently, high resolution multicolor banding (MCB) technique was developed. By producing changing florescence intensity ratios along the chromosomes, MCB approach allows the differentiation of chromosome region specific areas at the band and subband levels and is based on region specific microdissection libraries [##REF##10393418##8##]. MCB technique is a high resolution alternative suited to clarify the changes appearing in complex chromosomal rearrangements [##REF##11891523##22##]. We used MCB techniques for certain determination of the breakpoints on each chromosome and the detection of possible deletions. According to MCB results the proband has balanced complex chromosomal translocations. Liehr et al [2002] suggested that the MCB-technique is a high resolution alternative to other FISH based chromosome banding approaches and it suits to clarify the changes appearing in CCRs [##REF##11891523##22##].</p>" ]

[ "<title>Conclusion</title>", "<p>Further delineations are certainly required to provide more information about the relationships between balanced CCRs and their phenotypes. Determination of certain breakpoints is also important for counseling the patients. With these, correct prenatal diagnosis and efficient genetic counseling can be possible for the carriers of CCR. The couples with CCR should be also informed about the possible outcomes of the progeny and the fact that exact risk of malformation is still unknown and that phenotypically normal child can still have a high risk of reproductive problems. The carriers must be investigated with high resolution banding and molecular cytogenetic techniques in order to see whether the CCR is truly balanced or not and if balanced where these breakpoints are located. Finally, high resolution MCB techniques by themselves can be used as alternative methods to determine exact locations of the breakpoints.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Complex chromosomal rearrangements (CCRs) are defined as structural chromosomal rearrangements with at least three breakpoints and exchange of genetic material between two or more chromosomes. Complex chromosomal translocations are rarely seen in the general population but the frequency of occurrence is anticipated to be much higher due balanced states with no phenotypic presentation. Here, we report a severely mentally retarded fertile male patient in whom further delineation of CCR involving chromosomes 1, 4 and 2 was carried out by using high resolution multicolor banding (MCB) technique. As a FISH based novel chromosome banding approach, high resolution MCB allows for the differentiation of chromosome region specific areas at band and subband levels.</p>", "<title>Results</title>", "<p>Cytogenetic studies using high resolution banding of the proband necessitated further delineation of the breakpoints because of their uncertainty: 46,XY,t(1;4;2)(p21~31;q31.3;q31). After using high resolution MCB based on microdissection derived region-specific libraries, the exact nature of chromosomal rearrangements for chromosomes 1, 2 and 4 were revealed and these breakpoints were located on 1p31.1, 1q24.3 and 4q31.3 giving rise to a balanced situation.</p>", "<title>Conclusion</title>", "<p>Further delineations are certainly required to provide detailed information about the relationship between balanced CCRs and their phenotypes in order to offer proper counseling to the families concerned. Carriers must be investigated with high resolution banding and molecular cytogenetic techniques to determine the exact locations of the breakpoints. High resolution MCB is an alternative and an efficient method to other FISH based chromosome banding techniques and can serve in clarifying the nature of CCR.</p>" ]

[ "<title>List of abberations</title>", "<p>CCRs: comples chromosomal rearrengements; CGH: comparative genomic hybridization; FISH: fluoresence <italic>in situ </italic>hybridization; MCB: multi colour banding; WCP: whole chromosome painting.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>NK evaluated the family with examination, counseling and cytogenetically, and prepared the revised MS. KM and AW did the molecular cytogenetic analysis and interpretation of the MCB results. All authors' read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>Supported in parts by the Ernst-Abbe-Stiftung, the IZKF Jena (Start-up S16) and the Evangelische Studienwerk e.V. Villigst. The couple has informed this publication.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>shows image of the GTG banded metaphase</bold>. Image belongs to GTG banded metaphases from the father. Arrow indicates the breakpoints on each chromosome.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>shows MCB pattern of the cytogenetic result</bold>. It shows multicolor banding (MCB) applying probe-sets for chromosomes 1, 2 and 4 characterized the breakpoints as 1p31.1, 2q24.3 and 4q31.3. The corresponding results are shown here. For each chromosome depicted the MCB-pseudo-colors as well as the underlying fluorescence profiles are shown (for details of MCB evaluation see [##REF##17515301##22##]. The first of the 5 columns shows which probe set was used (MCB 1, 2 or 4). The second column shows the normal chromosomes #1, #2 and #4. The arrowhead shows the breakpoint as present in the derivative sister-chromosomes. Third to fifth columns show the derivative chromosomes (der) 1, 2 and 4. MCB 1 stains parts of der(1) and der(4), MCB 2 parts of der(1) and der(2) and MCB 4 parts of der(2) and der(4). Parts not stained by the corresponding MCB probe-sets are pseudo-colored in gray. Overall, the complex chromosomal rearrangement was balanced, according to molecular cytogenetics.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1755-8166-1-17-1\"/>", "<graphic xlink:href=\"1755-8166-1-17-2\"/>" ]

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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Neurofibromatosis type 1 (NF1) is a dominantly inherited multi-system disorder. Major features include pigmentary abnormalities, benign tumors of the nerve sheath (neurofibromas), malignant tumors, learning disabilities, and skeletal dysplasia. The <italic>NF1 </italic>gene functions as a tumor suppressor, but haploinsuffiency probably accounts for some aspects of the non-tumor phenotype. The protein product, neurofibromin, is a Ras GTPase-activating protein, and various Ras pathway inhibitors are being tested in preclinical models and clinical trials for effectiveness in treating NF1 complications. This month in <italic>BMC Medicine</italic>, a paper by Kolanczyk et al describes a preclinical mouse model for tibial dysplasia and provides evidence that the drug lovastatin – in use to treat cardiovascular disease – may be beneficial, opening the door to clinical trials in humans.</p>" ]

[ "<p>Patients with neurofibromatosis type 1 (NF1) are largely sustained by the hope that advances in research will result in new forms of treatment. The <italic>NF1 </italic>gene was identified in 1990 and was quickly found to encode a GTPase activating protein (GAP) whose target is the oncoprotein Ras. However, despite great progress in the dissection of cell signaling pathways involved in the disorder, there is still no definitive treatment to prevent or reverse the complications. The path from bench to bedside may be the best approach, but the challenges should not be underestimated. There are, however, recent signs that provide hope that this approach will pay off. The paper by Kolanczyk et al published this month in <italic>BMC Medicine </italic>describes a preclinical mouse model for one of the most difficult NF1 lesions to treat, tibial dysplasia, and provides evidence that an existing drug with a known safety profile in children, lovastatin, may be beneficial, opening the door to clinical trials in humans.</p>", "<p>NF1 poses a particular challenge given the complex and highly variable phenotype [##REF##12403555##1##]. The condition is one member of a group of disorders collectively referred to as 'neurofibromatoses', which includes NF1, NF2, and schwannomatosis. Each is associated with a distinct spectrum of nerve sheath tumors, and each is dominantly transmitted, due to mutation in a different gene. The hallmark lesion of NF1 is the neurofibroma, a benign tumor consisting of Schwann cells, fibroblasts, perineurial cells, and mast cells. Patients with NF1 may develop innumerable tumors on the skin, internal tumors, and large disfiguring tumors along major peripheral nerves. Other features include pigmentary lesions (café-au-lait macules, skin-fold freckles), learning disabilities, malignant tumors (gliomas and malignant peripheral nerve sheath tumors), and skeletal dysplasias.</p>", "<p>NF1 patients may suffer from both focal and generalized skeletal disorders. Among focal disorders, long bone dysplasia, most commonly involving the tibia, can be a major source of morbidity [##REF##10360395##2##]. Tibial dysplasia presents in infancy with bowing of the lower leg. The dysplastic bone is prone to fracture, and these fractures are very difficult to treat. Pseudoarthrosis ('false joint') may occur, and some patients still require amputation. Non-ossifying fibromas may also occur, sometimes leading to pain or even to fracture. The generalized dysplasia consists of decreased bone mineral density [##REF##15551055##3##, ####REF##15988556##4##, ##REF##17188620##5##, ##REF##17608828##6####17608828##6##], as well as slow healing following injury or orthopedic surgery.</p>", "<p>Tumors occur in NF1 patients by a classic two-hit model, indicating that the <italic>NF1 </italic>gene is a tumor suppressor [##REF##11115850##7##,##REF##16941471##8##]. A subset of Schwann cells within neurofibromas have mutations of both <italic>NF1 </italic>alleles, the first being the germline mutation and the second being acquired. Large plexiform neurofibromas probably acquire their second hits during development, whereas smaller dermal tumors may acquire theirs later in life. Malignant tumors likely occur upon accumulation of additional genetic changes. At least some non-tumor manifestations also involve a two-hit mechanism: <italic>NF1 </italic>homozygously mutated cells have been found in melanocytes from café-au-lait macules [##REF##17668375##9##] and in cells isolated from dysplastic tibial lesions [##REF##16773574##10##].</p>", "<p>Haploinsufficiency of <italic>NF1 </italic>may also play a pathogenetic role for some features. This appears to be the case for the cognitive phenotype, some aspects of which are modeled in mice rendered heterozygous for <italic>Nf1 </italic>mutation (<italic>Nf1 </italic>+/-) [##REF##9885821##11##,##REF##12403561##12##]. It might also explain the diffuse skeletal phenotype of osteopenia. The <italic>NF1 </italic>gene is expressed in chondrocytes, as well as osteoblasts, osteoclasts, and periosteal cells, and the Ras signaling pathway is hyperactive in these cells and in bone marrow-derived osteoprogenitors in <italic>Nf1 </italic>+/- mice [##REF##15125795##13##,##REF##15804420##14##]. The progenitor cells show increased proliferation and decreased ability to differentiate into osteoblasts [##REF##16893911##15##]. The <italic>Nf1 </italic>heterozygous mice do not develop tibial dysplasia, but Kolanczyk et al [##REF##17317783##16##] did obtain mice with tibial bowing by homozygous inactivation of <italic>Nf1 </italic>in developing limbs using a conditional knock-out system. Osteoblasts were found to display increased proliferation but impaired ability to mineralize [##REF##17317783##16##].</p>", "<p>Given the role of neurofibromin in stimulating the conversion of Ras-GTP to Ras-GDP, proteins involved in the Ras signal transduction pathway have been identified as potential therapeutic targets. Clinical trials are underway with several existing compounds [##UREF##0##17##], and several preclinical models have been used in drug testing. Both existing compounds with known action on this pathway, and newly identified compounds are of interest.</p>", "<p>The <italic>Nf1 </italic>+/- mouse model has been used, as already noted, to model learning disabilities. The phenotype is rescued by genetic crosses that reduce Ras-GTP in the brain, as well as by treatment with a farnesyl transferase inhibitor, which blocks Ras binding to the cell membrane [##REF##11793011##18##]. Recently, Li et al [##REF##16271875##19##] demonstrated that lovastatin also rescues the cognitive defect, presumably by virtue of its effect on membrane binding of Ras. This has opened the door to clinical trials in children with NF1, since the safety profile of statins in children is known. One negative statin trial has recently been reported [##REF##18632543##20##], and another using a different drug for a longer period of treatment is being planned.</p>", "<p>The paper by Kolanczyk et al in this volume of <italic>BMC Medicine </italic>[##REF##18671844##21##] represents the second report of a favorable response to lovastatin in a mouse model, and the first of preclinical treatment aimed at skeletal dysplasia. The conditional knock-out animals display tibial bowing, but do not spontaneously develop fractures, presumably because of different mechanical loads on mouse bones as compared with human. Kolanczyk et al have modeled the tibial fracture by drilling a small hole in the tibia and then monitoring the healing process. Wild-type animals completely heal within 28 days, whereas the knock-out mice display slower and less complete healing. Treatment of the animals with lovastatin resulted in markedly improved bone healing, and normalization of MAPK signaling as compared with untreated animals.</p>", "<p>Skeletal dysplasia may thus be another target of lovastatin therapy in clinical trials for patients with NF1. Improvement of bone healing in children with tibial dysplasia would be welcomed, given the enormous difficulty in the management of this complication and the severe associated morbidity. Whether statin treatment will have other benefits related to bone mineral density remains to be determined. Moreover, there is a possibility that these findings will have an impact on the management of bone disorders in non-neurofibromatosis-affected individuals as well. When patient-advocates argue to increase funding for research on neurofibromatosis, one of the potential benefits cited is that this work will shed light on common disorders such as cancer, learning disabilities, and osteoporosis. The NF1 gene clearly plays a role in normal bone development and metabolism, and there is indeed a possibility that pathogenetic and therapeutic insights gained from this rare disorder may someday benefit a much larger population.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1741-7015/6/22/prepub\"/></p>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Multiple myeloma (MM) is a plasma cell neoplasia characterized by profound genomic instability involving numerical and structural chromosomal aberrations [##REF##14989251##1##]. The availability of human MM cell lines (HMCLs) has been of critical importance in revealing many of the molecular and biological aspects of MM. Over the last few years, recurrent nonrandom genetic lesions have been identified that seem to correlate with the clinical course of MM and its response to therapy. Nearly half of MM tumors are nonhyperdiploid, and frequently show chromosome 13 deletion and constitutively activated <italic>CCND1 </italic>(11q13), <italic>CCND3 </italic>(6p21), <italic>MAF </italic>(16q24), <italic>MAFB </italic>(20q12), or <italic>FGFR3/MMSET </italic>(4p16.3) as a result of chromosomal translocations involving the immunoglobulin heavy chain locus (<italic>IGH@</italic>) on chromosome 14q32 [##REF##15755896##2##, ####REF##9616139##3##, ##REF##9354676##4##, ##REF##9949176##5####9949176##5##]. The remaining tumors are hyperdiploid, which are characterized by multiple trisomies of nonrandom odd chromosomes, and a low prevalence of <italic>IGH </italic>translocations and chromosome 13 deletions [##REF##14989251##1##].</p>", "<p>The recent discovery of microRNA (miRNA) genes encoding a class of small (17–25 base pairs) noncoding RNAs involved in the regulation of the cell cycle, survival, and differentiation programmes has added a further level of complexity to normal and cancer cell biology. Through complementary base pairing to specific protein-coding mRNA transcripts, miRNAs direct mRNA silencing by different mechanisms, including message degradation and translational repression [##REF##14744438##6##]. Several studies have reported that chromosomal abnormalities and/or epigenetic events contribute to miRNA deregulation; impaired miRNA expression has already been demonstrated in a number of solid tumors and, more recently, in some hematological disorders [##REF##16251535##7##, ####REF##17060945##8##, ##REF##17028600##9####17028600##9##]. To date, miRNA expression and deregulation in MM remain to be investigated; recently, it has been demonstrated that miR-21 can be induced by STAT3 and mediate IL-6-dependent HMCL survival [##REF##17496199##10##].</p>", "<p>Approximately one third of miRNAs are located within the intronic regions of coding transcription units [##REF##15701730##11##, ####REF##17465886##12##, ##REF##15364901##13####15364901##13##]. The expression of these miRNAs largely coincides with the transcription of the corresponding host genes, which suggests that they can share the same regulatory sequences as their host transcription units [##REF##15701730##11##] and can be cotranscribed with them under the regulation of the RNA polymerase II (PolII) following the coordinated processing of intronic miRNAs and cognate mRNA [##REF##17255951##14##]. However, the mechanism of intronic miRNA maturation remains to be fully understood, because miRNAs in an antisense orientation to their corresponding host gene may possess independent regulatory motifs [##REF##17465886##12##], and miRNAs located within genomic repetitive elements may be transcribed by RNA polymerase III (PolIII) [##REF##17099701##15##].</p>", "<p>In the present study, we investigated the expression of miRNAs located within transcription units found to be differentially expressed in a panel of HMCLs that we recently profiled using gene expression microarrays [##REF##17171682##16##]. This approach led to the identification of three miRNAs (miR-335, miR-342-3p, and miR-561) that were differentially expressed, along with their corresponding host genes, in the dataset of HMCLs. In addition, we found that overexpression of these miRNAs/host genes was recurrent in MM primary tumors compared with normal plasma cells. The data discussed here may suggest a possible role for deregulated intronic miRNA species in myeloma.</p>" ]

[ "<title>Methods</title>", "<title>Cell lines</title>", "<p>The HMCLs were obtained from DMSZ-German collection of Microorganisms and Cell Culture, Germany (NCI-H929, OPM2, JJN3, RPMI-8226, and KMS-12); or kindly provided by Dr. T. Otsuki, Kawasaki Medical School, Okayama, Japan (KMS-28, KMS-34, KMS-18, KMS-11, KMS-26, KMS-27, KMM-1 and KMS-20); Dr S. Iida, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan (KM4, FR4, and AMO1), and Dr. F. Malavasi, Department of Genetics, University of Torino, Italy (LP-1); or were established in our laboratory (CMA-01, CMA-02 and CMA-03) [##REF##16266902##17##]. They were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum at concentrations ranging from 3 × 10⁵to 8 × 10⁵, at 37°C in a 5% CO₂humidified atmosphere. CMA-01, CMA-02 and CMA-03 were cultured in presence of 20 U/ml recombinant human IL-6 (R&amp;D System, Minneapolis, MN, USA).</p>", "<title>Specific miRNA quantification by RealTime RT-PCR</title>", "<p>Total RNA was extracted from at least 2 × 10⁶purified plasma cells by using Trizol reagent. Quantitative assessment of the RNA was performed using Nanodrop ND-1000 Biophotometer (NanoDrop Technologies): the minimum OD_260/280ratio to be considered acceptable is 1.98–2.10. In the reverse transcription step, 50 ng total RNA was employed in RT reactions using reagents from the TaqMan^RMicroRNA RT kit (Applied Biosystems) and specific miRNA primers provided with the TaqMan^RMicroRNA Assays. 15 μl reactions were incubated in an Applied Biosystems 9700 Thermocycler. All reverse transcriptase reactions were run in duplicate. Real Time PCR was performed in triplicate using TaqMan^RMicroRNA Assays together with the TaqMan^RUniversal PCR Master Mix on an Applied Biosystems 7700 Sequence Detection System. All RNA samples were normalized based on the <italic>Z30 </italic>TaqMan^RMicroRNA Assays-Control. The threshold cycle (C_T) was defined as the fractional cycle number at which the fluorescence passes the fixed threshold. All signals with C_T≥ 40 were manually set to undetermined. Relative quantification of miRNA expression was calculated with the 2^-ΔCtmethod (Applied Biosystem User Bulletin N°2). Data were presented as relative quantity of target miRNA, normalized to <italic>Z30 </italic>housekeeping gene.</p>", "<title>Bioinformatic Analysis</title>", "<p>mRNA targets were predicted for the 3 miRNA of interest by querying four different bioinformatic algorithms which are miRanda [##UREF##0##18##,##REF##15502875##19##], TargetScan [##UREF##1##20##,##REF##15652477##21##], PicTar [##UREF##2##22##,##REF##15806104##23##], and Diana microT [##UREF##3##24##,##REF##15131085##25##].</p>", "<title>Genome-wide DNA profiling analysis</title>", "<p>Genome-wide DNA profiling was performed on Affymetrix GeneChip Human Mapping 250 k NspI arrays. To find the corresponding copy number (CN) values, we firstly extracted the raw data from the CEL files using the Affymetrix packages GTYPE 4.1 and Copy Number Analysis Tool 4.0.1 (Affymetrix, Santa Clara, CA, USA) and the Mapping Array 250 k NspI probe annotations released on July, 12 2007. In order to keep only the raw data and thus to avoid the CN inference facility of the latter software package, the Hidden Markov Model Genomic Smoothing window was set to 0. After the preprocessing, piecewise constant estimates of the underlying local DNA CN variation was calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS). In order to overcome scaling biases related to the greatly altered ploidy of HMCLs (reflected in a median value for SNP probes different from two in almost all samples) the median of the estimated profiles for each sample was scaled back to assign to a nominal multiplicity of two those values of probes mapped to regions for which FISH information was available and indicated the presence of exactly two alleles [##REF##17171682##16##]. After scaling, a k-means clustering algorithm was used to determine the interval values for inferring discrete CN values. As such, inferred CN higher than 1.73, 2.16, and 2.64 corresponded to two, three or more than four DNA copies, respectively; whereas CN below 1.73, and 1.37 to one copy or biallelic deletion, respectively. CN data of these HMCLs have been deposited in National Center for Biotechnology Information's Gene Expression Omnibus (GEO; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov/geo\"/>) and are accessible through GEO Series accession number GSE11522 [##UREF##4##26##].</p>", "<title>Gene expression profiling database</title>", "<p>Patients database included the proprietary database (4 normal samples, 12 MGUS, 132 MM, and 9 PCL), together with 20 normal samples, 22 MGUS, and 137 MM taken from 2 publicly available MM gene expression datasets [##REF##14989251##1##,##REF##17252022##27##] (GSE6477 and GSE6691, CEL files available at Gene Expression Omnibus [##UREF##4##26##]), all profiled on HG-U133A. The probe level data were converted to expression values using the Bioconductor function for the Robust Multi-Array average (RMA) procedure [##REF##12925520##28##], and the absence of outlier patients in the normalization process due to hybridization signals was verified by Expression Console tools (Affymetrix, Santa Clara, CA, USA). The supervised analyses were performed using SAM software version 3.0 [##UREF##5##29##,##REF##11309499##30##]. The cut-off for significance was determined by tuning the Delta parameter on the false discovery rate (FDR) and controlling the q-value for the gene list (at a q-value = 0). The selected lists were functionally analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Tool 2006 (U.S. National Institutes of Health [##UREF##6##31##]) and NetAffx [##UREF##7##32##].</p>" ]

[ "<title>Results</title>", "<title>Identification of intronic miRNAs deregulated in HMCLs</title>", "<p>We searched for potentially deregulated miRNAs mapping to the intronic, exonic, or 3' UTR regions of the most differentially expressed transcripts in a proprietary dataset of 20 HMCLs previously profiled on HG-U133A GeneChip arrays [##REF##17171682##16##].</p>", "<p>For each annotated probeset, we calculated the ratios of each individual expression value to the mean expression value of that probeset across the whole dataset. These ratios (or the inverse ratios whenever the individual expression value was lower than the mean) were then averaged to obtain the \"average fold change\" for each probeset, which is suitable as a measure of the variability of a transcript expression in a group of samples. The analysis revealed a subset of 1,032 most variable probes (specific to 799 transcripts), at an average fold change greater than 2 (2AVEFC probes). To identify which of these transcripts was a host gene for miRNA, we screened the miRNA Registry [##REF##15608160##33##] (Sanger database, miRBase Sequence, version 10.0, release 2007/08/01 [##UREF##8##34##]), which includes 533 human miRNA sequences, 300 of which map to the intronic, exonic, or 3' UTR regions of 240 transcripts. Of these, 137 (specified by 261 probes) are represented on HG-U133A GeneChip array (additional file ##SUPPL##0##1##). By merging these 261 probes with the 1,032 2AVEFC probes from the HMCLs dataset, we identified ten 2AVEFC probes specific to six host genes, all of which contain intronic miRNA (Table ##TAB##0##1##). Specifically, we found the following pairs of host genes and intronic miRNAs: <italic>CCPG1 </italic>(cell cycle progression 1) and miR-628; <italic>GULP1 </italic>(engulfment adaptor PTB domain containing 1) and miR-561; <italic>MEST </italic>(mesoderm specific transcript homolog, mouse) and miR-335; <italic>EVL </italic>(Enah/Vasp-like) and miR-342-3p; <italic>TACSTD1 </italic>(tumor-associated calcium signal transducer 1) and miR-559; and <italic>TNIK </italic>(TRAF2 and NCK interacting kinase) and miR-569 (details in Table ##TAB##0##1##). The expression levels of the six genes are shown in Fig. ##FIG##0##1##.</p>", "<title>Expression levels of miR-335, miR-342-3p, and miR-561 correlate with those of their corresponding host genes in HMCLs</title>", "<p>To verify whether the six intronic miRNAs were coordinately expressed with their host mRNAs in HMCLs, we investigated miRNA expression levels using quantitative-real time RT-PCR (Q-RT-PCR) assays (TaqMan^RmicroRNA assays) [##REF##16314309##35##]. Although we know that the levels of mature miRNAs are not always correlated to the corresponding precursors and that the measure of pri-miRNAs or pre-miRNAs would give a more complete and reliable information about the possibly coordinated transcriptional levels of miRNAs and their host genes, we chose to evaluate only mature miRNA expression, thus focusing our interest on the biologically functional molecule. The results, normalized for the expression of the housekeeping <italic>Z30 </italic>small nucleolar RNA, are reported in additional file ##SUPPL##1##2##. Specifically, we found appreciable levels of expression for miR-335, miR-342-3p, and miR-628, whereas mir-561 and mir-559 were moderately expressed. mir-569 was barely detectable among the HMCLs in the dataset, and thus it was excluded from further analysis. A regression analysis of Q-RT-PCR miRNA values and microarray expression levels of matching host genes, conducted with R packages/software, revealed a significant correlation with the corresponding host genes for miR-335, miR-342-3p, and miR-561 (R higher than 0.6 in all cases with a <italic>p </italic>value &lt; 0.005; see Fig. ##FIG##1##2##), but not for miR-559 (R = 0.12 at <italic>p </italic>value = 0.60) or miR-628 (R = 0.32 at <italic>p </italic>value = 0.15). As specified in Table ##TAB##0##1##, miR-335, miR-342-3p, miR-561, and miR-559 are all sense oriented, whereas miR-628 is in an antisense orientation with respect to its host gene.</p>", "<p>Based on these findings, we focused our study on the miRNAs/host genes that were coordinately expressed. As described in additional file ##SUPPL##2##3##, the bioinformatic target prediction for miR-335, miR-342-3p, and miR-561 suggests that they might play an important role in proliferation, cell cycle control, cellular migration, and angiogenesis.</p>", "<title>miR-335, miR-342-3p, and miR-561 deregulations are not associated with genomic alterations</title>", "<p>Because miRNA transcripts may be deregulated in cancer as a result of DNA CN variations [##REF##17028600##9##], we investigated whether the coordinated overexpression of the three miRNAs and host transcripts was associated with CN alterations in our dataset. To this end, we performed a genome-wide DNA profiling analysis on the entire panel of HMCLs using high-density 250K SNP-arrays (Affymetrix). By referring to the University of California Santa Cruz (UCSC) Genome Browser Database [##UREF##9##36##] annotations, we positioned <italic>MEST </italic>between telomeric SNP_A-1960494 and centromeric SNP_A-2263405, <italic>EVL </italic>between telomeric SNP_A-1927639 and centromeric SNP_A-2289968, and <italic>GULP1 </italic>between telomeric SNP_A-4201038 and centromeric SNP_A-1941986. The CNs associated with these SNP intervals were calculated using the DNAcopy Bioconductor package (see Methods). For each HMCL, the inferred CN values and the miRNA Q-RT-PCR expression data are reported in Fig. ##FIG##2##3##. Different CNs were not reflected by a corresponding modulation of miRNA expression levels.</p>", "<title>miR-335, miR-342-3p, and miR-561 are overexpressed in primary MM tumors</title>", "<p>We investigated the expression levels of the three miRNAs/host genes in normal plasma cells and MM primary tumors by querying a proprietary gene-expression-profiling (GEP) database including four normal samples, 12 monoclonal gammopathies of undetermined significance (MGUS), 132 MM, and nine plasma cell leukemias (PCLs), all profiled on HG-U133A. The MM cohort of patients was characterized for the presence of the main <italic>IGH </italic>chromosomal translocation, chromosome 13q deletion, 1q gain/amplification, and hyperdiploid status by fluorescence <italic>in situ </italic>hybridization (FISH) analyses, and stratified into the five molecular groups according to the proposed translocation/cyclin D expression (TC) classification [##REF##15090448##37##]. In addition, we included in the analyses 20 normal samples, 22 MGUS, and 137 MM from two publicly available MM gene expression datasets [##REF##14989251##1##,##REF##17252022##27##]. To identify samples showing correlated host gene/miRNA deregulation, we considered the entire database to establish a cut-off expression level for <italic>MEST</italic>, <italic>EVL</italic>, and <italic>GULP1 </italic>genes by calculating the mean expression value + three standard deviations (STDEV) derived from the 24 normal samples. In particular, we found nine MM and two PCL samples with <italic>MEST </italic>gene expression levels exceeding the cut-off value (\"positive\" patients); likewise, we identified four MGUS, 47 MM, and three PCL \"positive\" patients for <italic>GULP1</italic>, and one MGUS, ten MM, and one PCL for <italic>EVL</italic>, although for each of these three genes some of the samples displayed a gene expression level slightly above the cut-off value (Fig. ##FIG##3##4##). The distribution of the spiked expression of <italic>MEST</italic>, <italic>EVL</italic>, and <italic>GULP1 </italic>with respect to the major genetic characteristics of the 132 MM patients included in the proprietary dataset are specified in additional file ##SUPPL##3##4##. No significant associations were found between the subgroups of MM patients deregulating <italic>EVL </italic>or <italic>GULP1 </italic>and any of the genetic aberrations that occur frequently in MM. Concerning <italic>MEST</italic>, the limited number of positive samples precluded contingency analysis.</p>", "<p>To verify the correlated miRNA/host gene expression in primary tumors, we used Q-RT-PCR to test for specific miRNA expression in all of the available \"positive\" samples belonging to the proprietary database (Fig. ##FIG##3##4##). For each gene, a representative number of \"negative\" patients were also investigated (see results in additional file ##SUPPL##4##5##). Notably, although we tested only a limited number of samples, we found a significant correlation in expression with the corresponding host genes for miR-335, miR-342-3p, and miR-561 (R = 0.95 at <italic>p </italic>value = 5.03E-09, R = 0.7 at <italic>p </italic>value = 5E-03, and R = 0.78 at <italic>p </italic>value = 2E-04, respectively).</p>", "<title>Transcriptional profile associated with miR-335, miR-342-3p, and miR-561 overexpression</title>", "<p>To gain insight into the possible role of these three miRNAs deregulation in MM, we looked for a specific gene expression signature associated with MM patients displaying deregulated host gene/miRNA. For each host gene, we performed a supervised analysis grouping the 269 MM samples according to the specific cut-off expression values evaluated with normal plasma cells. We identified genes that were expressed differentially among the two classes using Significant Analysis of Microarrays software (SAM). Interestingly, in the nine multiple myeloma patients overexpressing <italic>MEST</italic>, 70 genes were significantly upregulated. Of these, 12 are involved in the cell cycle (<italic>p </italic>&lt; 0.001, DAVID tool 2006), particularly in the M phase of the cell cycle, and nine others are involved in actin polymerization and microtubule-based processes. With regard to miR-342-3p, solely the <italic>EVL </italic>itself resulted significantly upregulated in the ten samples overexpressing <italic>EVL</italic>. Finally, the 47 patients overexpressing <italic>GULP1 </italic>upregulated 35 probes specific to 29 genes with miscellaneous biological function. Overall the three supervised analyses did not identify downregulated transcripts in patient groups that overexpressed each miRNA; thus, no information was provided regarding putative direct targets regulated at the transcriptional level by the miRNAs themselves in MM. Full details of the differentially expressed genes resulting from the three supervised analyses are given in additional file ##SUPPL##5##6##.</p>" ]

[ "<title>Discussion</title>", "<p>Information concerning miRNA expression and deregulation in MM is still lacking. Based on the hypothesis that intronic miRNAs are coordinately expressed with host transcripts [##REF##15701730##11##,##REF##17255951##14##,##REF##15685193##38##], we sought to identify miRNAs potentially deregulated in MM by focusing on those mapping within the intronic regions of host genes that were significantly differentially expressed in a representative panel of HMCLs profiled with U133A gene expression chips.</p>", "<p>Following this approach, we identified six genes containing intronic miRNAs; all but one showed appreciable expression levels. For three miRNAs, miR-335, miR-342-3p, and miR-561, we demonstrated coordinated expression with their cognate protein-coding genes <italic>MEST</italic>, <italic>EVL</italic>, and <italic>GULP1</italic>; conversely, we did not find correlated expression of miR-628 and miR-559 and their host genes <italic>CCPG1 </italic>and <italic>TACSTD1</italic>. Notably, miR-335, miR-342-3p, miR-561, and miR-559, but not miR-628, are sense oriented with respect to the corresponding host gene. This finding is in agreement with the evidence that intronic miRNAs are usually oriented in the same direction as the pre-mRNA, and thus could be under the control of the same regulatory motifs as their host genes and processed from the same primary mRNA transcripts regulated by PolII. On the other hand, our data may also support the previous suggestion that even miRNAs in a sense orientation to annotated genes (e.g., miR-559) may have their own regulatory motifs that can be regulated by either PolII or PolIII [##REF##17465886##12##].</p>", "<p>The coregulation of these three miRNA/host gene pairs was also found in primary MM tumors. Specifically, our data showed that miR-335, miR-342-3p, and miR-561 were overexpressed in a fraction of the pathological samples with respect to normal plasma cells, without correlation to any of the known genetic alterations frequently found in MM; this finding may provide further evidence of the genetic complexity of this disease. In addition, despite the fact that miR-335 deregulation in melanoma and ovarian carcinoma was reported to be concordant with CN gain [##REF##16754881##39##], neither miR-335 nor miR-342-3p and miR-561 expression levels were significantly correlated with their corresponding locus CN in our panel of HMCLs tested by SNPs arrays. This finding indicates that DNA CN alterations may not be a critical factor affecting expression of these miRNA/host genes in MM, and suggest the occurrence of epigenetic mechanisms.</p>", "<p>Although determining the precise contribution of each miRNA to myelomagenesis was beyond the scope of this investigation, some evidence supports the potential involvement of deregulated miRNAs/host genes in myeloma. Interestingly, miR-335 and miR-342-3p were recently reported to be involved in human cancer; depleted expression of miR-335 was found to be associated with metastatic processes in human breast cancer. Specifically, miR-335 was shown to suppress metastasis by altering cell morphology and decreasing cell motility, which would limit metastatic migration [##REF##18185580##40##]. Notably, we found that the fraction of primary myeloma samples overexpressing miR-335/<italic>MEST </italic>also showed upregulation of genes implicated in actin polymerization and microtubule-based processes. In agreement with these data, bioinformatic tools predicted that a set of genes involved in actin cytoskeleton organization and biogenesis (<italic>DAAM1</italic>, <italic>ARPC5L</italic>, <italic>JAG1</italic>, <italic>MAP2</italic>, and <italic>RASA1</italic>) were putative miR-335 targets (additional file ##SUPPL##5##6##). Therefore, one can speculate that miR-335 may be involved in MM in the physiological mechanisms reported to be altered in breast cancer, possibly influencing different processes such as plasma cell homing into the bone marrow and/or interactions with the bone marrow microenvironment. Notably, MM patients overexpressing miR-335/<italic>MEST </italic>also upregulated genes promoting cell proliferation, a finding apparently in contradiction with the function of miR-335 as an apoptosis permissive factor and cell cycle suppressor, as demonstrated in cortical neurosphere cultures [##REF##17687032##41##]. One possible explanation might be that the cellular context and cooperation among multiple miRNAs play a key role in the final biological effect of miRNA. Alternatively, one cannot exclude a specific effect of <italic>MEST </italic>overexpression itself in promoting cell proliferation.</p>", "<p>With regard to miR-342-3p, it is usually expressed in a variety of human tissues, together with its host gene <italic>EVL</italic>. Notably, it is specifically silenced in the majority of colorectal cancers following methylation of CpG islands located upstream of <italic>EVL</italic>, although the functional consequences of its silencing in carcinogenesis remain to be elucidated [##REF##18264139##42##]. In addition, miR-342-3p was substantially downregulated in patients with primary myelofibrosis, polycythemia vera, or essential thrombocythemia [##REF##17976522##43##], and specifically upregulated in acute promyelocytic leukemia cell lines during retinoic acid-induced differentiation [##REF##17260024##44##]. Intriguingly, EVL is an actin-associated protein that is involved in a variety of processes related to cytoskeleton remodeling and cell polarity [##REF##14570581##45##]; among miR-342-3p predicted targets, we recognized genes involved in actin cytoskeleton organization and biogenesis (<italic>FHL3 </italic>and, again, <italic>RASA1</italic>). One can speculate that miR-342-3p and <italic>EVL </italic>deregulation may target plasma cell homing into the bone marrow and/or interactions with the bone marrow microenvironment, much the same as for miR-335.</p>", "<p>Finally, there is no information concerning the possible role of deregulated miR-561 and its cognate host gene <italic>GULP1 </italic>(which codes for an evolutionarily conserved adaptor protein required for efficient engulfment of apoptotic cells by phagocytes [##REF##11729193##46##]) in normal or tumor cells. Because of the high frequency of <italic>GULP1 </italic>overexpression in MM (34%) compared with normal plasma cells, both miR-561 and its cognate host gene warrant further investigation.</p>" ]

[ "<title>Conclusion</title>", "<p>Our data extend the current view of miRNA origins, provide further support for the hypothesis that intronic miRNAs and their host genes may be regulated dependently, and may contribute to the understanding of their biological role in cancer. In addition, to the best of our knowledge, this is the first evidence of putative deregulated miRNAs in MM and may lead the way to identifying new biomarkers and altered molecular pathways associated with the disease.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The role of microRNAs (miRNAs) in multiple myeloma (MM) has yet to be fully elucidated. To identify miRNAs that are potentially deregulated in MM, we investigated those mapping within transcription units, based on evidence that intronic miRNAs are frequently coexpressed with their host genes. To this end, we monitored host transcript expression values in a panel of 20 human MM cell lines (HMCLs) and focused on transcripts whose expression varied significantly across the dataset.</p>", "<title>Methods</title>", "<p>miRNA expression was quantified by Quantitative Real-Time PCR. Gene expression and genome profiling data were generated on Affymetrix oligonucleotide microarrays. Significant Analysis of Microarrays algorithm was used to investigate differentially expressed transcripts. Conventional statistics were used to test correlations for significance. Public libraries were queried to predict putative miRNA targets.</p>", "<title>Results</title>", "<p>We identified transcripts specific to six miRNA host genes (<italic>CCPG1</italic>, <italic>GULP1</italic>, <italic>EVL</italic>, <italic>TACSTD1</italic>, <italic>MEST</italic>, and <italic>TNIK</italic>) whose average changes in expression varied at least 2-fold from the mean of the examined dataset. We evaluated the expression levels of the corresponding intronic miRNAs and identified a significant correlation between the expression levels of <italic>MEST</italic>, <italic>EVL</italic>, and <italic>GULP1 </italic>and those of the corresponding miRNAs miR-335, miR-342-3p, and miR-561, respectively. Genome-wide profiling of the 20 HMCLs indicated that the increased expression of the three host genes and their corresponding intronic miRNAs was not correlated with local copy number variations. Notably, miRNAs and their host genes were overexpressed in a fraction of primary tumors with respect to normal plasma cells; however, this finding was not correlated with known molecular myeloma groups. The predicted putative miRNA targets and the transcriptional profiles associated with the primary tumors suggest that <italic>MEST</italic>/miR-335 and <italic>EVL/</italic>miR-342-3p may play a role in plasma cell homing and/or interactions with the bone marrow microenvironment.</p>", "<title>Conclusion</title>", "<p>Our data support the idea that intronic miRNAs and their host genes are regulated dependently, and may contribute to the understanding of their biological roles in cancer. To our knowledge, this is the first evidence of deregulated miRNA expression in MM, providing insights that may lead to the identification of new biomarkers and altered molecular pathways of the disease.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>DR conceived of the study and drafted the manuscript. ML performed the quantitative RT-PCR. LM performed the genome-wide DNA profiling analysis. LA participated in the genome-wide DNA profiling analysis. AA performed the statistical analysis. SF participated in the statistical analysis. GLD helped to draft the manuscript. AN participated in study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1755-8794/1/37/prepub\"/></p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) to AN, Associazione Italiana Leucemie (AIL), Ministero Italiano della Salute and Ministero dell'Istruzione (MIUR), Project FIRB 2006061851_004.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Host gene expression levels</bold>. Expression levels of <italic>GULP1</italic>, <italic>TNIK</italic>, <italic>MEST</italic>, <italic>EVL</italic>, <italic>TACSTD1</italic>, and <italic>CCPG1 </italic>assessed by DNA microarray analysis of HMCLs. The scaled values on the vertical axis represent the relative intensity levels as determined on HG-U133A arrays.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>miRNA and cognate host gene expression correlation analysis</bold>. Correlation analyses between host genes (GEP data, ordinate) and miRNA expression levels (Q-RT-PCR, abscissa) in 20 HMCLs. To compare these data, we converted gene expression and Q-RT-PCR (expressed as 2^-ΔCt) results between the interval values 0–1. Linear regressions, as well as the correlation coefficient R and the <italic>p </italic>values are indicated in each panel.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>miRNA and DNA CN correlation analysis</bold>. Correlation analysis between DNA CN variations and miRNA expression. The red lines represent mature miRNA expression, normalized to <italic>Z30 </italic>small nucleolar RNA, expressed as 2^-ΔCt, and converted between the interval values 0–1 (vertical axis on the right side). The spots represent the HMCL inferred DNA CNs (vertical axis on left side). Horizontal axis: HMCLs ordered according to increasing inferred CNs. The <italic>p </italic>value is indicated above each panel (Kendall's tau correlation test).</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Host gene expression level in primary tumors database</bold>. Expression levels of <italic>MEST</italic>, <italic>EVL</italic>, and <italic>GULP1</italic>, as assessed by DNA microarray analysis of a patient database including samples from 24 normal donors (N), 34 MGUS, 269 MM, and nine PCL. The scaled values on the vertical axis represent the fluorescence intensity of streptavidin-PE-stained biotinylated cRNA hybridized to specific probes set on HG-U133A arrays; the baseline was set at the cut-off value calculated as mean + 3STDEV in 24 normal samples. The samples are ordered and grouped on the horizontal axis. Samples above the cut-off level and analysed by Q-RT-PCR are coloured in red; for <italic>MEST</italic>, the gene expression value is also reported of the sample barely exceeding the cut-off and not analysed by Q-RT-PCR.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Selected HG-U133A probes with average fold-change higher than two in HMCLs</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>HG-U133A probe</bold></td><td align=\"left\"><bold>Host gene</bold></td><td align=\"left\"><bold>Host gene intron</bold></td><td align=\"left\"><bold>Intron length (bp)</bold></td><td align=\"left\"><bold>Strand direction</bold></td><td align=\"left\"><bold>miRNA</bold></td><td align=\"left\"><bold>Location</bold></td><td align=\"left\"><bold>Fold-change</bold></td></tr></thead><tbody><tr><td align=\"left\">222156_x_at</td><td align=\"left\"><italic>CCPG1</italic></td><td align=\"left\">intron 5</td><td align=\"left\">4,903</td><td align=\"left\">-</td><td align=\"left\">miR-628</td><td align=\"left\">15 q21</td><td align=\"left\">2.3</td></tr><tr><td align=\"left\">217838_s_at</td><td align=\"left\"><italic>EVL</italic></td><td align=\"left\">intron 3</td><td align=\"left\">25,879</td><td align=\"left\"><bold>+</bold></td><td align=\"left\">miR-342</td><td align=\"left\">14q32</td><td align=\"left\">2.7</td></tr><tr><td align=\"left\">204235_s_at</td><td align=\"left\"><italic>GULP1</italic></td><td align=\"left\">intron 1</td><td align=\"left\">90,649</td><td align=\"left\"><bold>+</bold></td><td align=\"left\">miR-561</td><td align=\"left\">2q32</td><td align=\"left\">4.3</td></tr><tr><td align=\"left\">204237_at</td><td align=\"left\"><italic>GULP1</italic></td><td/><td/><td/><td/><td/><td align=\"left\">4</td></tr><tr><td align=\"left\">215913_s_at</td><td align=\"left\"><italic>GULP1</italic></td><td/><td/><td/><td/><td/><td align=\"left\">2.5</td></tr><tr><td align=\"left\">202016_at</td><td align=\"left\"><italic>MEST</italic></td><td align=\"left\">intron 2</td><td align=\"left\">1,632</td><td align=\"left\"><bold>+</bold></td><td align=\"left\">miR-335</td><td align=\"left\">7q32</td><td align=\"left\">4.8</td></tr><tr><td align=\"left\">201839_s_at</td><td align=\"left\"><italic>TACSTD1</italic></td><td align=\"left\">intron 5</td><td align=\"left\">1,874</td><td align=\"left\"><bold>+</bold></td><td align=\"left\">miR-559</td><td align=\"left\">2p</td><td align=\"left\">9.7</td></tr><tr><td align=\"left\">211828_s_at</td><td align=\"left\"><italic>TNIK</italic></td><td align=\"left\">intron 21</td><td align=\"left\">5,584</td><td align=\"left\">-</td><td align=\"left\">miR-569</td><td align=\"left\">3q26</td><td align=\"left\">3</td></tr><tr><td align=\"left\">213107_at</td><td align=\"left\"><italic>TNIK</italic></td><td/><td/><td/><td/><td/><td align=\"left\">2.4</td></tr><tr><td align=\"left\">213109_at</td><td align=\"left\"><italic>TNIK</italic></td><td/><td/><td/><td/><td/><td align=\"left\">2.3</td></tr></tbody></table></table-wrap>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p><bold>240 host transcripts containing microRNAs</bold>. List of all transcripts containing intronic/exonic/3' UTR miRNA or cluster of miRNA, based on the Sanger database, miRBase Sequence, version 10.0.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p><bold>MiRNA expression values (expressed as 2^-ΔCt) by Q-RT-PCR analysis</bold>. Expression values of miRNAs 335, 342-3p, 559, 561, 569, and 628 evaluated by Q-RT-PCR (TaqMan^RMicroRNA assay) in HMCLs.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional file 3</title><p><bold>miRNA target bioinformatic prediction</bold>. List of the putative target genes of miRNAs 335, 342-3p, and 561, predicted by merging the results from the specified prediction algorithms. The last column reports the molecular function and biological process related to each putative miRNA target as described by DAVID Tool 2006 and NetAffx.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S4\"><caption><title>Additional file 4</title><p><bold>Distribution of the spiked expression of <italic>MEST</italic>, <italic>EVL</italic>, and <italic>GULP1 </italic>with respect to patients' genetic characteristics</bold>. The main genetic characteristics of the 132 MM patients of the proprietary GEP database. The distribution of MM samples in which one of the three host genes were deregulated (according to the cut-off evaluated on normal plasma cells) with respect to genetic abnormalities is also specified.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S5\"><caption><title>Additional file 5</title><p><bold>Host genes and miRNA expression values in primary tumors</bold>. Expression values of host genes <italic>MEST</italic>, <italic>EVL</italic>, and <italic>GULP1 </italic>(GEP data expressed as relative cRNA intensity level) and corresponding miRNAs 335, 342-3p, and 561 (Q-RT-PCR data expressed as 2^-ΔCt) in the analysed primary tumors.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S6\"><caption><title>Additional file 6</title><p><bold>Supervised analyses of MM patients with deregulated host genes/miRNAs versus MM samples with host gene/miRNA normal expression levels</bold>. The tables report all of the probes resulting from SAM analyses comparing MM patients overexpressing <italic>MEST </italic>(table 6a), <italic>EVL </italic>(table 6b), or <italic>GULP1 </italic>(table 6c) with respect to MM patients whose host gene expression levels were comparable with those of normal plasma cells. For each probe, the corresponding gene, chromosome location, involved pathway, and biological process (annotations from NetAffx), as well as the score and fold-change are specified.</p></caption></supplementary-material>" ]

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[ "<title>Background</title>", "<p>Neuroblastic tumours (NBTs) are one of the most common neoplasms in childhood, accounting for approximately 40% of solid tumours encountered in the first four years of life [##UREF##0##1##]. NBTs are heterogeneous in terms of their biological, genetic and morphological characteristics and exhibit marked diverse clinical behaviours.</p>", "<p>The biological bases of these processes are poorly understood. There is an apparent link between NBTs aggressiveness and specific genetic aberrations (i.e., MYCN amplification, chromosome deletions of 1p36, 11q23, 14q32 or 19q13.3; gain of 17q and near-diploid/tetraploid DNA content), indicating that specific genetic alterations are present in individual categories of NBTs and likely contribute to clinical outcome [##REF##12173347##2##, ####REF##2066755##3##, ##REF##6719137##4####6719137##4##].</p>", "<p>Abnormal cellular DNA content is ubiquitous in cancer and has been linked to the rate of cell proliferation, cell differentiation, and prognosis in a variety of tumour cell types. In contrast to most other tumours, hyperploidy confers a favourable prognosis in NBTs [##UREF##1##5##], acute lymphoblastic leukemia [##REF##2713510##6##], and rhabdomyosarcoma [##REF##7526971##7##]. Non-metastatic loco-regional NBTs (stages 1, 2 and 3) often show modal chromosomal numbers in the near-triploid range (58 to 80 modal chromosome number) and few structural aberrations [##UREF##1##5##]. On the other hand, karyotypes of metastatic NBTs are commonly near-diploid (44 to 57 chromosomes) or near-tetraploid (81–103 chromosomes) with structural changes [##UREF##1##5##].</p>", "<p>The presence of specific and recurrent chromosomal alterations in NBTs suggests that gene copy number abnormalities represent a major biologically relevant event, which contributes to NBT growth and survival. The aim of the current study was to gain further insight into the difference in gene expression of distinct biological entities within NBTs defined by the ploidy status.</p>" ]

[ "<title>Methods</title>", "<title>Patients and samples</title>", "<p>Forty-nine diagnostic primary NBT specimens (24 stages 1, 2, and 3; 7 stage 4s; and 18 stage 4) obtained from patients diagnosed and treated at MSKCC were selected for gene expression profiling (Table ##TAB##0##1##). Risk assessment was defined by the INSS staging classification, the MSKCC biological risk stratification criteria, and the COG clinical staging criteria. NBT stages 1, 2, 3 and 4s were treated without use of cytotoxic therapy, when possible, according to MSKCC protocols. Stage 4 NBTs patients were treated according to N5, N6 or N7 protocols. This study was approved by the MSKCC and HSJD Institutional Review Boards and informed consent was obtained before collection of all samples.</p>", "<p>Twenty-one samples (9 stages 1, 2, and 3; 1 stage 4s; and 11 stage 4) of the original MSKCC NBT cohort included in the gene profiling analysis and an independent set of 25 primary NBT specimens (12 stage 1, 2, and 3, 2 stage 4s, and 11 stage 4) obtained at diagnosis from 3 Spanish institutions (HSJD, Barcelona; Hospital La Paz, Madrid; and Department of Pathology, University of Valencia) were available for validation analyses (Table ##TAB##0##1##). Normal control DNA was obtained from the National DNA Bank of Spain.</p>", "<p>All tumour-specimens were evaluated by the same pathologists (WG and NP) to assess tumour cell content, only tumours with &gt; 70% were included in the study.</p>", "<title>DNA content analysis</title>", "<p>The modal DNA content was determined by flow cytometry DNA analysis on nuclei isolated from paraffin sections using the method of Hedley modified [##REF##2653738##8##]. DNA index (DI) was expressed as the ratio of tumour DNA content/standard DNA fluorescence; near-diploid DI = 0.90–1.20; near-triploid DI = 1.21–1.75; near-tetraploid DI = 1.76–2.20.</p>", "<title>Gene expression profiling</title>", "<p>Gene expression profiling was performed of 49 primary NBT samples (22 near-triploid, 23 near-diploid and 4 near-tetraploid) using Affymetrix GeneChip Human Genome U95 Set™ Arrays, as previously reported [##REF##12880970##9##]. Microarray data and sample annotations have been deposited in the caArray database <ext-link ext-link-type=\"uri\" xlink:href=\"http://caarraydb.nci.nih.gov/caarray/\"/>.</p>", "<title>Differential gene expression analysis</title>", "<p>Genes with high variability within samples were selected by pair-wise comparison analyses performed by adjusting the type-I error for multiple tests (Step-down permutation (SDP) [##UREF##2##10##], and False Discovery Rate (FDR) [##UREF##3##11##]), and with no type-I error adjustment (Raw method). The cut-off Family-wise error applied to select significant genes by means of the T-test for independent data, a univariate screening supervised procedure, was equivalent for all three methods: &lt; 0.1, &lt; 0.05 and &lt; 0.01. Hierarchical clustering analyses were performed for the differentially expressed genes for all the methods of adjustment of Type-I error and cut-off of P-values, using a multivariate unsupervised method, taking into account the relationship between gene expressions. Fisher's exact test and 95% bilateral confidence interval using Wilson method were used to evaluate the proportion with which chromosomes were represented in the selected gene sets in comparison to chromosome representation within the Affymetrix GeneChip U95Av2. Statistical analyses were performed using SAS 9.1 and JMP 5.1 (SAS Institute Inc) for Windows and CIA 2.1.1.</p>", "<title>Gene Ontology annotation categories</title>", "<p>Gene Ontology (GO) annotation categories were analyzed using <italic>explore </italic>GeneOntology (<italic>e</italic>GOn v2.0) in <italic>Gene Tools </italic>web service <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.genetools.no\"/> to create a biological profile of the differentially expressed genes. Overrepresented GO terms were determined statistically by Fisher's exact test (<italic>P </italic>&lt; 0.01) and adjusted FDR &lt; 0.01.</p>", "<title>Quantitative Real-time PCR (Q-PCR)</title>", "<p>Quantification of transcript levels using Q-PCR was performed of 13 genes located on chromosomes 1 and 17 (see Additional file ##SUPPL##0##1##). Concomitant quantification of gene copy number was performed for a set of these genes (see Additional file ##SUPPL##0##1##). MYCN gene copy number was analyzed by Q-PCR, and FISH when needed. Validation analyses were performed on 46 primary NBT specimens (see patients and samples).</p>", "<p>Q-PCR reactions and quantification, using the ΔΔC_Trelative quantification method, were performed on an ABI Prism 7000 Sequence Detection System using TaqMan^®Assay-on-Demand Gene Expression products, according to the manufacturer's protocols (Applied Biosystems, US). All experiments included no template controls and were performed in duplicate and repeated twice independently. Transcript levels were measured relative to 3 normal tissue samples (adrenal gland, lymph node and bone marrow) and normalized to TATA box binding protein (TBP), hypoxantine phosphoribosyltransferase 1 (HPRT1) and succinate dehydrogenase complex, subunit A (SDHA) expression values. Endogenous control genes were chosen on the basis of recent publications regarding accurate normalization of real-time quantitative RT-PCR in primary neuroblastoma [##REF##12184808##12##,##REF##15681479##13##]. These genes are reported within the most stable set of endogenous control genes. Gene copy number quantification was performed as reported previously [##REF##11850545##14##]. Gene copy number was calculated relative to placental DNA using the B-Cell maturation factor (BCMA) as reference gene. The validity of BCMA as reference gene in our cohort of NBTs was determined by copy number ratio: BCMA _{NB tumour test sample}/BCMA _{placenta calibrator sample}. The ratio measured was equal to 1.0016; (tumour DNA 1.0012 ± 0.13 SD)/(placental DNA 0.9996 ± 0.05).</p>", "<title>Fluorescent <italic>in situ </italic>hybridization (FISH)</title>", "<p>FISH was assayed on 4 μm sections of Tissue-Micro-Array (TMA) of formalin-fixed paraffin-embedded NBT samples corresponding to the validation set, and partially matching the MSKCC series described above. Tissue microarrays included only tumour areas showing &gt; 90% of tumour cells. Sections were washed with 2× SSC buffer and fixed in 4% paraformaldehyde in PBS. DNA-probes, CEP 17 Alpha (Ref: 32-112017;Vysis, IL, USA) LSI p53 (Ref:30-190008;Vysis) and/or LSI 1p36 (Ref:30-231004;Vysis), were denatured at 73°C, 5 min., applied to tissue sections and simultaneously denatured using the Hybridizer (DAKO) at 90°C, 4 min. Hybridization was performed for 16 h at 37°C in a humid chamber. Slides were then washed with Buffer post-hybridization (Master Diagnostica, Granada, Spain) and stained with DAPI (6-diamidino-2-phenylindole) and mounted with Vectashield H-1000 medium (Vector). One hundred nuclei were evaluated for each core. Results were recorded as percentage of nuclei present in the sample having each probe signal pattern. Cell populations &lt; 5% of abnormal cells were not scored as significant. Microscope Magnification ×1000.</p>", "<title>Array comparative genomic hybridization (aCGH)</title>", "<p>Whole genome BAC-aCGH studies were performed using the Sanger 1 Mb clone set (kindly provided by Dr. K. Szuhai LUMC, The Netherlands). BAC/PAC clones were added to increase resolution for regions of interest: full genomic coverage clones for chromosome 17 (CHORI) and chromosome 11 (BAC/PAC isolated DNA, kindly provided by Dr. J. San Miguel, CIC, Salamanca), and 19q13 enriched medium-coverage set (Invitrogen, CA, USA and kindly provided by Dr. JC Cigudosa, CNIO, Spain). BAC DNA was extracted, amplified by DOP and Aminolinking-PCR and spotted in triplicate onto Codelink slides (Amersham Biosciences, GE, USA).</p>", "<p>Tumour and reference DNA (an equimolar DNA pool from 40 healthy donors, obtained from the Spanish National DNA Bank) was Cy5/Cy3-dCTP (Amersham, GE) labelled using a non-commercial Random Priming kit composed by Random Octamers dissolved in Eppicentre Exo-Minus Klenow buffer, a dNTPs mix depleted in dCTP and Exo-Minus Klenow enzyme (Eppiocentre). Labelled DNA was purified through Illustra G-50 Microspin Columns, mixed and then precipitated along with Cot DNA (Roche). Hybridization was performed for 48 hours at 42°C and probe excess removed.</p>", "<title>Imaging acquisition and data analysis</title>", "<p>Log₂data was acquired using Axon 4000B scanner and GenePix software. Normalization was done with GenePix software using the mean of the median of ratios of all the autosomal features in the array, excluding those removed by the quality flagging scripts. Gpr files were subsequently processed with Bioconductor packages (CRAN) incorporating scripts for removing SD &gt; 0.2 and GenePix flagged spots. DNA copy algorithm and Merge Levels scripts (both implemented in snap CGH package) were applied for segmentation of the data. A graded colour code adjusted to the log₂rank of each individual plot was assigned to define the segments found by the applied algorithm. Universal threshold cut-off values for defining gain/loss were not applied because of subpopulation clonal heterogeneity, ploidy, and percentage of neuroblastic cells, which varied from one sample to another. Due to this, plots were evaluated independently by visual examination and results were depicted using a graded colour code adjusted to the log₂ rank of each plot, assigning a colour grade to every segment found by the segmentation algorithm.</p>" ]

[ "<title>Results</title>", "<title>Differential gene expression analysis</title>", "<p>Gene expression analysis was performed on a spectrum of 49 NBTs with varying DNA content (22 near-triploid, 23 near-diploid and 4 near-tetraploid). Owing to reduced number of near-tetraploid cases included in this study and taking into account the reported biological and clinical similarities with near-diploid NBTs [##UREF##4##15##,##REF##16682287##16##], near-diploid and near-tetraploid NBTs were combined in one group. Pair-wise comparison analyses of near-triploid (n = 22) <italic>versus </italic>near-diploid/tetraploid (n = 27) NBTs revealed small sets of differentially expressed genes when using a stringent correction for multiple sampling, (6 genes [FDR &lt; 0.01] and 12 genes [SDP &lt; 0.1]) (see Additional file ##SUPPL##1##2##). Interestingly, all genes showing a higher expression in the near-triploid group mapped to chromosome 17 (see Additional file ##SUPPL##1##2##). Less stringent multiple testing corrections selected a larger set of differentially expressed genes, (51 genes [FDR &lt; 0.05] (Fig. ##FIG##0##1##) and 254 genes [FDR &lt; 0.1] (see Additional file ##SUPPL##1##2##). Again, this resulted in a statistically significant proportion of genes mapping to chromosomes with described recurrent abnormalities in NBTs; chromosome 1 (p = 0.01) and chromosome 17 (p &lt; 0.0001) (Fig. ##FIG##0##1##). Chromosomal region specificity was observed since the majority of chromosome 1 and 17 differentially expressed genes spread over 1p36-p22.1 and 17p13-17q21 (Fig. ##FIG##0##1##; see Additional file ##SUPPL##1##2##). The majority showed higher expression in near-triploid NBTs; 92% (CI: 78% to 97%) of chromosome 1 genes and 91% (CI: 76% to 96%) of chromosome 17 (see Additional file ##SUPPL##1##2##). Only 8% (CI: 2% to 21%) probe sets for genes located on chromosome 1, ENO1 (1p36.2), CCT3 (1q23) and C1orf107 (1q32.2), and 9% (CI: 3% to 23%) for genes on chromosome 17, MAC30 (17q11.2) and NME1 (17q21.3), showed a higher expression within near-diploid/tetraploid NBTs.</p>", "<p>The Gene Ontology biological profile of genes with higher expression in near-diploid/tetraploid NBTs showed enrichment for genes related to protein, macromolecular and nucleic acid biosynthesis, such as, NME1, ATP5I, ATP5C1, NME4, TYMS and GMPS. Whereas, near-triploid tumours included genes involved in vesicle mediated transport, cell communication, signal transduction, nervous system development and regulation of small GTPase mediated signal transduction. A large portion of these genes mapped to chromosomes 1 and 17 (60–100%), among these RERE, CHD5, CLCN6, CDC42BPA, NTRK1, ARHGEF11, PMP22, VAMP2, GARNL4, MAP2K4 and FLOT2.</p>", "<title>Quantitative Real-time Polymerase Chain Reaction (Q-PCR)</title>", "<p>Quantification of transcript levels of 13 differentially expressed genes, located mainly on the chromosomal regions 1p36 and 17p13-q21, was performed on two separate groups of NBT specimens: 21 primary NBTs from the original MSKCC cohort as well as on an independent set of 25 NBTs (Table ##TAB##0##1##). Expression levels identified by Q-PCR confirmed the microarray data in both sets of NBTs (Fig. ##FIG##1##2A, B## and ##FIG##1##2C##).</p>", "<p>Four genes located on chromosomes 1 and 17 were further analyzed for gene copy number by DNA Q-PCR analysis in 27 cases (Tables ##TAB##1##2## and ##TAB##2##3##; see Additional file ##SUPPL##2##3##). Near-triploid NBTs (n = 13) showed, both for chromosome 1 and 17, fold values consistently higher (≥ 1.3-fold) than normal reference gene values, and were considered to represent a minimum trisomic gene copy number. Only case # 2 (Table ##TAB##1##2##; see Additional file ##SUPPL##2##3##) showed 0.8–1.1-fold values reflecting a possible loss of 1p36, subsequently confirmed by FISH and aCGH results. Near-diploid/tetraploid NBTs (n = 13) displayed a wider range of values (0.5–2.7-fold), indicative of losses and gains within a more heterogeneous clonal population, as shown by FISH results. Tumour clonal heterogeneity may often confound analyses performed on the bulk of the tumour specimen and could explain some discrepancies between ploidy and gene copy number.</p>", "<p>Comparison between DNA gene copy number and expression levels (Fig. ##FIG##2##3##) revealed an overall linear correlation for those analyzed genes that displayed in the microarray analysis higher expression levels in near-triploid NBTs. Conversely, NME1 gene, as from microarray results, showed low expression values, closer to the disomic reference sample expression, in near-triploid NBTs, and high fold increase in mRNA levels in near-diploid and tetraploid cases.</p>", "<title>Fluorescent <italic>in situ </italic>hybridization (FISH)</title>", "<p>Interphase FISH using the DNA probes LSI 1p36 and LSI 1q25 was performed on 13 primary NBTs drawn from the HSJD cohort; four cases were not evaluable (Table ##TAB##1##2##). According to chromosome 1 status, near-triploid and near-diploid/tetraploid NBTs were characterized by intratumoural heterogeneous cell population content. Only 1 case showed uniform distribution of probe signals within cells of the tumour specimen (case #10, Table ##TAB##1##2##). All but one of the near-triploid NBTs were constituted of clonal populations with two LSI 1p36 and LSI 1q25 signals (2:2) and/or three (3:3) DNA probe signal, ranging from 40–60% and 40–100% of the cells, respectively. Case # 2 was the only near-triploid NBT that exhibited a chromosome 1p36 loss in 30% of cells, confirmed by aCGH and Q-PCR. Even higher intratumoural heterogeneity was observed in near-diploid/tetraploid NBTs.</p>", "<p>Chromosome 17 FISH using centromeric CEP 17 and LSI p53 (17p13.1) DNA probes, was performed on 53 primary NBTs (13 cases from the HSJD cohort, Table ##TAB##2##3##, and 40 cases from MSKCC, Table ##TAB##3##4##). Based on chromosome 17 status, near-triploid tumours were constituted of two (2 CEP 17 and 2 LSI p53 signals, 2:2), three (3:3) and four (4:4) chromosome 17 signals clonal populations that ranged from 10–55%, 24–70% and 7–45% of the cells, respectively. Near-diploid/tetraploid NBTs were composed by a more heterogeneous cell population, with a high incidence of chromosomal structural abnormalities. In a large portion of these tumours, alongside with the two (2:2) DNA probe signal clonal populations (6%–100% of cells), the aneuploid cell population counterpart constituted a significant and heterogeneous portion of cell population (Tables ##TAB##2##3## and ##TAB##3##4##).</p>", "<p>Intratumoural clonal heterogeneity was observed in all the FISH analyses (Fig. ##FIG##3##4##).</p>", "<title>Array comparative genomic hybridization (aCGH)</title>", "<p>Genome array CGH was performed for 13 cases, drawn from the HSJD validation set of NBTs, with complete FISH and Q-PCR analyses (Tables ##TAB##1##2## and ##TAB##2##3##; Fig. ##FIG##4##5##). Near-triploid NBTs exhibited the highest incidence of specific chromosomal alterations, with consistent gain or loss of whole chromosomes, being chromosomes 7 and 17 the most frequently gained (83% and 100% cases, respectively), whilst, chromosomes 3, 4, 9, 14, 16 (50% cases), and 19 (67% NBTs) were among the most frequently lost, although the set of cases is not large enough for statistically significant results. Chromosome 1p loss was observed only in one case (case# 2, Table ##TAB##1##2##), a near-triploid stage 4s tumour.</p>", "<p>Specific near-diploid/tetraploid copy number alterations were characterized by a more heterogeneous pattern of chromosomal aberrations than those of near-triploid, being partial chromosomal segment alterations much more frequent than in near-triploid tumours (Fig ##FIG##4##5##; see Additional file ##SUPPL##3##4##). Partial loss of 11q and partial gain of 17q were only observed in near-diploid/tetraploid samples and never in near-triploid NBTs. Chromosome 20 showed a common pattern being one of the most frequent gains both in near-diploid and near-triploid NBTs. MYCN amplification was absent in near-triploid cases and shared by near-diploid/tetraploid cases.</p>", "<p>Further copy-number alterations that did not reach the maximum log₂values, but were clearly distinguishable in terms of segmentation algorithm, were detected in the array CGH plots and could reflect higher intratumoural clonal heterogeneity (data not shown).</p>" ]

[ "<title>Discussion</title>", "<p>Aneuploidy is ubiquitous in cancer and has been linked to cell proliferation, cell differentiation and prognosis. The karyotypes of most tumours are aneuploid, meaning that chromosomes, which carry thousands of genes, are structurally rearranged, duplicated, broken or entirely missing.</p>", "<p>Gain of chromosome 17 is one of the most frequent genetic abnormalities observed in NBTs, and may involve either the entire chromosome or partial gain of the distal segment 17q21-qter [##REF##9006325##17##]. Unbalanced translocations, characteristic of near diploid NBTs or tumours with structural rather than numerical chromosome aberrations, are thought to arise from DNA double strand breaks repaired erroneously, suggesting an impaired DNA maintenance or repair pathway [##REF##16294040##18##]. On the other hand, abnormalities in the mitotic segregation of chromosomes are thought to underlie the numerical aberrations characteristic of near-triploid, good prognostic, NBTs. Both mechanisms define the type of aneuploidy behind each of the subgroups of NBTs, determining the kind of genetic aberrations as well as the biological behaviour of each NBT subtype.</p>", "<p>Gene expression profiling of NBTs with different ploidy status, near-triploid or near-diploid/tetraploid, enabled us to identify distinct expression profiles associated with each subgroup. Interestingly, a statistically significant proportion of genes shown to be differentially expressed mapped to chromosomes described to be recurrently altered in NBTs, chromosomes 1 and 17 [##REF##9006325##17##]. Chromosomal region specificity was also observed for these differentially expressed genes since the majority spread predominantly over the chromosomal regions 1p36-p22.1 and 17p13-17q21. Besides, over 90% of these genes displayed higher expression levels in near-triploid tumours. Only two genes mapping to chromosome 17, MAC30 and NME1, exhibited a higher expression level in near-diploid/tetraploid NBTs. MAC30 gene encodes for a meningioma-associated protein, highly expressed in several types of tumours, but, with unknown clinicopathological and biological significance. The product of the NME1 gene, the nm23A protein, is a nucleoside diphosphate kinase, whose expression has been related to cell proliferative activity [##REF##1311721##19##]. Whereas reduced expression of NME1 is associated with a high potential for metastasis in some tumour types, like breast cancer and melanoma, its expression is increased in aggressive NBTs [##REF##15833843##20##].</p>", "<p>Genome array CGH, together with FISH and Q-PCR results, confirmed the association of specific chromosomal abnormalities with each of the NBTs subgroups. Therefore, it is not unreasonable to assume that these specific chromosomal alterations are associated with the observed gene expression profiles. The highly significant and strikingly persistent chromosomal localization of the differentially expressed genes made us hypothesize about which transcriptional regulation mechanisms can underlie these gene expression patterns. As a result of aneuploidy, cells possibly produce imbalanced expression of large sets of genes that are amplified or lost. Such gross imbalances would inevitably disrupt critical cellular circuits and destabilize regulatory pathways and cellular structures. It has been assumed that gene dosage effects may play a role in the pathogenesis of malignant diseases. Variations of the transcriptome due to alterations of the gene dosage have been described <italic>in vitro </italic>[##REF##15231742##21##], <italic>in vivo </italic>[##REF##12471051##22##] and in human pathologies such as trisomies 13 and 21 [##REF##9988266##23##]. In our hands, when comparing gene expression levels with gene copy number of a set of differentially expressed genes located at chromosomes 1p36 and 17q13-q21, we observed a concordance between copy number and mean expression values in all those analyzed genes that displayed in the microarray analysis higher expression levels in near-triploid NBTs. In contrast, NME1 gene, as from microarray results, showed low expression values, close to the disomic reference sample expression, in near-triploid NBTs, and high fold increase in mRNA levels in near-diploid/tetraploid cases. NME1 gene has been identified as one of the MYCN targets. Correlation between MYCN overexpression and upregulation of NME1 expression has been reported both in NBTs and neuroblastoma cell lines [##REF##11960382##24##]. In our experience, all MYCN amplified NBTs, displaying MYCN overexpression, as well as near-diploid cases with increased copy number of chromosome 17q, showed high NME1 expression levels. However, NME1 overexpression was also observed in 2 near-diploid MYCN single copy cases, with low MYCN expression and no 17q gain. This suggests that in NBTs NME1 gene expression is only partly dependent on gene copy number and MYCN expression, and therefore implies the existence of other mechanisms of NME1 transcriptional regulation.</p>", "<p>Recently, we reported that clonal ploidy heterogeneity is present in virtually every single loco-regional, near-triploid NBT, and detected the existence of clonal DNA content heterogeneity and evolution [##REF##11461074##25##,##REF##17243159##26##]. In this report our results underscore the clonal heterogeneity of all NBTs, with a marked complexity in the near-diploid/tetraploid tumours. Furthermore, clonal variations reflected in the array CGH plots as copy-number alterations with varying log₂values, could unveil the presence of subpopulations emerged during tumour development. These cellular subpopulations are likely to be the cause of the high cell heterogeneity also observed in the FISH analyses. These findings are important in emphasizing the cellular heterogeneity and karyotypic complexity (aneuploidy) generally associated with malignant tumours, but need a more detailed understanding of their significance.</p>" ]

[ "<title>Conclusion</title>", "<p>We have found that NBTs with different cellular DNA content display specific transcriptional profiles suggesting that near-diploid/tetraploid and near-triploid NBTs result from two different mechanisms of aneuploidy driving tumourigenesis. A large number of the differentially expressed genes participate in cell differentiation pathways and map to specific chromosomal regions recurrently involved in unbalanced translocations, gains and losses in NBTs. Our results demonstrate that these specific genetic abnormalities are complex, heterogeneous, and translate into a gene expression profile that defines the biological behaviour of each type of NBT.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status.</p>", "<title>Methods</title>", "<p>Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent <italic>in situ </italic>Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles.</p>", "<title>Results</title>", "<p>Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (<italic>P </italic>= 0.01) and 17p13-q21 (<italic>P </italic>&lt; 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours.</p>", "<title>Conclusion</title>", "<p>NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.</p>" ]

[ "<title>Abbreviations</title>", "<p>NBTs: neuroblastic tumours; MIBG: Meta-iodobenzylguanidine; LOH: loss of heterozygosity; MSKCC: Memorial Sloan-Kettering Cancer Center, New York; HSJD: Hospital Sant Joan de Déu, Barcelona; Children's Oncology Group: COG; CT: computed tomography; INSS: International Neuroblastoma Staging System; INPC: International NB pathology committee; CNS: central nervous system; Q-PCR: Quantitative real-time polymerase chain reaction; aCGH: array-Comparative Genomic Hybridization; FISH: Fluorescence <italic>in situ </italic>hybridization.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>CL and JM are responsible for the initial conception and overall hypothesis of this study. CL, IG and JM are responsible for the design of this manuscript, including the original draft and subsequent revisions and design of this manuscript. CdT assisted with the initial concept and was involved with the draft and revisions of this manuscript; provided guidance for many of the experiments. NKC and WLG are responsible for the procurement and cryopreservation of NBT tissue specimens derived from MSKCC. ER, IG, SA, HB and JM were responsible for the procurement and cryopreservation of NBT tissue specimens derived from the Spanish institutions. WLG and NP evaluated all tumour specimens for tumour staging classification and to assess tumour content. CL, NKC, WLG, and JM are responsible for patient clinico-biological database management and for microarrays studies. NKC and WLG were involved in the drafting and revision of this manuscript. IG and CL are responsible for the quantitative PCR experiments. CM and EdA are responsible for the FISH and aCGH analyses and were also involved with the interpretation of data, draft and revision of this manuscript. ET performed the flow cytometry DNA analysis. CL, GD, JR and IG performed the statistical analysis and interpretation of the data derived from all the samples. HB and SA assisted with valuable technical assistance for experiments associated with this manuscript. All were also involved in the drafting and revisions for this manuscript. All authors read and approved the final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1755-8794/1/36/prepub\"/></p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>This work was supported by: Career Development Award 2001 (to J. M.) from the American Society of Clinical Oncology (ASCO) and grants from the Spanish Ministry of Health (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, 2007; PI070286) (CL) and Spanish Society against Cancer (Asociación Española Contra el Cáncer, 2007) (JM and CL). The Developmental tumour biology laboratory, Hospital Sant Joan de Déu in Barcelona, is additionally supported by the Catalan government (AGAUR, Generalitat de Catalunya, 2005SGR00605; 2006FI00404), and the donation from Margarita del Pozo Fund. Supported in part by the National Cancer Institute grant CA106450 (NKC and WG), The Robert Steel Foundation (NKC), Hope Street Kids (NKC), and Katie's Find A Cure Fund (NKC) and the Government of Castilla y León (EdA).</p>", "<p>We would like to thank Dr. R. Noguera, Department of Pathology, University of Valencia; and Dr. J. Alonso and Dr. P. García Miguel, Hospital La Paz, Madrid, for kindly providing annotated samples, and T. Hernández (Centro de Investigación del Cáncer-IBMCC, Salamanca) for FISH hybridizations. Dr. J. San Miguel (Centro de Investigación del Cáncer-IBMCC, Salamanca) for kindly providing the BAC/PAC isolated DNA and Dr. JC Cigudosa (Centro Nacional de Investigaciones Oncológicas, Spain) for providing enriched medium-coverage set.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>A heatmap illustrating the distinct expression profiles of 49 NB primary tumours with varying ploidy status</bold>. Gene expression profiles visualized according to 51 differentially expressed genes [FDR &lt; 0.05]. <bold>(Right) </bold>Gene dendrogram is divided in 2 main gene clusters. Top cluster: genes displaying higher expression in near-triploid tumours; a statistically significant proportion of genes map to chromosome 1 (p = 0.01) and chromosome 17 (p &lt; 0.0001) (Blue). Bottom cluster: genes with higher expression in near-diploid/tetraploid NBTs. <bold>(Bottom) </bold>Filled in boxes: <bold>Ploidy: </bold>black = near-diploid, empty white boxes = near-triploid, grey = near-tetraploid NBTs; <bold>MYCN: </bold>black = amplified, white = not amplified; <bold>Age: </bold>black &gt; 12 months, white &lt; 12 months; <bold>INSS: </bold>black = Stage 4 NBTs, white = stages 1, 2, 3, and 4S.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Quantitative real-time PCR validation of microarray gene expression data</bold>. Comparison of gene expression levels of 5 representative genes located on chromosomes 1 and 17. <bold>A</bold>. Microarray gene expression data in 49 NBT from MSKCC. Gene expression data were log-transformed and normalized to TBP expression levels; <bold>B</bold>. Q-PCR gene transcript quantification in 21 NBTs from MSKCC; <bold>C</bold>. Q-PCR gene transcript quantification in 25 NBTs from Spanish institutions. Results were compared by two-tailed independent-sample <italic>t </italic>test using SPSS v.14.0 for Windows (SPSS, Chicago, IL). Expression data are shown as box plots (SPSS v.14.0).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Comparison between DNA copy number and gene expression levels analyses</bold>. Gene expression levels and gene copy number are exhibited as mean values in accordance with NBT ploidy subgroups. Correlation between DNA gene copy number and expression levels was observed in those analyzed genes that displayed in the microarray analysis higher expression levels in near-triploid NBTs.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>FISH analysis. Intra-tumoural cell heterogeneity, cancer cells exhibit different alterations of chromosome 17</bold>. FISH analysis using probes for chromosome 17 (red, LSI p53; green, CEP 17) showing different cellular populations within the same NBT in terms of probe signal numbers. In the panels are reported two representative NBT cases; <bold>A</bold>. near-triploid NBT; <bold>B</bold>. near-diploid case. Five signal cells in this sample were very rare populations (&lt; 5%) and are not displayed in Table 3.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Array-Comparative Genomic Hybridization (aCGH) results of 13 NBTs obtained from HSJD</bold>. Results are displayed according to tumour ploidy status. Chromosome alterations are visualized as a graded colour code adjusted to the log₂rank of each individual plot assigned to define chromosomal segment alterations. Filled boxes: from orange to pink colour shades represent increasing chromosomal copy number gains, whereas, from light blue to dark blue colour shades indicate chromosome losses. White colour boxes represent no detected chromosome change. Grey colour boxes represent not evaluable results.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Clinical and Biological characteristics of patients with Neuroblastoma evaluated according to tumour ploidy status.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Case number</td><td align=\"center\">ploidy</td><td align=\"center\">Age</td><td align=\"center\">INSS stage</td><td align=\"center\">MYCN amplification</td><td align=\"center\">Disease Status</td><td align=\"center\">Survival Status</td><td align=\"center\">microarray analysis</td><td align=\"center\">validation analysis</td></tr></thead><tbody><tr><td/><td/><td align=\"center\">&lt;12m=0; &gt;12m=1</td><td align=\"center\">1,2,3,4s=0; 4=1</td><td/><td/><td/><td/><td/></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\">1</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">2</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">3</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">4</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">5</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">6</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">7</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">8</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">9</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">10</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">11</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">12</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">13</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">14</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">15</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">16</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">17</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">18</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">19</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">20</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">21</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">22</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">23</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">24</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">25</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">26</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">27</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">28</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">29</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">30</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">31</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">32</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">33</td><td align=\"center\">near-3n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">34</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">35</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">36</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">37</td><td align=\"center\">near-3n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\">38</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">39</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">40</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">41</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">42</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">43</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">44</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">45</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">46</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">47</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">48</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">49</td><td align=\"center\">near-2n</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">50</td><td align=\"center\">near-2n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">51</td><td align=\"center\">near-2n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">52</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">53</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">A</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">54</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">55</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">56</td><td align=\"center\">near-2n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">57</td><td align=\"center\">near-2n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">58</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">59</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">60</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">61</td><td align=\"center\">near-2n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">62</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">63</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">64</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">65</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">66</td><td align=\"center\">near-2n</td><td align=\"center\">0</td><td align=\"center\">0</td><td align=\"center\">A</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">67</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">68</td><td align=\"center\">near-2n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td colspan=\"9\"><hr/></td></tr><tr><td align=\"center\">69</td><td align=\"center\">near-4n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">70</td><td align=\"center\">near-4n</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">71</td><td align=\"center\">near-4n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">NP</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">72</td><td align=\"center\">near-4n</td><td align=\"center\">0</td><td align=\"center\">1</td><td align=\"center\">NA</td><td align=\"center\">P</td><td align=\"center\">A</td><td align=\"center\">Y</td><td align=\"center\">.</td></tr><tr><td align=\"center\">73</td><td align=\"center\">near-4n</td><td align=\"center\">1</td><td align=\"center\">0</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr><tr><td align=\"center\">74</td><td align=\"center\">near-4n</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">A</td><td align=\"center\">P</td><td align=\"center\">D</td><td align=\"center\">.</td><td align=\"center\">Y</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Results of FISH, aCGH, Q-PCR analyses of chromosome 1, displayed in relation to NBTs ploidy status</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Case Number</bold></td><td align=\"center\"><bold>Ploidy</bold></td><td align=\"center\"><bold>MYCN</bold></td><td align=\"center\"><bold>FISH Chromosome 1</bold></td><td align=\"center\" colspan=\"3\"><bold>a CGH Chr. 1</bold></td><td align=\"center\" colspan=\"2\"><bold>Q-PCR Gene copy No. (fold change)</bold></td><td align=\"center\"><bold>Disease Status</bold></td><td align=\"center\"><bold>Survival Status</bold></td></tr></thead><tbody><tr><td/><td/><td/><td align=\"center\">Cell % (#DNA probe signals: LSI 1p36: LSI 1q25)</td><td align=\"center\">p</td><td align=\"center\">cen</td><td align=\"center\">q</td><td align=\"center\">GNB1 (1p36.33)</td><td align=\"center\">RERE (1p36.1)</td><td/><td/></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"center\">1</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">n.e</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">1.6</td><td align=\"center\">3.2</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">2</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">50 (2:2), 20 (3:3), 15 (1:3), 15 (2:3)</td><td align=\"center\">L</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">0.8</td><td align=\"center\">1.1</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">3</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">60 (2:2), 40 (3:3)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">1.5</td><td align=\"center\">2</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">4</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">5 (2:2), 95 (3:3)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">2.6</td><td align=\"center\">2.4</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">5</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">40 (2:2), 60 (3:3)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">1.4</td><td align=\"center\">1.3</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">6</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">50 (2:2), 50 (3:3)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">1.4</td><td align=\"center\">3</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"center\">7</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">n.e.</td><td align=\"center\">L</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">0.5</td><td align=\"center\">2.2</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">8</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">n.e.</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">1.6</td><td align=\"center\">1.5</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">9</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">95 (2:2), 5 (3:3)</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">0.7</td><td align=\"center\">0.5</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">10</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">100 (2:2)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">0.7</td><td align=\"center\">0.5</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">11</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">35 (2:2), 65 (1:3)</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">1</td><td align=\"center\">2.3</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"center\">12</td><td align=\"center\">near-4n</td><td align=\"center\">A</td><td align=\"center\">51 (1:2), 30 (2:2), 19 (1:3)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">G</td><td align=\"center\">0.5</td><td align=\"center\">0.6</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">13</td><td align=\"center\">near-4n</td><td align=\"center\">A</td><td align=\"center\">60 (2:2), 30 (3:3), 10 (4:4)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">1.3</td><td align=\"center\">2.7</td><td align=\"center\">P</td><td align=\"center\">D</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Results of FISH, aCGH, Q-PCR analyses of chromosome 17, displayed in relation to NBTs ploidy status</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Case Number</bold></td><td align=\"center\"><bold>Ploidy</bold></td><td align=\"center\"><bold>MYCN</bold></td><td align=\"center\"><bold>FISH Chromosome 17</bold></td><td align=\"center\" colspan=\"3\"><bold>a CGH Chr. 17</bold></td><td align=\"center\" colspan=\"2\"><bold>Q-PCR Gene copy No. (fold change)</bold></td><td align=\"center\"><bold>Disease Status</bold></td><td align=\"center\"><bold>Survival Status</bold></td></tr></thead><tbody><tr><td/><td/><td/><td align=\"center\">Cell % (# DNA probe signals: LSI 17p13.1: CEP 17)</td><td align=\"center\">p</td><td align=\"center\">cen</td><td align=\"center\">q</td><td align=\"center\">RUTBC1 (17p13.3)</td><td align=\"center\">NME1 (17q21)</td><td/><td/></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"center\">1</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">n.e</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">2.3</td><td align=\"center\">2.5</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">2</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">45 (2:2), 55 (3:3)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">1.5</td><td align=\"center\">1.5</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">3</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">45 (2:2), 55 (3:3)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">1.3</td><td align=\"center\">1.4</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">4</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">30 (2:2), 70 (3:3)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">3.6</td><td align=\"center\">2.2</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">5</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">50 (2:2), 50 (3:3)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">1.6</td><td align=\"center\">1.3</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">6</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">50 (2:2), 50 (3:3)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">1.4</td><td align=\"center\">1.5</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"center\">7</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">5 (1:1), 80 (2:2), 10 (3:3), 5 (4:4)</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">0.7</td><td align=\"center\">1.4</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">8</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">33 (1:1), 66 (2:2)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">0.8</td><td align=\"center\">0.7</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">9</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">7 (1:1), 7 (2:1), 60 (2:2), 20 (1:2), 6 (2:3)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">G</td><td align=\"center\">1.1</td><td align=\"center\">1.4</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">10</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">80 (2:2), 15 (2:3), 5 (3:3)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">G</td><td align=\"center\">1.2</td><td align=\"center\">1.5</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">11</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">28 (1:1); 11 (2:1), 56 (2:2), 5 (3:2)</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">n.e</td><td align=\"center\">1.1</td><td align=\"center\">0.9</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"center\">12</td><td align=\"center\">near-4n</td><td align=\"center\">A</td><td align=\"center\">45 (2:2); 55 (3:3)</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">G</td><td align=\"center\">1</td><td align=\"center\">1.4</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">13</td><td align=\"center\">near-4n</td><td align=\"center\">A</td><td align=\"center\">45 (2:2), 45 (3:3), 10 (4:4)</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">G</td><td align=\"center\">2.2</td><td align=\"center\">1.9</td><td align=\"center\">P</td><td align=\"center\">D</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Chromosome 17 Fluorescence <italic>in situ </italic>Hybridization results of 40 NBTs obtained from MSKCC, displayed in relation to NBTs ploidy status</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Case Number</bold></td><td align=\"center\"><bold>Ploidy</bold></td><td align=\"center\"><bold>MYCN</bold></td><td align=\"center\"><bold>FISH Chromosome 17</bold></td><td align=\"center\"><bold>Disease Status</bold></td><td align=\"center\"><bold>Survival Status</bold></td></tr></thead><tbody><tr><td align=\"center\" colspan=\"6\">Cell % (# DNA probe signals: LSI 17p13.1: CEP 17)</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"center\">1</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">23 (2:2), 44 (3:3), 33 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">2</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">50 (2:2), 50 (3:3)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">3</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">16 (2:2), 41 (3:3), 43 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">4</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">34 (2:2), 42 (3:3), 24 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">5</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">33 (2:2), 50 (3:3), 17 (4:4)</td><td align=\"center\">P</td><td align=\"center\">A</td></tr><tr><td align=\"center\">6</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">25 (2:2), 60 (3:3), 15 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">7</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">31 (2:2), 46 (3:3), 23 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">8</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">35 (2:2), 52 (3:3), 13 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">9</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">23 (2:2), 54 (3:3), 23 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">10</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">13 (3:3), 66 (3:4), 21 (5:5)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">11</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">16 (2:2), 48 (3:3), 36 (4:4)</td><td align=\"center\">P</td><td align=\"center\">A</td></tr><tr><td align=\"center\">12</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">35 (2:2), 58 (3:3), 7 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">13</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">46 (2:2), 24 (3:3), 30 (4:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">14</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">22 (3:3), 62 (4:4), 16 (4:5)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">15</td><td align=\"center\">near-3n</td><td align=\"center\">NA</td><td align=\"center\">10 (2:2), 29 (3:3), 45 (4:4), 16 (5:5)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"center\">16</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">5 (1:1), 65 (2:2), 5 (1:2), 10 (3:3), 5 (2:3), 5 (4:4), 5 (3:4)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">17</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">100 (2:2)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">18</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">95 (2:2), 5 (3:3)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">19</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">31 (CEP 2), 50 (CEP 3), 18 (CEP 4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">20</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">25 (1:1), 75 (2:2)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">21</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">100 (2:2)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">22</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">n.e</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">23</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">10 (CEP 1), 40 (CEP 2), 38 (CEP 3), 12 (CEP 4)</td><td align=\"center\">P</td><td align=\"center\">A</td></tr><tr><td align=\"center\">24</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">80 (2:2), 10 (1:2), 5 (3:3), 5 (2:2)</td><td align=\"center\">P</td><td align=\"center\">A</td></tr><tr><td align=\"center\">25</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">100 (2:2)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">26</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">25 (1:1), 75 (2:2)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">27</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">100 (2:2)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">28</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">5 (2:1), 74 (2:2), 5 (1:2), 5 (3:3), 6 (2:3), 5 (4:4)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">29</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">10 (2:1), 70 (2:2), 15 (1:2), 5 (3:3)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">30</td><td align=\"center\">near-2n</td><td align=\"center\">NA</td><td align=\"center\">n.e</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">31</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">20 (2:2), 32 (3:3), 12 (4:3), 20 (4:4), 16 (3:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">32</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">40 (1:1), 60 (2:2)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">33</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">n.e</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">34</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">100 (2:2)</td><td align=\"center\">P</td><td align=\"center\">A</td></tr><tr><td align=\"center\">35</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">35 (2:2); 5 (3:2), 20 (3:3), 5 (2:3), 30 (4:4), 5 (3:4)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">36</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">60 (2:2); 5 (3:2), 25 (3:3), 5 (4:3), 5 (4:4)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td align=\"center\">37</td><td align=\"center\">near-2n</td><td align=\"center\">A</td><td align=\"center\">10 (1:1), 90 (2:2)</td><td align=\"center\">P</td><td align=\"center\">D</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"center\">38</td><td align=\"center\">near-4n</td><td align=\"center\">NA</td><td align=\"center\">29 (2:2), 6 (3:3), 8 (4:3), 37 (4:4), 20 (3:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">39</td><td align=\"center\">near-4n</td><td align=\"center\">NA</td><td align=\"center\">49 (2:2), 37 (3:3), 9 (2:3), 5 (3:4)</td><td align=\"center\">NP</td><td align=\"center\">A</td></tr><tr><td align=\"center\">40</td><td align=\"center\">near-4n</td><td align=\"center\">NA</td><td align=\"center\">6 (2:2), 20 (3:3), 5 (4:3), 46 (4:4), 18 (5:5), 5 (4:5)</td><td align=\"center\">P</td><td align=\"center\">A</td></tr></tbody></table></table-wrap>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Quantitative Real-time Polymerase Chain Reaction Analysis. List of genes analyzed to determine expression levels and DNA copy number of genes located on chromosomes 1 and 17.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p>Gene expression profiling of NBTs with different ploidy status. List of differentially expressed genes identified applying different multiple testing corrections. Differentially expressed genes are displayed according to tumour ploidy and chromosomal location. <bold>A</bold>. List of 6 differentially expressed genes [FDR P &lt; 0,01]; B. List of 12 differentially expressed genes [SDP P &lt; 0,1]; C. List of 51 differentially expressed genes [FDR P &lt; 0,05];.D. List of 254 differentially expressed genes [FDR P &lt; 0,1].</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional file 3</title><p>Quantitative Real-time Polymerase Chain Reaction gene copy number analysis and array CGH analysis results. <bold>n.e </bold>= not evaluable results; <bold>n.d</bold>. = not done. MYCN amplification status: <bold>NA </bold>= not amplified, <bold>A </bold>= amplified. Disease status: <bold>NP </bold>= no disease progression, <bold>P </bold>= disease progression. Survival status: <bold>A </bold>= alive, <bold>D </bold>= dead. <bold>Q-PCR</bold>: gene copy number fold changes are determined by the ΔΔC_Trelative quantification method. Array CGH: <bold>p </bold>and <bold>q </bold>= chromosome arms, <bold>cen</bold>. = centromeric; <bold>G </bold>= chromosome gain, <bold>L </bold>= chromosome loss; <bold>- </bold>= no alteration observed.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S4\"><caption><title>Additional file 4</title><p>Array CGH images of NBT with different DNA content. <bold>A</bold>. Near-triploid NBT; <bold>B</bold>. Near-diploid tumour and <bold>C</bold>. Near-tetraploid NBT.</p></caption></supplementary-material>" ]

[ "<table-wrap-foot><p>MYCN amplification status: <bold>NA </bold>= not amplified, <bold>A </bold>= amplified. Disease status: <bold>NP </bold>= no disease progression, <bold>P </bold>= disease progression. Survival status: <bold>A </bold>= alive, <bold>D </bold>= dead. Microarray and validation analyses: Y = cases analyzed.</p></table-wrap-foot>", "<table-wrap-foot><p>Thirteen representative cases drawn from the HSJD cohort analyzed by FISH, aCGH and Q-PCR of chromosome 1. <bold>n.e </bold>= not evaluable results. MYCN amplification status: <bold>NA </bold>= not amplified, <bold>A </bold>= amplified. Disease status: <bold>NP </bold>= no disease progression, <bold>P </bold>= disease progression. Survival status: <bold>A </bold>= alive, <bold>D </bold>= dead. FISH: results are displayed as percentage of cells exhibiting the observed number of DNA probe signals, and exact number of signals for the DNA probes used: chromosome 1 (LSI 1p36 and LSI 1q25 DNA probes) and chromosome 17(LSI 17p13.1 and CEP 17 DNA probes). Array CGH: <bold>p </bold>and <bold>q </bold>= chromosome arms, <bold>cen</bold>. = centromeric; <bold>G </bold>= chromosome gain, <bold>L </bold>= chromosome loss. Q-PCR: gene copy number fold changes are determined by the ΔΔC_Trelative quantification method.</p></table-wrap-foot>", "<table-wrap-foot><p>Thirteen representative cases drawn from the HSJD cohort analyzed by FISH, aCGH and Q-PCR of chromosome 17. <bold>n.e </bold>= not evaluable results. MYCN amplification status: <bold>NA </bold>= not amplified, <bold>A </bold>= amplified. Disease status: <bold>NP </bold>= no disease progression, <bold>P </bold>= disease progression. Survival status: <bold>A </bold>= alive, <bold>D </bold>= dead. FISH: results are displayed as percentage of cells exhibiting the observed number of DNA probe signals, and exact number of signals for the DNA probes used: chromosome 1 (LSI 1p36 and LSI 1q25 DNA probes) and chromosome 17(LSI 17p13.1 and CEP 17 DNA probes). Array CGH: <bold>p </bold>and <bold>q </bold>= chromosome arms, <bold>cen</bold>. = centromeric; <bold>G </bold>= chromosome gain, <bold>L </bold>= chromosome loss. Q-PCR: gene copy number fold changes are determined by the ΔΔC_Trelative quantification method.</p></table-wrap-foot>", "<table-wrap-foot><p><bold>n.e </bold>= not evaluable results. MYCN amplification status: <bold>NA </bold>= not amplified, <bold>A </bold>= amplified. Disease status: <bold>NP </bold>= no disease progression, <bold>P </bold>= disease progression. Survival status: <bold>A </bold>= alive, <bold>D </bold>= dead. FISH: results are displayed as percentage of cells exhibiting the observed number of DNA probe signals, and exact number of signals for the DNA probes used: chromosome 1 (LSI 1p36 and LSI 1q25 DNA probes) and chromosome 17(LSI 17p13.1 and CEP 17 DNA probes). Array CGH: <bold>p </bold>and <bold>q </bold>= chromosome arms, <bold>cen</bold>. = centromeric; <bold>G </bold>= chromosome gain, <bold>L </bold>= chromosome loss. Q-PCR: gene copy number fold changes are determined by the ΔΔC_Trelative quantification method.</p></table-wrap-foot>" ]

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[ "<title>Background</title>", "<p>A whole-genome association study of major genetic determinants for host control of HIV-1 has identified two polymorphisms that explain nearly 15% of the variation among individuals in viral load during the asymptomatic set-point period of infection. One of these polymorphisms is located in the 5' region of the HLA-C gene, 35 kb away from transcription initiation and has been reported to be associated with differences in HLA-C expression levels [##REF##17641165##1##]. As a classical MHC class I gene, HLA-C has the potential to restrict HIV-1 by presenting epitopes to cytotoxic T cells (CTLs) [##REF##16882705##2##,##REF##9412709##3##], resulting in the destruction of infected cells. However, the potential ability of HLA-C to present epitopes to CTLs is severely limited by its poor expression at the cell surface (10-fold lower than either HLA-A or -B) [##REF##9870655##4##] and its tendency to accumulate as free heavy chains or heavy chains associated with β₂-microglobulin but free of peptides as a result of poor assembly [##REF##16181327##5##]. HLA-C has also the least diversity of the three classical MHC class I loci. Accordingly, an analysis of the class I restricted CD8+T cell responses against HIV-1 revealed that variation in viral set-point and absolute T cell count is strongly associated with particular HLA-B, but not HLA-A or HLA-C allele expression [##REF##15592417##6##]. In addition, HLA-Cw4/+ heterozygosity promotes rapid progression to AIDS illness, as does HLA-Cw4/Cw4 homozygosity [##REF##10073943##7##]. Interestingly, the virus has evolved a strategy to selectively down-regulate HLA-A and -B but not HLA-C, via the regulatory protein Nef [##REF##10403641##8##]. The immunity of HLA-C to Nef-mediated down modulation confers to the virus the capacity to escape NK cell attack since HLA-C is a dominant inhibitory ligand of NK cells [##REF##7769654##9##]. Thus, the overall trade-off of high HLA-C expression might be favourable to the virus, and not to the host. The relative importance of CTLs and NK cells <italic>in vivo </italic>is still unclear and the interpretation of genetic studies showing association to viral set-point is particularly complex.</p>", "<p>Like other MHC class I and II molecules, HLA-C is selectively incorporated into the HIV-1 envelope [##REF##11390619##10##,##REF##3542913##11##]. A study previously reported by our group [##REF##10546855##12##] demonstrated that virion-associated HLA-C molecules have a profound influence on the infectivity of HIV-1. MHC class I negative cell lines were non permissive for the replication of primary HIV-1 isolates and only partially permissive for the replication of T cell line adapted viruses. Transfection of HLA-Cw4 into these cell lines restored their capacity to support viral replication. The increased infectivity of viruses grown in the presence of HLA-Cw4 was associated with changes in viral envelope protein conformation, which included an enhanced expression of epitopes not normally exposed upon CD4 binding.</p>", "<p>Here we further investigate this phenomenon in a different experimental system where the expression of HLA-C was selectively silenced by small interfering RNA sequences (siRNA) and the infectivity-enhancement effect evaluated in fusion assays with cells expressing CCR5 and/or CXCR4 co-receptors. To overcome unknown effects of other viral gene products on viral infectivity, pseudotyped viruses expressing the same viral genome backbone, but different <italic>env</italic>, were used. The association of HLA-C with Env was tested using our previously reported technique for the detection of molecular complexes formed at the surface of cells during the fusion process (fusion complexes) [##REF##16702010##13##].</p>" ]

[ "<title>Methods</title>", "<title>Antibodies</title>", "<p>W6/32 is a mouse monoclonal antibody specific for HLA-A, -B and -C trimeric complex [##REF##87477##31##]. The L31 monoclonal antibody is specific for the α domain of HLA-C heavy chain [##REF##2446880##32##, ####REF##8316764##33##, ##REF##8727205##34####8727205##34##], not associated to β₂-microglobulin. Anti-gp120 human sera from HIV-positive patients were kindly provided by Dr. Lucia Lopalco, DIBIT-HSR, Milan, Italy. IgG were purified using Protein G Sepharose 4 Fast Flow (GE Healthcare Lifescience, Chalfont St. Giles, UK) following manufacturer's instructions.</p>", "<title>Cells</title>", "<p>HeLa (HLA-Cw12, [##REF##17331531##35##]) and HEK-293T (HLA-Cw07, [##REF##17331531##35##]) cells were obtained from the American Type Culture Collection (ATCC).</p>", "<p>HeLa-derived effector cell lines expressing the HIV-1 <italic>env </italic>gene of strains ADA, LAI [##REF##7903129##36##] and NDK [##REF##11576641##37##] and the indicator target cell line HeLa P4.2 [##REF##7520946##38##] were kindly provided by Dr. Mark Alizon and Dr. Uriel Hazan, Institut Cochin, Paris, France.</p>", "<p>NIH 3T3 cells expressing the HIV-1 receptor CD4 and the chemokine receptor CCR5 (3T3.T4.CCR5) or CXCR4 (3T3.T4.CXCR4) were obtained from the NIH AIDS Research &amp; Reference Reagent Program, division of AIDS, NIAID, Dr. Dan R. Littman [##REF##9230441##15##].</p>", "<p>The TZM-bl cell line [##REF##12019106##39##] was from the EU programme EVA/MRC, CFAR NIBSC, UK. This cell line expresses CD4, CCR5 and CXCR4 and contains HIV-1 LTR-driven <italic>E. coli </italic>β-galactosidase and firefly luciferase reporter cassette that are activated by HIV-1 Tat expression.</p>", "<p>CHO and CHO-gp120/gp41 [##REF##16702010##13##] cells were stably transfected with the vector pZeoSV2(+) (Invitrogen, Carlsbad, CA, USA) bearing the HLA-Cw4 gene, and the cell lines obtained were named CHO-HLA-C and CHO-gp120-HLA-C, respectively.</p>", "<p>CHO and CHO-HLA-C cell lines were transiently transfected with HIV-1 <italic>env </italic>genes from primary and laboratory isolates NDK, J500 (a primary X4 tropic isolate [##REF##9359702##40##]), 92UG024, 93MW965 and 91US005 [##REF##8627686##41##] cloned in the expression vector pCDNA3.1 (Invitrogen, Carlsbad, CA, USA).</p>", "<title>RNA silencing of HLA-C</title>", "<p>The HLA-C mRNA [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"NM_002117\">NM_002117</ext-link>] target sites for siRNA were determined by using the Dharmacon siGENOME software and synthesized by Dharmacon (Lafayette, CO, USA). The siRNAs targeted different regions of the HLA-C mRNA.</p>", "<p>In particular, siRNAs J-017513-06 (5'P-UAAUCCAUCAACGCUUCAUUU-3') and J-017513-08 (5'P-UUUGGAAGGUUCUCAGGUCUU-3') were found to be specific for HLA-C silencing, while siRNAs J-017513-05 (5'P-AUAGCGGUGACCACAGCUCUU-3') and J-017513-07 (5'P-ACUUCUAGGAAUUGACUUAUU-3') also silenced HLA-A and -B mRNAs.</p>", "<p>HeLa cells expressing <italic>env </italic>genes were transfected with 100 nmol/well of siRNA following manufacturer's instructions, using DharmaFECT 1 reagent (Dharmacon, Lafayette, CO, USA). The silencing of HLA-C protein expression was verified by Western blot after 72 hours.</p>", "<p>The absence of off-target effects was verified both by RT-PCR of HLA-A, -B, -C, β₂-microglobulin, HIV-1 <italic>env </italic>and GAPDH, and by ELISA analysis of gp120/gp41 expression using HIV-1 positive human sera.</p>", "<title>TZM-bl reporter gene assays</title>", "<p>The fusion process between gp120/gp41 effector cells (HeLa-ADA, HeLa-LAI, HeLa-NDK) and TZM-bl cells was assessed by measuring luciferase activity and by X-gal cell staining.</p>", "<p>TZM-bl cells (50.000 per well) were plated in 96 microtiter wells (Corning, NY, USA) to an equivalent number of effectors cells for 3 to 6 hours at 37°C. The luciferase activity resulting from fusion and transactivation was analyzed using the Brite Lite reagent following manufacturer's instructions and quantified by using a Victor 3 apparatus (Perkin Elmer, Waltham, MA, USA). All the assays were performed in triplicate.</p>", "<p><italic>In situ </italic>staining of fusing cells for β-galactosidase gene activation was performed in a 24-well plate format (Corning Life Sciences, Lowell, MA, USA) as reported [##REF##8095307##42##]. Blue-stained syncytia were photographed using a Nikon Eclipse 80 <italic>i </italic>microscope, counted and fusion efficiency determined by calculating the fusion index [##REF##6265470##43##].</p>", "<title>Cell fusion assays</title>", "<p>NIH 3T3.T4.CCR5 and 3T3.T4.CXCR4 were stained with the fluorescent lipophilic dye Vybrant DiI (Invitrogen, Carlsbad, CA, USA) following manufacturer's instructions. Cells were plated at 400,000 per well on a six-well plate (Corning Life Sciences, Lowell, MA, USA) and, 72 hours post siRNA transfection, co-cultivated at 1:1 ratio with HLA-C silenced and non-silenced HeLa-gp120/gp41 cells labeled with the fluorescent lipophilic dye Vybrant DiO (Invitrogen). After 6 hours, syncytia formation was analyzed using a fluorescence microscope (Nikon Eclipse <italic>80i</italic>) for green and red fluorescence and the double positive yellow syncytia counted [##REF##8858163##44##,##REF##16515685##45##].</p>", "<title>gp120 ELISA detection assay</title>", "<p>Ninety-six well plates (Nunc, Roskilde, Denmark) were coated with 50 μl/well of a solution of 2 μg/ml of the D7324 gp120 antibody (Aalto Bioreagents, Dublin, Ireland), and a 3 mg/ml solution of total protein from cell lysate samples was added as described [##REF##16702010##13##]. Positive controls consisted of a 100 ng/ml pool of 5 different gp120s obtained from EVA/MRC Centralised Facility for AIDS Reagents, NIBSC, UK (CN54, IIIB, MN, SF2 and W61D). Plates were washed and incubated with 1:200 diluted purified human IgG from sera of HIV-1 positive patients (25 mg/ml), washed and incubated with 1:500 diluted goat anti-human horseradish peroxidase conjugate (BioRad). Optical signal was developed with SigmaFast OPD solution (Sigma, St. Louis, MO, USA).</p>", "<title>RT-PCR</title>", "<p>Total RNA was extracted from 24 hours silenced and non-silenced cultured cells using the RNeasy Plus mini kit (Qiagen, Germantown, MD, USA) and treated with RNase-free DNase I (Sigma). Reverse transcription (RT) of 1 μg of total RNA was performed using the Quantitect Reverse Transcription kit (Qiagen) and random primers. For PCR amplification of HLA-A, -B and -C, primers and conditions were used as previously reported [##REF##10715516##14##]. Primers used to amplify HIV-1 <italic>env </italic>gene were: 5'-GGGCCACACATGCCTGTGTA-3' forward and 5'-CTAATTCCATGTGTACATTGTACTGTG-3' reverse; for β₂-microglobulin amplification 5'-GATGAGTATGCCTGCCGTGTG-3' forward and 5'-CAATCCAAATGCGGCATCT-3' reverse; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification 5'-GCATCCTGGGCTACACTGA-3' forward and 5'-TGACAAAGTGGTCGTTGAGG-3' reverse. PCR was performed for 32 cycles at 94°C, 60°C and 72°C for 1 min in each step. PCR products were analyzed on a 1% agarose gel and stained with Sybr Safe (Molecular Probes, Eugene, OR, USA). Images were acquired with an AutoChemi System apparatus (UVP, Cambridge, UK). Controls for genomic DNA contaminations consisted in RT reactions in which the polymerase was omitted.</p>", "<title>Western blot analysis</title>", "<p>Seventy-two hours after HLA-C siRNA transfection, cells were lysed, the total protein content of supernantant was measured using a colorimetric assay (DC protein assay, BioRad, Hercules, CA, USA) and used for western blot analysis.</p>", "<p>Equal amounts (30 μg/lane) of cell lysates were separated on 3 to 8% NuPAGE Tris-acetate acrylamide gels (Invitrogen, Carlsbad, CA, USA) and transferred onto polyvinylidene difluoride membranes (GE Healthcare Lifescience, Chalfont St. Giles, UK). Membranes were blocked in a Tris-buffered saline solution containing 5% non-fat dry milk and incubated with the L31 monoclonal antibody (1:200 dilution). Anti-mouse horseradish peroxidase-conjugated antibody (Dako, Carpinteria, CA, USA) was used as secondary antibody at 1:2,000 dilution and immunoreactive bands were visualized with the Opti-4CN detection kit (BioRad, Hercules, CA, USA).</p>", "<title>FACS analysis</title>", "<p>Cells were analyzed by immunofluorescent staining and cytofluorimetry on a FACScanto apparatus (Becton Dickinson, San Jose, CA, USA). After incubating 500,000 cells with the primary anti-HLA monoclonal antibodies W6/32 or L31, these were washed and incubated with a 1:200 dilution of the goat-anti mouse IgG-FITC secondary antibody (Becton Dickinson). The analysis was conducted using the FACSDiva software (Becton Dickinson). For L31 epitope unmasking through β₂-microglobulin stripping, cells were pre-treated with acidified medium as described [##REF##8827219##46##] and immediately analysed.</p>", "<title>Infectivity of pseudoviruses produced on HLA-C silenced cells</title>", "<p>HLA-C mRNA was silenced in 293T cells as previously described for HeLa cells. After 24 hours, silenced and non-silenced 293T cells were co-transfected with backbone (pSGΔenv) and <italic>env </italic>plasmids (subtype B isolates 6535.3 and pRHPA4259.7, subtype D isolate NDK, and Vescicular Stomatitis Virus (VSV) envelope protein G), as described [##REF##16051804##47##]. Pseudoviruses were collected after 48 hours and quantified for p24 content using a standard ELISA Kit following manufacturer's instructions (Innotest-HIV antigen mAb, Innogenetics, Gent, Belgium). Both normal and HLA-C silenced pseudoviruses were used at a p24 concentration of 150 pg/ml. Infections of TZM-bl cells were done in quadruplicate and luminescence measured after 4, 8, 24 and 48 hours of incubation using a Victor 3 luminometer (Perkin Elmer) as previously described.</p>", "<title>Fusion complex analysis</title>", "<p>Fusion complexes were fixed with paraformaldehyde, purified and analyzed as described [##REF##16702010##13##]. Briefly, CHO-gp120-HLA-C and CHO-CD4-CCR5 cells were co-cultivated 4 hours at 37°C, fixed and lysed. Cell lysates were passed over a snowdrop <italic>Galanthus nivalis </italic>lectin column and eluted in 1 M methyl α-D-mannopyranoside (Sigma). Fusing cells were also fixed with DTSSP (Pierce Biotechnology, Rockford, IL, USA), following manufacturer's instructions. Fusion complexes were purified and dissociated using 5% β-mercaptoethanol in SDS-PAGE sample buffer. Effector and target cells were also separately fixed prior purification. Paraformaldehyde fixed fusion complexes were analysed for HLA-C co-purification by dot-blot and DTSSP fixed complexes by Western blot with HLA-C specific mAb L31.</p>", "<title>Statistical analysis</title>", "<p>Data were analyzed by ANOVA and unpaired Student's t-test with Welch's correction, using the software GraphPad Prism 4.0c (GraphPad Software, Inc., CA, USA).</p>", "<title>Sequence analysis</title>", "<p>HIV-1 Env sequences (NDK [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"A34828\">A34828</ext-link>], LAI [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AF004394\">AF004394</ext-link>]; ADA [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"AY426119\">AY426119</ext-link>]; 92UG024 [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"U43386\">U43386</ext-link>]; 93MW965 [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"U08455\">U08455</ext-link>]: 91US005 [GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"U27434\">U27434</ext-link>]) were aligned and compared using CLC Sequence Viewer 4.6.2, developed by CLC bio A/S <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.clcbio.com\"/> for Apple Mac OSX.</p>" ]

[ "<title>Results</title>", "<title>Effects of HLA-C on the HIV-driven fusion process</title>", "<p>To assess the role of HLA-C in the fusion process we used a cell fusion assay between CHO cells expressing gp120/gp41, either alone or in combination with HLA-C and CHO cells expressing CD4-CCR5 (Table ##TAB##0##1##) [##REF##16702010##13##]. When CHO-gp120-HLA-C cells were co-cultivated with CHO-CD4-CCR5 cells, a dramatic increase (p &lt; 0.05) in the number and size of syncytia, as compared to those obtained with the same cells not expressing HLA-C, was observed (Fig. ##FIG##0##1A##). The increased fusion efficiency was not due to a higher expression level of gp120/gp41 in CHO-gp120-HLA-C cells, since they express on average 27% less gp120/gp41 than CHO-gp120/gp41 cells, when analyzed in ELISA using HIV-1 positive human sera (Fig. ##FIG##0##1B##).</p>", "<p>Similar results were obtained in a different cell fusion assay where CHO and CHO-HLA-C cells, transiently transfected with gp120/gp41 from different primary and laboratory HIV-1 isolates, were fused with TZM-bl cells and fusion quantified by luciferase transactivation. All gp120/gp41 tested (93MW965, 91US005, 92UG024) showed higher fusion efficiency when co-cultivated with TZM-bl cells if co-expressed with human HLA-C (Fig. ##FIG##1##2##). Only two X4-tropic isolates (J500 and NDK) failed to show a statistically significant fusion increase.</p>", "<title>HLA-C silencing of cells expressing gp120/gp41</title>", "<p>HeLa cells constitutively express HLA-C and HLA-A and, at lower levels, HLA-B [##REF##10715516##14##]. Various HeLa-derived cell lines, constitutively expressing HIV-1 Env, were silenced by HLA-C specific siRNAs (Table ##TAB##0##1##). The expression of gp120 in HeLa-ADA, -LAI and -NDK, as well as that of β₂-microglobulin and GAPDH genes was not affected.</p>", "<p>There was no unwanted off-target silencing of non HLA-C genes (Fig. ##FIG##2##3A##). The expression of HLA-C protein on HeLa-ADA and 293T cells was undetectable at 72 hours from siRNA transfection (Fig. ##FIG##2##3B##). Fusion efficiency, determined by counting the number of syncytia formed, was significantly lower (p &lt; 0.01) when HLA-C silenced cells expressing HIV-1 gp120/gp41 of the LAI strain were co-cultivated with HeLa P4.2 cells as target cells (Fig. ##FIG##3##4##). Fusion efficiency of HeLa-NDK cells was less affected by HLA-C silencing, confirming that the NDK gp120/gp41 has a lower sensitivity to the presence of HLA-C [##REF##10546855##12##]. When silencing was performed with siRNAs specific for HLA-C or with a pool of siRNAs silencing also HLA-A and -B, similar levels of reduction in fusion efficiency were observed.</p>", "<title>Syncytia formation using CCR5 or CXCR4 co-receptors</title>", "<p>To test the role of HLA-C in the fusion process with cells expressing CCR5 or CXCR4 co-receptors, we measured the fusion index in co-cultures of HeLa-ADA and 3T3.T4.CCR5 cells or HeLa-LAI and 3T3.T4.CXCR4 cells with or without siRNA silencing of HLA-C. In both cultures, the fusion index was significantly lower (p &lt; 0.01) in HLA-C-silenced cells than in the corresponding non-silenced controls (Fig. ##FIG##4##5##) showing that HLA-C increases the fusion efficiency of both CCR5 and CXCR4 tropic viruses.</p>", "<p>3T3.T4.CXCR4 cells express 2–3 times more CXCR4 than HeLa-P4.2 and TZM-bl cells. Similarly, 3T3.T4.CCR5 cells express about 10 times more CCR5 as compared to TZM-bl cells (data not shown). We observed that these cells allowed the fusion with cells expressing Envs with a different co-receptor tropism, although at lower level. The use of the heterologous co-receptor, already evident [##REF##9230441##15##] using pseudotyped viruses, is increased in fusion assays with Env-expressing cell lines, in particular for longer co-cultivation times. Under these experimental conditions, we investigated the role of HLA-C in modulating fusion efficiency in the presence of the heterologous co-receptor. We observed that the R5-tropic gp120/gp41 ADA was sensitive to HLA-C presence when fusing with 3T3.T4.CXCR4 cells whereas the X4-tropic LAI was not affected by HLA-C presence when fusing with 3T3.T4.CCR5 cells (Fig. ##FIG##4##5##). Also in these experiments, the NDK gp120/gp41 was found to fuse with the same efficiency with 3T3.T4.CXCR4 and, at lower levels, with 3T3.T4.CCR5 cells, when using HLA-C silenced or non-silenced HeLa-NDK cells (Fig. ##FIG##4##5##).</p>", "<title>Pseudovirus infection assay</title>", "<p>Pseudoviruses produced on normal and HLA-C silenced 293T cells were quantified for p24 content and used in transduction assays (Table ##TAB##0##1##). Pseudoviruses bearing subtype B 6535.3 and pRHPA4259.7 HIV-1 <italic>env </italic>genes showed a statistically significant reduction in infectivity when produced in HLA-C silenced 293T cells. Conversely, no significant differences were observed with either NDK subtype D <italic>env </italic>gene or control virus pseudotyped with the VSV-G protein (Fig. ##FIG##5##6A##).</p>", "<p>When the HLA-C insensitive NDK-pseudovirus was used at infectious doses that were 1/3 and 1/10 of the original inoculum, a significant infectivity difference between pseudoviruses produced in HLA-C silenced and non-silenced cells was noted. The HLA-C sensitive pseudovirus pRHPA4259.7 maintained its sensitivity to HLA-C also at lower m.o.i. (1/10 of the original inoculum, data not shown). When the m.o.i. of the pRHPA4259.7 pseudovirus was increased, the infectivity levels of pseudoviruses produced on normal and HLA-C silenced 293T cells was kept significantly different (Fig. ##FIG##5##6B##).</p>", "<title>HLA-C/gp120 association on cells before and after fusion</title>", "<p>In the previous study we provided evidence of a specific association between virionic HLA-C molecules and gp120 by co-immunoprecipitating the two molecules with the HLA-C-specific monoclonal antibody L31 and a gp120-specific antibody [##REF##10546855##12##]. In this work we looked for additional evidence of HLA-C-gp120 association occurring on cells taken after fusion using a previously described method that allows the isolation of CD4-CCR5-gp120/gp41 fusion complexes after fixation with paraformaldehyde or DTSSP and purification with <italic>Galanthus nivalis </italic>(<italic>GN</italic>) lectin, which specifically binds to gp120 [##REF##16702010##13##]. The presence of HLA-C molecules within the fusion complexes could be tested by dot blot with the antibody L31 which also recognizes the denatured protein [##REF##1711567##16##]. Fig. ##FIG##6##7## panel A shows a dot-blot with antibody L31 of total cell lysates or proteins eluted from <italic>GN </italic>lectin columns. L31-reactive molecules were detected in total cell lysates of CHO-HLA-C (lane c) and CHO-gp120-HLA-C cells (lane d) but not in the HLA-C negative CHO cell line (lane a) and the CHO-CD4-CCR5 fusion partner (lane b). The eluate of <italic>GN </italic>lectin columns loaded with a mixed extract of CHO-gp120-HLA-C and CHO-CD4-CCR5 cells which had been fixed before fusion, displayed a significant amount of L31 reactive molecules (lane g), showing that a specific association between HLA-C and gp120 occurred in cells co-expressing the two molecules, as previously described in LAI-infected 221-Cw4 cells [##REF##10546855##12##]. When the same cells were allowed to fuse before being fixed, the eluate of <italic>GN </italic>lectin purified cell extract displayed an increased amount of L31-reactive molecules (lane h) indicating that during the process of cell fusion additional HLA-C molecules are recruited within the fusion complexes. The lack of L31-reactive molecules in the eluate of <italic>GN </italic>lectin purified CHO-HLA-C cells (lane f) demonstrates that in this experimental setting HLA-C molecules are purified via their specific binding to gp120.</p>", "<p>To gain further evidence of the association between HLA-C and gp120, the same protein samples, after fixation with DTSSP and purification on <italic>GN </italic>lectin columns, were chemically reduced, separated on SDS-PAGE and blotted with L31 antibody which revealed a 45 kDa band corresponding to the HLA-C heavy chain. Also in this experiment a relatively higher amount of HLA-C was co-purified from cells which were allowed to fuse before fixation (fusion complex), as compared to non-fused cells (no fusion complex) (Fig. ##FIG##6##7B##). These results provide further evidence that HLA-C is associated to gp120 on the cell membrane and suggest that additional HLA-C is recruited within the fusion complex during cell fusion.</p>", "<title>Sequence analysis of HLA-insensitive Envs</title>", "<p>The sequence of the <italic>env </italic>gene of the HLA-C insensitive primary isolate J500 (clade B) was determined. When this was compared to the sequence of the other HLA-C insensitive isolate NDK (clade D), and to the sequences of the HLA-C sensitive Envs tested (93MW965, 91US005, 92UG024, ADA, LAI, 6535.3 and pRHPA4259.7), three identical aminoacid substitutions (N297K, N298Y and I318T, relative to the LAI <italic>env </italic>sequence) were identified in the V3 loop. <italic>Env </italic>sequence analysis of the Los Alamos HIV Reference Database showed that the I318T mutation is relatively uncommon, occurring in 92 out of the 1603 <italic>env </italic>sequences available (5.7%). Mutations N297K and N298Y are extremely rare, occurring only in 2 isolates reported in the database. In addition, the combination of these 3 mutations was found only in a single <italic>env </italic>sequence (isolate D.TZ.87.87TZ4622). Position 297 is associated with a potential N-glycosilation site [##REF##11181557##17##].</p>" ]

[ "<title>Discussion</title>", "<p>This work demonstrates that virion-associated HLA-C molecules, when present on cells expressing gp120/gp41, significantly enhance fusion efficiency and pseudovirus transduction. Our conclusions are supported by the following findings: a) CHO cells co-expressing HIV-1 gp120/gp41 and human HLA-C fuse more rapidly and produce larger syncytia than the original CHO-gp120/gp41 cells from which they are derived; b) transient transfection of gp120/gp41 from different primary isolates in CHO cells co-expressing HLA-C results in a significant increase in fusion; c) silencing of HLA-C in human cell lines expressing HIV-1 gp120/gp41 of R5 and X4 tropic strains, significantly suppresses fusion, d) pseudoviruses produced in HLA-C silenced 293T cells display a significant reduction of infectivity; e) the fusion enhancement property of HLA-C is specific for HIV-1 Env, since a virus pseudotyped with the G envelope protein of VSV is not influenced by the presence of HLA-C.</p>", "<p>The effect of HLA-C on fusion was observed with both exogenous HLA-C transfected into CHO cells and endogenous HLA-C after its silencing with siRNA in human cells.</p>", "<p>Some of the data point to the existence of HLA-C \"insensitive\" or \"less-sensitive\" variants since the fusogenic capacity of gp120/gp41 from two isolates, NDK and J500, was not different in HLA-C-silenced and non-silenced cells. However, we observed that HLA-C insensitivity is not an absolute feature, since there was a small difference, although not statistically significant, in the fusion efficiency of NDK and J500 in silenced and non-silenced cells. In addition, a relationship between the infectious dose and the HLA-C sensitivity of pseudoviruses was observed since when infections were performed at low infectivity ratios, the HLA-C insensitive NDK pseudovirus became HLA-C sensitive. Conversely, when high titers of an HLA-C sensitive pseudovirus were used, its infectivity remained dependant on the presence of HLA-C. The relative insensitivity of NDK to the presence of HLA-C could contribute to its reported higher cytopathicity and infectivity [##REF##2806917##18##] and could be the result of a variable infectivity degree of Env [##REF##10546855##12##] or of a lower level of incorporation of HLA-C [##REF##11390619##10##].</p>", "<p>The comparison of the <italic>env </italic>sequences of the two unrelated, HLA-C insensitive gp120/gp41s identified, NDK (clade D) and J500 (clade B), with the sequences of the HLA-C sensitive Envs, revealed 3 identical aminoacid substitutions in the V3 loop, which were absent in all other HLA-C sensitive Envs analyzed. This would suggest an involvement of these mutations in the V3 loop in the acquisition of the HLA-C insensitive phenotype. We analyzed an NDK-derived Env mutant, NDKm7 [##REF##11333929##19##], in which the KY mutations in position 297–298 reverted to NN. Virus particles pseudotyped with the NDKm7 <italic>env </italic>remained HLA-C insensitive as the original NDK <italic>env </italic>(data not shown), thus excluding the involvement of these mutations in reducing sensitivity to HLA-C presence. It is possible that other mutations, or their combinations, might directly affect the sensitivity to HLA-C by changing the pattern of interaction between HLA-C and gp120, as reported by other authors who studied mutations related to the acquisition of a CD4-independent tropism within gp120 [##REF##11333929##19##,##REF##10559349##20##].</p>", "<p>The data reported in this study confirm the physical association between HIV-1 gp120/gp41 and HLA-C, that was originally observed in experiments in which HLA-C and gp120 were co-immunoprecipitated from HIV-1 infected cells [##REF##10546855##12##]. HLA-C molecules could be co-purified and detected in fusion complexes in association with gp120/gp41, CD4 and the co-receptor. Such an association may induce conformational changes of gp120 favouring the exposure of cryptic functional epitopes [##REF##10546855##12##]. It has also been recently reported that viral particles carry more HLA molecules than gp120/gp41 trimers [##REF##17942547##21##]. The association between a gp120/gp41 trimer and multiple HLA-C molecules might reduce gp120 shedding, thus keeping more functional the trimeric gp120/gp41 complexes on the viral envelope and resulting in increased fusion efficiency.</p>", "<p>The increase in fusion and viral infectivity was observed using CHO cells transfected with HLA-Cw4, as well as HeLa cells which express constitutively HLA-Cw12 and pseudoviruses originating from 293T cells which express HLA-Cw7 (Table ##TAB##0##1##). Similar results were obtained with the HLA-Cw3 allele (L. Lopalco, DIBIT-San Raffaele, Milan, personal communication). Altogether, the Cw3, Cw4, Cw7, and Cw12 serological alleles include members of both groups of the known HLA-C dimorphism [##REF##8265660##22##] and account for almost 80% of all the common HLA-C serotypes. Due to the more limited polymorphism of HLA-C as compared to HLA-A and -B, this limited panel is inclusive enough to allow us to sample all the HLA-C-distinctive substitutions and most of the common allelic variations. Remarkably, most of these cluster around the binding groove, but the co-immunoprecipitation of env with HLA-C [##REF##10546855##12##] was observed by immunoprecipitating the complex with antibody L31, that binds on the alpha 1 domain alpha helix, e. g. in proximity to the sites at which essentially all the polymorphic HLA-C positions cluster. This suggests that HLA-C polymorphism is unlikely to influence this association, and that the residues important for co-immunoprecipitation reside within the relatively invariant HLA-C backbone. In line with this finding, we have observed the infectivity-enhancement effect with all the alleles tested so far, suggesting that most HLA-C alleles bind Env. We cannot however exclude the possibility that some HLA-C allelic variants may be more efficient than others in binding Env and enhancing viral infectivity.</p>", "<p>An implication of these findings is that HLA-C may be selectively involved in protective immunity. A protective effect was observed in HIV serodiscordant couples with unmatched HLA-C alleles [##REF##15220037##23##] and anti-HLA antibodies are frequent in exposed, but seronegative subjects [##REF##8568315##24##,##REF##10659050##25##]. It has also been reported that MHC class I concordance is associated with an increased risk of mother to child HIV-1 transmission [##REF##9498431##26##,##REF##12167265##27##]. Since early studies in primates were suggestive of anti-MHC antibodies being protective [##REF##7755909##28##], the possibility of using HLA molecules for a HIV-1 vaccine has long been debated [##REF##8303282##29##,##REF##8211133##30##]. Our data point to an association between HLA-C and Env in mature virions which may induce the expression of critical conformational epitopes [##REF##10546855##12##]. Since the few Env that showed lower sensitivity to HLA-C are X4 tropic, the inclusion of HLA-C in new immunogenic formulations may help eliciting broadly neutralizing antibodies that would be important for the <italic>in vivo </italic>host control of R5 tropic strains of HIV-1.</p>" ]

[ "<title>Conclusion</title>", "<p>HLA-C influences viral replication by at least three distinct and opposite mechanisms: induction of cytotoxic T cells (suppression), inhibition of NK cells (enhancement) and enhancement of virus infectivity. This last effect is associated to a specific association of virionic HLA-C molecules to Env. The immunity of HLA-C to the Nef-induced down-regulation confers to the virus not only the capacity to escape NK cells control but also a higher replicative capacity suggesting that high HLA-C expression is advantageous to the virus and not the host.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>A recently identified genetic polymorphism located in the 5' region of the HLA-C gene is associated with individual variations in HIV-1 viral load and with differences in HLA-C expression levels. HLA-C has the potential to restrict HIV-1 by presenting epitopes to cytotoxic T cells but it is also a potent inhibitor of NK cells. In addition, HLA-C molecules incorporated within the HIV-1 envelope have been shown to bind to the envelope glycoprotein gp120 and enhance viral infectivity. We investigated this last property in cell fusion assays where the expression of HLA-C was silenced by small interfering RNA sequences. Syncytia formation was analyzed by co-cultivating cell lines expressing HIV-1 gp120/gp41 from different laboratory and primary isolates with target cells expressing different HIV-1 co-receptors. Virus infectivity was analyzed using pseudoviruses. Molecular complexes generated during cell fusion (fusion complexes) were purified and analyzed for their HLA-C content.</p>", "<title>Results</title>", "<p>HLA-C positive cells co-expressing HIV-1 gp120/gp41 fused more rapidly and produced larger syncytia than HLA-C negative cells. Transient transfection of gp120/gp41 from different primary isolates in HLA-C positive cells resulted in a significant cell fusion increase. Fusion efficiency was reduced in HLA-C silenced cells compared to non-silenced cells when co-cultivated with different target cell lines expressing HIV-1 co-receptors. Similarly, pseudoviruses produced from HLA-C silenced cells were significantly less infectious. HLA-C was co-purified with gp120 from cells before and after fusion and was associated with the fusion complex.</p>", "<title>Conclusion</title>", "<p>Virionic HLA-C molecules associate to Env and increase the infectivity of both R5 and X4 viruses. Genetic polymorphisms associated to variations in HLA-C expression levels may therefore influence the individual viral set point not only by means of a regulation of the virus-specific immune response but also via a direct effect on the virus replicative capacity. These findings have implications for the understanding of the HIV-1 entry mechanism and of the role of Env conformational modifications induced by virion-associated host proteins.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>AM carried out siRNA silencing, RT-PCR, cell transfections, ELISA, Western blot and FACS analysis, cellular fusions and pseudovirus infections experiments. PR carried out sequencing, pseudovirus preparation and titration and fusion complexes preparation and analysis. MB isolated and cloned the HLA-C insensitive <italic>env </italic>sequence from the J500 primary isolate. AGS participated in the design and coordination of the study and drafted the manuscript. AB participated to study design, data analysis and gave a significant contribution in drafting and revising the manuscript. DZ produced Env-coding plasmids and stably transfected cell lines, did fusion complexes preparation and analysis, conceived the study and carried out its design, and, as corresponding author, carried out the drafting of the manuscript. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>We thank Prof. Umberto Bertazzoni (University of Verona, Italy) for helpful discussions and for critically reviewing the manuscript; Dr. Claudio De Santis (DIBIT-HSR, Milan, Italy) and Dr. Antonio Cosma (Helmholtz Zentrum München, Munich, Germany) for their continuous support and helpful discussions; Prof. Patrizio Giacomini, from the Regina Elena Cancer Institute in Rome, for helpful discussions and manuscript revision; Dr. Lucia Lopalco (DIBIT-HSR, Milan, Italy) for providing HIV-1-positive human sera; Dr. Gabriella Scarlatti (DIBIT-HSR, Milan, Italy) for providing the proviral DNA of the isolate J500; Prof. Uriel Hazan (Institut Cochin, Paris, France) for kindly providing some effector and target cell lines as well as NDK and NDKm7 Env expressing plasmids; Dr. Cettina Terranova (Section of Biology and Genetics, University of Verona, Italy) for designing PCR primers.</p>", "<p>Heartfelt thanks to Dr. Lucy Rasmussen (Stanford University, USA) for critically reviewing, editing and improving the manuscript.</p>", "<p>This study was supported by the Fondazione Cassa di Risparmio di Verona Vicenza Belluno e Ancona, Grant \"Salute e Ambiente\", by the VI Italian AIDS National Program of the Istituto Superiore di Sanità, Italy (ICAV grant 45G.41) and by the Europrise FP6 Network of Excellence, Life Sciences Programme, European Commission. Italfarmaco Spa provided a doctoral fellowship for M. B.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Fusion efficiency of CHO cells expressing HLA-C and HIV-1 Env</bold>. Panel A: Syncytia formation after co-cultivation of effector CHO cells expressing gp120/gp41 and HLA-C, or CHO cells expressing only gp120/gp41, with target CHO-CD4-CCR5 cells. The number and the extent of syncytia is significantly higher (p &lt; 0.05) when effector cells express HLA-C. Panel B: ELISA analysis of Env expression. CHO, negative control; CHO-gp120, cells stably expressing the Env gene of the R5 tropic HIV-1 isolate 91US005; CHO-gp120-HLA-C: CHO-gp120 cells stably expressing HLA-Cw4; gp120: positive control, consisting of a mixture of five different gp120s. The higher fusion efficiency of CHO-gp120-HLA-C cells is not due to an increased level of Env expression, since they express 27% less gp120 than CHO-gp120 cells.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Transient transfections of CHO cells expressing human HLA-C with different <italic>env </italic>sequences</bold>. CHO (-, grey bars) and CHO-HLA-C (+, black bars) cells transiently transfected with plasmids encoding Tat, Rev and Env from different primary and laboratory HIV-1 isolates and co-cultivated for 6 hours with TZM-bl target cells. After Tat driven transactivation of firefly luciferase expression, fusion efficiency was quantified and expressed as counts per second (CPS). Each value represents the average of four replicates. The gp120/gp41 of primary isolates 93MW965 (R5), 91US005 (R5) and 92UG024 (X4) are HLA-C sensitive (p &lt; 0.05) while isolates J500 (X4) and NDK (X4) are less sensitive to the presence of HLA-C (p not significant).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Specific silencing of HLA-C in human cell lines</bold>. Panel A: off-target effect analysis by RT-PCR in HLA-C silenced (+) and non-silenced (-) HeLa cells expressing HIV-1 gp120/gp41 (ADA). PCR was performed with primers specific for HLA (A, B, C), gp120, β₂-microglobulin and GAPDH. M: molecular weight marker. No off-target effect due to HLA-C mRNA silencing is affecting the mRNA levels of the other MHC class I genes, as well as β₂-microglobulin, HIV-1 gp120 or the housekeeping control gene GAPDH. Panel B: western-blot analysis of HLA-C protein expression. After 72 hours from siRNAs transfection, HLA-C is undetectable both in HeLa-ADA and in 293T cells.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Cell fusion of HLA-C silenced HeLa-Env cells with HeLa-P4.2 target cells</bold>. Analysis of syncytia formation by co-cultivating HLA-C silenced (+) and non-silenced (-) HeLa-LAI and HeLa-NDK cells with target HeLa-P4.2 cells, expressing CD4 and CXCR4. The number of syncytia formed is lower (p &lt; 0.01) using HLA-C silenced HeLa-LAI cells. Fusion efficiency of HeLa-NDK cells is not significantly affected by HLA-C silencing.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Comparison of the fusion efficiency of HLA-C silenced HeLa-Env cells with 3T3.T4.CCR5 and 3T3.T4.CXCR4 cells</bold>. HLA-C silenced (+, grey bars) and non-silenced (-, black bars) HeLa cells expressing gp120/gp41 of different HIV-1 isolates (ADA, LAI, NDK) co-cultivated with NIH 3T3.T4.CXCR4 and NIH 3T3.T4.CCR5 cells. Fusion efficiency of X4 tropic gp120 LAI is significantly lower (p &lt; 0.01) in HLA-C silenced cells when fusing with CXCR4 target cells. Similarly, fusion efficiency of the R5 tropic gp120 ADA is lower (p &lt; 0.01) in HLA-C silenced cells when fusing with CCR5 target cells. The fusion of ADA gp120 in HLA-C silenced cells with cells expressing CXCR4 is significantly (p &lt; 0.01) less efficient, while that of LAI gp120 with cells expressing CCR5 is similar, irrespective of HLA-C silencing. The NDK gp120 is HLA-C insensitive, when using either the CXCR4 or the CCR5 co-receptor.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Transduction efficiency of pseudoviruses produced in HLA-C silenced cells</bold>. Panel A: luciferase reporter gene assay analysis after transduction with pseudoviruses expressing subtype B HIV-1 <italic>env </italic>(6535.3 and pRHPA4259.7) or subtype D HIV-1 <italic>env </italic>(NDK), produced in HLA-C silenced (dashed line, open circles) and non silenced (continuous line, close squares) 293T cells. Each point (expressed as counts per second, CPS) represents average and standard deviation of four replicates. HLA-C sensitive pseudoviruses 6535.3 and pRHPA4259.7 show a significant lower infectivity (p &lt; 0.0001) when produced on HLA-C silenced cells. The NDK pseudovirus as well as a virus pseudotyped with the VSV-G envelope protein, do not show significant differences in infectivity when produced in HLA-C silenced or non silenced 293T cells. Panel B: analysis of the relation between pseudovirus infectious dose and HLA-C sensitivity. 1×, pseudovirus infectious titer giving a luciferase signal (expressed as counts per second, CPS) of 1000 at 16 hours post infection. When the HLA-C insensitive NDK pseudovirus was analyzed at lower infectious titers (0.3× and 0.1×), its infectivity was significantly increased by HLA-C. When the HLA-C sensitive pseudovirus pRHPA4259.7 was analyzed at higher infectious doses (3.3×, 10×), it remained sensitive to HLA-C presence.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p><bold>Co-purification of fusion complexes containing HLA-C molecules</bold>. Panel A: dot-blot analysis of purified fusion complexes for the presence of HLA-C. Lanes a, b, c and d: cell lysates before purification. Lanes e, f, g and h: cell lysates purified on <italic>Galanthus nivalis </italic>(GN) lectin columns. Panel B: western blot analysis to detect the presence of HLA-C in purified fusion complexes. Cells were treated with DTSSP, which fixes only proteins present on the cell membrane, and lysates purified on GN lectin columns. PC: positive control (HeLa cells expressing HLA-C); the arrow indicates HLA-C.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Summary of the HIV-1 envelopes tested in the different experimental models.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Experimental model</bold></td><td align=\"center\"><bold>Host cell</bold><break/><bold> (HLA-C allele)</bold></td><td align=\"center\"><bold>Env</bold><break/><bold> (tropism/subtype)</bold></td></tr></thead><tbody><tr><td align=\"center\">HLA-C siRNA silencing and cell<break/> fusion assays</td><td align=\"center\">HeLa (Cw12)</td><td align=\"center\">ADA (R5/B)<break/>LAI (X4/B)<break/>NDK (X4/D)</td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td align=\"center\">gp120/gp41 transient transfection<break/> and cell fusion assays</td><td align=\"center\">CHO-HLA-C (Cw4)</td><td align=\"center\">93MW965 (R5/C)<break/>91US005 (R5/B)<break/>92UG024 (X4/D)<break/>NDK (X4/D)<break/>J500 (X4/B)</td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td align=\"center\">Pseudovirus transductions</td><td align=\"center\">293T (Cw7)</td><td align=\"center\">pRHPA4259.7 (R5/B)<break/>6535.3 (R5/B)<break/>NDK (X4/D)<break/>m7NDK (X4/D)</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>HLA-C silencing was conducted on human cells (HeLa-derived) physiologically expressing HLA-C and stably expressing Env of different strains (ADA, LAI, NDK).</p><p>Transient transfections experiments with plasmids encoding different Envs were conducted on non-human CHO cells stably expressing HLA-C to directly compare the effect of HLA-C in the absence of other human MHC class I molecules.</p><p>Pseudoviruses were produced in HLA-C silenced 293T cells since this human cell line is the election host for efficient and quantitative production of pseudotyped virus particle. The Envs tested belong to a standard reference panel (NIBSC EVA CFAR ARP2066) except NDK.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>The overwhelming health benefits of physical activity are well documented [##UREF##0##1##,##REF##17671237##2##]. There is however mounting evidence that physical activity in clearly defined context is on the decline worldwide [##UREF##1##3##] and the physical environment is increasingly being recognized as a potential and promising determinant of physical activity behaviour [##UREF##2##4##, ####REF##9838979##5##, ##REF##14748314##6##, ##REF##16138933##7####16138933##7##]. The influence of the physical environment on physical activity behaviour is currently unknown among the African population and no specific measure exists for the assessment of environmental correlates of physical activity in the African environment. However, the influence of the environment on physical activity behaviours is particularly important because physical activity occurs in specific environmental settings [##REF##12113436##8##] and the environment that people build and inhabit provides potential opportunities for and barrier to engaging in a physically active lifestyle [##UREF##3##9##].</p>", "<p>Until recently, studies on environmental correlates of physical activity have focused on the narrower interpersonal and individual levels of intervention while neglecting the broader contextual framework of socioecological model [##REF##12471307##10##,##UREF##4##11##]. Research in this field are strengthened by utilizing the ecological model that recognizes the multiple levels of influence on health behaviours vis- social system, public policies and the physical environment [##UREF##5##12##]. This model has potential for explaining and facilitating better understanding of the influence of the environment on physical activity behaviors than the individual focus oriented model [##UREF##5##12##,##REF##11897464##13##].</p>", "<p>Sallis et al [##REF##9421846##14##] highlighted the necessity of first identifying reliable and valid measures of theoretically relevant environmental variables before the influence of the environment on physical activity can be adequately evaluated. Most of the studies that have evaluated the psychometric properties of environmental measures were conducted among Caucasians, especially in the United States [##REF##9421846##14##, ####REF##14499805##15##, ##REF##14998817##16##, ##REF##12948979##17####12948979##17##] and Europe [##REF##13677966##18##,##UREF##6##19##], with their findings reflecting few differences in the reliability coefficients of the environmental variables. For instance, while the European studies [##REF##13677966##18##,##UREF##6##19##] tend to identify items on neighborhood safety with lowest reliability coefficients, some American studies [##REF##14998817##16##,##REF##12948979##17##] implicated walking/cycling facilities and street/walking environments as items with the lowest reliability coefficients. There is therefore the potential possibility for the reliability of perceived environmental correlate items of physical activity to vary across countries and by cultures. Reliability studies from other continents may hence be necessary to fully compare and identify the international dimension and relevance of assessing environmental measures of physical activity behaviours.</p>", "<p>Various measures for assessing environmental correlates of physical activity are in existence [##REF##9421846##14##,##REF##12948979##17##,##REF##13677966##18##,##REF##15958168##20##]. Although, the development of these measures were based on the contextual framework of socioecological model, they are mostly lengthy, voluminous and yet to be assessed internationally. The International Physical Activity Prevalence Study (IPS) group in 2002 developed a shorter survey (IPAQ Environmental- Module), primarily for the assessment of environmental factors for bicycling and walking in the neighborhoods. The strengths of IPAQ E- module are its brevity and the inclusion of variables that have been shown to be associated with different levels of physical activity in different countries. Also, items on the E- module were considered to reflect current thinking in the field of environmental correlates of physical activity that are considered to be relevant to all countries regardless of their stage of economic development [##UREF##7##21##]. This assertion may however, need to be tested in African countries where the physical environment is distinct from that in other parts of the world.</p>", "<p>Evaluating the IPAQ E- module for reliability in an African environment is therefore necessary and may be a precursory step to identifying appropriate environmental correlates of physical activity behaviours among this population. Also, assessing the test- retest reliability of the IPAQ- E module in a cohort of African population may highlight differences, indicate cultural issues and espouse the environmental correlates that are contextually relevant to Africa. Since test- retest reliability is a useful means of assessing the reproducibility of a measure and hence the consistency and stability of an instrument over time [##UREF##8##22##], the purpose of this study was therefore to assess the test- retest reliability of the IPAQ E- module in an African population. The environment for the purpose of this study was defined as neighborhood characteristics.</p>" ]

[ "<title>Methods</title>", "<title>Sample</title>", "<p>Participants were undergraduate clinical students of the urban based premier University in Nigeria. They were selected from an ongoing larger study on environmental and sociodemographic determinants of physical activity among students of the University. A total of 298 male and female clinical students took part in the overall baseline survey consisting of 1006 students of the University. All the clinical students that were part of the baseline survey were invited to participate in the retest of the E-module questionnaire and about 69% of them (n = 103) agreed to participate in the retest study. Participants were given the second copy approximately 7 days after the first questionnaire was returned. The questionnaire was self administered and completed in the participants' rooms with the investigator in attendance in order to reduce items' misinterpretation. Socio-demographic information such as age, sex, height, weight, ethnic group, academic programs, years of study and religion were also sought from the participants. All participants provided an informed consent and the study was approved by the University of Ibadan/University College Hospital Joint Institutional Review Committee on Human Research (UI/EC/08/0004).</p>", "<title>Measurement of environmental characteristics</title>", "<p>Sixteen self- report items from the IPAQ- Environmental Module (IPAQ E- module) designed for measuring environmental correlates of physical activity in the neighborhood were assessed for test- retest reliability in this study. The IPAQ E- module was made up of 17- environmental items that are grouped as core, recommended and optional. All core items were mandatory to be asked, while as many of the recommended items as possible should be asked in any study utilizing the E- module survey [##UREF##7##21##] [see Additional file 1]. One item from the recommended items \"How many motor vehicles in working order are there in your household?\" was not assessed due to the nature of the sample in the present study. Specifically, the study sample comprised students living in the University hostel and neighborhood was defined as the campus environment rather than their various household environments.</p>", "<p>For the purpose of this study items on the IPAQ E- module were classified into seven categories [##UREF##6##19##]: residential density (one item), access to destinations (three items), neighborhood infrastructure (five items), aesthetic qualities (one item), social environment (one item), street connectivity (one item) and neighborhood safety (four items). These items have been shown to demonstrate moderate reliability coefficients among the Caucasians [##REF##12948979##17##, ####REF##13677966##18##, ##UREF##6##19####6##19##]. Responses to the IPAQ E- module were based on a 4- point likert scale ranging from strongly disagree to strongly agree as well as don't know or doesn't apply options for 15 of the questions. The only item with specific response option scale was the question assessing residential density (the main type of housing in my neighborhood).</p>", "<title>Data analysis</title>", "<p>Analysis for the test-retest reliability of each of the environmental variables was conducted overall and by gender using the one- way model intraclass correlation coefficient (ICC) along with 95% confidence interval (CI). ICC represents the total variance in the measure (subject variability and measurement error) that was due to true differences between participants (subject variability) [##REF##14499805##15##]. It accounts for the variability between, rather than within the participants. The agreement levels rating suggested by Landis and Koch: 0 – 0.2 (poor), 0.2 – 0.4 (fair), 0.4 – 0.6 (moderate), 0.6 – 0.8 (substantial) and 0.8 – 1.0 (almost perfect) was used to interpret the results [##UREF##6##19##]. Descriptive statistics of mean and percentage were used to describe the socio-demographic characteristics of the participants. Statistical analyses were performed with SPSS version 10.</p>" ]

[ "<title>Results</title>", "<p>One hundred and three participants completed the test- retest survey. The mean age and BMI of the participants were 24.24 ± 3.55 years and 23.24 ± 4.07 kg/m²respectively. About 51.5% were males and majority, (70.9%) was from the Yoruba ethnic group. More than half were clinical students of medicine (52.4%) and majority was of Christian religion (82.5%). The detailed general characteristics of the participants are shown in table ##TAB##0##1##.</p>", "<p>The result of the test- retest reliability for all respondents and by gender is presented in table 2 [see Additional file 2]. Overall, the one week ICC ranged from 0.43 – 0.91, with the lowest value recorded for question on crime during the day and the highest value for question on many interesting things to look at while walking. By gender, the ICC ranged from 0.11 – 0.96 for males and from 0.23 – 0.87 for females. Both males and females recorded the highest ICC on residential density item (main type of housing in the neighborhood), while two items on neighborhood safety that is, traffic against bicycling and traffic against walking demonstrated the lowest ICC among males and females respectively. When exploring the gender based findings, only items that differed by at least two categories on the rating of Landis and Koch were considered as meaningful and discussed as such.</p>", "<title>Reliability of items on residential density</title>", "<p>The only question that assessed residential density was the main type of housing in the neighborhood. This item demonstrated almost perfect agreement overall (ICC = 0.89) and among the male participants (ICC = 0.96), but exhibited moderate agreement among the female participants (ICC = 0.56).</p>", "<title>Reliability of items on access to destination</title>", "<p>The reliability of items on general access to destination ranged from moderate (ICC = 0.49) to substantial agreement (ICC = 0.74) overall. The lowest reliability was exhibited by the question \"it is within 10- to- 15 minutes walk to the bus stop\" and the highest reliability by the question \"there are many shops within walking distance of university\". The three items on access to destination differed meaningfully by gender. While males demonstrated poor agreement (ICC = 0.19) on the question \"many shops are within walking distance of home\", females tend to demonstrate almost perfect agreement (ICC = 0.80) on the question. Also, the question \"many places to go within easy walking distance\" was almost perfectly reliable (ICC = 0.89) among males but only moderately reliable (ICC = 0.59) among females. The question \"it is within 10- to- 15 minutes walk to a bus stop from home\" also demonstrated higher reliability (ICC = 0.60) among males than females (ICC = 0.27).</p>", "<title>Reliability of items on neighborhood infrastructure</title>", "<p>Overall the five items pertaining to neighborhood infrastructures demonstrated substantial (ICC = 0.66) to almost perfect agreement (ICC = 0.88). The highest reliability was found for question on well maintained and unobstructed places for bicycling infrastructure. No meaningful gender differences were found in this domain except for the question on well maintained and unobstructed sidewalks in the neighborhood where higher reliability was found among males (ICC = 0.86) than among females (ICC = 0.56).</p>", "<title>Reliability of items on aesthetic qualities</title>", "<p>The only question on aesthetic quality (many interesting things to look at while walking in the neighborhood) generated the highest reliability score overall (ICC = 0.91) and by female gender (ICC = 0.87). However, the reliability coefficients for both male (ICC = 0.94) and female (ICC = 0.87) fall within the same category (almost perfect).</p>", "<title>Reliability of items on social environment and street connectivity</title>", "<p>For the social environment, seeing many people being physically active demonstrated substantial agreement (ICC = 0.62) overall with reliability somewhat higher among males (substantial agreement) than females (moderate agreement). Also, item on street connectivity (there are many four way intersections in the neighborhood) demonstrated substantial agreement (ICC = 0.78) overall with higher reliability among the male participants (almost perfect agreement) than their female counterparts (substantial agreement).</p>", "<title>Reliability of items on neighborhood safety</title>", "<p>Apart from the question on crime rate at night (ICC = 0.83), all other items on neighborhood safety demonstrated moderate reliability overall, with the lowest reliability found for question on crime rate during the day (ICC = 0.43). Two items on neighborhood safety were meaningfully different by gender. Reliability was substantial (ICC = 0.69) among males but fair (ICC = 0.23) among females when assessing question on much traffic making it difficult or unpleasant to walk in the neighborhood. However, the reliability among females had moderate agreement (ICC = 0.45) while reliability among the males had poor agreement (ICC = 0.11) when assessing question on much traffic making it difficult or unpleasant to ride a bicycle.</p>" ]

[ "<title>Discussion</title>", "<p>This study evaluated the test- retest reliability of IPAQ E- module in an African population. The overall results indicate reliability to range from moderate agreement to almost perfect agreement. Few gender differences were observed in the reliability of some of the items on the E- module among the participants.</p>", "<p>The highest reliability coefficients were found for items on aesthetic qualities and residential density such as \"there are many interesting things to look at while walking in the neighborhood\" and \"the main type of housing in the neighborhood\". Items on neighborhood safety such as \"crime rate make it unsafe to go on walk during the day\" and \"so much traffic on the street makes it difficult or unpleasant to walk\" demonstrated low reliability coefficient. This result reflects the stability of question pertaining to more objective features of the environment as aesthetic (trees, flowers, landscaping view etc) and types of housing (residential, office buildings, apartments etc) than subjective environmental features such as crime and safety that are easily overtaken by time and events. It is possible that participants in this study found it more difficult to subjectively assess crime rate and safety in their neighborhood, thereby reducing the reliability of the items. There is substantial interest in perceived crime as a correlate of physical activity behaviour and studies to date have produced inconsistent results on the association between perceived crime and physical activity [##REF##16138933##7##,##REF##11897464##13##,##REF##13677966##18##,##REF##16159404##23##]. Future studies may need to utilize more objective measures of crime and safety to identify the important relationship that exists between neighborhood safety and physical activity behaviours.</p>", "<p>In a similar study [##UREF##6##19##] in Sweden, the main type of housing in the neighborhood was identified as the item with the highest reliability coefficient and neighborhood safety item \"the crime rate makes it unsafe to go on walk during the day\" demonstrated the lowest reliability coefficient. Also, in an American study [##REF##14998817##16##], items on residential density were found to demonstrate high reliability while lower reliability was found for the question on unsafe walking during the day due to crime. Somewhat consistent with the higher reliability found for the item on aesthetic quality in this study, the question on many interesting things to look at while walking was found to have the highest reliability coefficient within the domain of items assessing neighborhood aesthetic [##REF##14998817##16##]. The replication of these findings in an African population supports an international assumption that environmental variables pertaining to objective features like aesthetic qualities and housing type may be more reliable correlates of physical activity than subjective features like perceived crime and traffic.</p>", "<p>In this study, environmental items on neighborhood infrastructures demonstrated good reliability coefficients that ranged from substantial agreement to almost perfect agreement. This finding may suggest stability of items assessing neighborhood infrastructures. This is likely because participants in this study lived in an environment where infrastructures like sidewalks and recreational facilities can readily be perceived as available but infrastructures like bicycle facilities may not be readily perceived as available. This can increase consistency and stabilize variation between responses thereby influencing good reproducibility of the items assessing neighborhood infrastructures. Similarly to the finding in the present study, Alexander et al [##UREF##6##19##] found substantial agreement in the reliability of all items on neighborhood infrastructures, while Brownson et al [##REF##14998817##16##] found reliability of items assessing infrastructures for walking and cycling to vary from moderate agreement to substantial agreement. Somewhat consistently, another study from the United States [##REF##12948979##17##] reported moderate agreement for walking and cycling facilities but found the subscale to demonstrate the lowest reliability when compared with other subscales. Also, in a Belgium study [##REF##13677966##18##]; items on availability of sidewalks and bike lanes demonstrated almost perfect agreement. These findings suggest that variables assessing neighborhood infrastructures are ubiquitously reliable regardless of the geographical locations.</p>", "<p>Several studies have implicated access to destination as an important correlate of physical activity [##REF##14748314##6##,##REF##11897464##13##,##REF##12704009##24##]. In this study, the reliability of items on access to destination ranged from moderate agreement for the question \"it is within 10- to -15 minutes walk to a transit/bus stop\" to substantial agreement for the question \"there are many places to go within easy walking distance\". The lack of specificity in the time period given for the question \"it is within 10 to 15 minutes walk to a transit stop\" may reduce consistency between responses thereby lowering the reliability of the item. A previous study [##REF##13677966##18##] that used a specific time and narrower definition of the neighborhood reported higher reliability coefficient for the same item. Also, the non attribution of time frame to the question \"many places to go within walking distance\" may explain its higher reliability when compared to other questions with time dimension. Consistently, a reliability study of similar sample size [##REF##15958168##20##] reported comparable reliability coefficient for the question many places to go within walking distance (ICC = 0.63) to that of this study (ICC = 0.74). Similarly, the Sweden study [##UREF##6##19##] found reliability to range between (0.46 – 0.81) for items on access to destination, with particularly lower and higher reliability scores for the questions \"it is within 10- to- 15 minutes walk to a transit stop from my house and \"there are many places to go within easy walking distance\" respectively. Also, items on land use mix- access (store within walking distance and easy walking to transit stop) and land use mix diversity (how long from home to get to business or convenience facilities) were found to demonstrate substantial to almost perfect reliability in an American study [##REF##12948979##17##]. Replicating similar findings in an African population suggests an overwhelming importance of land use mix access as a reliable and viable environmental correlate of physical activity.</p>", "<p>Substantial agreement was also found for the only item on social environment (seeing many people being physically active) and street connectivity (many four way intersections in the neighborhood). Similarly, the Sweden study [##UREF##6##19##] found substantial reliability for the only item on street connectivity but lower (moderate) reliability coefficient for the item pertaining to seeing many people being physically active in the neighborhood. However, an American study [##REF##14998817##16##] found a somewhat lower reliability (ICC = 0.51) for the question on many four way intersections when compared to other studies. This was however not substantial since the value still falls within the moderate reliability found in these other studies [##REF##12948979##17##,##REF##13677966##18##].</p>", "<p>Like the previous studies [##UREF##6##19##,##REF##15958168##20##] that have assessed gender differences in the test- retest reliability of environmental measures of physical activity, few items demonstrated meaningful gender differences in the present study. Reliability was meaningfully higher among males than females on questions involving residential density, many places to go within easy walking distance, it is within 10- to- 15 minutes walk to the transit/bus stop and much traffic making it difficult or unpleasant to walk in the neighborhood. However, females had meaningfully higher reliability score on the questions \"many shops within walking distance\" and \"so much traffic making it difficult or unpleasant to ride bicycle in the neighborhood\" than males. Since males generally do not engage in shopping, it is plausible for them not to be consistent overtime in response to question on awareness of shops within walking distance of the university as compared to the females who may always be aware of shops because they regularly do shopping and hence answered more consistently, which consequently resulted in the meaningfully higher reliability of the item among females. While it may be difficult to draw any definite conclusion from these findings, it reflects the potential influence of gender on the reliability of some environmental correlate items. This is likely because evidence is now emerging that gender may exert a moderator link between perceived environment and physical activity behaviours [##REF##14748314##6##,##REF##13677966##18##,##REF##16159404##23##].</p>", "<title>Limitations</title>", "<p>This study has some important limitations. The generalization of this study is limited by the nature of the sample used which comprised clinical students of the University with potential higher comprehension and recall ability than may be found in the general population. The findings should therefore be interpreted with caution among other blacks of diverse educational level. Also, lower variation may exist in the limited environmental features available on campus than in the general community and this may lower reliability for the assessed environmental items in this study.</p>", "<p>Like all other studies involving recall, it is possible for participants in the present study to give vague rather than factual responses to perceived environmental features in their neighborhood, thereby affecting results and inferences from this study. Also, though quite unlikely because of the short time frame, actual changes might have occurred between the retest surveys thereby reducing the observed reliability coefficients. The 95% confidence intervals for some of the reliability estimates, especially for the gender based findings were not precise thus suggesting the possibility of a low sample size.</p>", "<p>The non utilization of objective environmental measures that would have served as the criterion for assessing the validity of IPAQ E- module in this study may constitute a limitation. Previous studies [##REF##13677966##18##,##REF##12726870##25##] have observed actual differences between objective and subjective data of environmental measures of physical activity. However, since no data presently exist in this field among the African population, the use of self- report measures of the environment may still hold sway for the time being in the African environment. This however does not suggest the non-importance of evaluating both subjective and objective environmental measures even at this early stage of this research type in African populations.</p>" ]

[ "<title>Conclusion</title>", "<p>This study has for the first time provided evidence on the psychometric properties of items on an environmental measure of physical activity in an African population. The test- retest reliability of IPAQ E- module ranged from moderate agreement to almost perfect agreement with few meaningful differences by gender. The reliability coefficients of environmental items among this population were mostly similar to that in other environments. Items on IPAQ E- module is therefore promising and may be useful for assessing environmental correlates of physical activity among the black population in Africa. Future studies should consider testing IPAQ E-module along with more objective environmental measures in a diverse African population.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>There is overwhelming evidence of the benefits of physical activity and the physical environment is increasingly recognized as a promising determinant of physical activity participation. The influence of the environment on physical activity has not been evaluated among black Africans and no specific measure exists for assessing environmental factors related to physical activity in an African environment. The IPAQ E- module was designed to assess environmental factors for physical activity participation and was considered to be relevant to all countries regardless of the stage of economic development. The objective of this study was to assess the test- retest reliability of IPAQ E- module in an African population.</p>", "<title>Methods</title>", "<p>One hundred and three clinical students of a University in Nigeria were invited to participate in the reliability testing of IPAQ E- module. Sixteen of the 17- items on the environmental measure were assessed for test- retest reliability using intraclass correlation coefficient (ICC) with 95% Confidence interval (CI) overall and by gender. The measure addressed items regarding residential density, access to destinations, neighborhood infrastructures, aesthetic qualities, social environment, street connectivity and neighborhood safety.</p>", "<title>Results</title>", "<p>Of the total respondents, 51.5% were males and 48.5% were females. Overall, the intraclass correlation coefficient (ICC) ranged from 0.43 to 0.91. The item regarding many interesting things to look at (aesthetic) produced the overall highest reliability score (ICC = 0.91, 95% CI = 0.86 – 0.94), while the item regarding safety from crime during the day (neighborhood safety) produced the lowest overall score (ICC = 0.43, 95% CI = 0.26 – 0.57). Reliability of items on neighborhood infrastructures ranged between substantial agreement to almost perfect agreement overall (ICC = 0.66 – 0.88) and by gender (male- ICC = 0.68 – 0.90 and female- ICC = 0.63 – 0.86). The access to destination items (ICC = 0.49 – 0.74), social environment (ICC = 0.62) and street connectivity (ICC = 0.78) all had acceptable reliability overall. Meaningful differences were found between males and females on two items on neighborhood safety and one item on access to destinations.</p>", "<title>Conclusion</title>", "<p>The test- retest of IPAQ E- module resulted in moderate to almost perfect agreement for most of the items with few meaningful differences by gender. Environmental items of physical activity in an African population exhibited reliability similar to that in other environments. These results suggest that IPAQ E- module may be a useful measure for assessing environmental correlates of physical activity among population in Africa.</p>" ]

[ "<title>Authors' contributions</title>", "<p>ALO conceived and designed the study, analyzed and interpreted the data, drafted the manuscript and gave approval for the final version. BOA was involved in study design, interpretation of data and drafting of the manuscript. AYO was involved in study design and drafting of the manuscript. BMF was involved in data acquisition and read the manuscript for final approval. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>We acknowledge Prof Arinola O, Sanya of the Department of Physiotherapy, University of Ibadan, Nigeria for providing support and motivation for conducting the study.</p>" ]

[]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characteristics of Participants</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Characteristics</td><td align=\"left\">Male</td><td align=\"left\">Female</td><td align=\"left\">Total</td></tr><tr><td/><td colspan=\"3\"><hr/></td></tr><tr><td/><td align=\"left\">n (%)</td><td align=\"left\">n (%)</td><td align=\"left\">n (%)</td></tr></thead><tbody><tr><td align=\"left\"><bold>Gender</bold></td><td align=\"left\">53 (51.5)</td><td align=\"left\">50 (48.5)</td><td/></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Age (Years)</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> 16 – 19</td><td align=\"left\">4 (3.9)</td><td align=\"left\">3 (2.9)</td><td align=\"left\">7 (6.8)</td></tr><tr><td align=\"left\"> 20 – 29</td><td align=\"left\">43 (41.7)</td><td align=\"left\">44 (42.7)</td><td align=\"left\">87 (84.5)</td></tr><tr><td align=\"left\"> 30 – 39</td><td align=\"left\">6 (5.8)</td><td align=\"left\">3 (2.9)</td><td align=\"left\">9 (8.7)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>BMI (kg/m²)</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> &lt; 18.5</td><td align=\"left\">5 (4.9)</td><td align=\"left\">5 (4.9)</td><td align=\"left\">10 (9.7)</td></tr><tr><td align=\"left\"> 18.5 – 24.9</td><td align=\"left\">33 (32.0)</td><td align=\"left\">32 (31.1)</td><td align=\"left\">65 (63.1)</td></tr><tr><td align=\"left\"> 25.0 – 29.9</td><td align=\"left\">11 (10.7)</td><td align=\"left\">10 (9.7)</td><td align=\"left\">21 (20.4)</td></tr><tr><td align=\"left\"> &gt; 30.0</td><td align=\"left\">4 (3.9)</td><td align=\"left\">3 (2.9)</td><td align=\"left\">7 (6.8)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Ethnic Group</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> Ibo</td><td align=\"left\">10 (9.7)</td><td align=\"left\">5 (4.9)</td><td align=\"left\">15 (14.6)</td></tr><tr><td align=\"left\"> Hausa</td><td align=\"left\">0 (0)</td><td align=\"left\">3 (2.9)</td><td align=\"left\">3 (2.9)</td></tr><tr><td align=\"left\"> Yoruba</td><td align=\"left\">33 (32.0)</td><td align=\"left\">40 (38.8)</td><td align=\"left\">73 (70.9)</td></tr><tr><td align=\"left\"> Others</td><td align=\"left\">7 (6.8)</td><td align=\"left\">5 (4.9)</td><td align=\"left\">12 (11.7)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Academic Programs</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> Physiotherapy</td><td align=\"left\">15 (14.6)</td><td align=\"left\">6 (5.8)</td><td align=\"left\">21 (20.4)</td></tr><tr><td align=\"left\"> Medicine</td><td align=\"left\">27 (26.2)</td><td align=\"left\">27 (26.2)</td><td align=\"left\">54 (52.4)</td></tr><tr><td align=\"left\"> Dentistry</td><td align=\"left\">11 (10.7)</td><td align=\"left\">17 (16.5)</td><td align=\"left\">28 (27.2)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Years of Study</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> 1^stClinical</td><td align=\"left\">20 (19.4)</td><td align=\"left\">23 (22.3)</td><td align=\"left\">43 (41.7)</td></tr><tr><td align=\"left\"> 2^ndClinical</td><td align=\"left\">19 (18.4)</td><td align=\"left\">15 (14.6)</td><td align=\"left\">34 (33.0)</td></tr><tr><td align=\"left\"> 3^rdClinical</td><td align=\"left\">14 (13.6)</td><td align=\"left\">12 (11.7)</td><td align=\"left\">26 (25.2)</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\"><bold>Religion</bold></td><td/><td/><td/></tr><tr><td align=\"left\"> Islam</td><td align=\"left\">9 (8.7)</td><td align=\"left\">9 (8.7)</td><td align=\"left\">18 (17.5)</td></tr><tr><td align=\"left\"> Christianity</td><td align=\"left\">44 (42.7)</td><td align=\"left\">41 (39.8)</td><td align=\"left\">85 (82.5)</td></tr></tbody></table></table-wrap>" ]

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[ "<title>Background</title>", "<p>Shortages of trained health personnel represent nothing less than a crisis of epidemic proportions in the developing world. In some of the poorest countries, health systems are in danger of collapse due to the lack of health staff to deliver services [##UREF##0##1##]. The global gap in the supply of health workers is estimated to be 4.3 million, with 57 countries depicted as 'countries with critical shortage' experiencing shortfalls of 2.4 million doctors, nurses and midwives. Human resource challenges manifest not only in shortages of health workers, but also in disparities in their distribution, poor training capacity, skills and skills mix deficits, and weak management and supervisory systems [##UREF##1##2##]. To aggravate matters, HIV and AIDS (even more so labour-intensive ART) are superimposing cumulative burdens on already overstretched health systems of those countries at the epicentre of the epidemic. Despite these challenges, human resource management systems remain weak and fragmented in most countries [##UREF##0##1##, ####UREF##1##2##, ##UREF##2##3##, ##REF##15807792##4##, ##UREF##3##5##, ##UREF##4##6####4##6##].</p>", "<p>The high demand for human resources for health applies also to South Africa, especially against the backdrop of a still growing HIV/AIDS epidemic and, since 2003, the introduction of a massive and expanding public sector ART programme. With more than five million South Africans infected, the 'Comprehensive Plan' intends to provide ART access to more than a million people by 2008 [##UREF##5##7##]. In addition to reducing morbidity and mortality from HIV/AIDS, a central aim of the Comprehensive Plan is \"to strengthen the national health care system overall\" [##UREF##4##6##]. In human resource terms, such systems strengthening is foreseen through the injection of large numbers of additional health care staff into the public health workforce, including 975 doctors, 2924 professional nurses, 661 pharmacists, 526 dieticians and 488 social workers by March 2008 [##UREF##5##7##].</p>", "<p>Concerns have been raised, however, as to where the large numbers of additional health professionals needed for public sector ART will come from in light of the international brain drain, unequal geographical and sectoral distributions of the health workforce, and high vacancy and turnover rates in the public health sector [##UREF##6##8##, ####UREF##7##9##, ##UREF##8##10##, ##UREF##9##11##, ##UREF##10##12####10##12##]. South Africa currently has fewer public health workers, including professional nurses, than it did ten years ago, both in absolute numbers and relative to population size [##UREF##9##11##,##UREF##11##13##]. To aggravate shortages, South Africa's national health system is structurally characterised by a deep public-private sector divide. In terms of human resources, this is reflected in large inequalities in the distribution of health personnel. In the case of professional nurses, in 2005, of the 99 534 professional nurses registered with the South African Nursing Council only 43 660 (43.9%) were employed in the public sector, despite the fact that this sector covers more than 80% of the population [##UREF##8##10##]. Against this backdrop, some have gone so far as to suggest that a badly managed introduction of ART \"could do more harm than good\" [##UREF##12##14##], and that the shortage of skilled personnel and inequities in human resource provisioning in the public sector might worsen, especially in the primary health care (PHC) system and particularly in poorer and rural districts.</p>", "<p>Calls are thus for ART scale-up to go hand-in-hand with comprehensive health systems development, upgrading of human resource capacity and realistic targets [##REF##15623853##15##,##REF##16713875##16##]. To accomplish this, policy makers and managers in high-burden countries have to devise new strategies and even make paradigm shifts to cope with human resource challenges at regional, national, sub-national and facility levels, either by exploring new human capacity or by utilising and managing the existing pools of personnel better [##REF##15807792##4##,##UREF##13##17##, ####REF##16514294##18##, ##UREF##14##19####14##19##]. Apart from staff retention and increased production strategies [##UREF##1##2##], a common response to human resource shortages in poor-resourced settings is the use of substitute health workers, i.e. mid-level, support and lay/community health workers, to relieve pressures on professional staff. The concepts of 'task shifting' and 'task delegation' are commonly used in reference to this response. Four such forms in African countries have been described: (1) indirect substitution or delegation of tasks to an existing but different profession (e.g. from doctors to nurses or pharmacists); (2) direct substitution or delegation of tasks from professionals to less-trained, mid-level health workers within the same profession (e.g. using nursing assistants in lieu of professional nurses); (3) delegating non-technical tasks to lower-trained (even lay) cadres of staff (e.g. employing administrative clerks and community health workers [CHWs] to support nurses); and (4) intra-cadre skills delegation or shifting of tasks to less-trained cadres from the same profession (e.g. from medical specialists to general practitioners) [##UREF##2##3##].</p>", "<p>After three years of programme expansion in South Africa, it is necessary to ask whether and how, firstly, the health system has thus far mobilised the essential human resources for ART, and, secondly, the extent to which the aim of overall system strengthening through the ART programme is being achieved. This paper reports on aspects of the human resource dynamics following the inception in 2004 of a public sector ART programme in one province (Free State) of South Africa. In particular, it examines whether the stated goal of strengthening human resource capacity through the establishment of extra posts, appointment of extra personnel, and the enhanced training of staff was achieved or in fact undermined. Core human resource management shortfalls and challenges and strategies to address these are highlighted.</p>" ]

[ "<title>Methods</title>", "<p>Facilities providing ART in the Free State comprise three types: treatment sites (referral hospitals), assessment sites (referring PHC clinics) and combined treatment-assessment sites (either community health centres or larger PHC clinics, which fulfil both assessment and treatment functions). The latter types were introduced in predominantly sparsely populated rural/small-town areas. PHC nurses assess patients for ART at assessment and combined sites, and patients who meet baseline criteria are referred to treatment sites (or doctors at the combined sites) where they are certified for ART by an ART-trained doctor [##UREF##10##12##,##UREF##15##20##]. During Phase I of ART roll-out in the province (May to December 2004), 4 treatment sites, 13 assessment sites and 3 combined sites were activated. In Phase II (2005/2006), a further 3 treatment, 8 assessment and 7 combined sites were introduced.</p>", "<p>The research reported here forms part of a larger, longitudinal evaluation of the roll-out of public sector ART in the Free State. The paper focuses on issues pertaining to staffing of the programme with professional nurses – regarded as the backbone of PHC in South Africa – in the first 38 ART sites of the five districts in the province.</p>", "<p>The study tracked and reconstructed human resource dynamics over a period of almost three years through assessments of the following:</p>", "<p>1. New staffing establishments created and recruitment patterns in the inception phases of the ART programme, in a staffing audit conducted in November 2004, i.e. after all 20 Phase I sites had been activated. Amongst others, the audit gives a count of the approved, filled and vacant professional nurse posts for the ART programme, as well as nurses trained for ART, at each facility at the time.</p>", "<p>2. The deployment, training and integration of new staff, and adaptations in task distribution through baseline and follow-up facility appraisals conducted in the first 20 (Phase I) ART sites. The baseline appraisal was conducted about one month prior to the introduction of the programme, and thereafter at two follow-up occasions: the first seven months after going operational, and the second a year thereafter.</p>", "<p>3. The number of approved, filled and vacant nurse posts per district and per facility at the 38 Phase I and II ART sites, extracted from the official government personnel database (PERSAL). These data were obtained twice, in March and August 2006. In November 2006, additional PERSAL data were retrieved on new appointments, promotions and lateral transfers of professional nurses servicing the ART programme.</p>", "<p>4. The training of professional nurses and other staff in ART during 2004, 2005 and 2006, obtained from the Directorate: Staff Development of the Free State Department of Health.</p>", "<p>The findings reported here apply to a specific province and to the ART programme as implemented in the facilities of this province at a particular point in time. They are thus not necessarily generalisable to other provinces of South Africa or other countries. However, given the shared nature of the human resource crisis across contexts and a degree of nationally determined standardisation of the ART programme, it is safe to claim that the study indeed highlights core problems and constraints in the rollout of the public ART programme in South Africa and, therefore, also furnishes important lessons for other provinces and countries.</p>", "<title>New staff establishments for ART and distribution of ART sites per district</title>", "<p>The professional staffing requirements per 500 patients on ART are stipulated as follows in the Comprehensive Plan: 1 medical officer; 2 professional nurses; 1 pharmacist; 1 dietician/nutritionist; 0.5 social workers. Non-professional full-time staff amount to 5 lay counsellors/CHWs and 2 administrative clerks/data capturers [##UREF##5##7##]. The Free State's initial staff establishments provided for three professional nurses at treatment sites, three (in some cases four) at combined treatment-assessment sites, and three (in exceptional cases four) at assessment sites [##UREF##10##12##,##UREF##16##21##]. In the Free State, 60 professional nurse posts were thus added to Phase I and a further 52 to Phase II sites, thus totalling 112 (later 115, after additions to some of the larger facilities). The large majority of these newly created professional nurse posts was based at PHC facilities hosting the ART programme. Relative to both patient populations (Table ##TAB##0##1##) and national norms, professional nurse staffing for the ART programme appears rather generous at the time, especially at those facilities in predominantly rural/small-town districts (see Table ##TAB##0##1##).</p>", "<p>During Phases I and II of the programme in the Free State (2004 to 2006), provincial policy was, firstly, to open an equal number of ART sites in each district and, secondly, to create uniform staff establishments with an equal number of staff allocated to the three ART facility types. Neither of these allocation strategies was premised on need, such as population sizes and densities of the districts, HIV prevalence rates, and demand for care. As a result, highly uneven professional nurse-patient ratios, as well as highly uneven workloads for nurses servicing the programme, emerged across the province (Table ##TAB##0##1##). In some districts, staff rapidly became overloaded without changes in staff establishments, while in other districts ART staff appeared underutilized.</p>", "<title>Recruitment of professional nurses into ART programme posts</title>", "<p>The creation of new posts does not necessarily translate into filled positions. The addition of personnel through actual appointments is thus a more reliable indicator of systems strengthening than the creation of posts. The filling of the new ART posts occurred against a backdrop of high general vacancy levels in the public health system. In 2003, only 59.3% (7176) of 12 104 health professional posts in the Free State were filled (i.e. a vacancy rate of 40.1%), well below the national average of 68.9% filled posts (or a 31.1% vacancy rate) [##UREF##8##10##,##UREF##10##12##,##UREF##18##23##]. By mid-2006, however, professional nurse vacancy rates in the ART programme were half (15.8%) that of the PHC system as a whole (37.1%) (Table ##TAB##1##2##). Although initial recruitment into the ART programme was difficult in some districts, by November 2006, 97 (85%) of the 115 professional nurse posts allocated to the ART programme had been filled (see Table ##TAB##1##2##).</p>", "<p>At inception of the ART programme, ART posts were possibly attractive due to the novelty and considerable political and bureaucratic attention focused on the programme, nationally and provincially [##UREF##15##20##]. This resulted in 'lateral transfers' of nurses from other programmes to the ART programme, which partly explains the differences in vacancy rates between the ART sites and the PHC system as a whole. These newly created posts in many cases also provided opportunities for promotion. In July 2005, all professional nursing posts for ART were upgraded to senior level. On the one hand, this led to promotion of nurses appointed in the programme (Table ##TAB##2##3##); on the other, it attracted nurses deployed in other programmes to posts into the ART programme for prospects of promotion and better remuneration. In two predominantly urban districts (Motheo and Lejweleputswa) promotions were the highest relative to the appointments made, which partly explain the low vacancy rates in the ART programme of these two districts relative to the other three districts.</p>", "<title>Sources of professional nurses recruited into ART programme and patterns of geographic mobility</title>", "<p>The origin of professional nurses recruited into the ART programme and their geographic mobility following the introduction of the ART programme were assessed through both facility appraisals and the review of PERSAL data.</p>", "<p>A staffing inventory in the facility appraisals found that, prior to the ART programme, a total of 147 professional nurses were employed at the 16 Phase I PHC facilities. After the introduction of the programme, i.e. at the first follow-up appraisals, this number had grown to 167 (including 40 ART nurses) and remained almost the same at the second follow-up – 165 (including 37 ART nurses). The net gain in these facilities at the second follow-up appraisal was thus only 18 professional nurses. Subtracting all the ART appointments (37) from the total number of professional nurses in these facilities, there was then a net loss of 19 professional nurses in the surrounding PHC system (i.e. the 16 PHC facilities). Given that in most of the facilities observed, the ART programme was provided in a vertical manner separate from other services, this suggests considerable displacement or drainage of professional nurses towards the ART programme.</p>", "<p>Across the Free State as a whole, one in every five (20.6%) of the 97 professional nurses appointed to the Phase I and II ART sites were genuinely new entrants to the province's staffing establishment, i.e. nurses entering the system as new graduates, or from the private and non-governmental sectors, or from other provinces and countries. The majority of professional nurses (43.2%) moved from a neighbouring (non-ART) facility in the same district to the ART providing facility (inter-facility mobility). One-quarter (24.7%) of new recruits came from another division/programme within the same facility (intra-facility mobility). Such intra-facility flow into the ART programme was also more obvious in the two predominantly urban districts (Motheo and Lejweleputswa). Little inter-district movement of nurses took place in filling the Free State's ART posts (11.3%) (see Table ##TAB##3##4##).</p>", "<title>Training of staff for ART</title>", "<p>During 2004, the Free State Department of Health developed its own training curricula on ART, and offered a series of five-day training courses for personnel working in the programme. The November 2004 staff audit showed that the majority of professional nurses appointed in the ART programme (37 of 42) had received such training in the months prior to the activation of the programme. Over and above these, an additional 23 nurses had been trained for ART at the time, thus securing a significant reserve workforce for the programme.</p>", "<p>A review of both the contents and process of ART training in 2005 resulted in a revised and integrated training model incorporating the themes of voluntary counselling and testing (VCT), integrated management of childhood illness (IMCI), prevention of mother-to-child transmission (PMTCT) and the syndromic management of STIs. This training model entails a combination of distance-based and on-site, theoretical and practical, initial and maintenance components [##UREF##10##12##]. Training for ART has been facilitated by the presence of a well-established distance-based training infrastructure offered via iCAM (Interactive Distance Communication and Management System), a television broadcasting medium which enables the Free State Department of Health to disseminate information and communicate with health workers from a central studio in the province's capital. The system reaches 38 receiving satellite sites spread over the entire province [##UREF##16##21##].</p>", "<p>The introduction of ART thus resulted in large-scale training of staff. The change in the mode of training to a more integrated approach in 2005 increased the numbers of staff trained for ART even further, especially professional nurses. After three years, this group represents and remained the largest category of all staff trained in ART (46.5%). Moreover, during the first three years, a significant extra pool of nurses has been trained in ART relative to the number of professional nurse posts approved for the programme – 580 as opposed to the 115 approved posts. However, a limited number of enrolled/staff nurses (two) and nursing assistants (five) were trained in ART (Table ##TAB##4##5##).</p>", "<p>A more general increase in training and skills development in certain HIV-related competency areas also took place in the months following the introduction of ART: between the baseline and first follow-up appraisal, of the 147 nurses employed at the assessment sites, 38 (26%) had received additional training in STI syndromic management, 32 (22%) in post-exposure prophylaxis, 28 (19%) in VCT, and 26 (18%) in PMTCT and child nutrition/growth monitoring, respectively.</p>", "<title>Task shifting, delegation and substitution</title>", "<p>To compensate for the general shortage of professional staff in the public health service, various forms of task shifting and task sharing have been observed in the Free State's ART programme. Firstly, after an initial period of relying strictly on doctors for decision-making on initiation of treatment, there is growing support – also due to the scarcity of doctors in certain areas – for a nurse-initiated and nurse-monitored model of ART at PHC facilities (a form of indirect substitution according to Dovlo [##UREF##2##3##]). Similarly, the general scarcity of pharmacists in the public sector, aggravated by the strong reliance of the ART programme on pharmaceutical services, necessitates the shifting of typical pharmacist tasks, i.e. filling and dispensing prescriptions, to the more generally available professional nurses (also a form of indirect substitution).</p>", "<p>There is little evidence in the Free State of direct substitution of professional nurses by enrolled nurses and nursing assistants, despite national guidelines advocating the use of these cadres on a larger scale. From the onset, the post establishments for the ART programme clearly favour professional nurses, and the resultant trend is that negligent numbers of enrolled nurses and nursing assistants have been recruited into the programme or trained on ART (Table ##TAB##4##5##), despite experiences elsewhere suggesting that a number of ART programme activities are readily amenable to task shifting [##UREF##21##26##].</p>", "<p>The appointment of additional administrative staff and the better deployment of lay health workers have been more prominent as manifestations of task shifting. These cadres are generously provided for in the ART programme. During Phases I and II of the ART programme, one administrative clerk and one data capturer were allocated to each ART facility. A total of 78 such posts were created, and their subsequent occupation shows negligible vacancy rates (less than 7%). Regarding lay health workers, the consecutive facility appraisals show increased strengthening of existing complements of lay health workers through multi-skilling and additional training. Initially, most lay health workers (82%) were trained either in lay counselling, home-based care or directly observed TB treatment; by the second follow-up the majority (85%) were trained in more than one area. In addition, lay counsellors are increasingly substituting professional nurses in providing aspects of drug readiness training on their own for ART patients, a task initially performed by professional nurses [##UREF##22##27##]. Midway into the ART programme, the participation of these lay workers was boosted by a standardised and expanded system of stipends, which amounted to nearly double the remuneration previously received and also making it possible to draw larger numbers of lay health workers into the ART programme.</p>" ]

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[ "<title>Discussion</title>", "<title>Strengthening the system</title>", "<p>Since its inception, it has been the explicit aim of the Comprehensive Plan to strengthen the overall health system through the ART programme. The provision of resources through ring-fenced national conditional grants reflects the programme's preoccupation with fulfilling this mandate and protecting the health system from unnecessary additional burdens. However, as the results in this study demonstrate, the general shortage of health professionals in the public health system is not immediately amenable to buy-out. Instead, the injection of new resources and the incentive structures they make possible can have the unintended consequence of weakening the overall health system by drawing essential human resources from other programmes and facilities. In many respects the human resource dynamics of the ART programme in the Free State thus reveal both the strengths and the weaknesses of human resource development and human resource management within the South African health system more generally. These relate to strategies of recruitment, deployment and retention of staff, human resource planning, decentralization of decision-making, human resource management and supervision, the role of vertical programmes, and the focus on and investment in training. The results of the study should sensitize policy makers and managers to these distorting effects.</p>", "<p>The ART programme established many new posts, primarily in the PHC system, and for both professional and non-professional cadres. In respect of professional nurses, who are at the core of both the PHC system and ART rollout, the filling of these new posts introduced a multi-faceted dynamic in the public health system, with potentially problematic effects on the larger provincial health system within which the programme is embedded. First, of the 97 professional nurses appointed to the Free State's programme, only one-fifth [##UREF##15##20##] were newly recruited into the system. The larger majority (more than 80%) came from within the Free State's own public health system, i.e. from within the very same facilities hosting the ART programme, from other facilities within the same district, and to a lesser extent from other districts in the province. Second, the flow of professional nurses towards the few facilities providing ART in the province was no doubt encouraged by prospects of promotion and better remuneration in the new and well-resourced ART programme. Given the high vacancy rates of professional nurse posts in provincial health facilities and persistent difficulties in filling newly vacated non-ART posts within the facilities providing ART, it is unlikely that the 77 professional nurse posts vacated elsewhere in provincial facilities in favour of the ART programme would have subsequently been filled or, due to slow administrative procedures in the public sector, speedily filled. Third, the filling of professional nurse posts in the ART programme was notably better in urban/large-town areas than in the rural/small-town areas, suggesting that there may have been a degree of flow away from rural areas and thus reinforcing national trends of migration away from underserved, poorer and rural areas [##UREF##23##28##].</p>", "<title>Implementing uniform staffing norms for districts and ART facilities</title>", "<p>Despite positive strides and many achievements with ART in the Free State, it is evident that the province's human resource allocation and distribution policies for the programme were not sensitive enough to the differential patient loads in the five districts. The staffing of ART sites adopted a uniform approach across the province and its districts, irrespective of differential AIDS burdens and needs among the five districts, or the varying functions performed by different types of ART sites. As a consequence, staff-patient ratios in the programme varied greatly across the province, while the presence of doctors in treatment sites meant that professional nurses were performing tasks way below their competency levels. On the other hand, however, ART facilities in rural/small-town areas generally struggled to attract sufficient professional nurses.</p>", "<title>More professional nurses for the public service</title>", "<p>Provincially the planning of the ART rollout and scale-up did not take sufficient account of the existing human resource shortages in the public sector, nor of the labour-intensive nature of the ART programme, and of the likely distorting impact of special incentives to attract staff to the ART programme on the PHC system as a whole. Although yet to translate into implementation at provincial level, there is national recognition of the need to increase the production and improve the conditions of service and retention of nurses in the public sector. One such development is the national human resources for health planning framework [##UREF##9##11##] which envisages a drastic raise in the annual production of professional nurses by more than 50% (from the recent 1896 to 3000 by 2011). It also recognises that the improvement of the conditions of service and remuneration for health professionals constitute the most urgent priority. To address this priority area, the occupational specific dispensation (OSD) was announced in 2007. In terms of the OSD, all professional nurses in the public service will be re-graded according to qualifications and years of experience, and remunerated accordingly. Significant improvements for professional nurses are thus being introduced which hold favourable prospects for retaining them in the public sector. However, the same common pool of professional nurses feeding the public sector also feeds the private and non-governmental sectors and one can, therefore, expect that competitive countermeasures will be instated to secure a sufficient professional nurse workforce for the private sector's local and overseas enterprises.</p>", "<p>A second development, commencing in 2008, is the extension of compulsory community service (CS) to professional nurses on completion of their training – a system already in place for other health professionals. About 2000 new nursing graduates will be deployed annually for one year in the public health system, especially in under-resourced areas. For the Free State this ensures an additional 65 professional nurses in the public service in 2008, and similar numbers to follow in years to come. This could compensate for some of the demands created by the ART programme.</p>", "<title>Integration, decentralization and centralization of service delivery</title>", "<p>Weaknesses in the planning and deployment functions in the ART programme are to some extent a product of the over-centralized and vertical nature of the ART programme. Minimum staff establishments for the ART programme were decided nationally and facilities required national accreditation before they could dispense ART to patients. This promoted an over-rigid design that left little room for provincial and even less for district-level innovation and adaptation. In addition, the ART programme was overly doctor- and pharmacist-centred. One of the effects was that within ART sites, the programme was implemented in a vertical manner with staff reserved for the ART programme only, even if these were at times under-utilised [##UREF##15##20##]. To counter these deficiencies, three notable human resource-related strategies have in time emerged to alleviate burdens, spread workloads more evenly, and address the general scarcity of professional nurses in the province. Firstly, and promoted by the broad-based training offered in the province, steps are being taken to initiate greater service and staff integration by rotating staff between programmes offered in the ART-hosting facility and thus spreading workload more equally within these PHC facilities. Secondly, decentralization of ART services to peripheral facilities not providing ART is being promoted with a view to spread the workload more equally among different facilities. Both such integration and decentralization hold promise for greater access and greater equity for patients entering the ART programme. With less central control, both these distributive mechanisms should be in the decision-making space of health managers at the district and facility levels, and both are likely to prove effective in alleviating the pressure on professional nurses in poor-resourced settings. Thirdly, centralization of service components of the ART programme has also proven to be useful in light of shortfalls of human resources of a specific kind. For example, in an attempt to counteract the general shortages of pharmacists in rural areas, a centralized ARV dispensary has been established in the Free State's capital to service a large rural district from a central, well resourced point. This initiative may also alleviate professional nurses of a dispensing workload.</p>", "<title>Task shifting and sharing</title>", "<p>After the initial prevalence of a doctor-centric model and heavy reliance on pharmacists to deliver ART, the provision of ART is increasingly shifted onto professional nurses at PHC facilities, i.e. nurses perform the typical tasks of doctors and pharmacists respectively, either by initiating treatment and prescribing ARV drugs, or by filling and dispensing prescriptions. In broader context, such task shifting initiatives will remain limited, unless more significant progress is made with redefining the scopes of the professions and scopes of practices to allow for the optimum use of human resources.</p>", "<p>The shifting of tasks from professional nurses to community health workers and administrative support staff to relieve nurses of non-clinical tasks and unwarranted workload is quite pronounced and perhaps the best developed form of delegation and substitution in the ART programme in the Free State. Major strides have been made in both forms of delegation by adding significant numbers of these cadres to the staff establishments of ART sites.</p>", "<p>Of concern, however, is the negligent utilization of semi-professional or mid-level nursing staff in the Free State's ART programme, despite the fact that this capacity is widely employed in the ART programmes in human resource-constrained settings, including other provinces in South Africa, but even more so in other African countries. Furthermore, this capacity remains largely unexplored in the province, despite national guidelines providing generously for the use of these lower level nurses in the ART programme: totals of 1255 enrolled nurses and 1255 assistant nurses were foreseen by 2008, i.e. for both these categories a ratio of 1:1.5 professional nurses is implied [##UREF##5##7##]. This imbalance in the Free State ART programme could be ascribed to the strict adherence to the initial norms for staffing the ART sites. A similar neglect, which perpetuates the existing pattern, is observed in the low numbers of mid-level nurses trained for the ART programme in the province. There is thus ample scope for employing enrolled nurses and nursing assistants to a larger extent in the Free State's ART programme in order to alleviate the pressure on professional nurses.</p>", "<title>Training of staff</title>", "<p>Strengthening of the health system is not only about more human resources. Strengthening also occurs through training and systems development, amongst others, through the upgrading of health facilities and infrastructure, laboratories, pharmacies, and IT systems. A key strength in the Free State was the rapid organisation and implementation of training on ART and related programme components, including the effective use of information technology for continuing education. In addition, the training initiatives stimulated by the ART programme also brought with it a wave of new training across the PHC care system, and going beyond the ART sites and ART themes. These factors may in part explain why nurses interviewed in the PHC system of the Free State have been more positive than negative about the ART programme, despite the additional demands made of them [##UREF##24##29##,##REF##17140716##30##]. However, on its own, training is not the sole solution. It is not uncommon for in-service training to become the centre of human resource development, rather than the other more complex tasks of supply, planning and management [##UREF##0##1##,##UREF##1##2##].</p>" ]

[ "<title>Conclusion</title>", "<p>As is the case with other PHC programmes, professional nurses form the backbone of the ART programme in South Africa. In the absence of sufficient numbers of other types of health professionals – doctors, pharmacists, dieticians and social workers – the onus is increasingly shifted onto the widely available professional nurses to fill emerging service gaps. However, in the long term, without concerted efforts to increase the supply of professional nurses and other health workers, neither goals of the Comprehensive Plan – universal access to ART and strengthening the health system – will be achieved. However, more than mere numbers of professional nurses and support staff for nurses are necessary for meeting the human resource challenges posed by the HIV/AIDS epidemic and the scale-up of ART. Equally important is the effective supervision, management and support of staff, better conditions of service and remuneration with a view to heightened work satisfaction, retention and productivity. In most recent years, several valuable strategies and tools have been developed with a view to achieve these human resource-related goals [##UREF##0##1##,##UREF##1##2##]. However, in the face of persistent shortages of professional health personnel and the growing pressures on them to deliver the needed services, it is imperative that the vital and increasing role of patients and community members in scale-up operations should be optimally explored and employed [##UREF##0##1##].</p>", "<p>To address the spectrum of human resource shortages and skills deficits in the sphere of ART programmes (but also in the health domain at large), policy makers and managers in heavy-burdened countries have to devise new strategies and even have to make paradigm shifts to cope with the challenges at regional, national, sub-national and facility levels, either by exploring new human capacity or by utilising and managing the existing, meagre pools of personnel better [##REF##15807792##4##,##UREF##13##17##,##REF##16514294##18##]. Much has indeed been accomplished in this direction with the ART programme in the Free State. However, it is also clear that much more new thinking is necessary to overcome the growing human capacity deficits in the light of the ever growing demands made on human resources for HIV care.</p>", "<p>Regarding further research, the study illustrates the value of recording information on the dynamics of human resources in general and as far as a specific programme is concerned. The study indeed gives an idea of what kind of data is important for research of this nature; at the same time it also shows what is lacking in the data presented, e.g. information on what happened to the vacancies generated by the movement of nurses to ART facilities.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>In common with other developing countries, South Africa's public health system is characterised by human resource shortfalls. These are likely to be exacerbated by the escalating demand for HIV care and a large-scale antiretroviral therapy (ART) programme. Focusing on professional nurses, the main front-line providers of primary health care in South Africa, we studied patterns of planning, recruitment, training and task allocation associated with an expanding ART programme in the districts of one province, the Free State.</p>", "<title>Methods</title>", "<p>Data collection included an audit of professional nurse posts created and filled following the introduction of the ART programme, repeated surveys of facilities providing ART over two years to assess the deployment of staff, and secondary data analysis of government personnel databases to track broader patterns of recruitment and training.</p>", "<title>Results</title>", "<p>Although a substantial number of new professional nurse posts were established for the ART programme in the Free State, nearly 80% of these posts were filled by nurses transferring from other programmes within the same facility or from facilities within the same district, rather than by new recruits. From the beginning, ART nurse posts tended to be graded at a senior level, and later, in an effort to recruit professional nurses for the ART programme, the majority (54.6%) of nurses entering the programme were promoted to a senior level. The vacancy rate of nurse ART posts was significantly lower than that of other posts in the primary health care (PHC) system (15.7% vs 37.1%). Nursing posts in urban ART facilities were more easily filled than those in rural areas, exacerbating existing imbalances. The shift of nurses into the ART programme was partially compensated for by the appointment of additional support staff, task shifting to community health workers, and a large investment in training of PHC workers. However, the use of less-trained, mid-level enrolled nurses and nursing assistants in the ART programme remained low.</p>", "<title>Conclusion</title>", "<p>The introduction of the ART programme has revealed both strengths and weaknesses of human resource development in one province of South Africa. Without concerted efforts to increase the supply of key health professionals, accompanied by changes in the deployment of health workers, the core goals of the ART programme – i.e. providing universal access to ART and strengthening the health system – will not be achieved.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>DvR and FS conceptualised the research, managed data collection and wrote the article. HS provided inputs into the design, interpretation and write-up phases. LL extracted, compiled and made available the relevant provincial data on human resources. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The generous financial support of the following donor agencies and institutions is gratefully acknowledged: IDRC (International Development Research Centre, Canada); JEAPP (The Joint Economics, AIDS and Poverty Programme), with support from AusAID, DFID, UNDP and USAID; and MRC (Medical Research Council of South Africa).</p>" ]

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[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>District characteristics, patient numbers and indicators of workload [##UREF##17##22##, ####UREF##18##23##, ##UREF##19##24##, ##UREF##20##25####20##25##]</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>District</bold></td><td align=\"center\"><bold>Lejwele-</bold><break/><bold>putswa</bold></td><td align=\"center\"><bold>Motheo</bold></td><td align=\"center\"><bold>Thabo </bold><break/><bold>Mofutsanyana</bold></td><td align=\"center\"><bold>Xhariep</bold></td><td align=\"center\"><bold>Fezile</bold><break/><bold> Dabi</bold></td><td align=\"center\"><bold>Free State</bold></td></tr></thead><tbody><tr><td align=\"left\">Population size</td><td align=\"center\">641 391</td><td align=\"center\">755 521</td><td align=\"center\">751 450</td><td align=\"center\">142 601</td><td align=\"center\">465 958</td><td align=\"center\">2 756 921</td></tr><tr><td align=\"left\">Antenatal HIV prevalence (2004)</td><td align=\"center\">33.0%</td><td align=\"center\">27.6%</td><td align=\"center\">27.1%</td><td align=\"center\">21.3%</td><td align=\"center\">32.2%</td><td align=\"center\"><bold>29.5%</bold></td></tr><tr><td colspan=\"7\"><hr/></td></tr><tr><td align=\"left\">Patient numbers:</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">- Enrolled</td><td align=\"center\">5 679</td><td align=\"center\">9 004</td><td align=\"center\">8 248</td><td align=\"center\">2 170</td><td align=\"center\">3 935</td><td align=\"center\"><bold>29 036</bold></td></tr><tr><td align=\"left\">- Eligible</td><td align=\"center\">2 629</td><td align=\"center\">3 262</td><td align=\"center\">3 530</td><td align=\"center\">785</td><td align=\"center\">1 348</td><td align=\"center\"><bold>11 554</bold></td></tr><tr><td align=\"left\">- On treatment</td><td align=\"center\">1 117</td><td align=\"center\">1 536</td><td align=\"center\">1 806</td><td align=\"center\">359</td><td align=\"center\">882</td><td align=\"center\"><bold>5 700</bold></td></tr><tr><td colspan=\"7\"><hr/></td></tr><tr><td align=\"left\">Approved nurse post: patient ratios:</td><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">- Enrolled</td><td align=\"center\">1:227</td><td align=\"center\">1:375</td><td align=\"center\">1:357</td><td align=\"center\">1:103</td><td align=\"center\">1:179</td><td align=\"center\"><bold>1:252</bold></td></tr><tr><td align=\"left\">- Eligible</td><td align=\"center\">1:105</td><td align=\"center\">1:136</td><td align=\"center\">1:153</td><td align=\"center\">1:37</td><td align=\"center\">1:61</td><td align=\"center\"><bold>1:100</bold></td></tr><tr><td align=\"left\">- On treatment</td><td align=\"center\">1:45</td><td align=\"center\">1:64</td><td align=\"center\">1:79</td><td align=\"center\">1:17</td><td align=\"center\">1:40</td><td align=\"center\"><bold>1:50</bold></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Professional nurses: trends in vacancy rates of ART posts (November 2004, to November 2006) as against all </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"4\"><bold>ART posts</bold></td><td align=\"center\"><bold>All PHC</bold><break/><bold>posts</bold><break/><bold> (August </bold><break/><bold> 2006)</bold></td></tr><tr><td colspan=\"5\"><hr/></td><td/></tr><tr><td align=\"left\"><bold>District</bold></td><td align=\"center\"><bold>November</bold><break/><bold> 2004</bold></td><td align=\"center\"><bold>March</bold><break/><bold> 2006</bold></td><td align=\"center\"><bold>August </bold><break/><bold>2006</bold></td><td align=\"center\"><bold>November </bold><break/><bold>2006</bold></td><td/></tr></thead><tbody><tr><td align=\"left\">Lejweleputswa</td><td align=\"center\">36.4%</td><td align=\"center\">8.0%</td><td align=\"center\">8.7%</td><td align=\"center\">8.0%</td><td align=\"center\">25.6%</td></tr><tr><td align=\"left\">Motheo</td><td align=\"center\">8.3%</td><td align=\"center\">4.6%</td><td align=\"center\">4.4%</td><td align=\"center\">0%</td><td align=\"center\">45.5%</td></tr><tr><td align=\"left\">Thabo Mofutsanyana</td><td align=\"center\">8.3%</td><td align=\"center\">4.6%</td><td align=\"center\">8.7%</td><td align=\"center\">13.0%</td><td align=\"center\">30.6%</td></tr><tr><td align=\"left\">Xhariep</td><td align=\"center\">46.2%</td><td align=\"center\">57.1%</td><td align=\"center\">42.9%</td><td align=\"center\">38.0%</td><td align=\"center\">54.4%</td></tr><tr><td align=\"left\">Fezile Dabi</td><td align=\"center\">45.5%</td><td align=\"center\">13.6%</td><td align=\"center\">18.2%</td><td align=\"center\">22.7%</td><td align=\"center\">35.9%</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Total posts</bold></td><td align=\"center\"><bold>28.8%</bold><break/>(of 59*)</td><td align=\"center\"><bold>16.8% </bold><break/>(of 113*)</td><td align=\"center\"><bold>15.8%</bold><break/>(of 114*)</td><td align=\"center\"><bold>15.7%</bold><break/>(of 115*)</td><td align=\"center\"><bold>37.1%</bold><break/>(of 1 664)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Promotion of ART nurses per district, November 2006 (PERSAL data)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>District</bold></td><td align=\"center\"><bold>Filled posts</bold></td><td align=\"center\"><bold>Promotions (% filled posts)</bold></td></tr></thead><tbody><tr><td align=\"left\">Motheo</td><td align=\"center\">24</td><td align=\"center\">17 (70.8%)</td></tr><tr><td align=\"left\">Lejweleputswa</td><td align=\"center\">23</td><td align=\"center\">15 (60.0%)</td></tr><tr><td align=\"left\">Thabo Mofutsanyana</td><td align=\"center\">20</td><td align=\"center\">6 (27.3%)</td></tr><tr><td align=\"left\">Xhariep</td><td align=\"center\">13</td><td align=\"center\">6 (28.6%)</td></tr><tr><td align=\"left\">Fezile Dabi</td><td align=\"center\">17</td><td align=\"center\">9 (40.9%)</td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td align=\"left\"><bold>Total</bold></td><td align=\"center\"><bold>97</bold></td><td align=\"center\"><bold>53 (54.6%)</bold></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Sources of professional nurses for the ART programme up to November 2006 (PERSAL data)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>District</bold></td><td align=\"center\"><bold>New</bold><break/><bold>appoint-</bold><break/><bold> ments</bold></td><td align=\"center\"><bold>From same</bold><break/><bold> facility</bold></td><td align=\"center\"><bold>From</bold><break/><bold>another </bold><break/><bold>facility in</bold><break/><bold> same district</bold></td><td align=\"center\"><bold>From a</bold><break/><bold>another</bold><break/><bold> district</bold></td><td align=\"center\"><bold>Total</bold><break/><bold>filled</bold><break/><bold> posts</bold></td></tr></thead><tbody><tr><td align=\"left\">Lejweleputswa</td><td align=\"center\">2 (8.7%)</td><td align=\"center\">10 (43.5%)</td><td align=\"center\">10 (43.5%)</td><td align=\"center\">1 (4.3%)</td><td align=\"center\">23</td></tr><tr><td align=\"left\">Motheo</td><td align=\"center\">3 (12.5%)</td><td align=\"center\">8 (33.3%)</td><td align=\"center\">11 (45.8%)</td><td align=\"center\">2 (8.3%)</td><td align=\"center\">24</td></tr><tr><td align=\"left\">Thabo Mofutsanyana</td><td align=\"center\">5 (25.0%)</td><td align=\"center\">3 (15.0%)</td><td align=\"center\">10 (50.0%)</td><td align=\"center\">2 (10.0%)</td><td align=\"center\">20</td></tr><tr><td align=\"left\">Xhariep</td><td align=\"center\">5 (38.5%)</td><td align=\"center\">1 (7.7%)</td><td align=\"center\">4 (30.7%)</td><td align=\"center\">3 (23.1%)</td><td align=\"center\">13</td></tr><tr><td align=\"left\">Fezile Dabi</td><td align=\"center\">5 (29.4%)</td><td align=\"center\">2 (11.8%)</td><td align=\"center\">7 (41.2%)</td><td align=\"center\">3 (17.6%)</td><td align=\"center\">17</td></tr><tr><td colspan=\"6\"><hr/></td></tr><tr><td align=\"left\"><bold>Total</bold></td><td align=\"center\"><bold>20 (20.6%)</bold></td><td align=\"center\"><bold>24 (24.7%)</bold></td><td align=\"center\"><bold>42 (43.2%)</bold></td><td align=\"center\"><bold>11 (11.3%)</bold></td><td align=\"center\"><bold>97</bold></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Number of staff trained in ART – 2004, 2005 and 2006 [##UREF##22##27##,##UREF##23##28##]</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Staff category</bold></td><td align=\"center\"><bold>2004</bold></td><td align=\"center\"><bold>2005</bold></td><td align=\"center\"><bold>2006</bold></td><td align=\"center\"><bold><italic>Total</italic></bold></td></tr></thead><tbody><tr><td align=\"left\">Professional nurses</td><td align=\"center\">127</td><td align=\"center\">172</td><td align=\"center\">281</td><td align=\"center\"><bold><italic>580</italic></bold></td></tr><tr><td align=\"left\">Enrolled/staff nurses</td><td align=\"center\">1</td><td align=\"center\">1</td><td align=\"center\">-</td><td align=\"center\"><bold><italic>2</italic></bold></td></tr><tr><td align=\"left\">Nursing assistants</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">5</td><td align=\"center\"><bold><italic>5</italic></bold></td></tr><tr><td align=\"left\">Other health professionals</td><td align=\"center\">87</td><td align=\"center\">160</td><td align=\"center\">154</td><td align=\"center\"><bold><italic>401</italic></bold></td></tr><tr><td align=\"left\">Non-professional staff (support staff,<break/> lay health workers, etc.)</td><td align=\"center\">107</td><td align=\"center\">69</td><td align=\"center\">231</td><td align=\"center\"><bold><italic>407</italic></bold></td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\"><bold><italic>Total</italic></bold></td><td align=\"center\"><bold><italic>322</italic></bold></td><td align=\"center\"><bold><italic>402</italic></bold></td><td align=\"center\"><bold><italic>671</italic></bold></td><td align=\"center\"><bold><italic>1 395</italic></bold></td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>Note: Although the cited patient numbers represent the official figures for the Free State, they are considered an undercount. A more accurate estimate of the total number of patients ever started on ART by end December 2006, was around 7105, based on the number of prescriptions dispensed by the provincial pharmaceutical services.</p></table-wrap-foot>", "<table-wrap-foot><p>PHC posts (August 2006), per district (PERSAL data)</p><p>Note: The numbers of professional nurses differ from the original 60 (Phase I) and 112 (Phase I and II) professional nurse posts created for the ART programme; these differences are ascribed to the creation of additional posts at some of the larger sites later during programme rollout.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Integration of synaptic signals during learning processes is critical to the function of cortical networks. This processing is achieved through various mechanisms that involve generation of coincident rhythmic activity, induction of properties of plasticity such as long-term potentiation (LTP), but also growth of new protrusions and remodeling of synaptic networks [##REF##8355787##1##–##UREF##0##5##]. The precise functional contribution of this structural remodeling to network properties remains unclear. In vitro experiments have demonstrated that LTP induction results during the next few hours in the growth of new filopodia and spines [##REF##10082466##6##–##REF##15572108##9##] which then rapidly become functional [##REF##16924105##10##] and show all characteristics of morphologically mature synapses over the course of 24 h [##REF##17652605##11##]. Also, work by several laboratories has shown that under in vivo conditions, spines and varicosities undergo a continuous turnover and replacement that vary in intensity as a function of development [##REF##12490942##12##–##REF##16543134##16##]. This process is further regulated by sensory activity, because under conditions of deprivation such as whisker trimming [##REF##16015331##17##] or unbalanced activity such as chessboard whisker trimming [##REF##12490942##12##,##REF##16791195##18##], spine turnover increases, new spines form synapses and become stabilized, and others are eliminated. These experiments therefore clearly demonstrated that stable synaptic contacts can be removed or created de novo through experience, raising the possibility that synapse remodeling, together with Hebbian forms of plasticity, could contribute to information processing and learning [##REF##15483599##3##,##REF##11970858##19##]. It remains unclear, however, whether and how sensory activity regulates this synaptic remodeling and whether it could actually affect signal integration by the neuron and/or the network. Also, the rules and mechanisms determining which synapse should be removed or restructured and where new synapses should be created are unknown. These are important issues because both the number and localization of spines may greatly affect the properties of integration of synaptic responses by a neuron. Recent studies have shown that spatiotemporal clustering of synaptic currents on small or remote dendrites represents a critical aspect for the expression of plasticity and the contribution to neuronal firing [##REF##17035526##20##–##REF##17206140##22##]. Identification of the mechanisms that underlie spine and synapse remodeling is therefore critical to a better understanding of the processing properties of synaptic networks. We investigated these issues, using a repetitive imaging approach applied to hippocampal slice cultures, and analyzed how precisely learning-related activity patterns affected the long-term behavior of identified spines.</p>" ]

[ "<title>Materials and Methods</title>", "<title>Slice cultures and transfection.</title>", "<p>Transverse hippocampal organotypic slice cultures (400 μm thick) from 6- to 7-d-old rats were prepared as described [##REF##1715499##38##] using a protocol approved by the Geneva Veterinarian Office (authorization 31.1.1007/3129/0) and maintained for 11–18 d in a CO₂ incubator at 33 °C. Transfection was done either with a pc-DNA3.1-EGFP or a pCX-mRFP1 [##REF##15996270##39##] plasmid using a biolistic method (Helios Gene Gun, Bio-Rad) 2–3 d before the first observation. Fluorescence usually started to be expressed after 24–48 h and then remained stable for at least 15 d.</p>", "<title>Electrophysiology.</title>", "<p>For electrophysiological recordings, slice cultures were maintained at 32 °C in an interface chamber under continuous perfusion as described [##REF##11943814##40##]. EPSPs were evoked by stimulation of a group of Schaffer collaterals and recorded in the stratum radiatum of the CA1 region with pipettes filled with medium. Potentiation was analyzed by measuring EPSP slopes expressed as percent of baseline values using an acquisition program written with Labview. LTP was induced by TBS (five trains at 5 Hz composed each of four pulses at 100 Hz, repeated twice at 10-s intervals). As controls, we used slice cultures stimulated at low frequency (0.3 Hz) and recorded in the same manner as well as slice cultures stimulated with TBS but in the presence of 100 μM D-AP5. In these experiments, D-AP5 was only applied for 30 min during application of TBS. Cch treatment was applied for 20–60 min at a concentration of 10 μM with or without concomitant application of D-AP5. The protein synthesis inhibitor was Ani applied 1 h before TBS or Cch treatment at a concentration of 25 μM and then maintained for 3 h.</p>", "<title>Confocal imaging.</title>", "<p>Short imaging sessions (10–15 min) of transfected slices were carried out with an Olympus Fluoview 300 system coupled to a single (Olympus) and a two-photon laser (Chameleon; Coherent) as described [##REF##17517683##25##]. Laser intensity in all these experiments was kept at the minimum and acquisition conditions maintained mostly unchanged over the different days of observation. Control experiments showed that transfection and repetitive confocal imaging of slice cultures did not alter cell viability over periods of weeks.</p>", "<p>We focused on dendritic segments of about 35 μm in length and located between 100 and 300 μm from the soma on secondary or tertiary dendrites using a 40× objective and a 10× additional zoom (final resolution: 25 pixels per micron; steps between scans: 0.4 μm; ##FIG##0##Figure 1##). We did not find differences in protrusion turnover within the limits of these dendritic locations. For calcium imaging of spine activity, transfected cells were additionally loaded with the cell-permeable calcium indicator Fluo-4 AM (F-14201, Invitrogen). For this, 50 μg of Fluo-4 AM was dissolved in 10 μl Pluronic (F-127, Invitrogen) and then diluted in 90 μl of standard pipette solution (150 mM NaCl, 2.5 mM KCl, 10 mM Hepes) for a final dye concentration of 500 μM. A standard patch pipette was then filled with 10 μl of dye solution and placed at a distance of about 10 μm from the soma of a mRFP1-expressing CA1 pyramidal cell. Dye was ejected by short pulses of pressured air at a frequency of three per minute during one-half hour. Calcium transients in 10–26 identified spines per dendritic segment were then recorded using line scans through the spine heads obtained during application of stimulation pulses to Schaffer collaterals. These pulses were of identical intensity and duration to those used for subsequent induction of LTP. Confocal aperture was set to the minimum during line scans, and matching with the mRFP fluorescence in the red channel was systematically checked. For each spine tested, calcium transients evoked by two or three consecutive stimulation pulses were recorded, and spines were determined as activated whenever the fluorescence signal increased by more than 20% over background in any of the recordings. In average, 36% of all spines tested corresponded to these criteria with the stimulation pulses used. To avoid biases, we then also verified that the size distribution of the spine heads did not differ between spines classified as activated and nonactivated (0.56 ± 0.02 μm versus 0.58 ± 0.02 μm, respectively; ##FIG##5##Figure 6##A).</p>", "<title>Image analysis.</title>", "<p>In this study we refer to protrusions, whenever analyses were carried out by considering filopodia and spines. Filopodia were defined as protrusions devoid of enlargement at the tip, while we classified as spines all protrusions exhibiting an enlargement at the tip. All turnover and stability analyses were carried out on single z-stacks of raw images (##FIG##0##Figures 1##E and ##SUPPL##0##S1##) using a plug-in specifically developed for OsiriX software (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.osirix-viewer.com\">http://www.osirix-viewer.com</ext-link>). The measures of turnover were carried out by analyzing all protrusions, i.e., filopodia and spines. We counted as new protrusions all new structures (spines or filopodia) appearing between two observations (5 or 24 h) and characterized by a length of &gt;0.4 μm. All filopodia were counted as separate protrusions. We also counted spines located behind each other on z-stacks whenever distinction was possible (##FIG##0##Figures 1##E and ##SUPPL##0##S1##). For disappearances, we counted all protrusions (spines and filopodia) that could no longer be identified on the next observation. Dubious situations due to possible changes in protrusion shape, size, or orientiation were discarded, but overall accounted for only a small number of cases (less than 1%). To further ensure reliability of analyses, all measurements of spine turnover and stability were carried out blind by two experimenters. Comparisons of the analyses made in this way showed variations in the results that were less than 3%. Furthermore, we used high numbers of <italic>n</italic> for both cells and spines, and labeled all new or lost protrusions directly on the raw data (##FIG##0##Figure 1##E) to allow multiple checks.</p>", "<p>Due to the lack of survival of filopodia on several days, stability analyses carried on 48 or 72 h periods only included pre-existing spines, i.e., spines present at the beginning of the experiment. For analyses of spine width, we measured the maximum diameter of the spine head on individual z-images, setting the fluorescence level on the levels obtained in the dendrite. Situations that did not allow a precise spine head width measurement (two spine heads overlapping each other on the same z sections) were excluded. Calcium fluorescence intensities were acquired and analyzed with Fluoview software (FV300, Olympus). Note that for illustration purposes, images presented in the figures are maximum intensity projections of z stacks, further treated with a Gaussian blur filter. All statistics are given with the standard error of the mean. Normality was tested for each distribution (D'Agostino and Pearson test), and α was set to 5% for all tests.</p>" ]

[ "<title>Results</title>", "<title>LTP Induction Results in a Lasting Increase in Protrusion Turnover</title>", "<p>Hippocampal slice cultures were transfected to express enhanced green fluorescent protein (EGFP) using a biolistic approach; we then monitored the behavior of identified protrusions (spines and filopodia) over several days following induction of learning-related activity patterns (##FIG##0##Figure 1##). For this, we used two different conditions that trigger LTP, a property believed to underlie learning mechanisms: first, we applied theta burst stimulation (TBS) to Schaffer collaterals, which triggers robust LTP, and second, we treated slice cultures for 20–60 min with carbachol (Cch, 10 μM), a cholinergic agonist, which, in the hippocampus and in slice cultures, triggers rhythmic activity in the theta and gamma range and induces a lasting synaptic enhancement (##FIG##1##Figure 2##C, inserts) [##REF##8355787##1##,##REF##17115043##23##]. In humans, these theta activities have been directly implicated in memory processes [##REF##17409248##24##]. Fluorescent cells and dendritic segments were then imaged repetitively and the changes in protrusion number and long-term spine stability monitored (##FIG##0##Figure 1##A–##FIG##0##1##C) through analysis of single z-stack images (##FIG##0##Figure 1##D and ##FIG##0##1##E; see criteria in <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>). Control experiments with propidium iodide staining showed that transfection and repetitive confocal imaging of slice cultures did not alter cell viability over periods of weeks.</p>", "<p>Analysis of protrusion turnover over periods of 3–8 d showed that the dynamics of synaptic networks is high at this developmental stage (11 d in vitro) with an average of 20.3% ± 1.1% new protrusions formed per 24 h and 20.8 ± 0.9% disappearing within the same period of time (##FIG##1##Figure 2##A and ##FIG##1##2##B). The other protrusions either remained stable without changes or underwent some sort of morphological transformations (16.2% ± 0.3% [##REF##17517683##25##]). These values are in the range of those reported in vivo in the cortex of very young mice [##REF##12490942##12##,##REF##15848798##14##,##REF##15664179##15##].</p>", "<p>Following theta burst activity we found that this basal turnover rate markedly increased. The effect was not short-lived [##REF##10331391##7##,##REF##15572108##9##], but the increase lasted for several days following a brief stimulation episode. This lasting increase in turnover rate was observed both following LTP induction by TBS (##FIG##1##Figure 2##C) and by Cch-induced rhythmic activity (10 μM; ##FIG##1##Figure 2##D). The insert in ##FIG##1##Figure 2##C shows the potentiation of the slope of evoked excitatory postsynaptic potentials (EPSPs) recorded in slice cultures following TBS. In ##FIG##1##Figure 2##D, we illustrate the spontaneous baseline activity of 1–3 Hz observed under control conditions, the increased 5–10 Hz field activity recorded in the stratum pyramidale of CA3 during application of 10 μM Cch and the synaptic enhancement observed in the cornus ammonis 1 (CA1) region. The proportion of new and lost protrusions, which includes spines and filopodia, increased markedly under both conditions to values of 34% ± 6% and 43% ± 6% of new protrusions and 33% ± 2% and 44% ± 3% of lost protrusions over the first 24 h for TBS and Cch, respectively (see also ##FIG##2##Figure 3##A). These changes reflected a similar increase in the formation of thin spines and filopodia, filopodia representing only a very small fraction of the new protrusions both under control conditions and after stimulation (4.3% ± 0.9%, <italic>n</italic> = 30 cells, control; 4.7% ± 1.4%, <italic>n</italic> = 17, LTP and 3.4% ± 1.6%, <italic>n</italic> = 17, Cch). Together, these experiments indicate a 70% and 115% increase in protrusion turnover rate following TBS or Cch treatment, respectively. To allow comparisons, the data obtained at the different observation times are expressed in ##FIG##1##Figure 2##C and ##FIG##1##2##D as percentage of the basal rate of protrusion formation or loss observed under control condition. To test for the specificity of the effect, we then carried out the same experiments, but applied the N-methyl-D-aspartate (NMDA) receptor antagonist D(−)-2-amino-5-phosphonopentanoic acid (D-AP5; 100 μM) during the stimulation protocol or during the application of Cch. As shown in ##FIG##1##Figure 2##C and ##FIG##1##2##D, D-AP5 specifically prevented the lasting increase in protrusion turnover under both conditions. As an additional control, we also analyzed hippocampal slice cultures stimulated in the same way at low frequency (0.3 Hz), but without induction of rhythmic activity. These controls showed no significant changes in turnover rate over time. Finally, we also tested whether this increase in protrusion turnover was dependent upon protein synthesis. For this, slice cultures were incubated in the presence of 25 μM anisomycin (Ani) and stimulated with either TBS or Cch. Under these conditions, both forms of potentiation were prevented (ratio of potentiation at 60 min: 1.13 ± 0.2, <italic>n</italic> = 6 and 1.08 ± 0.11, <italic>n</italic> = 3 for TBS and Cch, respectively) and, as shown in ##FIG##2##Figure 3##A, no significant increase in the rate of protrusion formation or loss could be observed over the next 24 h. Note also that Ani treatment of cultures for 5 h without TBS or Cch stimulation did not affect the rate of formation and loss of protrusions over 24 h. These results thus indicated that the changes in protrusion turnover associated with induction of LTP lasted several days and included formation and elimination of spines and filopodia.</p>", "<title>Increased Protrusion Turnover Promotes the Replacement of Pre-Existing Spines by New Ones</title>", "<p>To assess these results further and test for possible changes in spine stability and/or occurrence of populations of transient spines or filopodia, we next analyzed protrusion growth each day over a period of 5 h, a period during which most new events can be detected [##REF##17517683##25##]. Following LTP induction by TBS, the rate of protrusion formation expressed per 5 h and per 100 μm of dendritic segment increased by a factor of 2, and this for several days, an effect fully prevented by D-AP5 applied during the stimulation protocol (##FIG##2##Figure 3##B and ##FIG##2##3##C). We then also assessed spine stability, restricting the analysis to spines, since filopodia are essentially transient [##REF##17517683##25##] and mostly disappeared within 24 h. The stability of pre-existing spines, calculated as the proportion of spines still present on consecutive days, significantly decreased following LTP induction (##FIG##3##Figure 4##A), a change also dependent upon NMDA receptor activation. The stability of the new spines formed within the first 5 h following LTP induction was however not affected (##FIG##3##Figure 4##B) and remained particularly low as under control conditions. Thus, LTP induction promoted protrusion growth, but also destabilization of pre-existing spines. Altogether, these different effects approximately cancelled each other, so that the protrusion density did not greatly vary; actually, a significant increase was only observed transiently 2 d following LTP induction (##FIG##3##Figure 4##C). A similar situation was observed following Cch treatment. Protrusion growth increased in association with a decrease in stability of pre-existing spines and no effect on the process of new spine stabilization or on protrusion density (##FIG##3##Figure 4##D–##FIG##3##4##F). With both types of experiments, therefore, the net effect on several days of this increased turnover was to promote the replacement of existing spines by new ones.</p>", "<title>Differential Stabilization of Activated and Nonactivated Spines by Rhythmic Activity</title>", "<p>We then wondered how this increased spine remodeling could contribute to the specificity of the synaptic network and thus investigated whether it affected similarly activated and naive synapses. For this, we transfected pyramidal neurons with the red fluorescent dye monomeric red fluorescent protein (mRFP) [##REF##12060735##26##], to visualize the structural changes in spine morphology, and costained them 3 d later with Fluo-4 AM, a calcium indicator, to identify spines activated by single pulse and TBS stimulation protocols (##FIG##4##Figure 5##A–##FIG##4##5##C, see also <xref ref-type=\"sec\" rid=\"s4\">Material and Methods</xref>). ##FIG##4##Figure 5## illustrates the example of a dendritic segment with one spine that showed a clear increase in calcium fluorescence upon stimulation, while another one on the same segment remained silent. In all experiments carried out, we verified that spines activated by stimulation were always surrounded by other silent, nonactivated spines in order to exclude global activation effects. Also, we checked that analyses were done on spines of similar size (see ##FIG##5##Figure 6##) and that the maximum calcium signal perfectly coincided with the center of the spine head. We then assessed the stability of activated and nonactivated spines for the next 3 d. Overall, with the stimulation pulses used under these conditions, on average, 36% of all spines tested on analyzed dendritic segments were found to be activated (<italic>n</italic> = 349 spines, 18 cells or segments). TBS was then applied to the same synapses using the same stimulation pulses in ten cells (62 activated and 130 nonactivated spines analyzed), which resulted in a differential effect on spine stability: activated spines showed a striking increase in stability in comparison to nonactivated spines present on the same dendritic portions (##FIG##4##Figure 5##D; <italic>p</italic> &lt; 0.001). Nonactivated spines actually underwent pruning with regard to spines in nonstimulated slice cultures (##FIG##4##Figure 5##E; <italic>p</italic> &lt; 0.05). Interestingly, this differential stabilization was prevented by D-AP5 applied during TBS (##FIG##4##Figure 5##E). We also verified that simple activation of spines without TBS did not affect the long-term stability of spines (##FIG##4##Figure 5##E, squares).</p>", "<title>Spine Activation Coincides with Spine Enlargement and Stabilization</title>", "<p>Although for technical reasons we could not directly assess LTP in these stimulated spines, we found that most of them exhibited an enlargement of their head over the next 5 h. Several previous studies have indeed reported an enlargement of the spine head as a consequence of LTP induction [##REF##8755599##27##–##REF##16481433##29##] or used this criteria for identifying potentiated synapses [##REF##18097401##30##]. In the group of 272 activated and nonactivated spines analyzed before TBS, there was no difference in mean head width (##FIG##5##Figure 6##A). However, when analyzed 5 h after TBS, most activated spines now exhibited an enlargement of their head, an effect not observed with nonactivated spines (##FIG##5##Figure 6##B). Interestingly, we also found that this differential enlargement was transient, as most activated spines reversed their size after 24 h and the differences with nonactivated spines then became nonsignificant (##FIG##5##Figure 6##C). Note, in addition, that the head width of nonactivated spines tended to become smaller after TBS and that the size of spine heads, when analyzed individually, showed regular fluctuations over consecutive days for both activated and nonactivated spines. A robust effect, however, was the close correlation observed between activated spines, spines that showed an enlargement 5 h after stimulation, and spines that became stabilized by activity. When using spine enlargement as a criteria to analyze spine stability, we found, as for activity, that enlarging spines exhibited the same differential stabilization (##FIG##5##Figure 6##D). Thus LTP induction is very likely to promote a long-term stabilization of potentiated synapses.</p>", "<p>To verify whether Cch-induced rhythmic activity also produced the same selective stabilization process, we then analyzed how Cch treatment affected spine size. Analysis of 218 spines taken from nine dendritic segments showed that 34% of them exhibited enlargement of their head 5 h after Cch treatment. We then tested the stability of these spines over the next 2 d. As shown in ##FIG##5##Figure 6##E, spines that enlarged as a result of Cch-induced rhythmic activity also became significantly more stable, while nonenlarging spines tended to be eliminated, showing the same differential behavior as after TBS-induced potentiation.</p>", "<title>Hot Spots for Spine Growth around Activated Synapses</title>", "<p>We then asked how these mechanisms could affect spine organization and distribution and analyzed whether newly formed spines could appear at specific hot spots. As shown in ##FIG##6##Figure 7##A and ##FIG##6##7##B, we found that, indeed, newly formed spines tended to appear in close proximity to activated spines. In ##FIG##6##Figure 7##C, we analyzed the proportion of activated versus nonactivated spines that had a new protrusion formed within a distance of 1.5 μm in the next 48 h (defined as hot spot). As indicated, almost half of activated spines had a new spine growing close by, something that did not occur with nonactivated ones. As shown by ##FIG##6##Figure 7##D, we then examined all newly formed spines and asked how many actually grew close to an activated or a nonactivated spine. The results show that, again, about half of newly formed spines grew less than 1.5 μm from an activated spine, while only a small number of them grew close to a nonactivated spine, the others growing close to spines that could not be determined. The overall stability of newly formed spines was, however, not dependent on their localization (##FIG##6##Figure 7##E), because new spines generated close to or far from an activated spines showed the same probability of being present on subsequent days.</p>", "<title>New Spines Formed Consecutively to LTP Induction Are Functional</title>", "<p>We then tested whether these newly formed spines became functional. For this, TBS was applied to an mRFP-transfected neuron, and the new spines formed within the next 24 h monitored by repetitive imaging and their functionality tested through loading with Fluo-4 AM and stimulation trials of Schaffer collaterals. ##FIG##7##Figure 8##A shows an example of such a newly formed spine. Line scan analysis performed 24 h after TBS shows that this newly formed spine did indeed respond to stimulation through a calcium signal (##FIG##7##Figure 8##B and ##FIG##7##8##C), indicating that it was functional. Similar results were obtained in 30 spines out of 47 analyzed (<italic>n</italic> = 5 cells), indicating that a majority of them were functional. The mean ΔF/F₀ ratio (i.e., [fluorescence − basal fluorescence]/basal fluorescence) at the peak of the calcium signal recorded in these experiments was 4.3 ± 0.8 (<italic>n</italic> = 30). For the other spines, it remains unclear whether they were silent or whether we simply could not activate them. We then asked whether the new functional synapses were also likely to be more stable than those that did not exhibit any calcium signal in response to stimulation. Of the 47 newly formed spines analyzed here, we found that the probability to persist for 48 h was 82% ± 12% for the 30 functional spines (<italic>n</italic> = 5), but only 30% ± 10% for the 17 nonactivated spines (##FIG##7##Figure 8##D), indicating that activity is a major criteria for long-term stability. Together these results indicate that LTP induction favored a clustering of new functional spines around activated spines, promoting in this way possibilities of spatiotemporal interactions between them.</p>" ]

[ "<title> Discussion</title>", "<p>Together, these experiments provide evidence for an important new functional role of LTP-inducing activity in promoting a refinement of synaptic networks. Previous work in hippocampal slice cultures has shown that LTP induction is associated with two major types of structural remodeling. First, within minutes, potentiated synapses become larger and express larger and more complex postsynaptic densities [##REF##8755599##27##–##REF##18097401##30##], a change possibly associated with receptor expression and/or spine stabilization [##REF##12850432##31##]. Second, within minutes to hours, LTP induction also results in the growth of new filopodia and spines [##REF##10331391##7##,##REF##15572108##9##,##REF##14627649##32##], which then eventually become functional synapses [##REF##10586883##8##,##REF##16924105##10##,##REF##17652605##11##,##REF##17517683##25##]. These in vitro data are consistent with other in vivo experiments indicating that sensory deprivation or unbalanced activity does indeed affect cortical spine turnover and promote formation of new synapses [##REF##16015331##17##,##REF##16791195##18##,##REF##16892056##33##]. Here we add three new pieces of information providing a novel, important function for structural plasticity: namely, to operate as a selection process for the long-term stability of synaptic contacts and the promotion of spatiotemporal interactions between spines.</p>", "<p>First, we provide the first (to our knowledge) direct evidence that spines stimulated with LTP-inducing protocols are selectively stabilized over periods of several days. Although LTP could not be directly assessed together with repetitive imaging, we find that stabilization occurred specifically at spines stimulated with TBS and not at nonstimulated spines. Also, stabilized spines did exhibit an enlargement of the head at 5 h, a characteristic now demonstrated to be directly associated to LTP by several recent studies [##REF##8755599##27##–##REF##18097401##30##]. Finally, spine enlargement and spine stabilization were both D-AP5 sensitive and protein synthesis dependent. It seems therefore likely that the stabilization of stimulated synapses revealed here represents a central mechanism for the persistence of potentiated synapses.</p>", "<p>The second new feature uncovered by these experiments is that LTP is not only associated with a short-term increase in protrusion growth, but a lasting, enhanced turnover that affects pre-existing spine stability, probably through competition mechanisms. Consistent with previous data [##REF##10331391##7##,##REF##15572108##9##], protrusion growth initially tended to predominate over spine loss, leading to a transient increase in spine or protrusion density. However, all together, LTP mainly affected turnover, resulting not only in protrusion growth, but also in an increased loss and destabilization of spines, which, importantly, specifically affected nonstimulated spines. The net effect of LTP over several days was therefore to promote the replacement of nonactivated spines by new ones. This selective destabilization of nonactivated spines was quantitatively significant, because in these experiments more than 10% of the spines of the neurons were actually replaced. Accordingly, regular occurrence of activity susceptible to induce LTP works as a selection mechanism leading to a progressive stabilization of inputs showing coincident activity, increasing in this way the coherence of the synaptic information provided to the neuron and reducing background noise.</p>", "<p>The last important finding of these experiments is that newly formed protrusions do not appear just anywhere, but tend to cluster around activated spines. These new spines also become functional, and when functional, tend to remain stable. Together with the evidence that LTP induction is facilitated between spines located close to each other [##REF##18097401##30##], this result indicates that LTP will actually promote the creation of hot spots of functional synapses. This provides therefore a means to promote spatiotemporal clustering of synaptic signals, a property recently shown to be critical for determining the characteristics of plasticity and processing at synapses on small or remote dendrites [##REF##17035526##20##–##REF##17206140##22##].</p>", "<p>At the molecular level, an interesting implication of these results is that LTP mechanisms are likely to involve specific changes that could directly affect spine stability. Spine enlargement has been previously proposed to reflect this process [##REF##12850432##31##] and, consistent with this idea, we indeed found that activated spines did enlarge 5 h after stimulation. Curiously, however, this effect did not seem to remain stable over 24 h, and analyses of spine head width suggest that most spines regularly exhibit significant variations of their size [##REF##18097401##30##]. It could be, therefore, that stability is not only reflected in the size of the spine, but is linked to the expression of specific molecules. The current evidence indicating a contribution of protein synthesis to the long-term changes in synaptic strength and to the regulation of spine turnover as reported here could actually suggest such a mechanism [##REF##17018276##34##]. In order to become stable, activated spines would need to accumulate the machinery required for protein synthesis [##REF##12165474##35##] and/or express specific molecules conferring stability to the synaptic contact.</p>", "<p>Taken together, the mechanisms reported here provide a new framework for understanding how the specificity of cortical networks may progressively develop. These results might be particularly important during critical periods when refinement of connections represents a major process shaped by rhythmic activity and dynamic regulations between excitatory and inhibitory transmission [##REF##16261181##36##]. This network plasticity might, however, also contribute in the adult and provide the functional rules underlying the spine dynamics described in association with sensory activity [##REF##16791195##18##] or following brain damage [##REF##17428988##37##]. Together the synaptic mechanisms described here certainly point to the important role played by structural plasticity in association to Hebbian changes in synaptic strength for the refinement and specificity of cortical networks.</p>" ]

[]

[ "<p>Dendritic spines are the main postsynaptic site of excitatory contacts between neurons in the central nervous system. On cortical neurons, spines undergo a continuous turnover regulated by development and sensory activity. However, the functional implications of this synaptic remodeling for network properties remain currently unknown. Using repetitive confocal imaging on hippocampal organotypic cultures, we find that learning-related patterns of activity that induce long-term potentiation act as a selection mechanism for the stabilization and localization of spines. Through a lasting N-methyl-D-aspartate receptor and protein synthesis–dependent increase in protrusion growth and turnover, induction of plasticity promotes a pruning and replacement of nonactivated spines by new ones together with a selective stabilization of activated synapses. Furthermore, most newly formed spines preferentially grow in close proximity to activated synapses and become functional within 24 h, leading to a clustering of functional synapses. Our results indicate that synaptic remodeling associated with induction of long-term potentiation favors the selection of inputs showing spatiotemporal interactions on a given neuron.</p>", "<title>Author Summary</title>", "<title/>", "<p>In the central nervous system, excitatory contacts between neurons occur mainly on postsynaptic protrusions called dendritic spines. For decades, these structures have been considered static, and the adaptive properties of neuronal networks were thought to be only due to changes in the strength of neuronal connections. But recently, new imaging techniques used on living neurons revealed that spines and synapses are dynamic structures that undergo continuous turnover and can be formed or eliminated as a function of activity. The functional consequences of this structural remodeling, however, were still unknown. This work shows that application of learning related paradigms (such as induction of long-term potentiation or rhythmic activity) to hippocampal neurons allows them to operate a selection of synaptic inputs that show coincident activity. This is done through a competitive mechanism that promotes a selective stabilization of synapses activated by the learning paradigm and a replacement of non-activated inputs by new spines. Furthermore these new dendritic spines preferentially grow in close proximity to activated synapses and become functional. These findings provide evidence that learning related paradigms play a major role in shaping the structural organization of synaptic networks by promoting their specificity.</p>", "<p>In hippocampal neurons, the induction of learning related patterns of activity leads to a selective stabilization of activated synapses. Moreover, new dendritic spines, which are functional, cluster in close proximity to activated synapses.</p>" ]

[ "<title>Supporting Information</title>" ]

[ "<p>We thank Marlis Moosmayer, Lorena Jourdain, Irina Nikonenko, and Philippe Corrèges for technical support and Joël Spaltenstein for the developments of Osirix software plugins.</p>", "<p>\n<bold>Author contributions.</bold> MDR, PK, and DM conceived and designed the experiments. MDR and PK performed the experiments. MDR, PK, and DM analyzed the data. DM contributed reagents/materials/analysis tools. MDR, PK, and DM wrote the paper.</p>", "<p>\n<bold>Funding.</bold> The mRFP1 plasmid was a generous gift from AK Hadjantonakis and RY Tsien, University of California, San Diego. This work was supported by the Swiss Science Foundation, Boninchi Foundation, Velux Foundation, and European project Promemoria.</p>" ]

[ "<fig id=\"pbio-0060219-g001\" position=\"float\"><label>Figure 1</label><caption><title>Illustration of the Experimental Approach</title><p>(A) EGFP fluorescent CA1 pyramidal cell (arrow) in an 11 d in vitro organotypic hippocampal slice culture observed 3 d after transfection (scale bar: 100 μm).</p><p>(B) Low magnification view of a CA1 pyramidal cell imaged on the first (D1) and fourth day (D4) of observation (scale bar: 50 μm).</p><p>(C) 3D reconstructions of a dendritic segment imaged twice at 5-h intervals 24 h after LTP induction (see the axial rotation of the 3D reconstructed segments in ##SUPPL##1##Video S1##). Note the appearance of two new protrusions (yellow dots) and disappearance of one (red dot) within this 5-h interval (scale bar: 2 μm).</p><p>(D) Example of turnover analyses of z-stacks projections of raw images at observation day 1 (D1) and day 2 (D2). Color dots are placed on protrusions, one color per protrusion to facilitate their identification. At D2, three new protrusions are identified by a plus sign (+) and four lost protrusions by a minus sign (−) (scale bar: 1 μm).</p><p>(E) Series of images from the z-stacks shown in (D) but at different z depths to illustrate the possibility to detect protrusions in the three dimensions. Color dots from (D) and + and − signs are reported on the z plan that is the most relevant for each protrusion (highest brightness). Note that the relative position of each protrusion along the <italic>z</italic>-axis is well conserved between observations (scale bar: 1 μm).</p></caption></fig>", "<fig id=\"pbio-0060219-g002\" position=\"float\"><label>Figure 2</label><caption><title>Lasting Increase in Protrusion Turnover Induced by Rhythmic Activity</title><p>(A) Dendritic segments imaged on three consecutive days under control conditions (Ctrl, D1–D3) or before (D1) and following LTP induction (LTP, D1–D3; [+] new and [−] lost protrusions) (scale bars: 1 μm).</p><p>(B) Proportion of stable, new, and lost protrusions (spines and filopodia) per 24 h under control conditions (<italic>n</italic> = 30 cells/1,627 protrusions).</p><p>(C) Changes in protrusion turnover measured following LTP induction (TBS, circles; <italic>n</italic> = 17/916), or TBS + 100 μM D-AP5 (diamonds; <italic>n</italic> = 11/724), or low-frequency stimulation (0.3 Hz; squares; <italic>n</italic> = 6/183). Filled and empty symbols represent changes in new and lost protrusions, respectively. Data are plotted as percentage of control values with the shaded area representing the confidence interval. The insert shows the changes in EPSP slope measured in the CA1 area before and after LTP induction (filled circles; <italic>n</italic> = 13) and TBS + 100 μM D-AP5 (open circles; <italic>n</italic> = 7).</p><p>(D) Same as in (C) but following theta activity (shown in the insert) induced by treatment with 10 μM Cch (circles, <italic>n</italic> = 11/723). Diamonds: 10 μM Cch + 100 μM D-AP5 (<italic>n</italic> = 8/639; ***<italic>p</italic> &lt; 0.001, 2-way ANOVA with Bonferroni post-tests). The insert illustrates the spontaneous baseline activity of 1–3 Hz observed in one experiment and the increased 5–10 Hz activity induced by Cch. Below are shown the changes in EPSP slope measured during and after Cch treatment (<italic>n</italic> = 5).</p></caption></fig>", "<fig id=\"pbio-0060219-g003\" position=\"float\"><label>Figure 3</label><caption><title>Increased Activity-Induced Protrusion Formation Is Protein Synthesis Dependent and D-AP5 Sensitive</title><p>(A) Number of new protrusions (spines and filopodia) per 100 μm of dendritic length detected during the first 5 h and 24 h in control slice cultures (open columns), following TBS stimulation (black columns) or TBS applied in the presence of the protein synthesis inhibitor Ani (25 μM; grey columns; <italic>n</italic> = 6 cells/351 protrusions), as well as following Cch treatment (dark grey columns) and Cch together with Ani (light grey columns; <italic>n</italic> = 6 cells/307 protrusions). *<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01; ***<italic>p</italic> &lt; 0.001; two-way ANOVA with Bonferroni post-tests.</p><p>(B) The number of new protrusions formed per 5 h and per 100 μm of dendritic length was measured under control conditions (<italic>n</italic> = 15 cells), and at 1, 4, and 7 d after LTP induction (<italic>n</italic> = 8). **<italic>p</italic> &lt; 0.01, one-way ANOVA with Dunnett post-tests.</p><p>(C) Rate of protrusion formation measured 1 and 2 d after TBS applied in the presence of 100 μM D-AP5 (<italic>n</italic> = 7).</p></caption></fig>", "<fig id=\"pbio-0060219-g004\" position=\"float\"><label>Figure 4</label><caption><title>Changes in Spine Stability and Protrusion Density Induced by Theta Burst Activity</title><p>(A) Stability of pre-existing spines measured as the proportion of initial spines still present 5 h, 1, 2, and 3 d later in control slices (<italic>n</italic> = 20/557), in slices following LTP induction (<italic>n</italic> = 8/220) and in slices stimulated with TBS + D-AP5 (<italic>n</italic> = 7/301). Filopodia were not considered because they are essentially transient.</p><p>(B) Stability of the new spines formed during an interval of 5 h and still present 2 d later under control conditions, following LTP induction or TBS + D-AP5.</p><p>(C) Changes in protrusion density (spines and filopodia) measured after 2 and 3 d in control slices (open columns), following LTP induction (black columns) and following TBS + D-AP5 (grey column).</p><p>(D–F) Same as in (A), (B), and (C), respectively, but following Cch treatment (1 h, 10 μM; <italic>n</italic> = 11/328) or Cch + D-AP5 (<italic>n</italic> = 10/353).</p><p>*<italic>p</italic> &lt; 0.05, ***<italic>p</italic> &lt; 0.001; two-way ANOVA with Bonferroni post-tests.</p></caption></fig>", "<fig id=\"pbio-0060219-g005\" position=\"float\"><label>Figure 5</label><caption><title>Differential Stabilization of Activated and Nonactivated Spines following Rhythmic Activity</title><p>(A) Illustration of a dendritic segment with line scan analyses made on an activated (1) and a nonactivated (0) spine.</p><p>(B) Line scans showing the mRFP and Fluo-4 channels obtained by analysis of the activated and nonactivated spines (scale bars: 1 μm; 1 s, arrows: stimulation of Schaffer collaterals).</p><p>(C) Fluo-4 signals recorded in (B), expressed as ΔF/F₀.</p><p>(D) Differential long-term stability of activated (filled circles) and nonactivated spines (open circles) following LTP induction (<italic>n</italic> = 10 cells/62 activated and 130 nonactivated spines).</p><p>(E) Same as in (D) but with TBS + 100 μM D-AP5 (<italic>n</italic> = 4 cells/36 activated [filled circles] and 44 nonactivated spines [open circles]).</p><p>***<italic>p</italic> &lt; 0.001; two-way ANOVA with Bonferroni post-tests.</p></caption></fig>", "<fig id=\"pbio-0060219-g006\" position=\"float\"><label>Figure 6</label><caption><title>Differential Enlargement and Stability of Activated and Nonactivated Spines after Induction of Rhythmic Activity</title><p>(A) Box plot distributions (min to max) of the head width of activated (Act; <italic>n</italic> = 98) and nonactivated spines (N-Act; <italic>n</italic> = 174) measured before TBS.</p><p>(B) Proportion of activated and nonactivated spines that exhibited an enlargement 5 h after TBS (<italic>n</italic> = 4 cells/16 activated and 63 nonactivated spines). Spines were defined as enlarging if their head diameter increased by more than 0.1 μm in 5 h.</p><p>(C) Changes in spine width of activated and nonactivated spines and expressed as percent of initial values (<italic>n</italic> = 4 cells/16 activated and 63 nonactivated spines).</p><p>(D) Differential stability of spines that enlarged (filled circles) or did not enlarge (open circles) 5 h after LTP induction (<italic>n</italic> = 11 cells, 75 enlarging and 179 nonenlarging spines).</p><p>(E) Differential stability of spines that enlarged (filled circles) or did not enlarge (open circles) 5 h after carbachol treatment (<italic>n</italic> = 4 cells/21 enlarging and 58 nonenlarging spines).</p><p>*<italic>p</italic> &lt; 0.05, **<italic>p</italic> &lt; 0.01, ***<italic>p</italic> &lt; 0.001; Mann-Whitney U-test (B), two-way ANOVA with Bonferroni post-tests (C, D, and E).</p></caption></fig>", "<fig id=\"pbio-0060219-g007\" position=\"float\"><label>Figure 7</label><caption><title>Clustering of Newly Formed Spines around Activated Synapses</title><p>(A) Illustration of a dendritic segment with two spines (line scans, 1) that responded with a calcium signal to the stimulation and another spine (0) that did not.</p><p>(B) Same segment imaged 24 h later showing the formation of two new protrusions (+) in close proximity (&lt; 1.5 μm) of the activated spines (scale bar: 1.5 μm).</p><p>(C) Proportion of activated and nonactivated spines that exhibited a new protrusion formed at &lt; 1.5 μm (hot spot) within the next 48 h after TBS in seven experiments.</p><p>(D) Proportion of new spines formed during the 48 h that followed TBS and appeared close to (&lt; 1.5 μm) a nonactivated or an activated spine.</p><p>(E) Stability of new protrusions measured as the proportion of protrusions formed within 5 h after TBS in close proximity to an activated or nonactivated spine and still present 48 h later.</p><p>*<italic>p</italic> &lt; 0.05; **<italic>p</italic> &lt; 0.01, two-tailed unpaired <italic>t</italic>-test.</p></caption></fig>", "<fig id=\"pbio-0060219-g008\" position=\"float\"><label>Figure 8</label><caption><title>Functional Properties of Newly Formed Spines</title><p>(A) Newly formed spine detected 24 h after application of TBS (line scan; scale bar: 1.5 μm) and still present 48 h later (arrowhead).</p><p>(B) Line scan showing, superimposed, the mRFP and Fluo-4 channels obtained by analysis of the new spine illustrated in (A) and activated by electrical stimulation (arrow) 24 h after TBS (scale bars: 1 μm; 1 s).</p><p>(C) Fluo-4 signal recorded in (B) following electrical stimulation (arrowhead) and expressed as ΔF/F₀.</p><p>(D) Stability of newly formed spines that responded by a calcium signal upon stimulation (functional, black column) or failed to respond (failure, grey column) and measured over 48 h later (<italic>n</italic> = 5 cells; *<italic>p</italic> &lt; 0.05, Mann-Whitney U-test).</p></caption></fig>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"pbio-0060219-sg001\"><label>Figure S1</label><caption><title>Z-Stack Details from the Dendrite Illustrated in ##FIG##1##Figure 2##A</title><p>(A) Z-stacks projections of raw images obtained before (D1), 24 h (LTP-D2) and 48 h (LTP-D3) after LTP induction. Color dots or triangles are placed on protrusions, one color per spine, to facilitate protrusion identification. On day 2 (LTP-D2), three new protrusions are identified by a plus sign (+) and a color triangle; three lost protrusions by a minus sign (−). On day 3 (LTP-D3), two new protrusions are identified by a plus sign (+) and one lost protrusion by a minus sign (−).</p><p>(B) Series of images from the same stacks as in (A) but at different z steps to illustrate that scrolling through the <italic>z</italic>-axis allows a correct discrimination between protrusions. Color dots, triangles, and plus and minus signs from (A) are reported on the z level that is the most relevant for each protrusion (highest brightness) (scale bars: 1 μm).</p><p>(6.57 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pbio-0060219-sv001\"><label>Video S1</label><caption><title>3D Rotating Movie of the Dendritic Segment Illustrated in ##FIG##0##Figure 1##C</title><p>(2.38 MB MOV)</p></caption></supplementary-material>" ]

[ "<fn-group><fn id=\"ack2\" fn-type=\"COI-statement\"><p>\n<bold>Competing interests.</bold> The authors have declared that no competing interests exist.</p></fn></fn-group>" ]

[ "<graphic xlink:href=\"pbio.0060219.g001\"/>", "<graphic xlink:href=\"pbio.0060219.g002\"/>", "<graphic xlink:href=\"pbio.0060219.g003\"/>", "<graphic xlink:href=\"pbio.0060219.g004\"/>", "<graphic xlink:href=\"pbio.0060219.g005\"/>", "<graphic xlink:href=\"pbio.0060219.g006\"/>", "<graphic xlink:href=\"pbio.0060219.g007\"/>", "<graphic xlink:href=\"pbio.0060219.g008\"/>" ]

[ "<media xlink:href=\"pbio.0060219.sg001.tif\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pbio.0060219.sv001.mov\"><caption><p>Click here for additional data file.</p></caption></media>" ]

{ "acronym": [ "ΔF/F0", "Ani", "CA1", "Cch", "D-AP5", "EGFP", "EPSP", "LTP", "mRFP", "NMDA", "TBS" ], "definition": [ "(fluorescence − basal fluorescence)/basal fluorescence", "anisomycin", "cornus ammonis 1", "carbachol", "D(−)-2-amino-5-phosphonopentanoic acid", "enhanced green flourescent protein", "excitatory postsynaptic potential", "long-term potentiation", "monomeric red fluorescent protein", "N-methyl-D-aspartate", "theta burst stimulation" ] }

[ "<title>Background</title>", "<p>The prion protein disorder Creutzfeldt-Jakob disease (CJD) is a human transmissible spongiform encephalopathy (TSE) that leads to rapid decay of brain tissue. Human prion diseases are either idiopathic such as sporadic or spontaneous CJD (sCJD), genetic or familial (fCJD), acquired such as iatrogenic CJD (iCJD) or variant CJD (vCJD). Variant CJD is related to bovine spongiform encephalopathy (BSE), has clinical and pathological characteristics different from sCJD and has not been described in Norway. The most common form is sCJD, which accounts for about 85% of all known cases. Spontaneous CJD is a rare fatal disorder with rapid progression, an incidence of approximately 1/million/year [##REF##15883321##1##] and a mortality of more than 90% within one year. The etiology of sCJD is not known, but the pathogenesis is related to conversion of the normal membrane prion protein PrP^cto the pathological form PrP^Sc[##REF##15965195##2##]. Immunohistochemical demonstration of PrP^Scprovides a definite diagnosis of CJD at autopsy or by brain biopsy.</p>", "<p>In the Norwegian population of about 4.5 million, one would expect approximately 5 cases of sCJD per year. Due to the few specific pre-mortal diagnostic signs, it is difficult to separate sCJD clinically from rapidly progressing Alzheimer's disease (AD) and other rapidly progressing neurological diseases. As TSEs can cross species barriers, there is a public health concern about the ability of TSE to spread from other species to humans. Classical scrapie in sheep is endemic in Norway, and an atypical variant, Nor 98, has appeared [##REF##12956297##3##], but there is no evidence that scrapie has spread from animals to humans.</p>", "<p>The total concentration of tau protein (tTau) in CSF has been found to separate patients with CJD from those with AD [##REF##9147407##4##]. As an increased concentration of tTau is regarded to be a marker for degradation of neurons, the success of tTau as a marker for CJD depends on whether other neurological diseases have the same high amount and rate of neuronal degradation as CJD. A low concentration of the amyloid precursor protein (APP)-derived 42 amino acid peptide in CSF (Aβ₄₂), has been correlated with brain amyloid deposition in AD [##REF##12601108##5##]. Elevation of 181 phosphorylated tau protein (P-Tau) is also related to brain pathophysiology in AD, possibly as a result of interaction between amyloid and tau metabolism [##REF##11943163##6##]. As low CSF-Aβ₄₂and elevated P-tau are more specific for AD, one would expect the diagnostic specificity for CJD to rise when the markers are combined. In line with this, the ratio of tTau/P-tau has been described to separate CJD from other neurodegenerative diseases without overlap [##REF##12660807##7##,##REF##16633900##8##]. A separation of sCJD in two clinically different groups according to P-Tau level and a negative prognostic value of elevated P-Tau have been described for CJD [##REF##12082054##9##]. Spontaneous CJD patients with high P-Tau had a shorter disease duration (i.e. they died earlier), had earlier onset of akinetic mutism and a higher incidence of typical EEGs. The occurrence of elevated P-Tau values in CJD will decrease the value of combining tTau and P-Tau in the diagnosis of sCJD because the difference in tTau/P-Tau ratio between the groups would be reduced. The 14-3-3 proteins are evolutionarily conserved proteins present in the cytoplasm of brain neurons at a concentration of about 1% of the total protein content. It has been suggested that 14-3-3 protein as a <italic>pre-mortem </italic>immunoassay marker, may obviate the need for a brain biopsy in the diagnosis of CJD. Although 14-3-3 protein is known to be non-specific, it is included in the WHO criteria for CJD [##UREF##0##10##].</p>", "<p>We wished to investigate the utility of tTau, tTau/P-Tau ratio and 14-3-3 protein measurements in CSF as markers for sCJD. Other diagnostic characteristics for CJD, such as hyper intense magnetic resonance (MR) signals from the basal ganglia, sharp wave complexes in EEG and the examination of the CSF proteins, neuron specific enolase (NSE) and S100 have been evaluated elsewhere [##REF##16924018##11##]. Routine laboratory analyses of the three biological variables, tTau, P-Tau and Aβ₄₂, have been offered at the Department of Clinical Chemistry, Akershus University Hospital, since 2003. Routine analysis for 14-3-3 protein has been performed since November 2007. Neurological, psychiatric and outpatient departments in Norway have also had the opportunity to send CSF-samples for analysis.</p>" ]

[ "<title>Methods</title>", "<title>Patient selection and CSF analysis</title>", "<p>CSF samples (n = 691) were received by the Department of Clinical Chemistry, Akershus University Hospital between August 2005 and August 2007 from patients demonstrating symptoms of cognitive decline and possible neurodegenerative disease. This work was supported and approved by the Eastern Norway Regional Health Authority, RHA East, and approved by the Regional Committee for Medical Research Ethics, which also approved an exception from collecting informed patient consent.</p>", "<p>Total Tau, P-Tau and Aβ₄₂were analysed in CSF-samples with commercially available enzyme linked immunosorbent assay (ELISA) kits from Innogenetics (Gent, Belgium) adapted to a Tecan Robotic Microplate 150 Processor (Tecan AG, Switzerland). The analyses were performed approximately twice a month. The 14-3-3 protein was analysed by immunoblot with equipment from Invitrogen Corporation Ltd. (Paisley, U.K.) using an antibody against the γ-isoform, anti-14-3-3 gamma, clone CG31-2B (mouse monoclonal IgG₁, Upstate biotechnology, Lake Placid, NY, USA). The 14-3-3 protein results were assessed semi-quantitatively using arbitrary units. As standard we used dilutions of a homogenate from normal brain. For semi-quantification we performed image analysis using Fujifilm Multi Gauge software (Fujifilm Corporation, Tokyo, Japan). Thirty-nine patients studied retrospectively had tTau values &gt; 1200 ng/L (the highest standard of the kit). One patient was excluded because no additional sample was available to analyse tTau in dilution. Total Tau, P-Tau and Aβ₄₂were measured at the Akershus University Hospital between August 2005 and October 2007. 14-3-3 protein was analysed in seven of the 12 CJD patients and 25 of the 26 AD/VaD/OND patients in October and November 2007 in Akershus University Hospital. In one AD patient and in five CJD patients there was no sample available for analysis. Three of these CJD-patients had qualitative 14-3-3 protein results from laboratories outside Norway. Two CJD patients had no 14-3-3 protein determination. The maximum value for normal clinical values for tTau was 300 ng/L for patients &lt; 50 years, 450 ng/L for patients 50–70 years and 500 ng/L for patients &gt; 70 years [##REF##11568086##12##]. For Aβ₄₂, we have previously used values above 450 ng/L for normal levels [##REF##16966546##13##], but after comparison with the Neurochemistry laboratory at Sahlgrenska University Hospital, Gothenburg, Sweden (unpublished data), this has recently been revised to 550 ng/L. The maximum for normal clinical levels for P-Tau was 80 ng/L from the Neurochemistry laboratory in Gothenburg.</p>", "<title>Sample handling</title>", "<p>The samples for tTau, P-Tau and Aβ₄₂were initially frozen at -20°C. In February 2006 all old samples were transferred to -80°C and all new samples were kept at -80°C. The samples for 14-3-3 protein were stored for up to 2 years at -80°C before analysis in batches of six. The ELISA standards were run in duplicate and the samples were run singly. The analytical results were read from the corresponding standard curve for each run. No result exceeded the highest standard for P-Tau and Aβ₄₂, but for tTau all the patients in this study exceeded the highest standard point of 1200 ng/L. These samples were diluted and rerun.</p>", "<title>Patient diagnosis</title>", "<p>The patients were clinically diagnosed according to ICD-10 (International Classification of Diseases -10). The criteria were clinical suspicion, MR findings highly suggestive of CJD and three-phase sharp wave complexes on EEG also considered to be characteristic of CJD. Twelve patients had definite or probable CJD (Table ##TAB##0##1##); 21 patients had Alzheimer's disease (AD), vascular dementia (VaD), mixed dementia (AD/VaD), frontotemporal dementia (FTD) or unspecified dementia and five patients had other neurological diagnoses (OND, Table ##TAB##1##2##). Seven of the 12 CJD patients had short disease duration, mean 3.7 months (n = 7, SD = 1.5) and a further two patients had unspecified very rapid progression. Three patients had CJD with slower progression, disease duration 15, 22 and &gt;23 months.</p>", "<title>Data analysis</title>", "<p>Receiver operating characteristic (ROC) curve analysis was performed for tTau and tTau/PTau ratio with the statistical package Analyse-it (Analyse-it Software, Ltd. Leeds, U.K.). ROC-curve analysis was not performed for 14-3-3 protein because some of the results from other laboratories were qualitative only. The AD, VaD, AD/VaD and other dementia patients were treated as one group, AD/VaD. The lower limit chosen for the diagnosis of CJD was 3000 ng/L for tTau, for the ratio of tTau to P-Tau it was 60 and for 14-3-3 protein it was 0.75 arbitrary units. The limits, based on our test results, were set to obtain the best diagnostic performance for each marker. It could be argued that the cut-off for tTau and 14-3-3 protein could have been set slightly higher. In that case for tTau the sensitivity would decrease and the specificity would increase, and for 14-3-3 protein the sensitivity would also decrease and the specificity would increase to 100%. The sensitivity, specificity, positive and negative predictive values, and diagnostic efficiency for the three markers were calculated manually.</p>" ]

[ "<title>Results</title>", "<p>Table ##TAB##0##1## presents the characteristics for 12 patients who were diagnosed with CJD, two definite CJD and 10 probable CJD. Eight had MR findings suggestive of CJD. Generalized dysrhythmia and sharp wave complexes on EEG were found in nine patients and generalized dysrhythmia indicating diffuse cerebral pathology in the remaining three. Table ##TAB##1##2## presents the characteristics for patients with diagnoses other than CJD. Two patients had VaD or possible VaD, 11 had AD or possible AD, three had AD/VaD or possible AD/VaD, one had AD and Wernicke's encephalopathy, two had possible FTD and two had unspecified dementia. Five patients had OND.</p>", "<title>Total Tau</title>", "<p>The results for tTau by diagnostic group and lower limits for CJD are presented in Figure ##FIG##0##1A##. Eleven patients had tTau values above cut-off (3000 ng/L) and 27 below. Nine of the 12 patients with tTau-values above cut-off had CJD, one had OND (cerebral B-cell lymphoma) and one had possible FTD. Three patients with CJD of long duration had values (1343, 1880 and 2350 ng/L) which are below the chosen lower limit, 3000 ng/L. All but one of the OND patients and all but one of the AD/VaD patients had values below 3000 ng/L.</p>", "<title>Ratio of tTau to P-Tau</title>", "<p>The results for the ratio tTau/P-Tau by diagnostic group and chosen lower limit for CJD (60) are presented in Figure ##FIG##0##1B##. Ten patients had tTau/P-Tau ratios above the limit. Nine of these had CJD and one had cerebral B-cell lymphoma. The three patients with CJD of long duration, had values below cut-off (19, 25 and 26). All but one of the OND patients and all AD/VaD patients had values below the limit for CJD.</p>", "<title>P-Tau</title>", "<p>P-Tau results by diagnostic group and maximum for normal (80 ng/L) are presented in Figure ##FIG##1##2A##. Six of the 12 CJD patients had P-Tau above normal. In the AD/VaD-group (n = 21) 15 patients (71%) had P-Tau above and six below normal. The P-Tau results in the AD/VaD patients were distributed in two groups, range 27–92 and 200–287 ng/L. One of the OND patients (cerebral infarction) had P-Tau slightly above normal, 81 ng/L.</p>", "<title>Beta amyloid (1–42)</title>", "<p>The results for Aβ₄₂by diagnostic group and lower normal limit are presented in Figure ##FIG##1##2B##. Three of 11 patients (38%) in the CJD group (one missing value) and 11 of the 21 patients (52%) in the AD/VaD group had Aβ₄₂values below normal. The two CDJ patients with the lowest Aβ₄₂-values, 372 and 385, also had elevated P-Tau results, 149 and 211 ng/L.</p>", "<title>14-3-3 protein</title>", "<p>The lower limit for CJD diagnosis was set at 0.75 arbitrary units. Figure ##FIG##2##3## shows results from the home laboratory. Samples from 32 patients, seven from the CJD group, all five OND patients and 20 from the AD/VaD group, were available for analysis. Five of the seven CJD patients tested positive. Three of the 14-3-3 positive patients had been tested before and had positive results from laboratories outside Norway. Three of the five CJD patients not tested by us, had been tested before and had positive results. These were included in the estimations of diagnostic parameters (Table ##TAB##2##3##). Ten of the 12 patients with CJD had been examined for 14.3.3-protein by us, other laboratories or both. Eight of these were positive. One of the five patients with OND (cerebral B-cell lymphoma) tested weakly positive, and all the tested patients in the AD/VaD group were negative. Of the three CJD patients with long disease duration, two were negative.</p>", "<title>Diagnostic performance</title>", "<p>Calculations of sensitivity, specificity, positive predictive value, negative predictive value and diagnostic efficiency are presented in Table ##TAB##2##3##. The ROC curve analysis for the diagnosis of CJD showed that tTau was not significantly different from tTau/P-Tau for the diagnosis of CJD (area under curve 0.897 and 0.918, n.s.).</p>" ]

[ "<title>Discussion</title>", "<p>Although the ROC-curve analysis showed no significant difference between tTau and tTau/P-Tau, the tTau/P-Tau ratio did separate results around the cut-off value more clearly than t-Tau and the specificity and predictive values of a positive test for the CJD diagnosis were slightly better. Thus, our findings seem to agree with those of Riemenschneider <italic>et al </italic>[##REF##12660807##7##], who found that tTau/P-Tau ratio was a better marker for CJD than tTau. Buerger <italic>et al </italic>[##REF##16298235##14##] came to the opposite conclusion, but they measured P-Tau phosphorylated in the 232- position and not in the 181-position as measured here and by Riemenschneider <italic>et al</italic>. Our results suggest that 14-3-3 protein may be a slightly better marker than tTau and tTau/P-Tau.</p>", "<p>In contrast to P-Tau, Aβ₄₂did not contribute to providing a more definite diagnosis of CJD. Increased P-Tau was a more specific measure of AD/VaD than low Aβ₄₂, and contributed slightly to the diagnostic separation of CJD from the AD/VaD group. Similar values for Aβ₄₂in the CJD and AD/VaD groups suggest that comparable amyloid pathology was present in both groups. This indicates that some CJD patients may have acquired CJD in addition to an increased and possibly AD-related, amyloid pathology. The fact that the two CJD patients with the lowest Aβ₄₂values, also had elevated P-Tau is consistent with an interaction between prion protein and the AD pathological processes [##REF##15277640##15##]. The P-Tau values separated into two groups for AD/VaD patients (Figure ##FIG##1##2a##). The group with results above 200 ng/L was homogenous clinically because 11 of the 13 patients had the clinical diagnosis of AD, one had FTD/or possibly AD and one had AD/VaD. The group with results below 100 ng/L (n = 8) was clinically more heterogeneous (Table ##TAB##1##2##). Our results suggest that tTau, tTau/P-Tau and possibly 14-3-3 protein may only be good markers for sCJD of short duration and may not separate the CJD cases with longer duration from the other dementias and OND. This fact may be of considerable importance for the diagnosis of CJD and suggests that brain biopsy should be used more often in dementia cases. In addition, the use of autopsy should be encouraged.</p>", "<p>None of the patients in this study were tested for hereditary forms of CJD or AD, and the clinical information did not raise any suspicion about hereditary conditions. Seven patients in the AD/VaD group were &lt;65 years. None of them had a family history of dementia. Our results show that in addition to AD and VaD, other rapidly progressing neurological diseases may have the same tTau and P-Tau biomarker pattern as CJD. One patient with cerebral lymphoma had tTau and tTau/P-Tau above the cut-off values for CJD. Another patient with the same diagnosis was slightly positive for 14-3-3 protein. In spite of using both tTau, P-Tau and 14-3-3 protein for the diagnosis of CJD, our data suggest that there will still be a few patients with AD/VaD and OND that cannot be distinguished from CJD using the biomarkers tTau, P-Tau and 14-3-3 protein. Other investigations will usually establish the diagnosis in these cases by imaging or CSF-cytology.</p>", "<p>It can be argued that this study should have been performed prospectively, i.e. patients clinically suspected to have CJD should have been enrolled consecutively. This would have required a more active cooperation between the clinical centres. We were not able to undertake this task at that time, but it should ideally be done as a follow-up of the present study. Starting with high tTau patients was, however, a cost-effective way to find cases. The reason for not analysing 14-3-3 protein at the same time as the other markers was that the analysis was not set up by us until October 2007.</p>", "<p>Another criticism that could be raised against our study is the low frequency of histological verification. The reason for this is the right to refuse autopsy and the low autopsy frequency in Norway in general. The importance of obtaining histological verification should be stressed in future prospective CJD studies. Considering the low number of histological verifications, it is possible that some of the CJD patients were wrongly classified. We would argue that this is less likely as eight of the 12 patients (two proven histologically) had MR findings suggestive of CJD. Seven of these also had three-phase sharp wave complexes. Of the remaining four patients, all had non-specific MR changes and two had three-phase sharp wave complexes. The two remaining patients both had non-specific changes on EEG and the disease duration in one of them was four months, highly suggestive of CJD. The other patient had disease duration of 22 months and although 14-3-3 protein was positive, another type of degenerative brain disease cannot be totally excluded.</p>", "<p>This study also shows a higher prevalence of CJD in Norway than might be expected. We identified 12 cases during a period of two years. With a prevalence of one case per million inhabitants, approximately 10 cases would be expected, which is quite close to the observed number of 12. However, we do not expect that all the Norwegian cases in this period were known to us. The prevalence of CJD may therefore be higher than anticipated. This indicates a need for closer surveillance of human prion diseases in Norway.</p>" ]

[ "<title>Conclusion</title>", "<p>Total Tau, tTau/P-Tau ratio and 14-3-3 protein are useful, but not entirely sensitive and specific markers for the diagnosis of CJD in CSF. The results indicate that the diagnostic performance of 14-3-3 protein may be slightly better than tTau/P-Tau, which may be slightly better than tTau alone. There is clearly a need for a specific test for CJD, preferably for the misfolded prion protein (PrP^Sc) itself in readily obtainable biological samples such as blood and CSF. It remains to be seen whether a specific test for PrP^Scwould be as sensitive and specific as the markers used in this study.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The objective was to assess the utility of total tau protein (tTau), the ratio of (tTau)/181 phosphorylated tau protein (P-Tau) and 14-3-3 protein, as diagnostic markers in cerebrospinal fluid (CSF) for Creutzfeldt-Jakob disease (CJD).</p>", "<title>Methods</title>", "<p>CSF samples received from Norwegian hospitals between August 2005 and August 2007 were retrospectively selected from consecutive patients with tTau values &gt; 1200 ng/L (n = 38). The samples from patients clinically diagnosed with CJD (n = 12) were compared to those from patients with other degenerative neurological diseases: Alzheimer's/vascular dementia (AD/VaD, n = 21), other neurological diseases (OND, n = 5). Total Tau, P-Tau, and β-Amyloid (Aβ₄₂) were measured with commercial kits. Additionally, 14-3-3 protein was measured semi-quantitatively by immunoblot.</p>", "<title>Results</title>", "<p>The minimum cut-off limits for diagnosis of CJD were chosen from the test results. For tTau the lower limit was fixed at 3000 ng/L, for the tTau/P-Tau ratio it was 60, and for 14-3-3 protein it was 0.75 arbitrary units. For tTau and tTau/P-Tau ratio, all but three CJD patients had levels above the minimum, whereas almost all of the other patients were below. For the 14-3-3 protein, two CJD patients were below the minimum and five were above. Only one of the other patients was higher than the limit. The sensitivities, specificities and diagnostic efficiencies were: tTau 75%, 92%, and 87%; tTau/P-Tau 75%, 96%, and 89%; and 14-3-3 protein 80%, 96%, and 91%.</p>", "<title>Conclusion</title>", "<p>The results suggest that 14-3-3 protein may be the better marker for CJD, tTau/P-Tau ratio and tTau are also efficient markers, but showed slightly inferior diagnostic properties in this study, with tTau/P-Tau marginally better than tTau.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>AS designed the study, drafted the manuscript, and performed the statistical analysis. VS and TF participated in its design and coordination and helped to draft the manuscript. ASG performed ELISA tests, set up the immunoblot for 14-3-3 protein and contributed to the manuscript. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The authors wish to thank Lisbeth Johnsen, Randi Otterstad and Britt Skrogstad for technical assistance and medical writer Kari Skinningsrud (Limwric Ltd., Lillestoem, Norway) for critical review of the manuscript.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Data plots of Tau protein (tTau) (A) and tTau/P-Tau ratio (B) by diagnostic group Creutzfeldt-Jakob disease (CJD), other neurological diseases (OND) and Alzheimer's disease or vascular dementia (AD/VaD).</bold> Horizontal dashed lines show chosen lower limit for CJD: tTau: 3000 ng/L, and tTau/P-Tau ratio: 60.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Data plots of 181-phosphorylated tau protein (P-Tau) (A) and beta-amyloid (Aβ₄₂) (B) by diagnostic group CJD, OND and AD/VaD with upper (P-Tau) and lower (Aβ₄₂) normal limits used in clinical practice (dashed lines): P-Tau: 80 ng/L, Aβ₄₂: 550 ng/L.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Data plots of 14-3-3 protein by diagnostic group CJD, OND and AD/VaD with lower limit for CJD (dashed line): 0.75 arbitrary units (data from Akershus University Hospital only).</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characteristics and results for patients with Creutzfeldt-Jakob disease</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Sex/age (years)</td><td align=\"center\">Disease<break/>duration¹<break/>(months)</td><td align=\"left\">EEG</td><td align=\"center\">MR</td><td align=\"left\">Autopsy</td><td align=\"right\">tTau <break/>(ng/L)</td><td align=\"center\">P-Tau⁴<break/>(ng/L)</td><td align=\"center\">tTau/P-<break/>Tau<break/> (ng/L)</td><td align=\"center\">Aβ₄₂<break/>⁵(ng/L)</td><td align=\"left\">14-3-3<break/> protein</td></tr></thead><tbody><tr><td align=\"center\">Female/84</td><td align=\"center\">2</td><td align=\"left\">TSWC²</td><td align=\"center\">NC⁸</td><td align=\"left\">Denied</td><td align=\"right\">7,090</td><td align=\"center\">49</td><td align=\"center\">173</td><td align=\"center\">664</td><td align=\"left\">Not done</td></tr><tr><td align=\"center\">Male/75</td><td align=\"center\">6</td><td align=\"left\">TSWC</td><td align=\"center\">NC</td><td align=\"left\">Denied</td><td align=\"right\">13,830</td><td align=\"center\">211*</td><td align=\"center\">65</td><td align=\"center\">385*</td><td align=\"left\">Positive⁷</td></tr><tr><td align=\"center\">Male/62</td><td align=\"center\">4</td><td align=\"left\">GD⁶</td><td align=\"center\">NC</td><td align=\"left\">Not done</td><td align=\"right\">12,910</td><td align=\"center\">149*</td><td align=\"center\">83</td><td align=\"center\">372*</td><td align=\"left\">Not done</td></tr><tr><td align=\"center\">Female/58</td><td align=\"center\">22</td><td align=\"left\">GD</td><td align=\"center\">NC</td><td align=\"left\">Denied</td><td align=\"right\">1,880</td><td align=\"center\">92*</td><td align=\"center\">26</td><td align=\"center\">623</td><td align=\"left\">Positive⁷</td></tr><tr><td align=\"center\">Female/77</td><td align=\"center\">3</td><td align=\"left\">TSWC</td><td align=\"center\">HSC⁹</td><td align=\"left\">Positive³</td><td align=\"right\">3120</td><td align=\"center\">45</td><td align=\"center\">69</td><td align=\"center\">704</td><td align=\"left\">Positive⁷</td></tr><tr><td align=\"center\">Female/67</td><td align=\"center\">4</td><td align=\"left\">TSWC</td><td align=\"center\">HSC</td><td align=\"left\">No result</td><td align=\"right\">8532</td><td align=\"center\">66</td><td align=\"center\">129</td><td align=\"center\">526*</td><td align=\"left\">Positive⁷0.96</td></tr><tr><td align=\"center\">Female/64</td><td align=\"center\">Rapid</td><td align=\"left\">TSWC</td><td align=\"center\">HSC</td><td align=\"left\">Positive biopsy ³</td><td align=\"right\">14,060</td><td align=\"center\">84*</td><td align=\"center\">167</td><td align=\"center\">562</td><td align=\"left\">Positive⁷3.68</td></tr><tr><td align=\"center\">Male/74</td><td align=\"center\">2</td><td align=\"left\">GD</td><td align=\"center\">HSC</td><td align=\"left\">Not done</td><td align=\"right\">22,460</td><td align=\"center\">82*</td><td align=\"center\">274</td><td align=\"center\">946</td><td align=\"left\">2.42</td></tr><tr><td align=\"center\">Female/65</td><td align=\"center\">&gt;23</td><td align=\"left\">TSWC</td><td align=\"center\">HSC</td><td align=\"left\">Not done</td><td align=\"right\">1,343</td><td align=\"center\">72</td><td align=\"center\">193</td><td align=\"center\">M</td><td align=\"left\">0.30</td></tr><tr><td align=\"center\">Female/70</td><td align=\"center\">15</td><td align=\"left\">TSWC</td><td align=\"center\">HSC</td><td align=\"left\">Not done</td><td align=\"right\">2,348</td><td align=\"center\">93*</td><td align=\"center\">25</td><td align=\"center\">1051</td><td align=\"left\">0.35</td></tr><tr><td align=\"center\">Female/61</td><td align=\"center\">Rapid</td><td align=\"left\">TSWC</td><td align=\"center\">HSC</td><td align=\"left\">No result</td><td align=\"right\">11,038</td><td align=\"center\">48</td><td align=\"center\">230</td><td align=\"center\">755</td><td align=\"left\">1.75</td></tr><tr><td align=\"center\">Male/54</td><td align=\"center\">5</td><td align=\"left\">TSWC</td><td align=\"center\">HSC</td><td align=\"left\">No result</td><td align=\"right\">71,900</td><td align=\"center\">172*</td><td align=\"center\">97</td><td align=\"center\">1042</td><td align=\"left\">Positive⁷9.70</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Characteristics, results and diagnosis for patients with Alzheimer's disease, vascular dementia, mixed dementia and other neurological diseases</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Sex/age<break/> (years)</td><td align=\"center\">tTau¹<break/>(ng/L)</td><td align=\"right\">P-tau²<break/>(ng/L)</td><td align=\"right\">tTau/<break/>P-Tau</td><td align=\"right\">Aβ₄₂³<break/>(ng/L)</td><td align=\"center\">14-3-3 protein¹<break/>(arbitrary units)</td><td align=\"left\">Diagnosis</td></tr></thead><tbody><tr><td align=\"left\">Female/59</td><td align=\"center\">2120</td><td align=\"right\">244*</td><td align=\"right\">8</td><td align=\"right\">401*</td><td align=\"center\">M</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Female/64</td><td align=\"center\">2335</td><td align=\"right\">240*</td><td align=\"right\">10</td><td align=\"right\">347*</td><td align=\"center\">0.00</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Female/76</td><td align=\"center\">1400</td><td align=\"right\">220*</td><td align=\"right\">6</td><td align=\"right\">505*</td><td align=\"center\">0.00</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Female/79</td><td align=\"center\">2443</td><td align=\"right\">287*</td><td align=\"right\">9</td><td align=\"right\">517*</td><td align=\"center\">0.00</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Female/85</td><td align=\"center\">1350</td><td align=\"right\">49</td><td align=\"right\">27</td><td align=\"right\">746</td><td align=\"center\">0.59</td><td align=\"left\">MCI</td></tr><tr><td align=\"left\">Female/73</td><td align=\"center\">1380</td><td align=\"right\">217*</td><td align=\"right\">6</td><td align=\"right\">743</td><td align=\"center\">0.15</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Female/67</td><td align=\"center\">1410</td><td align=\"right\">199*</td><td align=\"right\">7</td><td align=\"right\">413*</td><td align=\"center\">0.00</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Female/62</td><td align=\"center\">1670</td><td align=\"right\">66</td><td align=\"right\">25</td><td align=\"right\">348*</td><td align=\"center\">0.41</td><td align=\"left\">AD/Wernicke</td></tr><tr><td align=\"left\">Female/51</td><td align=\"center\">1410</td><td align=\"right\">27</td><td align=\"right\">52</td><td align=\"right\">697</td><td align=\"center\">0.00</td><td align=\"left\">Possible FTD</td></tr><tr><td align=\"left\">Female/66</td><td align=\"center\">1410</td><td align=\"right\">238*</td><td align=\"right\">6</td><td align=\"right\">622</td><td align=\"center\">0.00</td><td align=\"left\">MCI/Possible AD</td></tr><tr><td align=\"left\">Female/64</td><td align=\"center\">1750</td><td align=\"right\">49</td><td align=\"right\">36</td><td align=\"right\">926</td><td align=\"center\">0.55</td><td align=\"left\">Possible VaD</td></tr><tr><td align=\"left\">Male/65</td><td align=\"center\">1910</td><td align=\"right\">79</td><td align=\"right\">24</td><td align=\"right\">1297</td><td align=\"center\">0.34</td><td align=\"left\">Unspecified dementia</td></tr><tr><td align=\"left\">Male/65</td><td align=\"center\">1280</td><td align=\"right\">57</td><td align=\"right\">22</td><td align=\"right\">1244</td><td align=\"center\">0.53</td><td align=\"left\">Possible AD/VaD</td></tr><tr><td align=\"left\">Female/62</td><td align=\"center\">3443</td><td align=\"right\">242*</td><td align=\"right\">14</td><td align=\"right\">312*</td><td align=\"center\">0.52</td><td align=\"left\">FTD/Possibly AD</td></tr><tr><td align=\"left\">Female/67</td><td align=\"center\">1540</td><td align=\"right\">238*</td><td align=\"right\">6</td><td align=\"right\">369*</td><td align=\"center\">0.00</td><td align=\"left\">Probably AD</td></tr><tr><td align=\"left\">Female/88</td><td align=\"center\">2720</td><td align=\"right\">90*</td><td align=\"right\">30</td><td align=\"right\">464*</td><td align=\"center\">0.00</td><td align=\"left\">AD/VaD</td></tr><tr><td align=\"left\">Male49</td><td align=\"center\">1350</td><td align=\"right\">81*</td><td align=\"right\">17</td><td align=\"right\">973</td><td align=\"center\">0.00</td><td align=\"left\">VaD</td></tr><tr><td align=\"left\">Female/72</td><td align=\"center\">1780</td><td align=\"right\">240*</td><td align=\"right\">7</td><td align=\"right\">533*</td><td align=\"center\">0.00</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Female/80</td><td align=\"center\">1280</td><td align=\"right\">239*</td><td align=\"right\">5</td><td align=\"right\">699</td><td align=\"center\">0.00</td><td align=\"left\">Probably AD</td></tr><tr><td align=\"left\">Female/76</td><td align=\"center\">1480</td><td align=\"right\">227*</td><td align=\"right\">7</td><td align=\"right\">516*</td><td align=\"center\">0.00</td><td align=\"left\">AD/VaD</td></tr><tr><td align=\"left\">Female/76</td><td align=\"center\">1360</td><td align=\"right\">207*</td><td align=\"right\">7</td><td align=\"right\">573</td><td align=\"center\">0.00</td><td align=\"left\">AD</td></tr><tr><td align=\"left\">Male/62</td><td align=\"center\">8,530*</td><td align=\"right\">75</td><td align=\"right\">114</td><td align=\"right\">1043</td><td align=\"center\">0.00</td><td align=\"left\">Cerebral lymphoma</td></tr><tr><td align=\"left\">Male/71</td><td align=\"center\">1,202</td><td align=\"right\">59</td><td align=\"right\">20</td><td align=\"right\">487*</td><td align=\"center\">0.00</td><td align=\"left\">Cerebral infarction</td></tr><tr><td align=\"left\">Female/69</td><td align=\"center\">1,992</td><td align=\"right\">83*</td><td align=\"right\">24</td><td align=\"right\">674</td><td align=\"center\">0.29</td><td align=\"left\">Cerebral infarction</td></tr><tr><td align=\"left\">Female/49</td><td align=\"center\">1,267</td><td align=\"right\">27</td><td align=\"right\">47</td><td align=\"right\">807</td><td align=\"center\">0.95*</td><td align=\"left\">Cerebral lymphoma</td></tr><tr><td align=\"left\">Female/32</td><td align=\"center\">1,433</td><td align=\"right\">49</td><td align=\"right\">29</td><td align=\"right\">960</td><td align=\"center\">0.00</td><td align=\"left\">Cerebellitis</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>A comparison between markers of the diagnostic performance for the diagnosis of CJD.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\"><bold>tTau</bold></td><td align=\"center\"><bold>tTau/P-Tau</bold></td><td align=\"center\"><bold>14-3-3 protein</bold></td></tr></thead><tbody><tr><td align=\"center\">Sensitivity</td><td align=\"center\">75</td><td align=\"center\">75</td><td align=\"center\">80</td></tr><tr><td align=\"center\">Specificity</td><td align=\"center\">92</td><td align=\"center\">96</td><td align=\"center\">96</td></tr><tr><td align=\"center\">Positive predictive value</td><td align=\"center\">82</td><td align=\"center\">90</td><td align=\"center\">89</td></tr><tr><td align=\"center\">Negative predictive value</td><td align=\"center\">89</td><td align=\"center\">89</td><td align=\"center\">92</td></tr><tr><td align=\"center\">Diagnostic efficiency</td><td align=\"center\">87</td><td align=\"center\">89</td><td align=\"center\">91</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>¹The time from disease recognition to death, ²Three-phase sharp wave complexes, ³Positive for immunochemical examination of pathological prion protein, PrP^Sc, ⁴Values above the normal limit used in clinical practice marked with *, ⁵Values below the normal limit used in clinical practice marked with*, ⁶Generalized dysrythmia, ⁷14-3-3 protein found positive in different laboratories outside Norway because some hospitals sent CSF samples for 14-3-3 protein analysis to laboratories outside Norway in parallel to sending samples to us. (Three patients both have semi quantitative results from us and a qualitative, positive result, from a foreign laboratory). ⁸NC, non-specific changes. ⁹HSC, highly suggestive changes of CJD, M = missing value.</p></table-wrap-foot>", "<table-wrap-foot><p>¹Values above lower limit for CJD marked with*, ²Values above maximum for normal in clinical practice marked with*, ³Values below minimum for normal used in clinical practice marked with*, M = missing value.</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1743-8454-5-14-1\"/>", "<graphic xlink:href=\"1743-8454-5-14-2\"/>", "<graphic xlink:href=\"1743-8454-5-14-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Viral hepatitis C is a global public health problem, and has been considered one of the major challenges in the third millennium [##REF##10422976##1##]. Injecting drug use is the main route of transmission, mediated by the sharing of injection equipment, especially needles and syringes but also spoons, cotton filters and other paraphernalia [##REF##9376782##2##]. In the UK, studies of prevalence rates of anti-HCV amongst Injecting Drug Users (IDUs) reported rates of 80%–86% in England (5 studies) and 37%–90% in Scotland (3 studies) [##REF##12010503##3##]. The association between injecting drug use and infection with HCV is mediated by sharing needles and syringes or other injecting paraphernalia. European studies showed rates of sharing needles and syringes and other injecting paraphernalia between 70% and 94% [##REF##10846954##4##].</p>", "<p>The recently introduced Hepatitis C Strategy for England [##UREF##0##5##] laid strong emphasis on preventing new cases of hepatitis C infection in IDUs by health promotion activities and the provision of needle exchange schemes. This is best achieved in the context of treatment for drug dependence complemented with information about hepatitis C and harm minimisation messages. However, this new policy falls short of recommending specific preventive interventions which are evidence based; hence the importance of this project which aims to evaluate a new preventive intervention for hepatitis C in IDU's.</p>", "<p>The aim of the present study was to evaluate the effectiveness of enhanced prevention counselling (EPC) in reducing HCV infection in HCV sero-negative patients. Our primary hypothesis was that EPC is more effective and cost-effective than simple educational counselling (SEC) in reducing the rate of HCV sero-conversion and its risk behaviour. Whilst we have also evaluated sexual risk behaviour in relation to the occurrence of HCV, we have not studied the prevalence and seroconversion rates of hepatitis B, HIV and other sexually transmitted diseases.</p>", "<p>However we were not able to prove the efficacy of EPC in comparison with SEC in the prevention of hepatitis C in IDUs. This was related to low recruitment and retention rates of the participants. Moreover there was a low adherence rate to EPC.</p>", "<p>In view of these reported negative findings, we have also provided an overview of the main problems that we faced and our attempts to overcome them, in the hope that it will guide future researchers in the field of prevention interventions in addiction [##UREF##1##6##].</p>" ]

[ "<title>Methods</title>", "<p>The study was conducted in 2 phases, a screening phase and an intervention phase.</p>", "<title>Screening phase</title>", "<p>Injecting drug users presenting to collaborating drug treatment services in South West London, North London and in Surrey were screened for eligibility in four steps: (1) the identification of IDUs, (2) distinguishing between those IDUs that have injected at least once in the last six months and those that have not, (3) assessment of IDUs for inclusion and exclusion criteria and (4) eligibility by testing for current HCV sero-positivity using a standard ELISA HCV antibody test [##REF##9305663##7##]. All IDUs who were confirmed to be HCV sero-negative by HCV antibody test were invited to attend a research interview conducted by the research workers. This occurred in the context of their ongoing care. Successive IDUs referred to community drug services were recruited to the trial as well those who were in treatment. The overall retention in treatment rate during the trial was 60% with no difference in retention rates between those receiving EPC and SEC.</p>", "<title>Inclusion Criteria were</title>", "<p>(1) male and female IDUs (2) age 18–70 years (3) ICD-10 diagnosis of mental and behavioural disorder due to the use of drugs (F11-F19) established by baseline research interview (4) willingness to nominate an independent informant to provide collateral information, and nominate a locator who can assist in tracing the subject at follow-up (5) stable place of residence (defined as having a domicile for which there is no imminent danger of eviction) and (6) living close enough to commute to the clinic.</p>", "<title>Exclusion Criteria were</title>", "<p>(1) current severe mental illness (e.g. bipolar effective disorder or schizophrenia), (2) severe physical illness that would preclude participation, (3) serious legal problems, including impending imprisonment, likely to interfere with treatment participation and/or follow-up and (4) severe brain damage or mental impairment. The inclusion and exclusion criteria were established by a combination of clinical assessment by service staff, and baseline research interview conducted by the research workers.</p>", "<p>Ethical approval for the research was sought and obtained from both the Multi-Centre Research Ethics Committee (MREC) and relevant Local Research Ethics Committees (LREC) where recruitment took place. The recruitment process, issues of information provision, consent, confidentiality, data protection, management of the research, and all other aspects of the Trial were modelled on the recommendations for good research and clinical practice provided by the MREC and LREC guidelines.</p>", "<title>Baseline assessment</title>", "<p>All drug users were assessed using the European Addiction Severity Index [##UREF##2##8##], Injecting Risk Questionnaire (IRQ) [##REF##10328042##9##], the HIV Risk Taking Behaviour Scale [##REF##2031690##10##] and Alcohol Use Disorders Identification Test (AUDIT) Questionnaire [##REF##8329970##11##]. Self-efficacy, outcome expectancies (situational confidence) were measured using an adapted version of the Situational Confidence Questionnaire [##UREF##3##12##]. Finally, stages of change in the \"readiness to change\" model [##UREF##4##13##] were assessed using the Readiness to Change questionnaire, and general knowledge on hepatitis C measured using a custom-designed questionnaire.</p>", "<title>Intervention phase</title>", "<p>After the completion of a baseline assessment, all clients were randomised to receive either the Enhanced Prevention Counselling (EPC) or Simple Educational Counselling (SEC) intervention. The therapists who administered the interventions were regular staff of community drug services that took part in the trial. All therapists received an intensive training programme in the administration of manualized EPC and SEC from PD who is an accredited clinical psychologist with national expertise in training clinicians in psychological interventions. The therapists received regular supervision from PD and other trained supervisors who completed the intensive training in EPC intervention and the techniques of supervision from PD. For quality control, all sessions were audio-taped to measure the fidelity of the EPC and SEC interventions.</p>", "<title>Enhanced prevention counselling</title>", "<p>This comprised four sessions of manual-guided intervention. The manual was based on a number of other treatment intervention manuals, and particularly on the Brief Intervention (Motivational Enhancement Therapy) used in Project Match (Project MATCH Research Group, 1998), and the manual established and evaluated for project RESPECT which was concerned with the reduction of high-risk sexual behaviour and the introduction of safer sex [##REF##8862164##14##]. In addition, exercises and elements were taken from substance misuse cognitive behavioural treatments [##UREF##5##15##], and elaborated in therapy manuals [##UREF##6##16##,##UREF##7##17##].</p>", "<p>The four sessions were intended to be completed within eight weeks of entry into the programme and were carried out by a drug clinic worker who was trained in delivering the intervention but who was not involved in collecting outcome data from participants. The aim of the intervention was to reduce risk behaviours associated with the acquisition of HCV infection in injecting drug users. HCV transmission risk behaviours include injecting drugs, the sharing of injecting equipment, not cleaning and reusing drug paraphernalia, alcohol misuse, cocaine use, unprotected sexual activity, multiple sexual partners, and non-compliance with methadone treatment.</p>", "<p>EPC as applied in this project utilises principles of motivational psychology, theories of behaviour change (particularly social cognitive learning theory), and health belief models, including the theory of reasoned action [##UREF##8##18##,##UREF##9##19##]. Changes in risk behaviour are hypothesised to take place through changes in outcome expectancies (expected consequences of a course of action, e.g. sharing injecting equipment) and self efficacy (confidence in one's ability to achieve a particular goal, e.g. avoidance of sharing injecting equipment). Motivational interventions have been applied to a variety of health behaviours in addition to addictive behaviour (reviewed in Miller and Rollnick [##UREF##10##20##]; [##REF##8616739##21##], and can be readily applied to health promotion in drug misusers. The aim of the intervention is to enhance the subject's self-perception of risk and facilitate the development of individual strategies to avoid engaging in HCV risk activities. The measurement of self-efficacy will allow assessment of the process of the intervention.</p>", "<p>All sessions last between 40–60 minutes and follow the format of the brief interventions described above. Session one has the aim of establishing rapport and a counselling relationship consistent with the principles of Motivational Interviewing, increasing knowledge and awareness of HCV risk behaviours and the consequences of HCV infection, introducing the rationale and structure of the intervention to the subject, and assessing the individual's risk behaviours, self efficacy and outcome expectancies. The remaining three sessions begin with a review of progress on targets set at the previous session, assessment of any difficulties in applying coping strategies, and assessment of the subject's motivational state. In the second session, if appropriate, targets for intervention are planned (e.g. unprotected sexual activity, injecting equipment sharing). Each session ends with a behavioural goal-setting exercise in which the participant arrives at a small behavioural risk-reduction step that could be achieved by the next session. At the end of the final session, a longer-term, individualised risk reduction plan is agreed upon. Various strategies are employed in EPC to foster compliance with the intervention, including the development of a therapeutic alliance, individual risk reduction plans to take home as a reminder of behavioural goals, appointment cards, combining sessions with regular clinic visits (e.g. for methadone prescriptions), and phoning the subject on the day of visits as a reminder.</p>", "<title>Simple Educational Counselling (SEC)</title>", "<p>This consisted of a ten-minute session of information-giving intervention about the nature and the risk factors of HCV, with advice on prevention including the need to reduce sharing of injecting equipment and safer injecting practices. It was intended to be a non-interactive intervention in order to contrast with the EPC, and clients were asked to direct any questions they might have to their key worker rather than the counsellor.</p>", "<title>Outcome Measurement</title>", "<p>Research follow up interviews were conducted at six months post randomisation, and blood tests for hepatitis C at both six months and twelve months. The primary outcome measure was the number of new cases of HCV infection by sero-conversion detected by HCV positive antibody at 6 and 12 months from recruitment. Secondary outcome measures were those administered at baseline.</p>", "<title>Sample size</title>", "<p>Power calculations were based upon rates of sero-conversion of 6% per hundred person years found in a research study that applied intensive preventative counselling to IDUs [##UREF##11##22##]. This was compared with the rate of sero-conversion obtained from research into the IDU population of 20% per hundred person years [##REF##9703523##23##]. Based on these figures, the difference was estimated to be around 14%, and so with a power of 0.8 and a difference proven at the 5% level using a two-tailed test, a followed-up sample of 180 IDUs was required.</p>", "<title>Randomisation</title>", "<p>Randomisation was stratified by two variables in order to provide a control for what were perceived to be potentially important influences. The stratification variables were the \"Treatment Centre\" that the client was recruited from, to control for differences in standard service at each of the recruitment sites, and \"Injecting Equipment Sharing Behaviour\", to control for a very important risk-factor predictor for contracting hepatitis C. Stratification was achieved in blocks of six within each variable, such that in each block of six half of the clients would receive SEC and half of the clients EPC. This method of stratified randomisation was chosen over true randomisation in order to ensure that allocation to either group was fairly constant throughout the life of the trial, helping to maximise therapist time by spreading the workload more evenly. It was also intended to act as a safeguard against bias to one intervention or the other if lower than expected levels of recruitment were achieved.</p>", "<p>The physical implementation of the randomisation scheme was accomplished by a custom-designed statistical computer program which generated stratified random integers between 1 and 2 in blocks of six; these were then transcribed onto cards and sealed in envelopes by a person unconnected with the Research Team, so that they never had any access to the randomisation scheme used. Envelopes were marked sequentially on the outside, and were opened by the Research Team upon completion of a baseline interview. Other features which contributed to the protection of the validity of the Trial and maximising the quality of the data included the written protocol that was followed, the manual-guided treatment interventions, and quality control of the interventions through the rating and assessing of a random sample of tapes from the sessions.</p>", "<title>Blinding</title>", "<p>Although it would have been preferable for the purposes of completely eliminating the potential for bias to make the Research Team blind to the therapy intervention allocated, due to the Research Team's need to co-ordinate and help facilitate the implementation of the intervention this was not possible. Once the therapy allocation was made, the Research Team had to locate a suitable therapist to conduct the intervention and liaise with them over the progress that they were making with the clients allocated to them, and this applied to both EPC and SEC therapists. Arrangements for the payment of travel expenses for clients attending the EPC sessions also had to be made (sometimes for both client and therapist), and the researchers also played a large role in chasing up clients who did not attend their sessions. Thus, although it may have been possible to implement a blinding procedure, it was felt that the benefits of doing so were outweighed by the increases in efficiency gained otherwise. It was also felt that not blinding would have minimal impact on the research outcomes as the primary outcome measure was rate of sero-conversion, which is not open to bias, and in addition majority of the interview measures were direct and quantitative rather than subjective and qualitative.</p>", "<title>Statistical Analyses</title>", "<p>Data analysis was conducted on intention to treat basis. The primary hypothesis was tested using chi-square for comparison of rates of sero-conversion and using analysis of covariance with risk-behaviour composite scores as dependent variables, controlling for baseline values on these measures, and treatment condition as the independent variable. Similar analysis was carried out on secondary outcome measures with calculations of differences between intervention groups and differences between the group who were and the group who were not followed up. Incidence of HCV was calculated using the person years method [##UREF##12##24##] among IDUs who were sero-negative for HCV antibody and who had repeated testing after 6 and 12 months from baseline testing. Analyses of covariance were performed for all relevant secondary outcome measures, using baseline scores as the first covariate to control for initial individual differences, and baseline score on the injecting subscale of the HIV Risk-Taking Behaviour Scale as the second covariate where appropriate, as this subscale was identified as being significantly different between the two interventions. Chi-square analyses were performed for all categorical data and Mann-Whitney U-Tests for ordinal data that were not normally distributed to apply parametric tests.</p>", "<title>Dried Blood Spot (DBS) testing for hepatitis C</title>", "<p>When recruitment began, it was found that far less testing of hepatitis C was happening at community drug teams than was reported, and this proved to be a major obstacle for the trial. It was suggested this could be overcome with the implementation of the Dried Blood Spot (DBS) test [##REF##12858408##25##] for hepatitis C, and after seeking approval from the Department of Health and the Trial Steering Committee the procedure was adopted at all recruitment centres. A study into the validity of the DBS test revealed it to have 100% sensitivity and 100% specificity [##REF##10325381##26##], and our own piloting work confirmed this. The introduction of the DBS test increased testing more than fourfold, greatly assisting the recruitment process.</p>" ]

[ "<title>Results</title>", "<title>Baseline analysis</title>", "<title>Participants</title>", "<p>A flow diagram detailing the number of IDUs at each stage of the recruitment process is presented in figure ##FIG##0##1##. As shown 95 subjects were recruited and 78 were followed up at 6 months and 62 were followed up at 12 months.</p>", "<title>Demographic characteristics</title>", "<p>The mean age of all those recruited was 32 years (SD 6.7). There were 70 males, 25 females, 10% were married, 42% had at least one child, 43% were unemployed and 48% had educational qualifications. There were no significant differences on these basic demographic characteristics between both those followed-up and those not followed-up and between those allocated to EPC and those allocated to SEC.</p>", "<title>Drug use and other characteristics</title>", "<p>Participants showed the following drug use characteristics (means and SDs): duration of drug use (11.4, 7.6 years), age of first drug injection (24.5, 6.3 years), duration of injecting drug use (5.9, 4.8 years), previous episodes of treatment (3.1, 2.9) and inpatient treatment episodes (1.8, 3.1). Every recruited client was currently receiving a prescription for a substitute drug with methadone being the most commonly reported drug at 85%, with the remainder prescribed buprenorphine. One of the main criteria for recruitment was having injected at least once in the past six months, but 49% of respondents reported having injected in the last thirty days. The vast majority were regular smokers of cigarettes (89%), and 70% drank alcohol at least once per week, 35% reported that a member of their immediate family had a history of alcohol problems, 31% reported a family history of drug problems, and 28% reported a family history of psychiatric problems, 41% reported a history of emotional abuse by significant others, 23% reported a history of physical abuse, and 11% reported incidences of sexual abuse.</p>", "<p>There were no significant differences between those followed-up and those not followed-up, or between those allocated to either intervention, on any of these measures.</p>", "<title>Standardised measures</title>", "<p>On the ASI just under 56% of clients had shared an item of injecting equipment in the last six months, with the mean number of people that they had shared with being 1.84 for the subgroup of those who admitted to any sharing at all, or 1.08 overall. Scores on the HIV Risk-Taking Behaviour Scale were expectedly high, particularly for injecting risk (mean 8.8, SD 5.7) as opposed to sexual risk (mean 4, SD 4.1), 37% were identified as having a probable drink problem by the AUDIT and 54% had used a needle exchange at least once in the last six months.</p>", "<p>The only significant differences on all measures between those followed-up and those not followed-up were on the legal and economic subscales of the ASI, with those not followed up exhibiting relatively higher degrees of problem on both measures than those followed-up. There was one significant difference between the intervention groups, with those allocated to EPC scoring more highly on the injecting subscale of the HIV Risk-Taking Behaviour Scale, although not scoring significantly differently overall. As this subscale is quite important, indicating a higher level of sharing behaviour that could have an impact on the outcome variables and the trial intervention, this measure was used as a covariate where appropriate in the outcome analysis.</p>", "<title>Psychological Measures</title>", "<p>Confidence at resisting the urge to inject, measured by the Drug Injecting Confidence Questionnaire, was on average 59%, but large variations were noted across subjects. Unpleasant emotions, urges and temptations, and social pressure to use were areas where respondents were least likely to resist the urge to inject, whilst circumstances of pleasant emotions was the area where patients were most confident that they would not inject. Knowledge of hepatitis C, as measured by our item true-or-false questionnaire was better than expected, with average scores of over 16 out of twenty. \"Stage of change\", measured by the Readiness to Change Questionnaire, revealed that the majority of subjects were at the \"Action\" stage of change, probably reflecting the locations from which they were recruited. There were no significant differences on these measures between those followed-up and those not followed-up, or between those allocated to either of the trial interventions.</p>", "<title>Outcome analysis</title>", "<p>As shown in Figure ##FIG##0##1##, out of 95 participants recruited, 78 (82%) were followed up at 6 months and 62 (65%) were followed up at 12 months. These follow up rates do not correspond to retention rates in treatment as some of the participants had dropped out from treatment but agreed to attend for the follow-up research interview and HCV testing. Moreover for SEC, 41 (79%) attended at 6 months and 29 (56%) at 12 months whilst for EPC, 37 (86%) attended at 6 months and 33 (77%) at 12 months.</p>", "<p>Table ##TAB##0##1## illustrates the number of participants who engaged for their allocated intervention, defined as either completing the SEC intervention or attending for at least one session of EPC of those followed-up. Table ##TAB##1##2## illustrates the number of completed sessions of EPC.</p>", "<p>There was a significant difference between the two groups in terms of their engagement with the therapy intervention (p &lt; 0.000). 78 participants were followed-up at six months (82.1%), and 62 at 12 months (65.3%). Overall, six-month follow-up data indicated a seroconversion rate of 9.0% in six months, or 18.0% per year. Twelve-month follow-up data indicated a seroconversion rate of 12.9% per year.</p>", "<p>The difference in seroconversion was not significant between the two interventions at either six months or twelve months, but it was however in the anticipated direction, with fewer of those allocated to EPC seroconverting compared to those that received SEC. The difference was even more pronounced (but still not significant) when only those who received at least one session of the intervention were included as no patients who received at least one session of EPC seroconverted at either six months or twelve months.</p>", "<p>There were no significant differences between the EPC and SEC groups on any of the secondary outcome measures (effect of treatment). However there were significant changes in a number of measures for both groups at 6 months follow-up (effects of time). Table ##TAB##2##3## shows significant changes for ASI alcohol use, medical subscale, economic subscale, satisfaction subscale and HIV-RTBS injecting risk, sexual risk behaviour and overall scores.</p>", "<p>Table ##TAB##3##4## shows non significant reduction in injecting behaviour, sharing, use of needle exchange and AUDIT in the last 6 months.</p>", "<p>Table ##TAB##4##5## shows significant changes in all DICQ scales indicating moderate increases in situational confidence in the ability to resist the urge to inject and increases in Hepatitis C knowledge questionnaire.</p>", "<p>Scores on the HIV Risk-Taking Behaviour Scale similarly reduced for both groups, with the EPC group in particular exhibiting a large reduction in injecting behaviour which put them at a similar level to the SEC group, eradicating the difference that was present at baseline. Baseline scores on the HIV Risk-Taking Behaviour Scale did not account for differences on any of the outcome measures.</p>" ]

[ "<title>Discussion</title>", "<p>We were not able to demonstrate the efficacy of EPC compared to SEC in the prevention of hepatitis C amongst injecting drug users. The main reasons for this were the lower than expected levels of recruitment, coupled with the lower than expected compliance with the experimental intervention. The EPC and SEC groups were well matched in their demographic characteristics, drug use and psychological characteristics, including measures of risk behaviour. Levels of injecting equipment sharing behaviour were similar to that reported in other studies, with around 60% of all users who had injected in the past six months reporting sharing behaviour over the same period [##UREF##0##5##]. Of note is the difference in follow-up rates of SEC (56%) and EPC (77%) groups at 12 months. It is not certain whether this difference in follow up rate had any effect on the trial's internal validity and we have no explanation for this finding. Moreover these follow up rates do not correspond to retention rates in treatment as some of the participants had dropped out from treatment but agreed to attend for the follow-up research interview and HCV testing.</p>", "<p>The difference in seroconversion was not significant between the two interventions at either six months or twelve months, but it was however in the anticipated direction, with fewer of those allocated to EPC seroconverting compared to those who received SEC. The difference was even more pronounced (but still not significant) when only those who received at least one session of the intervention were included as no patients who received at least one session of EPC seroconverted at either six months or twelve months. However, given the relatively low numbers of participants recruited and followed-up, and the differential rate of engagement in EPC and SEC and the even lower number of those who completed all 4 sessions of EPC therapy, no conclusions could be drawn and the efficacy of EPC in reducing new cases of HCV remains inconclusive.</p>", "<p>Notably, there were many significant changes on some of the secondary outcome measures from baseline values, indicating positive change and improvement for both groups. These reductions in drug use and risk behaviour may reflect the impact of their treatment in general rather than any specific effects of the interventions. The finding that only half of the initial sample had injected in the last month at baseline, would suggest a broad treatment effect i.e. injecting behaviour had already stopped in half of the participants at intake. A recently reported RCT of a brief behavioural intervention in comparison with standardised educational intervention for reducing HCV risk practices among IDUs showed a reduction in these practices for both interventions at one month follow-up [##REF##15317636##27##]. The study failed to demonstrate effectiveness of the brief intervention for a number of reasons: similarity between the interventions in duration and content, the short follow-up of one month and the inclusion of HCV positive IDUs.</p>", "<p>It was also worthy of note that 60% of this high-risk group had never been tested for HCV prior to this research, despite the cohort being engaged at local community drug teams. In addition, 10% of those who had been tested in the past, and who believed themselves to be HCV sero-negative, were found to be HCV sero-positive, emphasising the need for regular testing of IDUs.</p>", "<p>Possible reasons for the low overall incidence of 12.9 per 100 person years are the impact of treatment on risk behaviour [##UREF##13##28##], HCV screening and the provision of harm reduction approaches in the community. However there has been the notion that the impact of needle exchange programmes on the spread of HIV in IDUs has been limited in studies carried out in the US [##REF##9722812##29##] and in Canada [##REF##9110076##30##] with the conclusion that whilst needle exchange programmes are crucial for sterile syringe provision, they should be considered one component of a comprehensive programme including counselling, support, and education. Wright and Tompkins [##REF##16956393##31##] in a systematic review of the evidence for the effectiveness of primary prevention interventions for Hepatitis C among injecting drug users reported that needle exchange programmes reduced the prevalence of Hepatitis C though prevalence remains high. However, methadone maintenance treatment was found to be only marginally effective at reducing HCV incidence and limited evidence evaluating the effectiveness of behavioural interventions in reducing its incidence. The review concluded that a robust response to the global health problem of HCV would require the provision of new behavioural interventions in addition to needle exchange and methadone maintenance programmes. Indeed one study [##REF##11849904##32##] showed that in IDUs attending needle exchange schemes, brief intervention was effective in reducing alcohol use and probably attendant risk factors resulting in lower incidence of HCV in IDUs. Whilst the design of the present study has not enabled the examination of the contribution of treatment, needle exchange and HCV screening on the incidence of HCV, it is conceivable that both the SEC and the EPC interventions, in addition to the provision of treatment, together with the availability of needle exchange schemes for the present cohort of IDUs, has contributed to the low incidence rate of HCV in this population. Moreover, it is also conceivable that the testing regime instigated by the Research Team itself encouraged a change in risk behaviour: pre-test counselling in conjunction with the issues raised in the baseline interview, and a degree of self-selection. The impact of pre-test counselling alone is thus a potentially important mechanism of change in risk behaviour worthy of further investigation, and would indeed be a very encouraging development if it could be proven to be effective. In conjunction with the DBS, which has been shown to increase testing rates more than four-fold, the possibility of much greater testing taking place at primary care and other community settings, may help to monitor infection rates and help those who are not infected to remain so.</p>", "<title>Enhanced Prevention Counselling</title>", "<p>The EPC intervention described here is one of the first such interventions developed specifically for the prevention of HCV with injection drug users. A process evaluation suggested that EPC facilitated a positive therapeutic alliance compared with the SEC control intervention and was perceived as beneficial by the IDUs in helping reduce HCV-risks [##UREF##14##33##]. The intervention was deliberately designed to be an enhanced counselling intervention as opposed to a Brief Intervention (BI) of one session only. A major difficulty, however, with the intervention was in attendance for treatment sessions; the majority of participants who engaged only attended for one EPC session. Thus in retrospect it appears that enhanced counselling is unlikely to be more effective than a single session BI as participants attend one session (at least in this study) regardless of what is on offer. Participants attended as normal for standard key working and therefore one implication may be that only one session is offered and any further work from this therapy programme might be better placed within standard key working.</p>", "<p>A comparison with a group given no information or advice whatsoever is obviously not ethically possible and so the question as to whether any intervention, however brief, has any benefit (let alone knowing what the essential components of any intervention are) cannot be answered from the current study. From our clinical and field research experience, however, it seems likely that there are elements common to both interventions in this research that might be effective in helping prevent HCV infection. As with the Tucker [##REF##15317636##27##] study, it is possible that the essential components of prevention in this clinical population is the time spent with the health professional and researcher, completion of the standardised questionnaires and particularly discussion of risk behaviours and the heightened awareness of risk this produced. The EPC may be made more fit for purpose by reducing the number of sessions from 4 to one or 2 sessions the first of which could be grafted on the post-HCV counselling sessions: this would ensure its higher uptake by IDUs and enable its evaluation. Moreover preventive behavioural interventions should be informed by reference to IDUs experience and views using qualitative methods such as focus groups.</p>", "<title>Methodological issues</title>", "<p>The main problems encountered in the design, conduct and delivery of this research were the lower than expected levels of recruitment to the trial, and the low adherence to EPC but not the SEC. Retention of participants once recruited however was a lesser problem, as 65% of those recruited were followed-up, and adherence to the informational one brief session intervention was not a problem, as more than 90% of those randomised to the SEC informational intervention received it. The reasons for these two main problems can be grouped under issues relating to the participants' behaviour; issues relating to the service environment from which participants were recruited and issues relating to the trial design [##UREF##1##6##].</p>", "<title>Lessons learnt</title>", "<p>One of the main lessons learned in this project is that conducting research in UK treatment settings presents challenges that are very different from those encountered in US studies upon which research in addiction is often modelled, as was the case in this project. RCTs in the US are usually conducted against a background of higher funding which facilitates pilot work, the formation of larger research teams, and therapists who are dedicated to the trial rather than relying on service staff trained in delivering the experimental and control interventions. Research in the US also benefits from a well-established clinical research infrastructure, which aids the introduction of new interventions, increasing compliance from staff and users. Indeed, the development and fostering of a culture of research within the services involved in the present trial was a task that had to be instigated. There is also reason to believe that the clinical populations in the US are different to those in the UK, with those engaged in treatment being older, and more socially stable; this is of consequence because it is important that service users are well-engaged in standard drug treatment regimes before introducing further demands such as structured counselling sessions. Another of the lessons learnt is the need for piloting of the new intervention in an area of research that involves the development of new interventions amongst a difficult clinical population, with only limited guidance available from other research.</p>", "<p>One of the main policy implications for conducting trials of psychological interventions within addiction health care settings is for funding bodies to provide the necessary resources to improve the quality and comprehensiveness of treatment including the provision psychological interventions. This would provide the necessary infrastructure and capacity for the development of innovative interventions and thus offering opportunities for the evaluation of their effectiveness and cost-effectiveness in pragmatic clinical trials.</p>" ]

[ "<title>Conclusion</title>", "<p>We were not able to prove the efficacy of EPC in comparison with SEC in the prevention of hepatitis C in IDUs. Notwithstanding the negative findings of this research, we believe that the study has provided the benefits of developing and introducing specific behavioural primary preventive interventions (vide supra): the EPC and SEC interventions (manuals available from the authors) [##UREF##14##33##] and the DBS which hold promise and warrant further investigation. Moreover the main lessons learnt were that piloting of a new intervention is a crucial first step before conducting pragmatic RCTs of psychological interventions in the field of addiction; that an infrastructure and culture for psychosocial interventions is needed to enable applied research in the service environment, and research funding is needed for enabling the recruitment of dedicated trained therapists for the delivery of these interventions.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Aim</title>", "<p>To develop and evaluate the comparative effectiveness of behavioural interventions of enhanced prevention counselling (EPC) and simple educational counselling (SEC) in reducing hepatitis C viral (HCV) infection in sero-negative injecting drug users (IDU).</p>", "<title>Design</title>", "<p>Randomised controlled trial (RCT) of EPC intervention in comparison with simple educational counselling (SEC).</p>", "<title>Setting Specialised</title>", "<p>Drug services in London and Surrey, United Kingdom.</p>", "<title>Participants and Measurements</title>", "<p>Ninety five IDUs were recruited and randomised to receive EPC (n = 43) or SEC (n = 52). Subjects were assessed at baseline using the Addiction Severity Index (ASI), the Injecting Risk Questionnaire (IRQ), and Drug Injecting Confidence Questionnaire (DICQ). The primary outcome was measured by the rate of sero-conversion at 6 months and 12 months from baseline and by the ASI, IRQ and DICQ at 6 months from baseline. Hepatitis C testing was undertaken by the innovative test of the dried blood spot (DBS) test which increased the rate of testing by 4 fold compared to routine blood testing.</p>", "<title>Findings Seventy</title>", "<p>Eighty two subjects (82%) out of the 95 recruited were followed up at 6 months and 62 (65%) were followed up at 12 months. On the primary outcome measure of the rate of seroconversion, 8 out of 62 patients followed-up at twelve months seroconverted, three in the EPC group and five in the SEC group, indicating incidence rates of 9.1 per 100 person years for the EPC group, 17.2 per 100 person years for the SEC group, and 12.9 per 100 person years for the cohort as a whole. Analysis of the secondary outcome measures on alcohol use, risk behaviour, psychological measures, quality of life, showed no significant differences between the EPC and the SEC groups. However, there were significant changes on a number of measures from baseline values indicating positive change for both groups.</p>", "<title>Conclusion</title>", "<p>We were not able to prove the efficacy of EPC in comparison with SEC in the prevention of hepatitis C in IDUs. This was related to low recruitment and retention rates of the participants. Moreover there was a low adherence rate to EPC. The study provided the benefits of developing and introducing behavioural interventions of the EPC and SEC and the DBS screening for Hepatitis C. Moreover the main lessons learnt were that piloting of a new intervention is a crucial first step before conducting pragmatic RCTs of psychological interventions in the field of addiction; that an infrastructure and culture for psychosocial interventions is needed to enable applied research in the service environment, and research funding is needed for enabling the recruitment of dedicated trained therapists for the delivery of these interventions.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>MA was principal investigator, designed the study, had full responsibility for its overall management drafted and revised the article. PD designed, developed the manuals of the psychological interventions and trained and supervised the therapists. CD, NO, KC and HG contributed to the study design and methodology. PR, DM and CT were responsible for Hepatitis C testing and development of the Dried Blood Spot test. BC and CJ were the trial's co-ordinators and conducted statistical analyses under supervision of the biostatistician. CG was the health economist and designed the tools for measuring cost-effectiveness of the interventions. MD recruited participants from Surrey County. All authors reviewed drafts of the article and agreed the final manuscript and its revisions.</p>" ]

[ "<title>Acknowledgements</title>", "<p>This research was funded as part of the Department of Health Policy Research Programme. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health. The authors are grateful to the staff of all the services that took part in this research particularly those who assisted in recruitment and acted as therapists and supervisors. We are also grateful for the Steering Committee for their support and guidance.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Number of participants at each stage of the recruitment process.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Participants engaged, not engaged, sero converted and incidence of HCV in EPC and SEC groups</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\"><bold>Number</bold><break/><bold> Engaged</bold><bold>^a</bold></td><td align=\"left\"><bold>Number</bold><break/><bold> Not engaged</bold></td><td align=\"left\"><bold>Number</bold><break/><bold>Sero</bold><break/><bold> converted</bold></td><td align=\"left\"><bold>Total</bold></td><td align=\"left\"><bold>Incidence of HCV </bold><break/><bold> (per 100 person years)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>EPC</bold></td><td align=\"left\">17* (45.9%)</td><td align=\"left\">20 (54.1%)</td><td align=\"left\">3 ^b</td><td align=\"left\">37</td><td align=\"left\">9.1</td></tr><tr><td align=\"left\"><bold>SEC</bold></td><td align=\"left\">38 (92.7%)</td><td align=\"left\">3 (7.3%)</td><td align=\"left\">5 ^c</td><td align=\"left\">41</td><td align=\"left\">17.2</td></tr><tr><td align=\"left\"><bold>Total</bold></td><td align=\"left\">55 (70.5%)</td><td align=\"left\">23 (29.5%)</td><td align=\"left\">8</td><td align=\"left\">78</td><td align=\"left\">12.9</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Number of participants who completed or have not completed EPS and the number of sessions completed</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Number of</bold><break/><bold> EPC Sessions</bold></td><td align=\"left\"><bold>None</bold></td><td align=\"left\"><bold>One</bold></td><td align=\"left\"><bold>Two</bold></td><td align=\"left\"><bold>Three</bold></td><td align=\"left\"><bold>Four</bold><break/><bold>(course</bold><break/><bold> completed)</bold></td></tr></thead><tbody><tr><td align=\"left\">Number of<break/> participants</td><td align=\"left\">20</td><td align=\"left\">6</td><td align=\"left\">4</td><td align=\"left\">0</td><td align=\"left\">7</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Changes in addiction severity and risk behaviour</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Drug and Alcohol</bold></td><td align=\"center\" colspan=\"4\"><bold>Baseline</bold></td><td align=\"center\" colspan=\"4\"><bold>Six-Month follow-up</bold></td><td/><td/><td/><td/></tr></thead><tbody><tr><td/><td align=\"center\" colspan=\"2\"><bold>EPC n = 37</bold></td><td align=\"center\" colspan=\"2\"><bold>SEC n = 41</bold></td><td align=\"center\" colspan=\"2\"><bold>EPC n = 36</bold></td><td align=\"center\" colspan=\"2\"><bold>SEC n = 41</bold></td><td align=\"center\" colspan=\"2\"><bold>Effect of </bold><break/><bold>Treatment</bold></td><td align=\"center\" colspan=\"2\"><bold>Effect of</bold><break/><bold> Time</bold></td></tr><tr><td colspan=\"13\"><hr/></td></tr><tr><td align=\"left\"><bold>ANCOVA</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>F</bold></td><td align=\"center\"><bold>p</bold></td><td align=\"center\"><bold>F</bold></td><td align=\"center\"><bold>p</bold></td></tr><tr><td colspan=\"13\"><hr/></td></tr><tr><td align=\"left\">ASI – drug use</td><td align=\"center\">0.286</td><td align=\"center\">0.12</td><td align=\"center\">0.29</td><td align=\"center\">0.096</td><td align=\"center\">0.22</td><td align=\"center\">0.152</td><td align=\"center\">0.249</td><td align=\"center\">0.133</td><td align=\"center\">0.90</td><td align=\"center\">0.35</td><td align=\"center\">0.54</td><td align=\"center\">0.47</td></tr><tr><td align=\"left\">ASI – alcohol use</td><td align=\"center\">0.084</td><td align=\"center\">0.17</td><td align=\"center\">0.115</td><td align=\"center\">0.176</td><td align=\"center\">0.113</td><td align=\"center\">0.176</td><td align=\"center\">0.082</td><td align=\"center\">0.143</td><td align=\"center\">2.26</td><td align=\"center\">0.14</td><td align=\"center\">41.08</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">ASI – medical subscale</td><td align=\"center\">0.138</td><td align=\"center\">0.26</td><td align=\"center\">0.134</td><td align=\"center\">0.278</td><td align=\"center\">0.092</td><td align=\"center\">0.207</td><td align=\"center\">0.156</td><td align=\"center\">0.294</td><td align=\"center\">0.95</td><td align=\"center\">0.33</td><td align=\"center\">10.97</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">ASI – psychiatric subscale</td><td align=\"center\">0.208</td><td align=\"center\">0.26</td><td align=\"center\">0.174</td><td align=\"center\">0.204</td><td align=\"center\">0.204</td><td align=\"center\">0.241</td><td align=\"center\">0.159</td><td align=\"center\">0.207</td><td align=\"center\">0.34</td><td align=\"center\">0.56</td><td align=\"center\">3.74</td><td align=\"center\">0.06</td></tr><tr><td align=\"left\">ASI – Legal subscale</td><td align=\"center\">0.094</td><td align=\"center\">0.16</td><td align=\"center\">0.119</td><td align=\"center\">0.182</td><td align=\"center\">0.093</td><td align=\"center\">0.151</td><td align=\"center\">0.09</td><td align=\"center\">0.177</td><td align=\"center\">0.01</td><td align=\"center\">0.92</td><td align=\"center\">0.80</td><td align=\"center\">0.38</td></tr><tr><td align=\"left\">ASI – economic subscale</td><td align=\"center\">0.877</td><td align=\"center\">0.32</td><td align=\"center\">0.652</td><td align=\"center\">0.432</td><td align=\"center\">0.765</td><td align=\"center\">0.357</td><td align=\"center\">0.766</td><td align=\"center\">0.381</td><td align=\"center\">0.83</td><td align=\"center\">0.67</td><td align=\"center\">8.56</td><td align=\"center\">0.01</td></tr><tr><td align=\"left\">ASI – satisfaction subscale</td><td align=\"center\">0.289</td><td align=\"center\">0.31</td><td align=\"center\">0.235</td><td align=\"center\">0.27</td><td align=\"center\">0.222</td><td align=\"center\">0.334</td><td align=\"center\">0.154</td><td align=\"center\">0.266</td><td align=\"center\">0.65</td><td align=\"center\">0.42</td><td align=\"center\">5.72</td><td align=\"center\">0.02</td></tr><tr><td align=\"left\">ASI – family relationships</td><td align=\"center\">0.101</td><td align=\"center\">0.16</td><td align=\"center\">0.13</td><td align=\"center\">0.209</td><td align=\"center\">0.075</td><td align=\"center\">0.133</td><td align=\"center\">0.077</td><td align=\"center\">0.151</td><td align=\"center\">0.02</td><td align=\"center\">0.89</td><td align=\"center\">2.31</td><td align=\"center\">0.13</td></tr><tr><td align=\"left\">ASI – social relationships</td><td align=\"center\">0.104</td><td align=\"center\">0.19</td><td align=\"center\">0.085</td><td align=\"center\">0.133</td><td align=\"center\">0.03</td><td align=\"center\">0.102</td><td align=\"center\">0.039</td><td align=\"center\">0.084</td><td align=\"center\">0.13</td><td align=\"center\">0.72</td><td align=\"center\">2.07</td><td align=\"center\">0.16</td></tr><tr><td align=\"left\">IRQ – No. people shared IV<break/>equipment with in last 6<break/> months</td><td align=\"center\">0.81</td><td align=\"center\">0.88</td><td align=\"center\">1.32</td><td align=\"center\">1.4</td><td align=\"center\">0.22</td><td align=\"center\">0.64</td><td align=\"center\">0.37</td><td align=\"center\">0.97</td><td align=\"center\">1.1</td><td align=\"center\">0.31</td><td align=\"center\">0.00</td><td align=\"center\">0.98</td></tr><tr><td align=\"left\">HIV RTBS – Drug score</td><td align=\"center\">9.95</td><td align=\"center\">5.74</td><td align=\"center\">8.02</td><td align=\"center\">5.8</td><td align=\"center\">3.31</td><td align=\"center\">5.3</td><td align=\"center\">3.4</td><td align=\"center\">5.6</td><td align=\"center\">0.4</td><td align=\"center\">0.55</td><td align=\"center\">10.0</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">HIV RTBS – Sex score</td><td align=\"center\">4.5</td><td align=\"center\">4.2</td><td align=\"center\">3.1</td><td align=\"center\">3.4</td><td align=\"center\">4.7</td><td align=\"center\">4.2</td><td align=\"center\">3.9</td><td align=\"center\">4.14</td><td align=\"center\">0.1</td><td align=\"center\">0.78</td><td align=\"center\">8.2</td><td align=\"center\">0.01</td></tr><tr><td align=\"left\">HIV RTBS – Overall</td><td align=\"center\">13.8</td><td align=\"center\">7.45</td><td align=\"center\">11.2</td><td align=\"center\">6.98</td><td align=\"center\">8.0</td><td align=\"center\">8.12</td><td align=\"center\">7.3</td><td align=\"center\">6.86</td><td align=\"center\">0.0</td><td align=\"center\">0.84</td><td align=\"center\">11.4</td><td align=\"center\">0.00</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Changes in risk behaviour and alcohol misuse</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Drug and Alcohol</bold></td><td align=\"center\" colspan=\"4\"><bold>Baseline</bold></td><td align=\"center\" colspan=\"4\"><bold>Six-Month follow-up</bold></td><td/><td/></tr></thead><tbody><tr><td/><td align=\"center\" colspan=\"2\"><bold>EPC n = 37</bold></td><td align=\"center\" colspan=\"2\"><bold>SEC n = 41</bold></td><td align=\"center\" colspan=\"2\"><bold>EPC n = 36</bold></td><td align=\"center\" colspan=\"2\"><bold>SEC n = 41</bold></td><td align=\"center\" colspan=\"2\"><bold>Effect of</bold><break/><bold> Treatment</bold></td></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">χ²<bold>analysis</bold></td><td align=\"center\"><bold>N</bold></td><td align=\"center\"><bold>%</bold></td><td align=\"center\"><bold>N</bold></td><td align=\"center\"><bold>%</bold></td><td align=\"center\"><bold>N</bold></td><td align=\"center\"><bold>%</bold></td><td align=\"center\"><bold>N</bold></td><td align=\"center\"><bold>%</bold></td><td/><td align=\"center\"><bold>p</bold></td></tr><tr><td colspan=\"11\"><hr/></td></tr><tr><td align=\"left\">IRQ – injected at all in last<break/> six months</td><td align=\"center\">37.0</td><td align=\"center\">100.0</td><td align=\"center\">41.0</td><td align=\"center\">100.0</td><td align=\"center\">20</td><td align=\"center\">55.6</td><td align=\"center\">24</td><td align=\"center\">58.5</td><td align=\"center\">0.07</td><td align=\"center\">0.79</td></tr><tr><td align=\"left\">IRQ – Shared any IV<break/>equipment at all in last<break/> 6 months</td><td align=\"center\">21.0</td><td align=\"center\">56.8</td><td align=\"center\">27.0</td><td align=\"center\">65.9</td><td align=\"center\">6</td><td align=\"center\">16.6</td><td align=\"center\">8</td><td align=\"center\">19.5</td><td align=\"center\">0.10</td><td align=\"center\">0.75</td></tr><tr><td align=\"left\">Used needle exchange in <break/>the last six months</td><td align=\"center\">21.0</td><td align=\"center\">56.8</td><td align=\"center\">23.0</td><td align=\"center\">56.1</td><td align=\"center\">16</td><td align=\"center\">44.4</td><td align=\"center\">17</td><td align=\"center\">41.5</td><td align=\"center\">0.07</td><td align=\"center\">0.79</td></tr><tr><td align=\"left\">AUDIT (score of 8 or more)</td><td align=\"center\">11.0</td><td align=\"center\">29.7</td><td align=\"center\">16.0</td><td align=\"center\">39.0</td><td align=\"center\">8</td><td align=\"center\">22.2</td><td align=\"center\">7</td><td align=\"center\">17.1</td><td align=\"center\">0.32</td><td align=\"center\">0.57</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Changes in situational confidence, hepatitis C knowledge and readiness to change measures</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Measure</bold></td><td align=\"center\" colspan=\"4\"><bold>Baseline</bold></td><td align=\"center\" colspan=\"4\"><bold>Six-month Follow-up</bold></td><td/><td/><td/><td/></tr></thead><tbody><tr><td/><td align=\"center\" colspan=\"2\"><bold>EPC n = 37</bold></td><td align=\"center\" colspan=\"2\"><bold>SEC n = 41</bold></td><td align=\"center\" colspan=\"2\"><bold>EPC n = 36</bold></td><td align=\"center\" colspan=\"2\"><bold>SEC n = 41</bold></td><td align=\"center\" colspan=\"2\"><bold>Effect of </bold><break/><bold>Treatment</bold></td><td align=\"center\" colspan=\"2\"><bold>Effect of </bold><break/><bold>Time</bold></td></tr><tr><td colspan=\"13\"><hr/></td></tr><tr><td align=\"left\"><bold>ANCOVA</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>Mean</bold></td><td align=\"center\"><bold>sd</bold></td><td align=\"center\"><bold>F</bold></td><td align=\"center\"><bold>p</bold></td><td align=\"center\"><bold>F</bold></td><td align=\"center\"><bold>p</bold></td></tr><tr><td colspan=\"13\"><hr/></td></tr><tr><td align=\"left\">DICQ – Unpleasant emotions</td><td align=\"center\">49.27</td><td align=\"center\">30.1</td><td align=\"center\">54.51</td><td align=\"center\">30.06</td><td align=\"center\">56.86</td><td align=\"center\">28.8</td><td align=\"center\">62.85</td><td align=\"center\">31.56</td><td align=\"center\">0.38</td><td align=\"center\">0.54</td><td align=\"center\">14.44</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Physical discomfort score</td><td align=\"center\">56.76</td><td align=\"center\">27.2</td><td align=\"center\">61.9</td><td align=\"center\">28.26</td><td align=\"center\">64.11</td><td align=\"center\">24.09</td><td align=\"center\">66.27</td><td align=\"center\">30.85</td><td align=\"center\">0.01</td><td align=\"center\">0.92</td><td align=\"center\">10.07</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Pleasant emotions</td><td align=\"center\">74.16</td><td align=\"center\">25.3</td><td align=\"center\">79.17</td><td align=\"center\">21.67</td><td align=\"center\">77.44</td><td align=\"center\">24.62</td><td align=\"center\">78.51</td><td align=\"center\">25.73</td><td align=\"center\">0.03</td><td align=\"center\">0.87</td><td align=\"center\">15.38</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Testing personal control</td><td align=\"center\">57.76</td><td align=\"center\">30.7</td><td align=\"center\">60.44</td><td align=\"center\">31.51</td><td align=\"center\">64.53</td><td align=\"center\">28.76</td><td align=\"center\">63.1</td><td align=\"center\">32.42</td><td align=\"center\">0.05</td><td align=\"center\">0.82</td><td align=\"center\">11.54</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Urges and temptations</td><td align=\"center\">49.27</td><td align=\"center\">30.2</td><td align=\"center\">56.98</td><td align=\"center\">29.4</td><td align=\"center\">58.22</td><td align=\"center\">28.41</td><td align=\"center\">59.61</td><td align=\"center\">31.64</td><td align=\"center\">0.06</td><td align=\"center\">0.81</td><td align=\"center\">15.08</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Conflict with others</td><td align=\"center\">59.27</td><td align=\"center\">30.3</td><td align=\"center\">65.24</td><td align=\"center\">28.36</td><td align=\"center\">67.61</td><td align=\"center\">26.35</td><td align=\"center\">68.59</td><td align=\"center\">30.84</td><td align=\"center\">0.07</td><td align=\"center\">0.79</td><td align=\"center\">20.54</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Social pressure to use</td><td align=\"center\">49.43</td><td align=\"center\">32.9</td><td align=\"center\">52.68</td><td align=\"center\">34.48</td><td align=\"center\">48.5</td><td align=\"center\">35.9</td><td align=\"center\">56.54</td><td align=\"center\">34.49</td><td align=\"center\">0.92</td><td align=\"center\">0.34</td><td align=\"center\">20.63</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Pleasant times with others score</td><td align=\"center\">59.14</td><td align=\"center\">27.5</td><td align=\"center\">63.49</td><td align=\"center\">28.64</td><td align=\"center\">67.5</td><td align=\"center\">27.02</td><td align=\"center\">68.68</td><td align=\"center\">29.12</td><td align=\"center\">0.00</td><td align=\"center\">0.99</td><td align=\"center\">13.93</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">DICQ – Overall score</td><td align=\"center\">56.08</td><td align=\"center\">26.8</td><td align=\"center\">61.76</td><td align=\"center\">26.14</td><td align=\"center\">61.06</td><td align=\"center\">25.14</td><td align=\"center\">65.73</td><td align=\"center\">28.2</td><td align=\"center\">0.22</td><td align=\"center\">0.64</td><td align=\"center\">21.68</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">Hepatitis-C Knowledge Questionnaire</td><td align=\"center\">16.22</td><td align=\"center\">2.29</td><td align=\"center\">16.2</td><td align=\"center\">1.79</td><td align=\"center\">17.44</td><td align=\"center\">1.89</td><td align=\"center\">17.61</td><td align=\"center\">1.66</td><td align=\"center\">0.08</td><td align=\"center\">0.78</td><td align=\"center\">16.46</td><td align=\"center\">0.00</td></tr><tr><td align=\"left\">Readiness to change stage</td><td align=\"center\">2.54</td><td align=\"center\">0.73</td><td align=\"center\">2.54</td><td align=\"center\">0.78</td><td align=\"center\">2.58</td><td align=\"center\">0.73</td><td align=\"center\">2.56</td><td align=\"center\">0.78</td><td align=\"center\">0.00</td><td align=\"center\">0.99</td><td align=\"center\">0.57</td><td align=\"center\">0.45</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>* Chi-squared test χ²= 20.43, p &lt; 0.000</p><p>(EPC) Enhanced Prevention Counselling</p><p>(SEC)Simple Education Counselling</p><p>a Number of participants completing EPC sessions: none 20; one session 6; two sessions 4; three sessions none and four sessions 7.</p><p>b Three participants seroconverted at 12 months: none engaged with EPC: HCV incidence 9.1 per100 person years</p><p>c Five particpants seroconverted at 12 months: 4 engaged with SEC: HCV incidence 17.2 per 100 person years</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1477-7517-5-25-1\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>The burden of malaria in many parts of peninsular Malaysia has decreased substantially due to malaria control activities. The reported incidence of malaria in Malaysia has decreased to 5,294 cases in 2006 compared to 12,705 cases in 2000. In 2006 there were only 852 cases in peninsular Malaysia compared with 3918 cases in 2000 (Annual Report, Ministry of Health Malaysia). However, a fifth species, <italic>Plasmodium knowlesi </italic>that was originally described as a malaria parasite of the long-tailed macaque monkeys [##UREF##0##1##] is now occurring here. The first case was reported in peninsular Malaysia in 1965 [##REF##14332847##2##]. However, since 2004 there have been reports of P. <italic>knowlesi </italic>infecting humans in the Southeast Asia region [##REF##15051281##3##, ####REF##15663864##4##, ##REF##16866152##5##, ##REF##18171245##6##, ##REF##18439370##7##, ##REF##18439369##8####18439369##8##].</p>", "<p>The accidental discovery that <italic>Plasmodium cynomolgi </italic>could be transmitted to humans <italic>via </italic>mosquito bites in the laboratory [##REF##13821129##9##] stimulated great interest and thus, extensive studies were carried out in peninsular Malaysia to determine the distribution, prevalence and species of malaria parasites in monkeys and apes and the vectors of monkey malaria in nature and to determine whether monkey malaria infection transmissible to man existed in Malaya [##REF##13784726##10##, ####REF##14006429##11##, ##REF##14166986##12##, ##REF##5857000##13##, ##REF##4392806##14####4392806##14##]. From their studies, several new species of simian malaria parasites were described [##UREF##1##15##, ####UREF##2##16##, ##REF##14084190##17####14084190##17##].</p>", "<p>After the first human case of <italic>P. knowlesi</italic>, a study that was initiated in the state of Pahang to investigate whether malaria was a zoonosis, concluded that simian malaria in humans was an extremely rare event [##REF##4231980##18##,##UREF##3##19##]. This was based on studies in which blood samples were collected from more than 1100 local residents, the samples were pooled and injected them into rhesus monkeys. However, none of the monkeys contracted malaria.</p>", "<p>In most instances, infection of the human host with the malaria parasite begins with the bite of an infected <italic>Anopheles </italic>mosquito that inoculates sporozoites into the host. In the early works carried out on the vectors of simian malaria in peninsular Malaysia, it was postulated that <italic>Anopheles leucosphyrus </italic>complex are involved in simian malaria transmission. In a study conducted in the coastal area of Selangor in peninsular Malaysia, <italic>Anopheles hackeri </italic>was incriminated as a vector of <italic>P</italic>.<italic>knowlesi </italic>[##REF##13784726##10##]. In the inland hill forest area of Selangor, (Hlu Lui) a simian parasite, <italic>Plasmodium inui </italic>was isolated from <italic>Anopheles latens </italic>(= <italic>An. leucosphyrus</italic>) [##REF##14006429##11##]. A year later in 1963 <italic>Plasmodium cynomolgi </italic>was isolated from <italic>Anopheles introlatus </italic>(= <italic>An. balabacensis introlatus</italic>) [##REF##14166986##12##]. In the monsoon rain forest of northern Malaysia in the state of Perlis <italic>An</italic>. <italic>cracens </italic>(= <italic>An. balabacensis balabacensis</italic>) was the vector of both <italic>P. inui </italic>and <italic>P. cynomolgi </italic>[##REF##5857000##13##].</p>", "<p>In 2004, a large focus of human <italic>P. knowlesi </italic>infection was reported in the Kapit Division of Sarawak [##REF##15051281##3##]. Thus, it was pertinent to deduce if knowlesi malaria was currently occurring in peninsular Malaysia and to elucidate the vectors and to study the parasites in macaques in the areas around human cases. This paper reports the preliminary results of the study.</p>" ]

[ "<title>Methods</title>", "<title>Human blood samples</title>", "<p>Blood samples or Giemsa stained blood films were sent to the Parasitology Unit of the Institute from hospitals and health centres that wanted a confirmation of <italic>Plasmodium malariae </italic>or in some cases to rule out <italic>P. knowlesi</italic>.</p>", "<title>DNA extraction from whole blood</title>", "<p>DNA was extracted from whole blood using the Qiagen D Neasy Blood Tissue Kit (Hilden, Germany), following the manufacturer's recommendations.</p>", "<title>DNA extraction from blood films</title>", "<p>In some cases only Giemsa stained blood films were provided. Before the extraction of the DNA, the slides were first cleaned with chloroform to remove oil. Fifty microliters of TE buffer was then pipetted on to the thin blood film. Two discs were punched out from a Whatman 1 filter paper (Whatman USA) using a pre flamed paper puncher. The discs were placed on the slide to soak up the buffer. Using a clean, flamed forceps, at least half of the smear was completely wiped off the slide with the filter paper and was transferred to 1.5 ml centrifuge tubes (Axygen). The DNA was extracted using the Qiagen D Neasy Blood Tissue Kit (Hilden, Germany).</p>", "<title>Nested Polymerase Chain Reaction</title>", "<p>Nested PCR assays [##REF##15051281##3##,##REF##10348249##20##], based on the <italic>Plasmodium </italic>DNA sequence of the small subunit ribosomal RNA (SSUrRNA) genes, were used to detect and identify the species of malaria parasites found in the blood. The maximum number of samples processed at any one time was not more than 10. Positive controls for <italic>P. falciparum</italic>, <italic>P. knowlesi, P. malariae </italic>and <italic>P. vivax </italic>were included for all nested PCR species assays. A negative control was also included for each batch of assays. Nest 1 reaction was carried out in a 50 μl reaction mixture containing 1× reaction buffer (5× Green Go Taq Flexi Buffer, Promega Madison USA) 3 mM MgCl₂(Promega), 200 mM of each deoxynucleoside triphosphate (Promega), 300 nM of each primers and 1.25 U of Go Taq DNA polymerase (Promega) and 5 μl of DNA template was used for each reaction.</p>", "<p>Nest 2 PCR amplification was done in a 20 μl reaction mixture containing 1× reaction buffer (5× Green Go Taq Flexi Buffer Promega) 2 mM MgCl₂(Promega,), 200 mM of each deoxynucleoside triphosphate (Promega,), 300 nM of each primers and 0.5 U Go Taq DNA polymerase (Promega,) and 2 μl of the nest 1 PCR products were used as DNA templates. All PCR reactions were carried out using thermal cycler (Techne TC 152 -Barloworld Sci Ltd UK). Ten microliters of the nest 2 amplicons were analyzed by agarose gel.</p>", "<title>Sequencing of the Plasmodium csp genes</title>", "<p>The <italic>csp </italic>genes of malaria parasites from all human isolates positive for <italic>P. knowlesi </italic>were amplified with primers, PKCSP-F and PKCSP-R [##REF##15051281##3##]. However, for the mosquito and monkey isolates, primers PKCSPF2 (5' TACAAGAACAAGATGARGAAC 3') and PKCSPR2 (5' TCAGCTACTTAATTGAATAATGC 3') were used since many non- specific bands were obtained with PKCSP-F and PKCSP-R.</p>", "<p>PCR was carried out in a 20 μl reaction volume containing 1× Phusion HF buffer (Finnzymes), 200 mM/l of each dNTP (Finnzymes Finland) and 250 nM/l of each primer, and 0.02 U of Phusion DNA Polymerase (Finnzymes Finland). The PCR was carried out using a thermal cycler (Techne TC 152 -Barloworld Sci Ltd UK). The PCR conditions were as follows: initial denaturation at 98°C for 30 sec followed by 40 cycles of amplification at 94°C for 7 sec, 51°C for 20 sec, 72°C for 20 sec followed by a final extension step of 10 min. The expected size of the PCR products is approximately 1.2 kb and amplicons from each isolate were excised from the gel and purified using Perfectprep gel cleanup kit (Eppendorf, Germany), following the manufacturer's recommendation. The purified products were cloned as previously described [##REF##15051281##3##]. At least 20 of transformants from each PCR were screened using the <italic>csp </italic>primers mentioned above. Amplification was done in a 20 μl reaction mixtures containing 1× reaction buffer (5× Green Go Taq Flexi Buffer, Promega Madison USA) 2 mM MgCl₂(Promega), 200 mM of each deoxynucleoside triphosphate (Promega), 300 nM of each primers and 0.5 U Go Taq DNA polymerase (Promega,). PCR conditions were as follows: initial denaturation of 94°C for 10 min followed by 30 cycles of amplification at 94°C at 1 min, annealing at 53°C at 1 min, extension at 72°C for 1 min 20 sec, followed by a final extension step at 72°C at 5 min. Ten microliters of the amplicons were digested with <italic>Eco</italic>R1 (Promega) and analyzed by gel electrophoresis. Plasmids from clones having the correct inserts were extracted using S.N.A.P. plasmid extraction kit (Invitrogen, USA) following the manufacturer's protocol. Purified plasmids was sent to Solgent Company Limited (Daejeon, South Korea) for sequencing to obtain the entire <italic>csp </italic>gene sequence of <italic>P. knowlesi</italic></p>", "<title>Analysis of sequence data</title>", "<p>Analysis of the csp genes was performed as previously described [##REF##15051281##3##]. Sequences of the 456 nucleotide that encodes non – repeat N- terminal (first 195 nucleotides of coding sequence) and C- terminal (the last 261 nucleotides of the <italic>csp </italic>gene coding sequence) region of the protein were aligned by CLUSTAL W using Megalign (Lasergene, DNASTAR, USA). Regions of the protein were aligned using Megalign software (Lasergene). The <italic>csp </italic>gene sequences from patients, mosquitoes and monkey samples were compared with those obtained from the GenBank data base. Phylogenetic trees were performed by the neighbour-joining (NJ) [##UREF##4##21##] and Bayesian methods [##REF##9214744##22##]. The NJ method was analyzed using the Kimura-2 parameter with 1000 bootstrap replicates and was carried out using the MEGA version 4.0 software [##UREF##5##23##]. On the other hand, the Bayesian method was analysed using the Hasegawa-Kishino-Yano (HKY) model with the following parameters: the search was performed at 1,000,000 generations, sampling every 100 generations and the first 2000 trees were discarded in the burn-ins. The analysis was carried using the Mr. Bayes 3.1 software [##REF##12912839##24##].</p>", "<p>Nucleotide sequences reported in this study have been deposited in Gen Bank under accession numbers: <ext-link ext-link-type=\"gen\" xlink:href=\"EU687467\">EU687467</ext-link>–<ext-link ext-link-type=\"gen\" xlink:href=\"EU687470\">EU687470</ext-link>, <ext-link ext-link-type=\"gen\" xlink:href=\"EU708437\">EU708437</ext-link> (human samples), <ext-link ext-link-type=\"gen\" xlink:href=\"EU821335\">EU821335</ext-link> (mosquito sample), <ext-link ext-link-type=\"gen\" xlink:href=\"EU821336\">EU821336</ext-link> (monkey sample). The other malaria <italic>csp </italic>gene sequences used were obtained from GenBank: <italic>P. knowlesi </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"M11031\">M11031</ext-link>), <italic>P. knowlesi </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"K0082\">K0082</ext-link>), KH35 (<ext-link ext-link-type=\"gen\" xlink:href=\"AH013332\">AH013332</ext-link>), KH43 (<ext-link ext-link-type=\"gen\" xlink:href=\"AH013333\">AH013333</ext-link>), KH50 (<ext-link ext-link-type=\"gen\" xlink:href=\"AH013334\">AH013334</ext-link>), KH107 (<ext-link ext-link-type=\"gen\" xlink:href=\"AH013336\">AH013336</ext-link>), KH115 (<ext-link ext-link-type=\"gen\" xlink:href=\"AH013337\">AH013337</ext-link>), <italic>P. coatneyi </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"AY135360\">AY135360</ext-link>), <italic>P. cynomolgi </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"M15104\">M15104</ext-link>), <italic>P. simiovale </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"U09765\">U09765</ext-link>), <italic>P. simium </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"L05068\">L05068</ext-link>), <italic>P. inui </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"FJ009512\">FJ009512</ext-link>) <italic>P. vivax </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"M34697\">M34697</ext-link>), <italic>P. malariae </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"U09766\">U09766</ext-link>), <italic>P. malariae </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"J03992\">J03992</ext-link>), <italic>P. falciparum </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"K02194\">K02194</ext-link>) and <italic>P. vinckei lentum </italic>(<ext-link ext-link-type=\"gen\" xlink:href=\"AF162331\">AF162331</ext-link>).</p>", "<title>Trapping of monkeys</title>", "<p>Monkeys were trapped in the vicinity of Kuala Lumpur, Selangor State and Kuala Lipis in Pahang State with help from the wild-life department. The captured monkeys were anesthetized by intramuscular injection with ketamine hydrochloride. Ten ml blood was collected, after which the monkeys were tagged with an electronic identification system (Trovan Ltd London). After they recovered they were released into the deep forest.</p>", "<title>Preparation of blood films</title>", "<p>Both thick and thin blood films were prepared followed by staining with Giemsa. Microscopic examinations were carried out using compound microscope under oil immersion under 100× magnification.</p>", "<title>DNA extraction from whole blood, PCR and sequencing</title>", "<p>DNA was extracted from the whole blood using the Dneasy Tissue Extraction Kit (Qiagen, Germany) following the manufacturer's recommendation. All monkeys were screened for malaria parasites using primers rPLU3 and rPLU4 [##REF##10348249##20##], and for <italic>P. knowlesi </italic>using the primers Pmk8 and Pmk9 [##REF##15051281##3##]. Nested PCR, cloning and sequencing of all samples positive for <italic>P. knowlesi </italic>were performed as described above.</p>", "<title>Study Sites for mosquito collection</title>", "<p>The study site for mosquito collection was in Kuala Lipis district in the State of Pahang. Pre-surveys for mosquito collections were carried out to determine suitable sites for long term study based on presence of monkeys and occurrence of cases. Based, on pre-surveys two sites were selected for the study. One is Serunai Mela village [4° 7.0'N, 102° 11.9'E ] and the other is a fruit farm in Sungai Ular [4° 15.7'N, 102°4.8'E ]. In Serunai Mela, a case of <italic>P. knowlesi </italic>had occurred and the case house is situated at the forest fringe. Sungai Ular was selected as a patient had reported that he visited the farm before falling ill and it was also frequented by monkeys</p>", "<title>Mosquito collections</title>", "<p>All night mosquito collections using bare-leg catch method [##REF##8629075##25##] were performed from July 2007 to November 2007. In each area 4 nights of collection were carried out every month by three men working outdoors from 18.00 to 06.00 hours.</p>", "<title>Monkey-baited-trap</title>", "<p>In order to compare the number of mosquitoes attracted to humans and monkeys, a monkey- baited – trap was constructed in Serunai Mela Village as previously described [##REF##14000207##26##,##UREF##6##27##].</p>", "<title>Mosquito identification and dissection</title>", "<p>All mosquitoes were identified morphologically in the field laboratory. The keys of Reid [##UREF##7##28##] were used for the identification of <italic>Anopheles </italic>mosquitoes and keys of Sallum [##REF##15958025##29##] were used for <italic>leucosphyrus </italic>group in particular. <italic>Anopheles </italic>mosquitoes were dissected to extract ovaries to determine parity and the midguts and salivary glands were examined for oocysts and sporozoites, respectively. All the positive salivary glands were placed in 1.5 microcentrifuge tubes (Axygen, USA) containing absolute alcohol and were labeled accordingly.</p>", "<title>DNA extraction, PCR and sequencing</title>", "<p>Ethanol used in the preservation of the salivary glands were allowed to evaporate completely by placing the tubes in a Thermomixer (Eppendorf, Germany) set at 70°C. DNA was then extracted using the Qiagen D Neasy Blood Tissue Kit (Hilden, Germany) as described above. PCR and sequencing were also carried out as mentioned above.</p>", "<title>Ethical clearance</title>", "<p>This project was approved by the Institute for Medical Research &amp; Ethical Committee Ministry of Health Malaysia and the Animal Use Committee of the Institute.</p>" ]

[ "<title>Results</title>", "<title>Humans</title>", "<p>A total of 111 samples were received for PCR (from July 2005–March 2008). Of these 77 (69.37%) were positive for <italic>P. knowlesi </italic>(Table ##TAB##0##1##). By microscopy, 93 (83.78%) of the slides were reported as <italic>P. malariae </italic>of which by nested PCR 62 (55.86%) were positive for <italic>P. knowlesi </italic>and 11 (9.91%) were mixed infection with <italic>P. knowlesi </italic>and other human malaria parasites (Table ##TAB##0##1##). Positive <italic>P. knowlesi </italic>cases were observed in all states in peninsular Malaysia with the exception of four – Johore, Negeri Sembilan, Perlis and Terengganu (Fig. ##FIG##0##1##). Pahang has the highest number of <italic>P. knowlesi </italic>cases (50.65%).</p>", "<title>Monkeys</title>", "<p>Until December 2007 a total of 145 monkeys had been trapped. Of these 143 (98.62%) were <italic>Macaca fascicularis </italic>and one (0.69%) each of <italic>M. nemestrina </italic>and <italic>Presbytis melalophos</italic>. Seventy five of the monkeys were trapped from Kuala Lipis and of these 73 (97.33%) were positive for malaria parasites by microscopy and 10 were positive for <italic>P. knowlesi </italic>by PCR. In Kuala Lumpur, 2 (6.90%)were positive for malaria parasites out of the 29 that were trapped but none was positive for <italic>P. knowlesi</italic>. In Selangor, all 41 monkeys examined were negative.</p>", "<title>Mosquitoes</title>", "<p>A total of 339 <italic>Anopheles </italic>mosquitoes belonging to 12 species were caught biting humans and monkeys in the five months period as shown in Table ##TAB##1##2##. <italic>Anopheles cracens </italic>was the predominant mosquito comprising 62.2% of the total collection. This was followed by <italic>Anopheles maculatus</italic>. The two species attracted to monkeys were <italic>An. cracens </italic>and <italic>Anopheles kochi</italic>.</p>", "<title>Sporozoite rate</title>", "<p>A total of two <italic>An. cracens </italic>were positive for sporozoites from Serunai Mela of which one was positive for oocyst as well. Thus, the sporozoite rate in Serunai Mela was 1.7. PCR results found them to be positive for <italic>P. knowlesi </italic>and this was confirmed by the sequencing of the CSP gene.</p>", "<title>Biting cycles</title>", "<p><italic>Anopheles cracens </italic>were early biters coming to bite man as early as 19.00 hours and the peak biting time was 19.00 to 21.00 hours. In the forest, the biting was reduced after 22.00 hours but in the fruit orchard they continued to bite throughout the night.</p>", "<title>Sequencing analysis of the <italic>csp </italic>genes</title>", "<p>The <italic>csp </italic>genes of malaria parasites, from <italic>P. knowlesi-</italic>positive isolates, were successfully amplified, cloned and sequenced. The size of the PCR product ranges from 1050 to1128 bp. Phylogenetic analysis inferred from the NJ method (Figure ##FIG##1##2##) showed that malaria parasites isolated from these samples clustered with the reference <italic>P. knowlesi </italic>obtained from Gen bank and with those reported by others [##REF##15051281##3##]. When the flanking regions of the <italic>csp </italic>genes, from isolates obtained in this study, were compared with the reference <italic>P. knowlesi </italic>Nuri strain (M11031), the pairwise identity ranges from 97.1% to 99.6% (data not shown). Furthermore, a clone from a mosquito isolate shared an identical sequence with KH115, which was isolated in Sarawak, East Malaysia. The topology obtained using the Bayesian method (Fig ##FIG##2##3##) is comparable to the NJ tree, which strongly supports the results obtained using the NJ method.</p>" ]

[ "<title>Discussion</title>", "<p>From these preliminary data it is evident that the fifth human malaria parasite <italic>P. knowlesi </italic>is present in most states of peninsular Malaysia. With better molecular diagnostic techniques one can differentiate between <italic>P. knowlesi </italic>and <italic>P. malariae</italic>. The extent of the problem would depend on the cohabitation of humans, non-human primates and the presence of the potential vectors, which are simio-anthropophagic as shown in this study. In a laboratory setting it has been shown that <italic>P. knowlesi </italic>was transmitted by mosquito bites from monkey to monkey, from monkey to humans, from human to human and from human back to monkeys [##UREF##8##30##]. At that time it was postulated that when human malaria cases reaches a low level, the possibility of reseeding the human population with simian malaria parasites could be highly significant [##UREF##8##30##]. This perhaps is due to the declining anti-plasmodial immunity in humans leading to increased susceptibility to simian parasites. Most of these cases are occurring in malaria-free areas which are not subject to control activities. There have also been reports of mortality associated with knowlesi malaria infection in humans [##REF##18171245##6##]. This shows that simian malaria could pose a serious problem to public health.</p>", "<p>This study has shown that there is a link between <italic>An. cracens</italic>, humans and monkeys. Human cases of <italic>P. knowlesi </italic>are occurring in areas purportedly free from the four human malarias, but where <italic>An. cracens </italic>is found and where monkeys are positive for <italic>P. knowlesi</italic>. A previous study showed that <italic>An. cracens</italic>, an important vector of human malaria, was also positive for <italic>P. inui</italic>, another simian malaria [##UREF##9##31##]. This mosquito was found feeding on monkeys at the canopy and humans on the ground [##UREF##9##31##]. In Malaysia, <italic>An. cracens </italic>was first reported in Perlis, the Northern most state bordering Thailand and a later study showed that it was also present in the state of Terengganu [##REF##5857000##13##,##REF##15958025##29##]. This is the first report of the presence <italic>An. cracens </italic>in Kuala Lipis, Pahang and it has now been incriminated as the vector of <italic>P. knowlesi</italic>. Both positive mosquitoes had more than 1,000 sporozoites showing that they are efficient vectors.</p>", "<p><italic>Anopheles maculatus </italic>is the most important vector of human malaria in peninsular Malaysia [##UREF##7##28##]. It has been shown to be susceptible to simian malaria in the laboratory [##REF##6035726##32##,##REF##9645855##33##] and coming to monkey bait in the canopy [##UREF##1##15##,##REF##8629075##25##]. However, in our study areas <italic>An. maculatus </italic>has been found only in small numbers biting humans and none in a monkey- bait-trap.</p>", "<p>Earlier workers in Malaysia [##UREF##1##15##] felt strongly that simian malaria will not be easily transmitted to humans. They must have based their conclusions on the fact that in the coastal areas, where monkeys were heavily infected with <italic>P. knowlesi</italic>, the vector <italic>An. hackeri </italic>was highly zoophilic. In the same area, they dissected <italic>An. lesteri </italic>which was found feeding on humans and macaques but were negative by dissection although the macaques were heavily infected. Based on these findings, the chances of humans being infected with simian malaria were deemed remote. In the early days, perhaps the monkeys were not living in close association with humans. Currently with development and deforestation the macaques have come close to human habitation and those in the semi-urban areas were positive for malaria parasites.</p>", "<p>Long-tailed macaques are also found in close association with humans in the urban areas but fortunately those found in the urban areas are free of malaria parasites. This could be due to the absence of competent vectors that could transmit malaria parasites.</p>", "<p>The rain forests of Southeast Asia occupy hilly areas over parts of Indochina, Thailand, Myanmar, Indonesia and the Philippines and in most of these areas human cases of <italic>P. knowlesi </italic>have been reported [##REF##15663864##4##,##REF##16866152##5##,##REF##18439369##8##]. Natural hosts of <italic>P. knowlesi </italic>such as the long and pig-tailed macaques abound in this region and so does the <italic>An. leucosphyrus </italic>group of mosquitoes. Thus, in the future it will be of no surprise if more human cases of <italic>P. knowlesi </italic>malaria are reported in these areas, contrary to what was perceived by the earlier scientists.</p>", "<p>In Sarawak, Malaysian Borneo, <italic>An. latens </italic>has been incriminated as the vector [##REF##16725166##34##] and the biting ratio of monkey to human was 1:1.3 (25). While <italic>An</italic>. <italic>cracens </italic>biting ratio of monkey to humans was 1: 5.6. This shows that <italic>An. cracens </italic>prefer to bite humans compared to monkeys, and could probably explain for fewer cases in peninsular Malaysia. <italic>Anopheles kochi </italic>was found biting monkeys in monkey-baited traps but was not found biting humans. Thus, the possibility is there for it to maintain the infection in the natural hosts.</p>", "<p>From the current study, ninety seven percent of the macaques from Kuala Lipis were positive for malaria parasites and the presence of vectors in the area demonstrate that simian malaria in humans may pose to be a public health problem in the near future. Some human cases of P. <italic>knowlesi </italic>in Kuala Lipis were infected in the vicinity of their houses or in the fruit orchards. Sporadic collections of mosquitoes in the case areas revealed the presence of <italic>An. cracens </italic>and macaques were also sighted in those areas. A recent study in Thailand [##REF##18385364##35##] failed to detect <italic>P. knowlesi </italic>in macaque populations in the area where the first case was reported [##REF##15663864##4##].</p>" ]

[ "<title>Conclusion</title>", "<p>This study has established that <italic>An. cracens </italic>is the vector of <italic>P. knowlesi </italic>in Kuala Lipis, Pahang and cases of <italic>P. knowlesi </italic>have occurred in humans in areas where long tailed macaques have also been found infected with <italic>P. knowlesi</italic>. These findings are important for the planning and control of malaria strategies in the future, especially in the elimination of malaria.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Since a large focus of human infection with <italic>Plasmodium knowlesi</italic>, a simian malaria parasite naturally found in long-tailed and pig tailed macaques, was reported in Sarawak, Malaysian Borneo, it was pertinent to study the situation in peninsular Malaysia. A study was thus initiated to screen human cases of <italic>Plasmodium malariae </italic>using molecular techniques, to determine the presence of <italic>P. knowlesi </italic>in non- human primates and to elucidate its vectors.</p>", "<title>Methods</title>", "<p>Nested polymerase chain reaction (PCR) was used to identify all <italic>Plasmodium </italic>species present in the human blood samples sent to the Parasitology laboratory of Institute for Medical Research. At the same time, non-human primates were also screened for malaria parasites and nested PCR was carried out to determine the presence of <italic>P. knowlesi</italic>. Mosquitoes were collected from Pahang by human landing collection and monkey-baited-traps situated on three different levels. All mosquitoes were identified and salivary glands and midguts of anopheline mosquitoes were dissected to determine the presence of malaria parasites and nested PCR was carried out on positive glands. Sequencing of the csp genes were carried on <italic>P. knowlesi </italic>samples from humans, monkeys and mosquitoes, positive by PCR.</p>", "<title>Results and Discussion</title>", "<p><italic>Plasmodium knowlesi </italic>was detected in 77 (69.37%) of the 111 human samples, 10 (6.90%) of the 145 monkey blood and in 2 (1.7%) <italic>Anopheles cracens</italic>. Sequence of the csp gene clustered with other <italic>P. knowlesi </italic>isolates.</p>", "<title>Conclusion</title>", "<p>Human infection with <italic>Plasmodium knowlesi </italic>is occurring in most states of peninsular Malaysia. <italic>An. cracens </italic>is the main vector. Economic exploitation of the forest is perhaps bringing monkeys, mosquitoes and humans into increased contact. A single bite from a mosquito infected with <italic>P. knowlesi </italic>is sufficient to introduce the parasite to humans. Thus, this zoonotic transmission has to be considered in the future planning of malaria control.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>IV, NA, LHS conceived the study, IV, CHT were responsible for the preparation of the manuscript, IV, NAY, AIJ, YY, AAH, NA were responsible for field collection, supervision, identification and processing of mosquitoes and collection of blood from monkeys. IV, NAY, AIJ, CHT were responsible for the molecular work, CHT analysed sequence data. All authors have read and approved the manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The authors wish to thank Dr Rahimi Hassan and staff of Vector Borne Disease Control Program Kuala Lipis for the their help in collection of mosquitoes, Mr S Subramaniam Entomology Unit, IMR for his contribution in dissecting the mosquitoes, staff of Parasitology Unit IMR for help in field and laboratory work, Director General of Wild Life Department Malaysia for permission to trap monkeys, Head of Parasitology Unit for her support, Director General of Health Malaysia and Director of IMR for permission to publish. This project was supported by a grant from the National Institute of Health Malaysia \"06 CAM 04–06\"</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Map of Malaysia showing cases of <italic>P. knowlesi</italic> by PCR in P. Malaysia.</bold> Denominator indicates the total number of samples for each state.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Phylogenetic tree based on the non-repeat region of the circumsporozoite (<italic>csp</italic>) genes of malaria parasites produced by the neighbor-joining method.</bold> Figures on the branches are bootstrap percentages based on 1000 replicates and only those 70 and above shown. </p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Phylogenetic tree based on the non-repeat region of the circumsporozoite (<italic>csp</italic>) genes of malaria parasites produced by the Bayesian method.</bold> Figures on the branches are the posterior probabilities from the Bayesian analysis.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Results of blood samples of malaria obtained by microscopy and PCR</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"/><td align=\"center\" colspan=\"5\">Cases detected by microscopy</td><td align=\"left\"/></tr><tr><td/><td colspan=\"5\"><hr/></td><td/></tr><tr><td align=\"left\">PCR results</td><td align=\"left\">Pf</td><td align=\"left\">Pv</td><td align=\"left\">Pm</td><td align=\"left\">Pf+Pm</td><td align=\"left\">Pm+Pv</td><td align=\"left\">Cases detected by PCR</td></tr></thead><tbody><tr><td align=\"left\">Pf</td><td align=\"left\">4</td><td align=\"left\">1</td><td align=\"left\">1</td><td/><td/><td align=\"left\">6</td></tr><tr><td align=\"left\">Pv</td><td/><td align=\"left\">6</td><td align=\"left\">3</td><td align=\"left\">1</td><td align=\"left\">1</td><td align=\"left\">11</td></tr><tr><td align=\"left\">Pm</td><td/><td/><td align=\"left\">16</td><td/><td/><td align=\"left\">16</td></tr><tr><td align=\"left\">Pk</td><td align=\"left\">2</td><td align=\"left\">1</td><td align=\"left\">62</td><td/><td/><td align=\"left\">65</td></tr><tr><td align=\"left\">Pf+Pm</td><td/><td/><td align=\"left\">1</td><td/><td/><td align=\"left\">1</td></tr><tr><td align=\"left\">Pf+Pk</td><td/><td/><td align=\"left\">2</td><td/><td/><td align=\"left\">2</td></tr><tr><td align=\"left\">Pv+Pk</td><td/><td align=\"left\">1</td><td align=\"left\">5</td><td/><td align=\"left\">1</td><td align=\"left\">7</td></tr><tr><td align=\"left\">Pk+Pm</td><td/><td/><td align=\"left\">3</td><td/><td/><td align=\"left\">3</td></tr><tr><td align=\"left\">Total</td><td align=\"left\">6</td><td align=\"left\">9</td><td align=\"left\">93</td><td align=\"left\">1</td><td align=\"left\">2</td><td align=\"left\">111</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Mosquitoes collected from two sites in Kuala Lipis, Pahang from July to November 2007.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><italic>Anopheles </italic>species</td><td align=\"center\" colspan=\"2\">Serunai Mela</td><td align=\"left\">Sg Ular</td><td align=\"left\">Total (%)</td></tr><tr><td/><td colspan=\"4\"><hr/></td></tr><tr><td/><td align=\"left\">BLC</td><td align=\"left\">MBT</td><td/><td/></tr></thead><tbody><tr><td align=\"left\"><italic>An. aconitus</italic></td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">4</td><td align=\"left\">4 (1.2)</td></tr><tr><td align=\"left\"><italic>An. barbirostris </italic>gr</td><td align=\"left\">2</td><td align=\"left\">2</td><td align=\"left\">2</td><td align=\"left\">6 (1.8)</td></tr><tr><td align=\"left\"><italic>An. cracens</italic></td><td align=\"left\">107</td><td align=\"left\">19</td><td align=\"left\">85</td><td align=\"left\">211 (62.2)</td></tr><tr><td align=\"left\"><italic>An. hyrcanus </italic>gr</td><td align=\"left\">8</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">9 (2.7)</td></tr><tr><td align=\"left\"><italic>An. kochi</italic></td><td align=\"left\">1</td><td align=\"left\">15</td><td align=\"left\">0</td><td align=\"left\">16 (4.7)</td></tr><tr><td align=\"left\"><italic>An. maculatus</italic></td><td align=\"left\">4</td><td align=\"left\">0</td><td align=\"left\">67</td><td align=\"left\">71 (20.9)</td></tr><tr><td align=\"left\"><italic>An. phillippinensis</italic></td><td align=\"left\">3</td><td align=\"left\">0</td><td align=\"left\">3</td><td align=\"left\">6 (1.8)</td></tr><tr><td align=\"left\"><italic>An. pujutensis</italic></td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">1 (0.3)</td></tr><tr><td align=\"left\"><italic>An. separatus</italic></td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">5</td><td align=\"left\">5 (1.5)</td></tr><tr><td align=\"left\"><italic>An. tessellatus</italic></td><td align=\"left\">4</td><td align=\"left\">0</td><td align=\"left\">3</td><td align=\"left\">7 (2.1)</td></tr><tr><td align=\"left\"><italic>An. umbrosus</italic></td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">1 (0.3)</td></tr><tr><td align=\"left\"><italic>An. vagus</italic></td><td align=\"left\">1</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">2 (0.6)</td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\">Total</td><td align=\"left\">131</td><td align=\"left\">39</td><td align=\"left\">169</td><td align=\"left\">339</td></tr></tbody></table></table-wrap>" ]

[]

[]

[]

[]

[]

[]

[ "<table-wrap-foot><p>Pf = <italic>Plasmodium falciparum</italic>; Pv = <italic>Plasmodium vivax</italic>, Pm = <italic>Plasmodium malariae</italic>, Pk = <italic>Plasmodium knowlesi</italic></p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1756-3305-1-26-1\"/>", "<graphic xlink:href=\"1756-3305-1-26-2\"/>", "<graphic xlink:href=\"1756-3305-1-26-3\"/>" ]

[]

{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Ulcerative colitis (UC) is a disorder characterized by chronic mucosal inflammation of the large intestine. It is frequently associated with various extraintestinal manifestations. The inflammation may be limited to the rectum (proctitis), but mucosal lesions often continue more proximally (left-sided UC) or additionally embrace the transverse colon (extensive colitis) or the entire large bowel (pancolitis). The immune and cellular (non-immune) response is dysregulated in both the acute and the chronic phase of UC [##REF##9649475##1##,##REF##15790845##2##]. In Scandinavia, UC has been found to affect individuals of all ages, with an annual incidence of about 15 per 100 000 [##REF##1985033##3##,##REF##8726304##4##] and a prevalence of about 300 per 100 000 inhabitants [##REF##16702848##5##].</p>", "<p>The pathogenesis and pathophysiology of UC are still under investigation [##REF##17075348##6##]. We can tentatively say that the cause and onset of the disease is polygenic with environmental interaction; that is, there is a genetic predisposition [##REF##8841195##7##, ####REF##12512026##8##, ##REF##11181568##9####11181568##9##] in combination with eliciting environmental factors which may precipitate the phenotype of UC [##REF##9178675##10##]. In addition, interaction between the colonic epithelium and microbiological flora as well as a disintegrated mucosal barrier function may be important factors in the onset and development of UC [##REF##17075348##6##]. The use of microarray technique analyses on mucosal specimens obtained from both patients with established UC and controls has allowed identification of candidate genes, which are valuable in research on UC pathogenesis. However, these UC candidate genes must be carefully selected, since recent evaluations of microarray data have revealed considerable divergence after examination of similar tissues [##REF##14645736##11##, ####REF##14500831##12##, ##REF##11074008##13####11074008##13##]. Such divergent results are commonly presented in studies using pooled patient samples. In the present study however, the transcripts selected are based on our earlier individual whole-genome microarray screening and quantitative real-time PCR (RT-PCR) in patients with UC [##REF##16954802##14##], where five changed genes/transcripts were identified; aldolase B, elafin, MST-1, simNIPhom (similar to NIP homolog), and SLC6A14. The pathophysiological properties of SimNIPhom have not yet been clarified, but the other transcript products have potential importance in secretion [##REF##10446133##15##,##REF##955981##16##], anti-microbiological activity [##REF##10386612##17##], and cell-mediated immune response [##REF##17200145##18##].</p>", "<p>The primary aim of the present study was to define differences in the mucosal expression of five selected transcripts, retrieved from two different colonic locations in UC, by using a quantitative RT-PCR technique. We also aimed to evaluate the influence of ongoing anti-inflammatory treatment as well as the importance of the colonic UC extension and the severity class.</p>" ]

[ "<title>Methods</title>", "<title>Patients and tissue specimens</title>", "<p>Before the colonoscopy procedure, consecutive male and female subjects (UC patients and controls, &gt;18 y) were recruited to the present study. The UC diagnosis was based on the medical history, endoscopic findings, histological examination, laboratory tests, and the clinical disease presentation. The extent of UC and the clinical activity were classified in accordance with the Montreal Classification [##UREF##0##19##]. In brief, the colonic inflammatory involvement is defined as extension (letter E) combined with a number between 1–3 (E1 denotes proctitis, E2 left-sided UC, and E3 extensive colitis). In addition, the clinical severity grade (letter S) is defined. The S-score ranges from clinical remission (S0) to severe UC (S3). Mucosal biopsies were obtained from the rectum (10–15 cm proximal from anal verge) and caecum from all participants.</p>", "<p>No uses of corticosteroids, aminosalicylates, or immunosuppressants were registered in the control group, while 9 patients (18%) in the UC group were treated systemically with corticosteroids (prednisolone 10–20 mg). Twenty-seven UC patients (55%) were treated systemically with aminosalicylates (mesalazine 1.600–2.400 mg/24 h). Among these, two patients had additional ongoing therapy with aminosalicylate (mesalazine 500 mg QD) enemas and three patients were treated with corticosteroid enemas (prednisolone 37.5 mg QD or BID). Seven patients had stable (&gt;3 months) ongoing immunosuppressant treatment (Azathioprine, 1.8–2.2 mg/kg bw).</p>", "<p>The remaining demographic and clinical data are presented in Figure ##FIG##0##1##.</p>", "<title>RNA isolation</title>", "<p>The biopsy specimens were immediately stored in RNA-later solution for isolation of RNA. The RNA-later-preserved biopsies were homogenized in a lysis buffer from the GenElute Mammalian Total RNA kit (Sigma, St. Louis, MO.) and total RNA was isolated according to the manufacturer's instructions. The RNA concentration was measured spectrophotometrically.</p>", "<title>Quantification by real-time polymerase chain reaction (RT-PCR)</title>", "<p>Two μg of total RNA from each sample were converted into cDNA. The cDNA synthesis was performed as described previously [##REF##15013762##20##]. Oligonucleotide primers purchased from MWG-BIOTECH AG (Ebersberg, Germany) were used for the relative quantification (ABI-7500 system, software version 1.3) (Table ##TAB##0##1##). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene in all experiments. The expression level in each sample was compared with a calibrator by using the ΔΔC_T-formula (ΔC_{T(calibrator)}- ΔC_T(sample)).</p>", "<title>Statistical analysis</title>", "<p>Descriptive statistics and the Wilcoxon signed rank test (SAS, Statview^®) were used. Median values are presented.</p>", "<title>Ethics</title>", "<p>The study was approved by the local research ethical committee. All patients were given oral and written information before entering the study. Informed consent was obtained from all patients and controls.</p>" ]

[ "<title>Results</title>", "<p>The mean duration of UC was 9.3 years (proctitis 9.6 years, left-sided colitis 9.5 years and extensive colitis/pancolitis 9.2 years). Neither age nor gender was matched between the UC group and the control group.</p>", "<p>In order to evaluate any differences in transcript expressions within the control group (n = 67) with respect to background diagnoses (anaemia, diverticulosis, irritable bowel disease and polyposis), statistical analysis of each background diagnosis were compared to the remaining group of controls. No significant differences (p &gt; 0.05) were detected for any of these diagnoses.</p>", "<p>Significantly higher transcript expressions of aldolase B and SimNIPhom and significantly lower transcript expressions of elafin, MST-1, and SLC6A14 were found in caecal biopsies in comparison to rectal biopsies from the control group (Figure ##FIG##1##2##). The only significant differences between rectal and caecal transcript expressions in UC patients were the decreased transcript expressions of elafin and SLC6A14 in caecal biopsies in comparison to rectal biopsies.</p>", "<p>Comparison of rectal biopsies from controls (n = 67) with rectal biopsies from UC patients with inflammatory activity in accordance with Montreal classifications S1–S3 (n = 28) showed significant elevations (p &lt; 0.05) in UC patients of all transcript expressions with the exception of MST-1, which showed significantly (p &lt; 0.05) decreased expression in UC patients. The same analysis of caecal biopsies from controls and patients with S1–S3 UC (n = 16) showed significantly elevated transcript expressions of aldolase B and SLC6A14 only. Distal biopsies from controls, compared with UC patients without inflammatory activity (S0), showed increased transcript expression in aldolase B only (median -1.62 vs. 1.0, p = 0,012). All other transcript analyses from both locations showed no significant differences (p &gt; 0.05).</p>", "<p>All transcript analysis with respect to UC extension showed that left-sided (E2) and total colitis (E3) differed significantly from controls (p &lt; 0.05); this was not the case for proctitis (E1).</p>", "<p>Statistical analysis concerning the influence of anti-inflammatory treatment on the transcript expressions within the UC cohort showed no statistical differences (p &gt; 0.05) when comparing UC patients with ongoing corticosteroids (n = 9) or azathioprine (n = 7) respectively with the remaining UC patients. However, the 27 patients treated with mesalazine show a significant increase in aldolase B (median 0.48 vs. 3.02, p = 0.035) in comparison to the remaining UC patients.</p>" ]

[ "<title>Discussion</title>", "<p>Genetic predisposition, psychological stress, nutritional and environmental influences, intestinal pathogens and disturbed intestinal barrier function have all been proposeas pathogenetic factors in UC [##REF##17075348##6##]. However, current knowledge about the pathogenesis and pathophysiology of UC [##REF##16819502##21##] is incomplete. Moreover, with the exception of a few general serological inflammatory activity biomarkers, even less information is available regarding mucosa-associated transcript changes and their potential pathogenetic and pathophysiological role in UC [##REF##17217454##22##]. This lack of knowledge may sometimes lead to uncertainty in diagnosis, judgement of prognosis and clinical management of UC patients.</p>", "<p>On the basis of exsisting knowledge of the biological functions of the transcripts investigated in this study, it is reasonable to believe that the demonstrated alterations might be related to predisposition and/or the pathophysiological response in UC.</p>", "<p>It is intriguing that aldolase B and SLC6A14 were up-regulated in rectal as well as caecal mucosa in UC compared to controls. Aldolase B is known to be mainly expressed in the intestinal villus cells and it has a central role in the glycolytic pathway. It also participates in regulation of intestinal secretion [##REF##955981##16##]. Since SLC6A14 is also known to encode a Na⁺/Cl^-driven amino acid transporter B(0+) [##REF##10446133##15##], the up-regulation of aldolase B and SLC6A14 might be a common pathophysiological response, aimed at counteracting the exaggerated loss of fluid seen in UC. Theoretically, the up-regulation of these two transcripts could be a local response to the increased feacal/fluid stream, where bioactive molecules comprise ability to regulate transcript expression. Additionally, since the inflammatory activity and load of fluid over time is usually most pronounced in the distal part of the colon, the registered changes in aldolase B and SLC6A14 may reflect long-term inflammatory activity. Our finding that aldolase B from distal biopsies is significantly elevated during the remission phase (S0) indicates that the regulation of this transcript not only is secondary to the inflammatory activity.</p>", "<p>The involvement of the microflora and its importance in the onset, development and preservation of UC has been discussed [##REF##17075348##6##]. The SLC6A14 transcript expression is therefore also interesting in this respect, since it is involved in the host's antibacterial response [##REF##16819502##21##]. In addition, the defensine-like epithelium associated antimicrobial molecule elafin, antagonizing human neutrophil elastase preventing tissue injury via inhibition of excessive release of proteolytic enzymes from inflammatory cells is interesting in this context [##REF##17200145##18##]. The present results confirm an elafin transcript enhancement in caecal as well as rectal biopsies from patients with UC. Thus, the combined elevation of elafin and SLC6A14 may contribute to an amplified defence reaction aimed to restoration and maintenance of the mucosal integrity. This finding may indicate a pathogenetic role of the microflora in UC.</p>", "<p>MST-1 was included in the present study due to its alterations in UC, as shown in our previous experiment [##REF##16954802##14##], although it was excluded from that publication due to deviation in its control group. MST-1 is known to be capable of inhibiting cell-mediated immune responses via down-regulation of IL-12 production and subsequently inhibition of macrophage activation [##REF##14734766##23##]. Consequently, the observed down-regulation of MST-1 in rectal specimens may contribute to an enhanced cellular immune response in UC. A reasonable explanation of the concomitant decreased MST-1 transcript expression and increased aldolase B, SLC6A14, and elafin transcript expression is that the changes describe a pathophysiological response to a more pronounced inflammatory and, possibly, an exaggerated microbial load in at least the rectal part of the colon mucosa.</p>", "<p>The fifth identified significantly up-regulated transcript (in rectum only) SimNIPhom (similar to the numb-interacting homolog), encodes a hypothetical protein, at present of unknown pathophysiological importance.</p>", "<p>Our results supports that specimens from the rectal mucosa are more suitable for further analysis of the selected transcripts, due to the more predictable inflammatory involvement in the rectum and its availability for direct inspection and easy biopsy sampling.</p>", "<p>Our data can not answer whether the observed changes in expressions of the five selected transcripts may be present in e.g. other inflammatory, infectious or autoimmune conditions since this study uniquely focused on UC patients compared to non-inflamed controls.</p>" ]

[ "<title>Conclusion</title>", "<p>The five changed gene transcript expressions have relation to UC, its extension and clinical severity. Whether the presented results will contain discriminative potential of importance for the medical care of patients with UC in future clinical practice remains to be elucidated.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The cause and pathophysiology of ulcerative colitis are both mainly unknown. We have previously used whole-genome microarray technique on biopsies obtained from patients with ulcerative colitis to identifiy 5 changed mucosal transcripts. The aim of this study was to compare mucosal expressions of these five transcripts in ulcerative colitis patients vs. controls, along with the transcript expression in relation to the clinical ulcerative colitis status.</p>", "<title>Methods</title>", "<p>Colonic mucosal specimens from rectum and caecum were taken at ambulatory colonoscopy from ulcerative colitis patients (<italic>n </italic>= 49) with defined inflammatory activity and disease extension, and from controls (<italic>n </italic>= 67) without inflammatory bowel disease. The five mucosal transcripts aldolase B, elafin, MST-1, simNIPhom and SLC6A14 were analyzed using quantitative real-time PCR.</p>", "<title>Results</title>", "<p>Significant transcript differences in the rectal mucosa for all five transcripts were demonstrated in ulcerative colitis patients compared to controls. The grade of transcript expression was related to the clinical disease activity.</p>", "<title>Conclusion</title>", "<p>The five gene transcripts were changed in patients with ulcerative colitis, and were related to the disease activity. The known biological function of some of the transcripts may contribute to the inflammatory features and indicate a possible role of microbes in ulcerative colitis. The findings may also contribute to our pathophysiological understanding of ulcerative colitis.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>AE designed the study, preformed sampling of biopsies, analyzed the data, and prepared the manuscript. C-FF analyzed the data. AL preformed sampling of biopsies. EK coordinated the study. SL designed the study, analyzed the data and prepared the manuscript. All authors read and approved final manuscript.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1471-230X/8/34/prepub\"/></p>" ]

[ "<title>Acknowledgements</title>", "<p>This work was supported by grants from the Swedish federal government under the LUA/ALF agreement, (grant no. 7157). The RT-PCR analysis was performed by Index Pharmaceuticals, Stockholm, Sweden. We thank Professor Sven Wallerstedt for critical reading of the manuscript.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Demographic and clinical data from the control and UC group respectively.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>RT-PCR result (ΔΔCt(=ΔCt_target-ΔCt_calibrator)) for controls (filled dots) and UC patients (▲) presented as median values and 25^thand 75^thpercentil (bars).</bold> * = <italic>p </italic>&lt; 0.05, ** = <italic>p </italic>&lt; 0.01, *** = <italic>p </italic>&lt; 0.001, n.s. = non significant. Reference gene: GAPDH.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Primers used for real-time polymerase chain reaction.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Gene</bold></td><td align=\"left\"><bold>Forward primer</bold></td><td align=\"left\"><bold>Reverse primer</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Aldolase B</bold></td><td align=\"left\">5'-aaggctgcaaacaaggaggcaacc-3'</td><td align=\"left\">5'-tgaagagcgactgggtggaagcag-3'</td></tr><tr><td align=\"left\"><bold>Elafin</bold></td><td align=\"left\">5'-tgtgaaggctcttgcgggatgg-3'</td><td align=\"left\">5'-agggcagcagggacttaggaccag-3'</td></tr><tr><td align=\"left\"><bold>SimNIPhom</bold></td><td align=\"left\">5'-cgccagacagctaggggagtgaag-3'</td><td align=\"left\">5'-gcatttctgatattttgtgaccacgcac-3'</td></tr><tr><td align=\"left\"><bold>SLC6A14</bold></td><td align=\"left\">5'-gctgcttggttttgtttctccttggtc-3'</td><td align=\"left\">5'-gcaattaaaatgccccatccagcac-3'</td></tr><tr><td align=\"left\"><bold>MST-1</bold></td><td align=\"left\">5'-aaccaggagtgtaacatcaagcaccgag-3'</td><td align=\"left\">5'-cagttgtgggtaaagcaggcaagtgg-3'</td></tr><tr><td align=\"left\"><bold>GAPDH</bold></td><td align=\"left\">5'-gagcaccaggtggtctcctctgacttc-3'</td><td align=\"left\">5'-gccaaattcgttgtcataccaggaaatg-3'</td></tr></tbody></table></table-wrap>" ]

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[ "<graphic xlink:href=\"1471-230X-8-34-1\"/>", "<graphic xlink:href=\"1471-230X-8-34-2\"/>" ]

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[ "<title>Background</title>", "<p>Cerebral infarction induced by cardiogenic embolism is observed in about 20% of stroke patients. Of those patients, atrial fibrillation is responsible for over 50% of the cardiogenic emboli, while myxomas are observed in only 0.5% of emboli [##REF##7477198##1##]. Atrial myxomas are a very rare source of cardiogenic embolism. Although they are usually asymptomatic, myxomas can develop lethal complications without warning because of their ability to embolize. This report describes a patient who presented with a left-sided hemiparesis. The cause of the patient's right cerebral infarction was a left atrial myxoma which was detected by transesophageal echocardiography (TEE).</p>" ]

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[ "<title>Discussion</title>", "<p>A myxoma is the most common primary tumor of the heart. Primary cardiac neoplasms are rare, with incidences ranging from 0.001–0.3% in autopsy series. Benign tumors account for 75% of primary neoplasms and malignant tumors account for 25%. Myxomas compromise 30–50% of primary cardiac tumors [##REF##7477198##1##]. The majority of myxomas are sporadic, but 7% of patients have a genetic mutation that is inherited in an autosomal dominant manner. Familial myxoma has been well-described as the Carney complex, characterized by hyperpigmentation, cutaneous myxomas, and endocrine adenomas. This tumor is three times more common in females than in males and generally occurs between the third and sixth decades, with an average age of presentation at 43 years [##REF##12493135##2##].</p>", "<p>Myxomas originate from the mesenchymal cells of the septal endocardium. They are gelatinous with a smooth or lobulated surface and are usually white, yellow, or brown in color. They can present as villous, papillary, sessile, or pedunculated-type growths. Approximately one-half of the cases of myxomas are pedunculated tumors, and these are irregular and more likely to result in emboli because of the mobility of this type of tumor [##REF##17059948##3##]. Sixty to 75% of cardiac myxomas develop in the left atrium, most of which are from the atrial septum near the fossa ovalis. Most other myxomas develop in the right atrium. Fewer than 20 cases of myxomas arising from the right or left ventricle have been reported [##REF##16327954##4##]. Myxomas produce a vascular endothelial growth factor that stimulates angiogenesis and tumor growth and an increased expression of interleukin-6 [##REF##17024671##5##].</p>", "<p>A myxoma may be completely asymptomatic until it grows large enough to obstruct the mitral or tricuspid valve or fragments that give rise to emboli. Because they are intravascular and friable, myxomas account for most cases of tumor emboli [##REF##7477198##1##]. Embolism occurs in about 30–40% of patients with myxomas. The site of embolism is dependent upon the location of the myxoma (left or right atrium) and the presence of an intracardiac shunt. This is not surprising, given the degree of motion that can be seen on echocardiography and angiography, as the myxoma swings on a small pedicle with each cardiac contraction [##REF##16055401##6##]. Intermittent acute obstruction of the mitral orifice has been reported to produce syncope and even sudden death. Some myxomas produce generalized symptoms resembling an autoimmune disorder, including fever, weight loss, digital clubbing, myalgias, and arthralgias. These patients may have an immune reaction to the neoplasm, as elevated levels of interleukin-6 and elevated levels of antimyocardial antibodies have been described [##REF##17024671##5##].</p>", "<p>The emboli that occur are either a tumor fragment that is released from the myxoma or a blood clot that is formed on the surface of the myxoma. These resulting emboli can result in infarction, as occurred in our patient. More precisely, it has been reported that 45% of patients with myxomas have neurologic manifestations resulting from embolization [##REF##454248##7##]. This embolization includes pulmonary embolism, myocardial infarction, mesenteric infarction, retinal artery occlusion, spinal cord ischemia, and stroke [##REF##12493135##2##, ####REF##17059948##3##, ##REF##16327954##4##, ##REF##17024671##5##, ##REF##16055401##6##, ##REF##454248##7####454248##7##]. Right-sided myxomas cause pulmonary embolization, but left-sided myxomas usually cause systemic embolization. Ischemic infarction of the brain is responsible for the majority of cases of systemic embolization. The MCA is frequently affected by this type of infarction because of the MCA's dominant blood flow [##REF##1553039##8##]. In cases in which frontal or parietal infarction is suspected in a patient with myxoma, the MCA territory should be thoroughly investigated.</p>", "<p>Usually, the diagnosis is readily established by two-dimensional echocardiography, which is considered the gold standard. TEE may be useful when transthoracic findings are equivocal or confusing. MRI has been of value in diagnosis, providing excellent cardiac definition. Cardiac catheterization is not necessary in the majority of cases, but may be necessary when other cardiac disease is suspected or if other diagnostic studies are equivocal. TTE has a sensitivity of 95% and the sensitivity is nearly 100% [##REF##7486462##9##]. Whether performing TTE or TEE, echocardiography is able to evaluate the location, size, shape, and movement of myxomas. TTE or TEE may also show other cardioembolic sources, such as a patent foramen ovale, mitral valve calcification, or aortic atherosclerosis. Prompt resection is required after the diagnosis, even in asymptomatic patients. It is important that myxomas should be excised with negative margins because any remnant can aggravate an infarction. The recurrence rate is 1~3% after surgery [##REF##14609975##10##]. Therefore, all patients with myxomas are recommended to undergo long-term follow-up with echocardiography. This patient described herein, who was morbidly obese with a BMI of 48.8 kg/m², is representative of a growing medical problem in the United States. With stroke patients, physicians use TTE routinely when they search for cardiogenic embolic sources. But, in using TTE exclusively, myxomas in the obese will frequently be missed.</p>" ]

[ "<title>Conclusion</title>", "<p>This case demonstrates the importance of investigating the possibility of cardiogenic source in stroke, as our patient developed cerebral infarction that was caused by an atrial myxoma. It is important that clinicians consider using echocardiography in stroke patients. Treating the atrial myxoma can prevent a cardioembolic stroke and its complications. In conclusion, TEE, as compared to TTE, has many more advantages when physicians search for a cardiogenic embolic source in obese stroke patients. In addition, because obesity has sharply increased in the United States, the importance and use of TEE will increase over time as physicians encounter obese patients with cardiogenic emboli.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>A myxoma is the most common primary tumor of the heart. It has been reported as the source of a cardiogenic embolism. Therefore, it is important for clinicians to detect the myxoma early via echocardiography to prevent complications, such as syncope, sudden death, and cerebral embolic ischemic stroke. This report presents the case of a 54-year-old female whose clinical manifestation of atrial myxoma was an ischemic stroke. Atrial myxoma was later confirmed as the cause of her symptoms via transesophageal echocardiography.</p>" ]

[ "<title>Case presentation</title>", "<p>A 54-year-old Caucasian female presented to the emergency room with a 4 day history of left-sided weakness. The patient stated that she was at home when she suddenly felt a sharp pain in her left hand that radiated to her neck. She then lost consciousness and collapsed to the floor. It was not until 4 days later that her friend convinced her to go to the hospital. The patient had a medical history of longstanding hypertension, obstructive sleep apnea, hypothyroidism, and depression. She had been a smoker for 25 years. Her mother also had hypertension and her father had a myocardial infarction at the age of 56. Her height was 161.5 cm and her weight was 127.3 kg. The patient's vital signs were as follows: blood pressure, 153/104 mm Hg; heart rate, 101 beats/minute; respiratory rate, 18/minute; and body temperature, 36.6°C (98.0°F). She was alert and oriented, had left facial paralysis, slight dysarthria and right-sided tongue deviation, but no dysphasia. On cardiac examination, the carotid impulse was normal without a bruit. Her heart had a regular rate and rhythm, and there were normal S₁and S₂heart sounds without murmurs. An EKG showed a normal sinus rhythm. Range of motion was limited to 30° for the left upper and lower extremities. She had 1/5 motor strength on the left side, but 5/5 motor strength on the right side. The deep tendon reflexes were 2+ bilaterally. Her sensation was intact bilaterally. The Babinski and Hoffman signs were both negative. All her laboratory results were normal. A chest X-ray showed a normal cardiac silhouette with no signs of pulmonary edema. A non-contrast computed tomography (CT) scan of the brain revealed multiple low density areas in the right frontal and parietal lobes. Our stroke team started her on intravenous heparin. Metoprolol (Toprol-XL) and furosemide (Lasix) were administered to stabilize her blood pressure. The following day, magnetic resonance imaging (MRI) of the brain demonstrated an acute infarction in the distribution of the right middle cerebral artery (MCA; Figure ##FIG##0##1##). On the third day of hospitalization, the patient underwent a TEE. A TEE was chosen since the less invasive transthoracic echocardiography (TTE) showed negative imaging for a cardiogenic embolic source. In addition, the patient was obese and the TTE did not provide a comprehensive image. The TEE identified a 4.3 cm × 1.3 cm mass in the left atrium. A cardiac catheterization showed no significant coronary artery disease. The patient was thus diagnosed with a right MCA ischemic infarction and a left atrial myxoma. On the 13^thhospital day, the patient underwent successful surgical excision of the myxoma (Figure ##FIG##1##2##). The biopsy confirmed the diagnosis of myxoma. The patient recovered without any complications and was discharged on the 20^thday of hospitalization.</p>", "<title>Abbreviations</title>", "<p>MCA: Middle Cerebral ArteryP; CT: Computed Tomography; MRI: Magnetic Resonance Imaging; TEE: Transesophageal Echocardiography; TTE: Transthoracic Echocardiography.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>MY and DG were involved in the clinical assessment and writing the case report. All authors read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Full written consent was received for the manuscript to be published.</p>" ]

[ "<title>Acknowledgements</title>", "<p>I would like to extend my thanks to Michael A. Wait, M.D., the surgeon who excised the myxoma. I would also like to express my appreciation to Craig Litz, M.D., the pathologist who performed the analysis of the myxoma biopsy.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Axial T₁-weighted MRI shows acute infarcts in the right caudate body, and the frontal and parietal lobes.</bold> (A), The diffusion weighted image (DWI) presents an abnormal signal corresponding to the restricted diffusion in the right hemisphere. (B), MRA shows no aneurysm, stenosis, or abnormal flow in the visualized vessels of the Circle of Willis, the carotid arteries, and the vertebral arteries. (C).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Transesophageal echocardiography shows a mobile mass in the left atrium, which does not obstruct the mitral valve</bold>. (A), After performing cardiopulmonary bypass, the retractor allows visualization of the left atrium, and the atrium is then opened by a blade. The myxoma is attached from the atrial septum. (B), LA myxoma: tan and jelly-like tissue with an aggregate measurement of 6 cm × 1.5 cm. (C).</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1757-1626-1-96-1\"/>", "<graphic xlink:href=\"1757-1626-1-96-2\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Gastritis Cystica Profunda is a well recognized entity occurring several years after previous gastric surgery [##REF##1139487##1##]. It is suggested that ischaemia and chronic inflammation along with the effects of surgery and the presence of suture material may have a role in the pathogenesis of GCP [##REF##7286916##2##]. A correlation seems to exist between gastritis cystica profunda and gastric ulcer [##REF##18291267##3##].</p>" ]

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[ "<title>Discussion</title>", "<p>Gastritis cystica profunda (GCP) is a condition characterized by benign, cystic down growth of gastric glands into the sub mucosa of the stomach [##REF##16564886##4##]. This is a well described entity following previous gastric surgery [##REF##1139487##1##]. The pathogenesis of GCP is probably due to chronic ischaemia and inflammation occurring at the suture site of previous gastroenterostomy [##REF##7286916##2##]. Disruption of integrity of muscularis mucosa causes the migration of epithelial contents into the sub mucosa with subsequent atrophic gastritis, intestinal metaplasia and cystic dilatation of gastric glands [##REF##7286916##2##]. This is a precursor of cancer of the stomach [##REF##1139487##1##]. Gastritis cystica profunda has been described in a patient with a history of gastric ulcer that was treated with H₂blockers [##REF##428290##5##]. This indicates that GCP can occur after exposure of gastric mucosa to chronic mucosal inflammation, which subsequently leads to hyperplastic and metaplastic changes and increased risk for progression towards carcinoma</p>", "<p>Littler and Gleibermann proposed that mucosal prolapse and subsequent inflammation play a role in development of GCP [##REF##5007382##6##]. Histologically two stages are evident:</p>", "<p>Stage1: with cystic glands limited to mucosal layer (gastritis cystica superficialis).</p>", "<p>Stage 2: with gastric glands spreading into the sub mucosa (gastritis cystica profunda).</p>", "<p>Gastritis cystica superficialis shows wide cystic glands superficial to muscularis mucosae lined by both columnar and flattened mucous producing epithelium with basophilic cytoplasm [##REF##5007382##6##]. The characteristic feature is the presence of wide cystic glands of pyloric type, together with epithelial lining in the cysts identical to that of crypts. The muscularis mucosae is thickened and further penetration of cystic glands into sub mucosal layer gives them the name gastritis cystica profunda [##REF##5007382##6##], and not dissimilar to colitis cystica profunda where in dilated cystic glands are noted in the bowel wall and this condition has a strong association with inflammatory bowel disease [##REF##8313829##7##].</p>", "<p>In their series of 18 patients with GCP, Franzin <italic>et al</italic>. reported that the interval from gastric surgery to the formation of GCP ranged from 3 to 40 years (mean 16.2) and the risk increase with time [##REF##3984043##8##]. In our case report the patient had gastric surgery for gastric ulcer over 28 years ago and presented with severe upper GI bleeding from GCP. There are only three previous cases reported where a patient underwent previous gastric surgery and years later developed haemorrhage from GCP near the anastomotic site [##REF##16564886##4##].</p>" ]

[ "<title>Conclusion</title>", "<p>We suggest that patients who are diagnosed with gastritis cystica profunda should be regularly followed up as this is a premalignant condition.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Gastritis Cystica Profunda is a well recognized entity which may occur several years after previous gastric surgery. This is a premalignant condition and may lead on to carcinoma of the stomach.</p>", "<title>Case presentation</title>", "<p>We report a case of a 50-year-old man with epigastric pain and haematemesis. 28 years ago he had undergone partial gastrectomy and gastroenterostomy for benign gastric ulcer. An Upper gastrointestinal endoscopy showed a possible bleeding vessel on the anterior wall lesser curve of the stomach. The lesion was injected with adrenaline 1 in 100,000. In spite of the intervention he continued to have haemetemesis with significant haemodynamic impairment. At exploratory laparotomy, an oedematous ridge on the posterior wall with a bleeding point on the posterior gastric wall. Histology showed features consistent with gastritis cystica profunda. He made an excellent post-operative recovery.</p>", "<title>Conclusion</title>", "<p>We suggest that patients who are diagnosed with gastritis cystica profunda should be regularly followed up as this is a premalignant condition.</p>" ]

[ "<title>Case presentation</title>", "<p>A 50-year-old man presented as an emergency with epigastric pain and haematemesis. He was continuously vomiting out copious amount of fresh blood. He had a history of heavy alcohol intake and used to consume 10 units of alcohol per day and smoked 20 cigarettes/day. The patient had undergone partial gastrectomy and gastroenterostomy for benign gastric ulcer 28 years ago.</p>", "<p>General examination revealed anaemia with no jaundice, clubbing or lymphadenopathy. The pulse rate was 108 beats per minute and blood pressure was 80/50 mm Hg. His laboratory tests revealed Hb 8.9 g/dL and MCV 80.2. Chest and abdominal x-rays were normal. He was transfused four units of packed red blood cells. An urgent upper gastrointestinal endoscopy revealed large amount of fresh blood and clots in the fundus of stomach. A possible bleeding vessel was identified on the anterior wall lesser curve high in the fundus. The lesion was injected with adrenaline 1 in 100,000. In spite of the intervention he continued to have haemetemesis with significant haemodynamic impairment. The patient underwent an exploratory laparotomy which revealed an oedematous ridge on the posterior wall with a bleeding point on the posterior gastric wall.</p>", "<p>This was overrun with a Vicryl 2/0 stitch which controlled the bleeding. A biopsy of the oedematous ridge was obtained.</p>", "<p>Histology of the oedematous ridge revealed partly duodenal mucosa and partly a mixture of body and antral-type gastric mucosa, the latter showing areas of intestinal metaplasia. There was widespread disruption of the muscularis mucosa. The sub mucosa contained numerous glandular structures, lined mainly by antral-type gastric mucosa, showing marked cystic dilatation. These findings were consistent with gastritis cystica profunda (Fig ##FIG##0##1##).</p>", "<p>The patient was discharged from hospital in ten days without any post-operative complications. Arrangements were made for him to be followed up as an outpatient. He was also advised to stop smoking and consuming alcohol and was referred to support groups.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>VI helped in acquisition of data and preparation of the first draft, IHM was responsible for conception of the idea, overall preparation and revision of the manuscript, PJM was responsible for management of the patient and revising the manuscript for important intellectual content. All authors read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The authors would like to thank Carol Hunt, Pathologist at Scunthorpe Distict General Hospital for her assistance with histopathology slides.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Histology showing features of gastritis cystica profunda: Widespread disruption of the muscularis mucosa</bold>. The sub mucosa contained numerous glandular structures, lined mainly by antral-type gastric mucosa, showing marked cystic dilatation.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1757-1626-1-85-1\"/>" ]

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{ "acronym": [], "definition": [] }

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[ "<title>Discussion</title>", "<p>The kidneys in children are proportionally larger and have much less perirenal fat and costal protection than in adults which predisposes them to injury after blunt abdominal trauma (10% of cases) [##REF##15351596##1##,##REF##11490296##2##]. Prompt attention to these injuries is vital to allow for optimal recovery and to minimise long term sequelae. The management of renal trauma ranges from an emergency laparotomy for haemodynamic compromise to observation without intervention in minor lacerations. As with all solid organ injuries in children there has been a shift to conservative management where possible [##REF##15351596##1##,##REF##11490296##2##]. Increasingly minimally invasive techniques are being adopted to manage significant renal trauma where there is no haemodynamic compromise [##REF##11490296##2##, ####REF##17499798##3##, ##REF##9895260##4##, ##REF##17085141##5####17085141##5##]. This approach results in a lower incidence of nephrectomy and has few long term complications.</p>", "<p>However the management of grade IV renal injury in children is controversial, particularly with regard to the management of urinary extravasation. Grade IV trauma is defined as parenchymal laceration extending through the corticomedullary junction and into the collecting system [##REF##11988661##6##]. This results in urinary extravasation with the potential for large urinomas. Traditionally these injuries have been treated aggressively with open surgery with the justification that urinomas may lead to perirenal fibrosis with complications of obstruction, infection and hypertension. However much of this evidence is from the adult population and is not necessarily applicable to paediatric practice [##REF##11988661##6##]. The options for treating urinomas are; open drainage, with or without surgical repair, ureteric stents, percutaneous drains or just observation.</p>", "<p>Grade 1V renal injuries with complete fracture and separation of the poles but with intact blood supply constitutes a special group with a decreased likelihood of spontaneous resolution and where early intervention may be necessary [##REF##15351596##1##]. Endourological stenting or percutaneous drainage and failing this, open surgery, are options described in the management these patients. Endourological stenting on its own has not gained widespread popularity because stent calibre is not thought to be sufficient for optimal drainage [##REF##15351596##1##].</p>", "<p>There is currently no consensus on the optimal timing of these interventions. However from the experience of this case where a policy of early aggressive management with a combination of internal and external drainage was successfully utilised would lead us to support this approach to improve renal salvage. It would appear that reconstitution of the distracted but viable renal poles into a solitary functioning unit is possible provided the urinoma is adequately managed. Successful drainage also ensured that there was no significant residual scarring on late DMSA scanning.</p>", "<p>The expectant management of isolated high grade renal injuries with complete fracture in the haemodynamically stable patient is evolving and both internal and percutaneous drainage are crucial to the success of this approach. It seems the advances in interventional radiology are facilitating a less invasive approach to the management of these significant injuries in the paediatric population. The experience of this case reinforces a strategy which is leading to kidney salvage with minimal complications and is advocated.</p>" ]

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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>The expectant management of high grade renal injuries in hemodynamically stable children has gained increasing acceptance amongst paediatric surgeons. However, patients with grade 1V injury with complete renal transection have been identified as a subgroup with a poor outcome that may benefit from early operative intervention.</p>", "<p>Interestingly, both internal and external drainage have been independently utilised as part of the expectant approach. The former is more widely practiced and was first suggested by Haas et al who used it successfully in 5 patients with grade 1V renal trauma. Yet to be clearly established in this context is the value and timing of external drainage, particularly, when used in combination with internal stenting.</p>", "<p>Described is a child with complete renal transaction who was successfully managed with a combination of internal and external drainage.</p>" ]

[ "<title>Case presentation</title>", "<p>A 5 year old Caucasian male patient was admitted to a tertiary paediatric surgical centre within 1 hour of coming off a quadbike, sustaining blunt trauma to the right chest and the right upper quadrant of abdomen. Initial assessment confirmed hypovolaemic shock which responded to aggressive resuscitation as well as a tender right flank with macroscopic haematuria. Once stabilised a Computarised Tomography (CT) scan and IVP were performed which confirmed contusion of the right lower lung, fracture of several overlying ribs and complete right renal fracture with distraction of upper and lower poles (Grade1V), (Figure ##FIG##0##1##). Also documented were contrast extravasation from the pelvico-calyceal system and a substantial perinephric haematoma. Significantly, some contrast was noted in the distal right ureter confirming ipsilateral pelvico-ureteric continuity.</p>", "<p>The patient was subsequently admitted to the intensive care unit where he was managed with bed rest, broad spectrum antibiotic cover and regular haemoglobin checks with transfusion top ups as necessary.</p>", "<p>Over the next day the patient developed an ileus with progressive abdominal distended and an ultrasound scan was performed. This confirmed an expanding right perinephric collection which was managed by sonar guided placement of a percutaneous pigtail catheter – maintained on free drainage.</p>", "<p>Twenty four hours later with worsening abdominal distension and high output drainage from the pigtail catheter, the child was taken to theatre where under a general anaesthetic and fluoroscopic guidance a 4.5 French double J stent was passed retrogradely into the right renal pelvis. (Fig ##FIG##1##2##) This had the immediate effect of diverting urine from the pigtail catheter whose output dropped significantly. Macroscopic haematuria and the ileus improved gradually over the next 10 days allowing for the resumption of oral feeding and withdrawal of total parental nutrition.</p>", "<p>The pigtail catheter was removed on cessation of urine drainage at 3 weeks. A duplex scan at this point demonstrated resolution of the perinephric collection with greater approximation of the renal poles. A repeat ultrasound scan at 5 weeks with the double J stent still in situ showed radiological evidence of a reconstituted kidney. The patient was discharged at this stage and readmitted 3 weeks later for removal of the ureteric stent.</p>", "<p>His subsequent recovery was uneventful and at 4 years post injury he remains normotensive with equitable renal function noted on Dimecaptosuccinic acid (DMSA) scan (Fig ##FIG##2##3##)</p>", "<title>Consent</title>", "<p>Consent was obtained from both parents and the involved institutions for publication of the case report.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>CH prepared the manuscript, MH provided the clinical details of the case, AM managed the case and edited the paper.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Polar diastasis and urinary extravasation as seen on IVP.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Concomitant percutaneous pigtal and internal double J drainage of right kidney.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>DMSA Scan demonstrating a slightly elongated but otherwise normally functioning right kidney.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1757-1626-1-129-1\"/>", "<graphic xlink:href=\"1757-1626-1-129-2\"/>", "<graphic xlink:href=\"1757-1626-1-129-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Management of the burn patient is the most challenging condition for the medical staff as the fate of the patient depends on the quality of the management provided during hospital stay and after discharge. Even if the patient recovers from the burn injuries, the development of the deformities overshadow the earlier management. This post burn reconstructive surgery and physiotherapy consultation needs to be made compulsory in the burn units.</p>" ]

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[ "<title>Discussion</title>", "<p>In the review of literature (Medline search), there are number of publications on the perineal burn and its management in the children [##REF##2401686##1##, ####REF##8056817##2##, ##REF##8360241##3##, ##UREF##0##4####0##4##]. As compared to this, we have found only one publication on perineal contracture leading to anal stricture and mega rectum in a three years old child [##REF##8360241##3##]. In comparison to this report, in our case there was no involvement of anus or rectum. The intestinal obstruction was due to the post burn contracture in the gluteal fold which lead to the obstruction beyond the anus. This contracture was released and patient had complete recovery without any sequel. One more interesting fact in this case was that the patient sustained burn injury in gluteal area by sitting on the 'Chullah' (Figure ##FIG##2##3##), an earthen made stove, in which wood is used as fuel, a very common practice for the cooking in rural area of our country.</p>" ]

[ "<title>Conclusion</title>", "<p>Although perineal and gluteal burns are rare even in the rural areas of our country, as people are now using natural gases for the cooking etc but this rare case report emphasises on the critical burn care, post burn care, physiotherapy and regular follow up to the hospital E. Ye [##UREF##0##4##] has also given emphasises on the meticulous preoperative and post operative care in patients with chronic obstruction due to peri-anal contractures.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Peri-anal contracture lead to intestinal obstruction whenever there is involvement of anal orifice. In this case anus and peri-anal skin up to two cm was normal; however both gluteal folds were fused because of post burn scar leaving a very small opening which lead to faecal impaction and sub acute intestinal obstruction.</p>" ]

[ "<title>Case report</title>", "<p>A two and half year old male child was admitted with complaint of progressive difficulty in passing stools along with progressive distension of abdomen, for last one year. There was history of vomiting, off and on for the last fortnight. Patient had history of sustaining 10% thermal burns over perineum, gluteal region and left foot about one and half year back.</p>", "<p>On examination, there was mild distension of abdomen and occasional visible peristalsis movement with exaggerated bowel sounds. Examination of perineum showed that both the gluteal folds were fused because of post burn scar and there was a small opening approximately three mm in diameter in the centre (Figure ##FIG##0##1##). Rectal examination could not be carried out through this opening. The general physical and other systemic examination was normal.</p>", "<p>The blood investigations were normal and x-ray abdomen showed few air fluid levels. The child was operated under general anaesthesia. The contracture was released and both gluteal folds were separated. Raw area was grafted with split thickness skin graft. When contracture was released, it was found that anal verge along with peri-anal skin up to two cm was normal (Figure ##FIG##1##2##). It was fusion of gluteal folds due to post burn scar which led to sub acute intestinal obstruction. There was faecal impaction in the rectum. Post operative recovery was uneventful and graft was well taken.</p>", "<title>Consent</title>", "<p>The written informed consent of the patient has been obtained for the publication of this case report and accompanied images.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>JST has designed and written the article and is the principal contributor, CGS was involved with the management of the patient, conception, design and review of the article, VKD was involved in the management of the patient, conception and critical review of the article, AT was involved in acquisition of the data, review of the literature and critical review of the article. All the authors have read and given final approval for this article</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Peri-anal post burn contracture obstructing normal view of anal area.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Intraoperative view with skin graft in place.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>'<bold>Chullah' a traditional stove</bold>.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1757-1626-1-117-1\"/>", "<graphic xlink:href=\"1757-1626-1-117-2\"/>", "<graphic xlink:href=\"1757-1626-1-117-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Giant cell tumour is a benign but locally aggressive tumour. The classification and definition of giant cell lesions was first proposed by Jaffe and Lichtenstein[##UREF##0##1##]. 20% of all benign bone tumours and 5% of all tumours are giant cell tumours. It is more common in young adults between 20 and 40 years of age [##UREF##1##2##, ####UREF##2##3##, ##REF##1728946##4####1728946##4##]. It is more common in females with the rate of growth enhanced in pregnancy[##UREF##3##5##].</p>", "<p>Appearance before epiphyseal plate closure is rare[##UREF##1##2##,##UREF##4##6##,##REF##6833323##7##]. It occurs commonly in the distal femur, the proximal tibia, the distal radius and the sacrum[##UREF##1##2##, ####UREF##2##3##, ##REF##1728946##4####1728946##4##]. Giant cell tumours (GCT) usually prefers the epiphyses of long bones. The involvement of the metaphysis or diaphysis without epiphyseal extension is quite uncommon[##UREF##0##1##]. Often the tumour extends to the articular subchondral bone, However it seldom crosses the joint or its capsule. If the GCT appears prior to epiphyseal, it is likely to be found in the metaphysis[##UREF##5##8##,##REF##9564283##9##]. A diaphyseal GCT is almost unheard of A literature search brought forth very few reported cases[##REF##5805420##10##, ####REF##3212492##11##, ##REF##8490899##12##, ##REF##2664056##13##, ##UREF##6##14####6##14##]. This is perhaps the eighth case of diaphyseal GCT reported in the literature. It recurs from time to time and rates between 25–50% have been reported[##UREF##1##2##,##UREF##4##6##,##REF##11005533##15##]. In very rare cases, a malignant change may occur[##UREF##6##14##,##REF##3025132##16##,##REF##2732265##17##]. Taking this into account, it is essential that a correct diagnosis of GCT should be made. It is essential that we are aware of the rare existence of giant-cell tumours in areas other than the epiphysis. We may miss a few if we are not [##REF##5805420##10##].</p>" ]

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[ "<title>Discussion</title>", "<p>Most GCTs are present in the epiphyseal or epimetaphyseal end of the long bones.</p>", "<p>If the epiphysis is not involved, a diagnosis of GCT is dubious. The radiographic findings are helpful but cannot clinch the diagnosis. Histological examination is still the gold standard for diagnosis.</p>", "<p>To distinguish metaphyseal and diaphyseal GCT from other lesions is a challenge for the pathologist. Several lesions like aneurysmal bone cyst, giant cell-rich osteosarcoma, fibrous cortical defect, solitary (unicameral) bone cyst, and giant cell lesion of hyperparathyroidism (brown tumour) are more common in these sites than are true GCT. This extensive range of lesions with the exception of giant cell lesion of hyperparathyroidism, usually appears in the first two decades of life.</p>", "<p>Aneurysmal bone cyst is clinically benign It commonly appears in the metaphysis. It contains prominent blood-filled spaces. The more solid zones within them exhibit fibrogenesis and osteoid trabeculae[##REF##8490899##12##]. The stroma between the spaces contains hemosiderin laden macrophages, chronic inflammatory cells and broad seams of reactive osteoid[##UREF##3##5##]. Multinucleated giant cells are often conspicuous. Mitoses may be numerous, but anaplasia is absent.</p>", "<p>Osteosarcoma is a lesion in the metaphysis and contains numerous benign giant cells. The stroma reveals cells with ananplasia and irregular size and shape. Also presence of cartilage is not uncommon[##REF##8490899##12##].</p>", "<p>Fibrous cortical defect is a benign lesion which regresses spontaneously. Radiology shows a characteristic eccentric zone of rarefaction with well-defined scalloped margins[##REF##8490899##12##]. The microscopic picture reveals a mixture of collagen and fibroblasts with irregular cluster of histiocytes filled with lipid and hemosiderin. Multinucleated giant cells may be found[##UREF##3##5##].</p>", "<p>A simple bone cyst generally touches the epiphyseal growth plate. It is benign lesion and shows radiolucence with fine trabeculation. It contains fibrous tissue with few giant cells[##REF##8490899##12##].</p>", "<p>A giant cell lesion of hyperparathyroidism appears in the metaphysis. Many giant cells scattered in a fibrogenic stroma may be present. A serum hypercalcemia and hypophosphatemia is characteristic[##REF##8490899##12##].</p>", "<p>Giant-cell reparative granulomas demonstrate an appearance which suggests previous injury and inflammation with subsequent fibrosis. These lesions characteristically have an appearance which suggests previous injury and inflammation with subsequent fibrosis. Giant cells are found in the vicinity of old areas of hemorrhage, though not dispersed throughout the lesion. Mandible is a common site for these lesions[##REF##5805420##10##].</p>", "<p>The case we have reported did not contain areas of hemorrhage, hemosiderin pigment, osteoid, bone, or significant amounts of collagen. The lesion also did not have the fibrotic, scarred appearance of a fibrous cortical defect, reparative granuloma, or brown tumor of hyperthyroidism. The patient had no detectable parathyroid dysfunction. The clinical and histological features of other osseous lesions which may contain giant cells were not present.</p>", "<p>The patient's age, the location of the lesion, its roentgenographic appearance, and the gross and microscopic appearances are crucial to unravel the mystery of an osseus lesion. However, the final diagnosis depends on the tumour's histological appearance only[##REF##5805420##10##]. As Jaffe has mentioned 'A bone lesion may be uncharacteristic in all other respects, but if it exhibits the cytological pattern of a giant cell tumour, it should be recognised as a GCT' [##REF##13114840##18##].</p>" ]

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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>We present a case of a 35 yrs old female who presented with swelling over her forearm. This is a rare case of a giant cell tumour in a nonepiphyseal region.</p>", "<title>Methods</title>", "<p>Case report and presentation of clinical, radiological and histological data on single case of giant cell tumour of diaphysis of radius.</p>", "<title>Results</title>", "<p>Although age, clinical features and radiological features are helpful, it is still the histology that helps to clinch the diagnosis.</p>", "<title>Conclusion</title>", "<p>A thorough literature search and an exhaustive online search using various search engines revealed seven reported cases of giant cell tumours in the diaphysis of long bones. We reiterate the fact that irrespective of the location, a giant cell tumour should be diagnosed based on its histology.</p>" ]

[ "<title>Case Report</title>", "<p>A 35 years old Asian school teacher was admitted in our hospital with a complaint of swelling over her left forearm. The swelling had increased gradually over the preceding year. She also complained of occasional pain over the inside of her forearm. She did not sustain any kind of trauma or suffer from any fever in her last few months. Examination revealed a diffuse fusiform swelling over the middle third and outer aspect of her left forearm. The overlying skin was tense. No signs of inflammation were visible. On palpation, there was tenderness over the swelling, especially over the lateral aspect. The swelling was soft in consistency with a feeling of 'egg shell crackling'. Movement at all the joints was full in range and was painless. There was no neurovascular deficit. The calcium, phosphorus and parathyroid levels in the serum were within normal limits. A radiograph of the forearm showed an expansile lesion of 10 cms × 5 cms over the middle third of the radius (Fig ##FIG##0##1##). The lesion was lytic and ballooned. The cortical margins were thinned out and breached. The chest radiograph was normal. A fine needle aspiration biopsy was done.</p>", "<p>To our surprise, it was reported as a giant cell tumour It was decided to excise the tumour. At surgery, the tumour was reddish brown, ovoid in shape and soft in consistency. Frozen section was done to know the extent. It extended from the metaphyseal-diaphyseal junction area of the distal radius to the proximal fourth. It was removed cleanly. The ulna was centralised over the third metacarpal and the wrist was arthrodesed with a dynamic compression plate (Fig ##FIG##1##2##).</p>", "<p>Histology revealed large number of scattered giant cells with centrally placed nuclei. The tumor was composed of plump spindle shaped cells. It was also reported as a giant cell tumour. The tumour did not recur two years after the surgery and the patient is in good health.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>SS drafted the manuscript and operated on the patient. SPN, YK and PS drafted the manuscript, performed a literature search and participated in the management. All the authors read and approved the final manuscript.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient for publication of this case report and accompanying images.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Radiograph of diaphyseal giant cell tumour</bold>.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Radiograph-two years postoperative.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"1757-1626-1-106-1\"/>", "<graphic xlink:href=\"1757-1626-1-106-2\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Since the introduction of antibiotics, group A streptococcal infection has been an uncommon disease in neonates. Thirty-nine patients with severe neonatal disease caused by group A streptococcus (GAS) have been described since 1966 [##REF##14872185##1##]. Meningitis caused by GAS is reported in only 15 neonates during same period [##REF##5945530##2##, ####UREF##0##3##, ##REF##789841##4##, ##REF##389176##5##, ##REF##6336834##6##, ##REF##6356881##7##, ##REF##6146901##8##, ##REF##3293424##9##, ##REF##9563422##10##, ##REF##10530572##11##, ##REF##10530575##12##, ##REF##10867852##13####10867852##13##]. This case report describes a neonate with GAS meningitis seen at a university hospital and provides an overview of well-documented cases in the English-language literature over past 40 years.</p>" ]

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[ "<title>Discussion</title>", "<p>In the pre-antibiotic era, GAS was a major cause of neonatal sepsis and puerperal infections. Meningitis accounted for 10 – 20% of these infections with fatality rate of 95%. Since the advent of antimicrobial therapy, meningitis caused by GAS is rarely reported in adults or children, with less than 1% of all cases of bacterial meningitis [##REF##6336834##6##].</p>", "<p>Since the1980's an increase in the incidence of invasive infections caused by GAS has been noted. A review of GAS meningitis in children beyond the neonatal period describes only 31 well-documented patients in world literature in the past 30 years [##REF##11753187##14##].</p>", "<p>The present case included a MEDLINE search of the English literature from 1966 revealed only 16 neonates with GAS meningitis [Table ##TAB##0##1##]. All but two patients in the current review developed late onset (&gt; 7 days of age) neonatal meningitis. Associated conditions included sepsis, erysipelas, necrotizing fasciitis, necrotizing enterocolitis, cellulitis, and respiratory infection. No associated or preceding illness was reported in 6 (40%) patients.</p>", "<p>A 2004 review of GAS invasive infection in the neonates has described 39 patients since 1966 in the world literature [##REF##14872185##1##]. Vertical transmission accounted for the majority of invasive early onset GAS disease in these neonates. Sixty percent of the mothers who delivered infants with early onset of GAS disease developed puerperal sepsis, toxic shock-like syndrome or both in the peripartum period. The mode of transmission in the majority of invasive late onset cases is unknown. Vertical transmission or postnatal acquisition of focal GAS infection such as pharyngitis and episiotomy abscess is a probable source of transmission. There was no a recognizable underlying illness or predisposing factor of GAS meningitis in the present case. However, a family member had developed symptomatic pharyngitis 10 days prior to the onset of the disease, which may have been the source of infection.</p>", "<p>GAS is sensitive to a variety of antibiotics administered either alone or in combination. Penicillin was the most commonly prescribed antibiotic for GAS invasive infection in children [##REF##11753187##14##,##UREF##1##15##]. Burnett et al in his case series reported a favorable outcome when combind clindamycin with beta lactam penicillin [##UREF##1##15##]. In neonates with GAS meningitis penicillin was used in (77%). Other antibiotics used included amino glycosides, second and third generation cephalosporin, and vancomycin [table ##TAB##0##1##].</p>", "<p>The choice of empiric antibiotic treatment for neonates with meningitis usually involves ampicillin and gentamicin, or ampicillin and cefotaxime.</p>", "<p>The clinical course of GAS neonatal meningitis was associated with major complications including seizures in 8 patients (60%) disseminated intravascular coagulopathy (DIC), cardio respiratory insufficiency, hepatitis, and Waterhouse-Frederichsen syndrome. Brain abscess developed in 3 patients [table ##TAB##0##1##].</p>", "<p>Group A streptococcus is an uncommon cause of brain abscess in children and adults. Etiology of brain abscess in the reported cases includes meningitis, contiguous spread from a middle ear infection, facial furuncles and hematogenous spread from distant site.</p>", "<p>In neonates brain abscesses are very rare. It is usually occurs as complications of bacterial meningitis or bacteremia. Maternal factors include mastitis and genitourinary tract infection can be an important source of neonatal brain abscesses. They are most often caused by gram negative organisms. The abscesses are often large and may be multiple. Symptoms of seizures, signs of sepsis and bulging fontanels are frequently seen in neonatal brain abscesses.</p>", "<p>Eight (50%) patients with neonatal GAS meningitis died. This is higher than the mortality rate reported in neonates with meningitis caused by several other types of pathogens, and the mortality rate of GAS meningitis reported in children beyond the neonatal period [##REF##11753187##14##]. No neurological sequelae were reported in 4 patients on whom follow up data is available.</p>" ]

[ "<title>Conclusion</title>", "<p>GAS meningitis remains an uncommon but serious disease affecting mostly older neonates. Parent and siblings of the patients constitute the source of infection and may unknowingly infect the neonates. Since invasive infection is on the increase, clinicians should always consider GAS in the differential diagnosis of neonatal sepsis and meningitis. Prompt and appropriate treatment may reduce complications and mortality.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Group A streptococcus is a rare cause of neonatal meningitis. A review of MEDLINE database since1966 revealed only 15 documented cases of group A streptococcal meningitis in neonates.</p>", "<title>Case report</title>", "<p>A previously healthy 28 days old male neonate presented with a history of irritability, fever, and focal seizures. Cerebrospinal fluid analysis and culture confirmed the diagnosis of group A streptococcal meningitis. The clinical course was complicated by the development of brain abscess. The patient made full recovery following a surgical drainage of the abscess and a 6-week total course of antibiotics.</p>", "<title>Conclusion</title>", "<p>Although it is an uncommon organism, clinician should always consider group A streptococcal infection and its potential complications in the differential diagnosis and management of neonatal meningitis.</p>" ]

[ "<title>Case report</title>", "<p>A 28 days old boy was presented to the Emergency Department with a history of fever, irritability, poor feeding of 1 week duration and right-sided focal seizures on the day of presentation. He was born after an uncomplicated pregnancy and delivery. An elder brother had developed symptomatic pharyngitis with fever, sore and congested throat 10 days prior to the neonate's disease. This pharyngitis, however, was not microbiologically investigated. He was recovered after a one week course of antibiotic. Upon examination, the patient looked ill, and irritable. Vital signs were stable. Anterior fontanel was normal and there was no neurological deficit. The peripheral blood white cells (WBC) were 33,600/mm³with differential count 61% Neutrophils, 18% Lymphocytes, 6% Monocytes and 10% Eosinophils. C-reactive protein was positive. Cerebrospinal fluid (CSF) was turbid and showed 23,520 WBC/mm³with 82% Neutrophils, 18% Lymphocytes and 3 red blood cells/mm³. CSF protein was 502 mg/dl, Glucose 5 mg/dl (simultaneous blood glucose 98 mg/dl). Latex antigen test was negative for Hemophillus influenzae B, Neisseria meningitides, Escherichia Coli, Streptococcus group B and Streptococcus pneumoniae. Gram positive cocci were seen in the deposit. He was treated with intravenous cefriaxone and vancomycin. The CSF culture yielded group A beta hemolytic Streptococci. Sub-typing of the organism was not available. Vancomycin was discontinued after results of culture. Given the history of focal seizures, head MRI, performed on the seventh hospital day, and showed a large lobulated abscess in the left parieto-occipital region [Figure ##FIG##0##1##]. The patient underwent drainage of the abscess. No organisms grew in the culture of the pus (presumably owing to the preceding antibiotic treatment). The child was discharged after 6 weeks of treatment. At the age of 13 month, hearing test and neurological examination were normal.</p>", "<title>Consent</title>", "<p>Written informed consent was obtained from the patient's parent for publication of this case report and accompanying image. A copy of the written consent is available for review by the Editor-in Chief of this journal.</p>", "<title>Competing interests</title>", "<p>The author declares that they have no competing interests.</p>" ]

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[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Coronal section of brain MRI with contrast showing a large lobulated collection in the parieto-occipital region.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Group A streptococcal meningitis in neonates.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Case No.</bold></td><td align=\"left\"><bold>Age/Sex</bold></td><td align=\"left\"><bold>Associated Condition</bold></td><td align=\"left\"><bold>Complication</bold></td><td align=\"left\"><bold>Management</bold></td><td align=\"left\"><bold>Outcome/Sequelae</bold></td><td align=\"left\"><bold>Reference</bold></td></tr></thead><tbody><tr><td align=\"left\">1</td><td align=\"left\">6 days/M</td><td align=\"left\">Erysipelas</td><td align=\"left\">None</td><td align=\"left\">Penicillin + Kanamycin</td><td align=\"left\">Recovered/NA*</td><td align=\"left\">2</td></tr><tr><td align=\"left\">2</td><td align=\"left\">1 Mo./F</td><td align=\"left\">None</td><td align=\"left\">Seizures</td><td align=\"left\">NA</td><td align=\"left\">Died</td><td align=\"left\">3</td></tr><tr><td align=\"left\">3</td><td align=\"left\">8 days/M</td><td align=\"left\">None</td><td align=\"left\">Seizures</td><td align=\"left\">NA</td><td align=\"left\">Died</td><td align=\"left\">3</td></tr><tr><td align=\"left\">4</td><td align=\"left\">17 days/F</td><td align=\"left\">Umbilical sepsis</td><td align=\"left\">None</td><td align=\"left\">Penicillin</td><td align=\"left\">Recovered/NA</td><td align=\"left\">4</td></tr><tr><td align=\"left\">5</td><td align=\"left\">3 days/F</td><td align=\"left\">Necrotizing fasciitis, septicemia</td><td align=\"left\">None</td><td align=\"left\">Penicillin</td><td align=\"left\">Recovered</td><td align=\"left\">5</td></tr><tr><td align=\"left\">6</td><td align=\"left\">14 days/F</td><td align=\"left\">Erysipelas</td><td align=\"left\">Seizures, D.I.C*.</td><td align=\"left\">Penicillin</td><td align=\"left\">Recovered</td><td align=\"left\">6</td></tr><tr><td align=\"left\">7</td><td align=\"left\">14 days/NA</td><td align=\"left\">None</td><td align=\"left\">None</td><td align=\"left\">Cefotaxime + Ampicillin</td><td align=\"left\">Recovered</td><td align=\"left\">7</td></tr><tr><td align=\"left\">8</td><td align=\"left\">26 days/M</td><td align=\"left\">Paronychiae, porencephalic cyst</td><td align=\"left\">Seizures</td><td align=\"left\">Penicillin + Cefuroxime+ Gentamycine</td><td align=\"left\">Recovered/Hydrocephalus</td><td align=\"left\">8</td></tr><tr><td align=\"left\">9</td><td align=\"left\">13 days/F</td><td align=\"left\">Cellulitis of both feet</td><td align=\"left\">Seizures, Hepatitis</td><td align=\"left\">Penicillin + Cefuroxime</td><td align=\"left\">Died</td><td align=\"left\">8</td></tr><tr><td align=\"left\">10</td><td align=\"left\">1 Mo./F</td><td align=\"left\">None</td><td align=\"left\">Multiple brain abscess, seizures</td><td align=\"left\">Penicillin</td><td align=\"left\">Recovered/NA</td><td align=\"left\">9</td></tr><tr><td align=\"left\">11</td><td align=\"left\">1 Mo./F</td><td align=\"left\">Sepsis +RSV* Infection</td><td align=\"left\">Sepsis, Waterhouse-Friderichsen syndrome</td><td align=\"left\">NA</td><td align=\"left\">Died</td><td align=\"left\">10</td></tr><tr><td align=\"left\">12</td><td align=\"left\">33 days/F</td><td align=\"left\">Pneumonia</td><td align=\"left\">NA</td><td align=\"left\">Penicillin + Gentamycin</td><td align=\"left\">Died</td><td align=\"left\">11</td></tr><tr><td align=\"left\">13</td><td align=\"left\">34 days/NA</td><td align=\"left\">None</td><td align=\"left\">NA</td><td align=\"left\">Penicillin + Gentamycin</td><td align=\"left\">Recovered/NA</td><td align=\"left\">11</td></tr><tr><td align=\"left\">14</td><td align=\"left\">1 Mo./NA</td><td align=\"left\">NEC*+ Septicemia</td><td align=\"left\">NA</td><td align=\"left\">Penicillin + Gentamycin</td><td align=\"left\">Died</td><td align=\"left\">12</td></tr><tr><td align=\"left\">15</td><td align=\"left\">24 days/F</td><td align=\"left\">None</td><td align=\"left\">Seizures, brain abscess, cardiorespiratory Insufficiency</td><td align=\"left\">NA</td><td align=\"left\">Died</td><td align=\"left\">13</td></tr><tr><td align=\"left\">16</td><td align=\"left\">28 days/M</td><td align=\"left\">None</td><td align=\"left\">Seizures, brain abscess</td><td align=\"left\">Ceftriaxone + Vancomycin</td><td align=\"left\">Recovered</td><td align=\"left\">This case</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>*<bold>D.I.C</bold>-Disseminated intravascular coagulation, <bold>NA</bold>-Data not available, <bold>NEC</bold>-Necrotizing entercolitis <bold>RSV</bold>-Respiratory syncetial virus</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1757-1626-1-108-1\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>During the last thirty years, there has been a marked increase in the incidence of adenocarcinoma close to the esophagogastric junction whilst the incidence of squamous cell carcinoma of the esophagus has remained relatively unchanged [##REF##1995976##1##]. Surgical resection of tumors in the esophagus and esophagogastric junction has been based upon the concept that, if all neoplastic tissue can be removed, a worthwhile period of survival and possibly cure can be achieved. Despite oncological advances, surgical resection is the only treatment that has repeatedly been shown to prolong survival, albeit in only 30% of patients [##REF##12049068##2##].</p>", "<p>Transhiatal esophagectomy is often advocated as the preferred surgical approach in patients with benign disease or early tumors or those patients with more advanced disease who would not tolerate a thoracotomy. This approach has been criticized because of the lack of a formal two field lymphadenectomy and the failure to completely resect the tumor under direct vision [##REF##12049068##2##]. Transhiatal esophagectomy has been the favoured operative approach in our institution for managing both carcinoma of the oesophagus below the level of the carina and type I and II tumours of the esophagogastric junction. It has also been utilised for benign lower oesophageal disease including high grade dysplasia. This study evaluates our experience and outcomes with transhiatal esophagectomy in an era in which the use of neoadjuvant chemotherapy became more prevalent.</p>" ]

[ "<title>Methods</title>", "<title>Study population</title>", "<p>Between January 2000 and January 2007, 215 patients with benign or malignant disease of the intrathoracic esophagus and type I and II tumours of the esophagogastric junction underwent transhiatal esophagectomy at our institution. Prospective data on these 215 consecutive patients was collected from consultant databases supplemented by cancer registry data and case note review. A further 152 patients underwent transthoracic esophagectomy during the same time period and were excluded from analysis. Ethical committee approval was obtained for this study and the need for individual patient consent was waived.</p>", "<title>Preoperative evaluation and treatment</title>", "<p>Routine preoperative evaluation involved upper gastrointestinal endoscopy with biopsy, endoscopic ultrasound and computed tomography of the neck, chest and abdomen. Staging laparoscopy and PET scanning were performed on a selective basis. Operative risk analysis included standard blood examination, electrocardiography, echocardiography, pulmonary function tests and cardiopulmonary exercise tests (in higher risk patients). Surgery was offered to medically fit patients following discussion at a multidisciplinary meeting.</p>", "<p>90 patients in the study group (42%) received preoperative chemotherapy based upon the presence of T3 disease or positive lymph nodes on preoperative staging. The preferred chemotherapy at our institution consisted of three cycles of combination epirubicin, cisplatin and 5-fluorouracil each given over three weeks, following the MAGIC trial protocol [##REF##16822992##3##].</p>", "<title>Operative technique</title>", "<p>All patients underwent subtotal esophagectomy and proximal gastrectomy by the transhiatal technique as described in detail by Orringer. [##REF##15927654##4##, ####REF##11338022##5##, ##REF##15895452##6####15895452##6##] An initial laparotomy was performed through a rooftop incision to confirm tumour resectability. After abdominal exploration and gastric mobilisation had been performed, the esophageal hiatus was enlarged by splitting the diaphragm anteriorly and retractors were positioned to facilitate exposure of the intrathoracic esophagus up to the level of the carina. This enabled en bloc resection of the esophagus and paraesophageal tissue including the crura and pleura (if indicated) under direct visualisation. Standard lymph node dissection involved lymph nodes in the lower mediastinum, around the esophagogastric junction and along the lesser curvature of the stomach. A radical lymph node dissection was performed at the origins of the left gastric and common hepatic arteries; lymph nodes at the celiac axis were included when enlarged and resectable. A less radical resection was performed for patients with benign disease. Gastrointestinal continuity was re-established with a narrow gastric tube vascularized by the right gastroepiploic artery in all cases, positioned within the posterior mediastinum. An end to side hand sewn single layer esophagogastric anastomosis was fashioned in the neck through a left sided cervical incision. Transmediastinal chest drains and placement of a feeding jejunostomy were performed in all patients.</p>", "<title>Pathological examination</title>", "<p>Pathology specimens were processed by three dedicated esophagogastric pathologists according to Royal College of Pathologists' guidelines. [##UREF##0##7##] Tumors of the esophagogastric junction were categorized according to Siewert's classification based upon macroscopic tumor location, irrespective of the presence of Barrett mucosa. [##REF##9823902##8##] Type I adenocarcinoma of the esophagogastric junction was staged according to esophageal pTNM classification whilst type II adenocarcinoma of the esophagogastric junction were staged according to gastric pTNM classification. [##UREF##1##9##] To ensure standardized histopathology results, all early specimens were re-categorized according to the latest guidelines.</p>", "<title>Follow up</title>", "<p>During the immediate postoperative period, patients were kept intubated and ventilated until the following morning. Following extubation, patients were monitored on a surgical High Dependency Unit until well enough to be managed on a surgical ward. Oral nutrition was recommenced if a water soluble contrast swallow examination failed to demonstrate an anastomotic leak on the seventh day.</p>", "<p>After discharge, patients were routinely followed up at 3–6 monthly intervals. Patients were offered either adjuvant chemotherapy (up to a maximum of 6 cycles) or chemoradiotherapy (if any margins were positive) based upon analysis of the pathological specimen and the histologically determined response to any preoperative treatment. Additional diagnostic procedures were only performed if indicated by the development of any new symptoms suggestive of recurrent disease. In the presence of recurrent disease, further oncological or palliative options were considered. The median duration of postoperative follow up was 26 months (range = 1–82 months) for all patients and 36 months (range = 2–82 months) for those alive at final follow up.</p>", "<title>Statistics</title>", "<p>Overall survival was defined as the time interval from the date of operation until the date of death or most recent follow up. Disease free survival was defined as the time interval from the date of operation until the date of disease recurrence or most recent follow up. Survival curves were calculated according to the Kaplan-Meier method. Univariate group comparisons were calculated using the log rank test. Categorical variables were assessed using Fisher's exact test and continuous variables were assessed by student's t test [##UREF##2##10##]. A p value &lt; 0.05 was regarded as statistically significant. Statistical analysis was performed with Graphpad Prism v3.0 and Instat v2.0 (GraphPad Software, San Diego California USA).</p>" ]

[ "<title>Results</title>", "<title>Preoperative features</title>", "<p>The demographic details of the 215 patients undergoing transhiatal esophagectomy are shown in Table ##TAB##0##1##. Dysphagia and weight loss were present in 73% and 48% of patients respectively with preoperatively confirmed malignant tumours. Twenty two patients (10%) had an asymptomatic cancer or high grade dysplasia detected during endoscopic surveillance of Barrretts oesophagus. Three patients (1%) underwent urgent transhiatal esophagectomy following endoscopic tumor perforation. According to the American Society of Anesthesiologists (ASA) classification [##REF##697077##11##], operative risk was scored as ASA-I (n = 15), ASA-II (n = 125), ASA-III (n = 72) or ASA-IV (n = 3).</p>", "<title>Intraoperative surgical findings</title>", "<p>Only one patient required intraoperative conversion to a right posterolateral thoracotomy due to tumor adherence at the carina and difficulties in achieving macroscopic tumor clearance through the esophageal hiatus. Macroscopic tumor clearance could not be achieved in one patient due to the presence of extensive left gastric and celiac axis lymphadenopathy. The median operative time was 151 minutes (range = 93–276 minutes).</p>", "<title>Postoperative course</title>", "<p>There were two in-hospital deaths during this study (&lt;1%). One patient, a 74 year old man, with a past medical history including pneumonectomy for lung cancer and a previous myocardial infarction, developed respiratory failure requiring prolonged ITU admission and respiratory support; he died from myocardial infarction on day 44. The second patient, a 70 year old man, died from a pulmonary embolus on day 13 in ITU following admission with multiorgan failure secondary to chest sepsis.</p>", "<p>Major postoperative complications are listed in Table ##TAB##1##2##. All 12 patients with clinically apparent anastomotic leaks were managed conservatively with opening the cervical wound to allow adequate wound drainage and reduction of oral intake combimed with jejunostomy tube feeding. None of these patients required re-operation for their anastomotic leaks. 10 patients (5%) required re-operation in the early post-operative stage for: bleeding (n = 4), bowel obstruction (n = 3), chyle leak (n = 2) and wound dehiscence (n = 1). Unplanned ITU admission was required in 29 patients (14%), most commonly for respiratory failure. The median ITU stay in this group was 7 days (range 2–44 days). Overall median length of hospital stay was 14 days (range 8–95 days). All patients were discharged directly home and the in-patient stay reflects the need for sufficient mobility and tolerance of an adequate oral diet prior to discharge.</p>", "<title>Oncological outcomes</title>", "<p>Histopathological analysis of the operative specimens in the 215 patients revealed the following tumor types: adenocarcinoma (n = 169), squamous cell carcinoma (n = 22), high grade dysplasia (n = 17), adenosquamous carcinoma (n = 3), benign strictures only (n = 3) and spindle cell tumor (n = 1). In 3 patients, all initially diagnosed with adenocarcinoma, there was a complete pathological response to neoadjuvant chemotherapy whilst, in a further 2 patients, there was residual adenocarcinoma in lymph nodes only. The type of esophagogastric junctional tumour in 169 patients with adenocarcinoma was classified as follows: type I (n = 93), type II (n = 70) or type III (n = 6). All 6 patients with type 3 tumors had been preoperatively staged as type 2 tumours.</p>", "<p>Macroscopic tumour clearance was achieved in 193 out of 194 patients with pathological evidence of invasive malignancy. Residual microscopic disease was found at the proximal or distal resection margins in 11 patients (5%), all in association with positive circumferential resection margins and involved lymph nodes. Eighty eight patients (46%) were subsequently found to have tumor cells at or within 1 mm of the esophageal adventitia or the gastric serosal surface.</p>", "<p>The radicality of resection in relation to tumour infiltration and involved lymph nodes is shown in Table ##TAB##2##3##. The median lymph node yield in all patients was 12 (range 1–52). Both tumour stage and radicality of resection were independent predictors of overall survival on univariate analysis (Figures ##FIG##0##1## &amp;##FIG##1##2##).</p>", "<title>Recurrence and survival</title>", "<p>All patients undergoing transhiatal esophagectomy for benign disease remain alive on follow up. Excluding the two in-hospital deaths, 79 patients (40%) who underwent esophagectomy for invasive malignancy have died on follow up. The causes of death are as follows: locoregional recurrence (n = 14), systemic metastases (n = 27), combination of locoregional recurrence and systemic metastases (n = 29), medical causes (n = 5), ongoing surgical complications (n = 1) and cause unable to be identified (n = 3). In total, 39% of patients developed recurrent disease during the period of study. The median survival for all patients undergoing transhiatal esophagectomy for invasive malignancy was 43 months and the one year and five year survival rates were estimated at 81% and 48% respectively (Figure ##FIG##2##3##). There was no difference in overall or disease free survival between patients with type I and II adenocarcinoma of the oesophagogastric junction.</p>" ]

[ "<title>Discussion</title>", "<p>This study has demonstrated that transhiatal esophagectomy can be associated with a low morbidity and a mortality of less than 1%. Although other units have reported similar results for transhiatal esophagectomy, several multicentre studies and national audits have shown that the mortality for all types of esophagectomy may exceed 10% [##REF##10493486##12##, ####REF##11803954##13##, ##REF##14630753##14##, ##REF##16038824##15##, ##REF##15620945##16####15620945##16##]. It is recognised that high volume centres with a concentration of surgical, critical care and interventional radiological expertise achieve better outcomes. [##REF##11301408##17##, ####REF##15569369##18##, ##REF##17443856##19####17443856##19##] The rationale for a transhiatal esophagectomy is the avoidance of a thoracotomy, thereby reducing the incidence of pulmonary complications, and the fashioning of a cervical anastomosis so that the clinical consequences of any anastomotic leak are minimized [##REF##10493486##12##,##REF##11803954##13##]. Critics of the transhiatal approach argue that there is a risk of blind intrathoracic injuries such as massive bleeding from the azygous vein, tracheal injury and episodes of cardiac instability resulting from retraction and surgical manipulation within the mediastinum. Case selection for transhiatal esophagectomy is crucial to prevent these problems and also to ensure adequate macroscopic tumor clearance for more proximally located esophageal tumors. It is the authors' policy that only patients with subcarinal tumors identified on preoperative imaging and confirmed by transhiatal dissection to above the proximal macroscopic extent of the tumor are suitable for the transhiatal approach. In the current series, only one patient required intraoperative conversion to a thoracotomy to obtain tumor clearance and 2 patients (1%) required reoperation for bleeding (both of these patients had active intrathoracic bleeding although none were associated with an azygous vein injury). Clinically apparent anastomotic leaks occurred in 6% of patients and all were managed successfully with conservative treatment. The data from this study supports the concept that a transhiatal esophagectomy in appropriately selected patients is safe and feasible.</p>", "<p>Surgeons who advocate a transthoracic approach argue that neglecting to perform a mediastinal lymphadenectomy risks leaving behind residual tumour, resulting in higher rates of locoregional recurrence and worse overall survival. [##REF##11685019##20##, ####REF##16571425##21##, ##REF##11573045##22####11573045##22##] However, the additional value of formal mediastinal lymph node dissection remains controversial in Western patients, especially with the concept that lymph node involvement may reflect systemic micrometastatic disease and that extended resections will not alter the natural history of this disease. Reported differences in recurrence and survival may merely represent a stage migration effect due to an increased accuracy of histological staging. [##REF##12049068##2##,##REF##11465217##23##,##REF##15570202##24##] Portale et al recently suggested that extended en bloc transthoracic resections were significantly associated with better survival rates of up to 50% compared to transhiatal resections and that this could not be ascribed to a stage migration effect. [##REF##16571425##21##] R0 status (defined in this study as clear circumferential and longitudinal margins) is a recognized independent prognostic factor for survival. Advocates of a transthoracic esophagectomy have suggested that the transhiatal approach limits the ability to achieve an R0 resection [##REF##11685019##20##, ####REF##16571425##21##, ##REF##11573045##22####11573045##22##]. Macroscopic tumour clearance was achieved in all but one patient in the current study. Longitudinal margin involvement, especially at the proximal margin, has been shown to independently impact on survival via increased loco-regional recurrence. The rate of positive longitudinal margins in this study was 5% which is in keeping with other published series [##REF##2042594##25##]. The problem of a positive gastric resection margin at transhiatal esophagectomy has recently been addressed by DiMusto and Orringer [##REF##17532385##26##]. They achieved a negative gastric margin in 98% of over 1000 patients treated. In the few patients who had a positive gastric margin, they found that 80% die with distant metastases, which would not be influenced by more extensive gastric resection, and, in about 20%, local tumor recurrence in the intrathoracic stomach was usually asymptomatic. They also demonstrated that adjuvant therapy for a positive gastric margin was usually unhelpful. A similar picture was seen in the current study with all five patients with involved distal resection margins developing systemic metastases.</p>", "<p>The role of circumferential resection margin (CRM) involvement is more controversial. Khan et al concluded that a positive CRM did not influence outcome. [##REF##12771920##27##], but this has been disputed by other studies which suggested that it may independently predict survival [##REF##16504455##28##]. One of these was performed by Maynard and colleagues who recently studied 242 patients undergoing esophagectomy and reported higher rates of local recurrence in patients with a positive CRM. Interestingly, there was no difference in CRM positivity when comparing different operative approaches [##REF##17948302##29##].</p>", "<p>In our population, CRM involvement was encountered in 46% of patients with malignant disease, predominantly affecting those with T3 tumours, and this was the main limiting factor in achieving an R0 resection. R0 resection rates varied from 97–100% with T0/1 tumours to 0–17% for T3–4 tumours. In keeping with previous studies, R0 resections were significantly associated with improved overall survival and hence the group benefiting most from this operative approach would appear to be those patients with early (T1–2) tumours. [##REF##11685019##20##, ####REF##16571425##21##, ##REF##11573045##22####11573045##22##]</p>", "<p>Advocates of more radical en-bloc transthoracic strategies argue that their approach may reduce rates of CRM involvement although this is yet to be proven [##REF##16504455##28##]. Regardless of the operative technique, it is often difficult to obtain circumferential clearance due to the proximity of vital structures and the lack of any fascial boundaries. [##REF##11803954##13##,##REF##16504455##28##] The local recurrence rates in this study compare favourably to previous studies of both transhiatal and transthoracic esophagectomy [##REF##11685019##20##,##REF##16571425##21##,##REF##17030271##30##,##REF##10945357##31##]. Furthermore, the predominant pattern of recurrence was haematogenous metastatic disease (present in 70% of patients with disease relapse), mirroring the patterns seen with more radical en-bloc strategies [##REF##11953876##32##]. These patterns of early systemic relapse were also noted by Orringer in his analysis of 2000 esophagectomy patients [##REF##17717440##33##].</p>", "<p>To date, there has been only one randomised controlled trial comparing transthoracic and transhiatal approaches and this failed to show any significant differences in radicality of surgery or survival at the cost of increased postoperative morbidity in the transthoracic group. [##REF##12444180##34##] Recent five year survival data from this trial have again failed to demonstrate a survival benefit for the transthoracic approach although a sub-group of patients with oesophageal cancer and 1–8 involved lymph nodes appear to have improved disease-free survival. This study did not include chemotherapy and overall five year survival rates were 34% (Transhiatal) and 36% (Transthoracic) with in-hopsital mortality of 2% and 7% respectively [##REF##18043101##35##]. Other meta-analyses have attempted to compare the two approaches and have favoured the transhiatal approach in terms of early morbidity and mortality with no long term survival disadvantage [##REF##11573045##22##,##REF##18222237##36##]. Despite this evidence, it remains difficult preoperatively to select the appropriate operative approach for individual patients.</p>", "<p>Over the last few decades, the survival rates following esophagectomy have significantly improved, largely as a result of improvements in postoperative mortality. The one year survival rate of 81% in the current study for patients with invasive malignancy compares very favorably with the Western standard from the 1990s of 61%. [##REF##15286953##37##] Furthermore, quality of life data suggests patients undergoing a transhiatal approach have fewer physical symptoms and better activity levels in the short term compared to the transthoracic approach although these differences become less evident by 1 year. [##REF##15483031##38##] Several authors have emphasized the central role of surgery in achieving five year survival rates of approximately 50%. [##REF##16571425##21##,##REF##17030271##30##] It is increasingly recognized that there is an important role for oncological treatments in the perioperative management of esophageal and esophagogastric junctional cancer. The survival advantages associated with chemotherapy in both the MRC OEO2 and MRC MAGIC trials have significantly influenced surgical decision making in the UK. [##REF##16822992##3##,##REF##17329193##39##,##REF##12049861##40##] The current series, which combined transhiatal esophagectomy with neoadjuvant chemotherapy in 42% of patients, has achieved equivalent five year survival results to Portale et al but with a greater preponderance of AJCC stage II and III disease. A complete pathological response was seen in 4% of patients receiving neoadjuvant chemotherapy and for many patients, there was little or no histological evidence of response. This emphasizes the need to identify potential responders prior to treatment, and also for the development of new chemotherapeutic agents. [##REF##16571425##21##]</p>", "<p>The development of high volume centres within the UK and the increasing use of (neo)adjuvant therapies have undoubtedly improved both the short term surgical results as well as the long term oncological outcomes of these patients. In summary, we have shown that transhiatal esophagectomy is a safe approach in appropriately selected patients. Radical resections, postoperative complication rates and survival results were in line with data reported for traditional transthoracic approaches. Some units restrict transhiatal esophagectomy to patients deemed unfit for thoracotomy or to patients with very early tumours or, conversely, locally advanced tumours where the benefits of more radical resections may be limited. However, the authors suggest that transhiatal esophagectomy is at least a viable alternative with certain advantages in terms of post-operative recovery, and ever improving oncological outcomes especially when combined with chemotherapy.</p>" ]

[]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The optimal operative approach for carcinoma at the lower esophagus and esophagogastric junction remains controversial. The aim of this study was to assess a single unit experience of transhiatal esophagectomy in an era when the use of systemic oncological therapies has increased dramatically.</p>", "<title>Study Design</title>", "<p>Between January 2000 and November 2006, 215 consecutive patients (182 males, 33 females, median age = 65 years) underwent transhiatal esophagectomy; invasive malignancy was detected preoperatively in 188 patients. 90 patients (42%) received neoadjuvant chemotherapy. Prospective data was obtained for these patients and cross-referenced with cancer registry survival data.</p>", "<title>Results</title>", "<p>There were 2 in-hospital deaths (0.9%). Major complications included: respiratory complications in 65 patients (30%), cardiovascular complications in 31 patients (14%) and clinically apparent anastomotic leak in 12 patients (6%). Median length of hospital stay was 14 days. The radicality of resection was inversely related to T stage: an R0 resection was achieved in 98–100% of T0/1 tumors and only 14% of T4 tumors. With a median follow up of 26 months, one and five year survival rates were estimated at 81% and 48% respectively.</p>", "<title>Conclusion</title>", "<p>Transhiatal esophagectomy is an effective operative approach for tumors of the infracarinal esophagus and the esophagogastric junction. It is associated with low mortality and morbidity and a five survival rate of nearly 50% when combined with neoadjuvant chemotherapy.</p>" ]

[ "<title>Authors' contributions</title>", "<p>AD was primary author of the manuscript. MF performed some of the surgery, set up the database and assisted in data collection as well as drafting of the paper. AK, VP and AN were the primary data collectors and also performed the statistical analysis. DS helped conceive the study, performed some of the surgery and assisted in data collection. RM was the consultant in charge, performed the majority of the surgery and made alterations to the final draft prior to submission. All authors read and approved the final manuscript.</p>" ]

[]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Survival curves comparing overall survival for p (and yp) T0–2 tumours versus p (and yp) T3–4 tumours.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Survival curves comparing overall survival for R0 and R1–2 resections. There was only one R2 resection</bold>.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Kaplan Meier survival curves for overall survival of 21 patients with benign disease and 194 patients with invasive malignancy undergoing transhiatal esophagectomy.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Demographic data on 215 patients undergoing transhiatal esophagectomy.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Demographics</bold></td><td align=\"center\"><bold>n</bold></td></tr></thead><tbody><tr><td align=\"left\">Sex (M:F)</td><td align=\"center\">182:33</td></tr><tr><td align=\"left\">Age (range)</td><td align=\"center\">65 years (29–83 years)</td></tr><tr><td/><td/></tr><tr><td align=\"left\" colspan=\"2\"><bold>Preoperative indication</bold></td></tr><tr><td align=\"left\">Adenocarcinoma</td><td align=\"center\">162 (75%)</td></tr><tr><td align=\"left\">Squamous cell carcinoma</td><td align=\"center\">23 (11%)</td></tr><tr><td align=\"left\">Other malignant tumours</td><td align=\"center\">3 (1%)</td></tr><tr><td align=\"left\">Benign tumours</td><td align=\"center\">1 (0.5%)</td></tr><tr><td align=\"left\">High grade dysplasia</td><td align=\"center\">23 (11%)</td></tr><tr><td align=\"left\">Benign strictures</td><td align=\"center\">3 (1%)</td></tr><tr><td/><td/></tr><tr><td align=\"left\" colspan=\"2\"><bold>Preoperative staging </bold>(in 188 patients with preoperatively confirmed malignant tumours)</td></tr><tr><td align=\"left\">T1</td><td align=\"center\">28 (15%)</td></tr><tr><td align=\"left\">T2</td><td align=\"center\">48 (26%)</td></tr><tr><td align=\"left\">T3</td><td align=\"center\">108 (57%)</td></tr><tr><td align=\"left\">T4</td><td align=\"center\">4 (2%)</td></tr><tr><td align=\"left\">N0</td><td align=\"center\">113 (60%)</td></tr><tr><td align=\"left\">N+</td><td align=\"center\">75 (40%)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Major postoperative complications</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Complication</bold></td><td align=\"right\"><bold>n (%)</bold></td></tr></thead><tbody><tr><td align=\"left\">Clinical anastomotic leak</td><td align=\"right\">12 (5.6)</td></tr><tr><td align=\"left\">Respiratory^a</td><td align=\"right\">65 (30)</td></tr><tr><td align=\"left\">Cardiovascular</td><td align=\"right\">31 (14)</td></tr><tr><td align=\"left\">Recurrent laryngeal nerve neuropraxia</td><td align=\"right\">6 (3)</td></tr><tr><td align=\"left\">Wound infection</td><td align=\"right\">22 (10)</td></tr><tr><td align=\"left\">Renal failure</td><td align=\"right\">6 (3)</td></tr><tr><td align=\"left\">Chyle leak</td><td align=\"right\">5 (2)</td></tr><tr><td align=\"left\">Deep vein thrombosis/pulmonary embolism</td><td align=\"right\">3 (1)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Pathology results from 194 patients undergoing transhiatal esophagectomy for invasive malignancy.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\"><bold>N0</bold></td><td align=\"center\"><bold>N+</bold></td><td align=\"center\"><bold>R0</bold></td><td align=\"center\"><bold>R1</bold></td><td align=\"center\"><bold>R2</bold></td><td align=\"center\"><bold>% R0 resections</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>T0</bold></td><td align=\"center\">3</td><td align=\"center\">2</td><td align=\"center\">5</td><td/><td/><td align=\"center\">100%</td></tr><tr><td align=\"left\"><bold>T1</bold></td><td align=\"center\">35</td><td align=\"center\">7</td><td align=\"center\">41</td><td align=\"center\">1</td><td/><td align=\"center\">98%</td></tr><tr><td align=\"left\"><bold>T2</bold></td><td align=\"center\">23</td><td align=\"center\">38</td><td align=\"center\">41</td><td align=\"center\">20</td><td/><td align=\"center\">68%</td></tr><tr><td align=\"left\"><bold>T3</bold></td><td align=\"center\">25</td><td align=\"center\">52</td><td align=\"center\">19</td><td align=\"center\">57</td><td align=\"center\">1</td><td align=\"center\">25%</td></tr><tr><td align=\"left\"><bold>T4</bold></td><td align=\"center\">1</td><td align=\"center\">5</td><td align=\"center\">1</td><td align=\"center\">6</td><td/><td align=\"center\">17%</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>^aRespiratory complications are defined as respiratory failure, lower respiratory tract infection and symptomatic pleural effusion requiring drainage.</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1477-7819-6-88-1\"/>", "<graphic xlink:href=\"1477-7819-6-88-2\"/>", "<graphic xlink:href=\"1477-7819-6-88-3\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Under its Humanitarian program Australia accepts approximately 13,000 refugees each year [##UREF##0##1##]. Most of these will have experienced torture and trauma, forced migration and family separation, commonly the result of prolonged war or civil conflict [##UREF##1##2##,##REF##9822406##3##]. Many will have had limited or disrupted access to medical care and will have spent long periods in refugee camps in environments that are extremely unsafe, where sanitation is poor and where access to safe drinking water and a nutritional diet is limited [##UREF##2##4##]. As a result, refugees often arrive in Australia with a complex mix of physical and mental health problems many of which are rarely seen in Australia [##UREF##3##5##, ####UREF##4##6##, ##UREF##5##7####5##7##]. The recent focus of the Australian Humanitarian Program has been to resettle those who have endured protracted refugee situations and who have originated from regions of very low socio-economic development. This has seen a large increase in the number of African refugees arrivals over the past 10 years – from 16% of the total annual intake in 1998/99 [##UREF##6##8##] reaching a peak of 70% in 2004/05 [##UREF##0##1##]. SA, which receives approximately 10% of the intake, has seen a similar change in the pattern of refugee resettlement [##UREF##7##9##]. Evidence is emerging that current refugee arrivals experience significantly poorer health status in addition to even greater resettlement challenges [##REF##17181502##10##, ####REF##17181501##11##, ##UREF##8##12##, ##REF##12919501##13##, ##REF##12534878##14##, ##REF##16884406##15##, ##UREF##9##16##, ##UREF##10##17####10##17##].</p>", "<p>Despite the potential for high levels of morbidity, refugees undergo only a limited health assessment prior to their arrival in Australia. For the majority this includes a medical examination, a Chest X-ray for those 11 years and over and an HIV test for those 15 years and over [##UREF##11##18##]. Although more recently some refugees have received an additional health check a few days before departure, this is primarily to assess their 'fitness to fly' and a number of high prevalence infectious diseases and nutritional deficiencies are not included in this assessment [##REF##17181495##19##]. As a result, refugees will often arrive with health conditions not previously identified [##UREF##12##20##] and there is a general consensus nationally[##UREF##1##2##,##REF##17181501##11##,##REF##17181495##19##,##UREF##13##21##] that all newly arrived refugees should undergo a voluntary comprehensive health assessment. Such an assessment would focus both on psychological as well as physical health needs in addition to providing information about illness prevention and health promotion activities and an introduction to the Australian health care system.</p>", "<p>Until recently in SA, the State funded refugee health service along with two community health centres (CHCs) with specific refugee health expertise, performed comprehensive health assessments on a large proportion of newly arrived refugees to SA. With changes to the Department of Immigration and Citizenship (DIAC) funded Integrated Humanitarian Settlement Service (IHSS) contract in 2005, the responsibility of providing this initial care in SA was passed to General Practitioners (GPs) in private practice. Whilst this is the path taken in SA, each Australian state and territory has a different model for the provision of initial health care services with varying levels of involvement of specialist refugee and community health services [##REF##17181495##19##], although GPs in private practice still provide a large proportion of this care. Recognising that GPs require extra assistance to do this work, the Federal Government introduced a new Medicare item number in May 2006 to better remunerate GPs who perform initial refugee health assessments.</p>", "<p>There has been surprisingly little written documenting the experiences of GPs who provide initial care to refugees, both in Australia and overseas, including the challenges they might face and hence their capacity to do this work is uncertain. Some general review articles have been written by health practitioners based on their own experiences providing health care to refugees and challenges listed include those related to language, [##REF##9822406##3##,##UREF##14##22##] the time consuming nature of the work [##UREF##14##22##,##REF##16217566##23##], cultural differences [##UREF##14##22##,##UREF##15##24##] and the special health care needs of refugees [##UREF##15##24##]. Additional challenges found in a limited number of empirical studies overseas include GPs being unaware what previous screening or treatments refugees had undergone [##REF##8292426##25##,##REF##10232324##26##], that there were a lack of targeted services for refugees, [##REF##8292426##25##] that refugees had greater heath care needs compared to non-refugees and that GPs lacked familiarity with the management of conditions unique to refugees [##REF##16048533##27##].</p>", "<p>There are limitations, however, in the applicability of these studies to the Australian context given differing national health systems and special service entitlements for refugees and the fact that it was unclear to what extent these studies related to the experiences of GPs providing care to refugees from similar backgrounds to those currently arriving in Australia. A review of the Australian literature found only one published study addressing this issue. In a report to the Victorian Department of Human Services, the Victorian Foundation for Survivors of Torture (VFST), drawing on their experiences as well as interviews with 19 GPs performing initial health assessments for newly arrived refugees in Melbourne, identified a number of similar challenges for GPs to those already listed [##UREF##1##2##]. Additional challenges included those related to the Australian Medicare fee-for-service system of remuneration which provided only a limited incentive for GPs to offer longer consultations or participate in 'extra consultation' activities often required when providing care to refugees. Challenges were also encountered relating to problems refugees experienced navigating the health system (eg. participating in follow-up GP care or attending referrals) and GPs faced difficulties sustaining their involvement because of the stressful nature of the work.</p>", "<p>Whilst the VFST study provides valuable insight into the experiences of GPs in the Australian context, much of the initial health care for refugees in Victoria is performed by GPs working in State funded community health centres where they are more likely to be salaried and have better access to supports to assist them to manage patients with multiple and complex health needs. By contrast, in SA all newly arrived refugees are referred directly to GPs in private practice for initial health care and there has been no research to date assessing their experiences providing care to refugees in SA.</p>", "<p>Given that current refugee arrivals are likely to carry a greater disease burden combined with the recent increased responsibility of GPs in private practice in SA to provide initial care, the aims of this study were: (i) to document the existence and nature of challenges for GPs who do this work in SA, (ii) to explore the ways in which these challenges could be reduced and (iii) to discuss the policy implications of this in relation to optimising the initial health care for refugees.</p>" ]

[ "<title>Methods</title>", "<title>Design and participants</title>", "<p>Given that the nature of this study was exploratory, a qualitative approach was taken in order to gain a deeper understanding of the challenges faced by GPs in private practice when providing care to refugees. Semi-structured interviews were conducted with 12 GPs providing care to refugees in private practice as well as the Medical Directors of three of the Divisions of General Practice in metropolitan Adelaide with high levels of current or proposed refugee settlement. The study was approved by the University of Adelaide Human Research Ethics Committee.</p>", "<p>To recruit GPs, potential participants were identified via a database of GPs (held by the state funded refugee health service) who were either currently accepting or had accepted refugee referrals in the past. One of the authors (DJ) also used his personal knowledge of GPs known to provide care to refugees through his previous work at the SA specialist refugee health service and through formal and informal networks in the refugee health sector. Additional GPs were also identified following the Division interviews. An introductory letter with a fax-back reply was sent to 77 potential GP participants. After the initial mail out, six GPs agreed to participate. Follow-up phone calls were subsequently made to another ten GPs in order to recruit the remaining six GP participants. GPs were recruited from most regions of the Adelaide metropolitan area although there were none from the southern Adelaide region (resettlement of refugees to this region had only occurred relatively recently). The twelve GPs represented eight separate practices – two groups of three GPs were recruited from the same practices. Two thirds of participants had longstanding involvement in providing care to refugees whereas the remainder had become more recently involved with increasing numbers of African refugees resettling close to their practices. Whilst African refugees made up a large proportion of newly arrived refugees seen in the past twelve months, GP participants also reported providing care to large numbers of refugees from the Middle East as well as a small number of refugees from the Former Yugoslavia.</p>", "<p>To recruit the Medical Directors of Divisions, five were contacted by email with two agreeing to participate. Follow-up telephone calls led to the recruitment of one further Division.</p>", "<title>Data collection</title>", "<p>Given that a number of potential challenges were identified from the literature as well as there being much anecdotal evidence of challenges for GPs to do this work, a semi-structured interview format was chosen to examine these specifically whilst at the same time allowing any previously unknown challenges to emerge. Different interview schedules were used for the GPs and the Divisions respectively. Although the questions for each group focused on the broad aims of the study they addressed slightly different aspects of the issue. The GP interviews generally explored the challenges GPs face when working with refugees whilst the Division interviews focused on the current or potential role of Divisions to support GPs in private to work with refugees and well as the identification of potential structural impediments for GPs doing this work.</p>", "<p>The interviews were conducted between April 2006 and July 2006. Each participant was interviewed once with interviews ranging from 40 to 70 minutes. Individual interviews were conducted with nine of the GP participants and a small group interview was conducted with the remaining three GPs. The three Divisions were each interviewed individually. The interviews were tape recorded and transcribed verbatim. In both the GP and Division interviews data saturation was reached.</p>", "<title>Analysis and reporting of results</title>", "<p>A template analysis approach was adopted [##UREF##16##28##,##UREF##17##29##] where a coding template was developed which included <italic>a priori </italic>themes in addition to new themes identified from initial reading and analysis of the transcripts. Final thematic templates for both the GP and Division transcripts were agreed upon by the Project Team and then all data was coded according to these themes, with DJ undertaking the bulk of the coding. Two transcripts were also independently coded by the other members of the Project Team. Following this, comparisons were made and a consensus reached on how the remaining data was to be coded. Coding numbers were randomly assigned to protect the confidentiality of the participants where direct quotes were reported in the results.</p>" ]

[ "<title>Results</title>", "<p>GPs in this study reported a range of challenges when providing care to refugees. In many cases these challenges were explicitly linked to performing initial assessments whilst at other times GPs spoke of challenges in the broader context of providing care to refugees – but which can be assumed to be operating when providing initial care. The challenges fell into three main categories: (i) refugee health issues; (ii) GP/refugee interaction; and (iii) the structure of general practice. There was a great deal of overlap, however, between these categories and a very strong theme to emerge was not having enough time to do the work required which related to any issue that made a consultation with a refugee longer. Further, these challenges did not appear to be related to how long GPs had been providing care to refugees or to the intensity of their involvement other than that more experienced GPs had a greater awareness of available interpreter services. The Divisions also reported challenges assisting GPs to provide care relating to a limited awareness of refugee numbers settling in their divisions and which GPs needed extra support as well as a lack of specific Commonwealth funding to do this work. Finally, whilst participants suggested ways these challenges could be reduced, overall strong support was provided for initial health care to be provided via a specialist health service.</p>", "<title>Challenges for GPs</title>", "<title>Refugee health issues</title>", "<p>Challenges for GPs providing health care to refugees that related to refugee health issues included GP knowledge of previous health assessments, GP awareness of and experience managing health conditions unique to refugees, and the multiple and complex nature of refugee health conditions.</p>", "<title>GP knowledge of previous health assessments</title>", "<p>A number of GPs and Divisions expressed uncertainty regarding what health assessments refugees had received prior to arrival in Australia:</p>", "<p>I don't know if there is some sort of system that they go through, or some sort of protocol that they, medically, have to go through before they are granted visas... (Dr 1)</p>", "<p>There was also uncertainty regarding what health assessments were carried out after arrival in Australia with some GPs assuming that the MHS still performed this work. Uncertainty regarding previous assessments did not relate to how long GPs had been providing care to refugees.</p>", "<p>For some GPs this resulted in confusion over their role in detecting and managing health conditions unique to refugees:</p>", "<p>So we have got the clinical exotica; we have got very little understanding of what has happened to these people before and where the responsibility stops and starts for who should be following up all these things. (Dr 7)</p>", "<title>GP awareness of and experience managing health conditions unique to refugees</title>", "<p>Only one GP reported using guidelines to assist screening for exotic conditions. It was likely that many conditions unique to refugees were not being detected as indicated by one GP:</p>", "<p>I haven't personally come across anything unusual that would be something that was quite rare... I'm sure I will. I'm sure I have probably missed heaps, too. Slipped through that I haven't seen or recognised. (Dr 1)</p>", "<p>Concern was expressed, however, that refugee screening guidelines would just be another one of many such guidelines that GPs were expected to know about and follow.</p>", "<p>Even if GPs were detecting conditions unique to refugees, there was concern that they did not necessarily have the experience to manage them:</p>", "<p>I guess it is out of our comfort zone, because our medical experience doesn't include the exotic illnesses that they front up with... (Dr 7)</p>", "<p>Again, these challenges were not dependent on how long GPs had been providing care to refugees as many of these challenges related to more recently arrived African refugees.</p>", "<p>The expectation that GPs develop this expertise was also questioned as this was seen to compete with what was thought to be the important broader generalist role of general practice:</p>", "<p>...we are supposed to be highly trained now in mental health and refugee health...when actually we are general practice. We are not specialty people...I think it is important that we stay that way, because you start going down into specialty areas too much and you start to miss the bigger picture... (Dr 1)</p>", "<p>In terms of mental health problems, most GPs were aware of previous traumatic experiences of refugees and that combined with the stresses of resettlement meant that psychological problems often resulted. This also meant, however, that the time it took to build rapport and trust when seeing a refugee was far greater than with a non-refugee patient and this affected the ability to gather information about their background and past medical history.</p>", "<title>The multiple and complex nature of refugee health conditions</title>", "<p>Most GPs described providing care to refugees as demanding, with refugees more likely to have multiple and complex health needs:</p>", "<p>...usually the refugees or migrants that I have seen have got multiple needs. It is usually not just one simple thing. (Dr 10)</p>", "<p>This often meant the nature of the work was time consuming:</p>", "<p>Often at the beginning there are so many issues to get through that I think it takes quite a number of long consultations before you really even have a clear idea about who this person is and what their experiences are. To get all the health issues on the table, I think, takes a really long time. (Dr 11)</p>", "<title>GP-refugee interaction</title>", "<p>A number of challenges relating to the interaction between GP and refugee were raised including issues related to culture and language as well as refugee knowledge of the Australian health care system.</p>", "<title>Issues related to culture</title>", "<p>A number of GPs observed that refugees often had a different understanding of disease causation when compared to a Western model:</p>", "<p>...the way people behave around their health and their illness is very culturally determined. To try and understand what is going on, I can't just impose my framework, because the way they will express themselves is really different about what they are feeling. (Dr 11)</p>", "<p>As a result, GPs reported having to spend much longer than normal explaining Western health concepts to refugees. This included screening activities such as Pap smears and organising referrals for mammograms, when giving a diagnosis of an illness such as Hepatitis B or when referring refugees for pathology tests. Other challenges attributed to cultural differences included uncertainty over cultural appropriateness of examination, gender related issues such as decision-making over birth control and gaining consent for invasive procedures:</p>", "<p>...one woman came in and consented to a Pap-smear and thankfully, we managed to get around it that she actually had one before and was okay to do it, but I thought if she had never had one, how was I going to explain to this woman what I was going to do. (Dr 8)</p>", "<p>Different naming practices also sometimes presented challenges in locating the correct patient file. Although GPs with longer standing involvement in refugee care may have been more aware of cultural differences, all GPs reported challenges related to this issue.</p>", "<title>Issues related to language</title>", "<p>A number of challenges relating to language resulted from the need to use interpreters. These included not being able to adequately provide explanations via an interpreter, difficulties dealing with mental health problems and the extra time required to both conduct a consultation with an interpreter as well as organise an interpreter when one was not pre-booked. Communication challenges were also experienced when contacting refugee patients for follow up of test results or to book an appointment. Although most GPs were aware of the need to use an interpreter when refugees were not fluent in English, those GPs with more recent involvement in refugee care were more likely to be unfamiliar with all the services offered by the Commonwealth Translating Interpreting Service (TIS), such as the doctor's priority phone line (where an interpreter can usually be made available on demand):</p>", "<p>The times that I have needed it they have been – appointments have been booked well in advance. How do you book an interpreter when someone rings up at lunchtime and sees you two hours later for something that is minor or insignificant? (Dr 1)</p>", "<p>One GP was confused about who paid for TIS believing that the practice was billed for an interpreting service when it was booked and the patient did not attend the appointment. A number of GPs also talked about difficulties contacting refugee patients for follow up of test results or to book an appointment:</p>", "<p>Communication, when you know they can't speak English, so you can't phone them, and when you know that they are quite a mobile group of people, so that when you send a letter to their address, they might have moved on. (Dr 6)</p>", "<title>Refugee knowledge of the Australian healthcare system</title>", "<p>Almost all GPs mentioned difficulties resulting from refugees' lack of familiarity with the Australian health care system. This related to missed appointments, which meant no remuneration for GPs, or refugees arriving late:</p>", "<p>That is a difficult problem with them... they will turn up really late for an appointment with no sort of seeming reference to the timeslot that they were given... it does sometimes make it difficult for us if we are then on the back foot for the rest of the session. (Dr 1)</p>", "<p>GPs also reported that refugees' lack of understanding of the Australian health system resulted in challenges for GPs when they were referring refugees to other agencies or specialists and also when writing prescriptions. As a result GPs reported spending more time providing explanations about how the health system worked:</p>", "<p>I take more time with them...Because the health care system here is much different... I try to make it easier for them to understand the system and how it works here. (Dr 5)</p>", "<title>Structure of general practice</title>", "<p>A number of challenges relating to the structure of general practice were identified including GP workforce shortages, a lack of organised referral pathways for refugees to general practice as well as a lack of remuneration and infrastructure support required to perform initial assessments.</p>", "<title>GP workforce shortages</title>", "<p>As a result of the demand for GP services outstripping supply in some regions of Adelaide, providing appointments for any new patients, whether they were refugees or not, was often difficult. Three GPs in this study, with high loads of patients with complex health care needs including refugees, had closed their books to new patients and another GP described potential difficulties accepting new patients:</p>", "<p>We are having trouble accepting new patients full stop... freely accepting new patients irrespective of whether they are a refugee or not, it is difficult to actually accommodate everybody. (Dr 1)</p>", "<p>Further, as highlighted by one Division, with GP shortages most acute in socioeconomically disadvantaged areas, refugees were more likely to be affected given that they are often settled in areas where housing was cheaper:</p>", "<p>The cheaper areas for housing [are] where the workforce is the worst, so you can end up in this vicious circle where the practices go \"Ah, we are closing our books, we are just not seeing anyone new\". (Div 2)</p>", "<title>Referral systems</title>", "<p>Overall GPs reported that there was usually no clear referral pathway for refugees to private general practice. This was perceived as a problem because it meant that GPs were not able to control the numbers of refugee patients they saw when there were limits to the amount of work they could do with patients with multiple and complex needs such as refugees:</p>", "<p>... because it is primary care you are expected to just take everybody that walks through the door. That doesn't work... lots and lots of agencies would like to refer here. We have to somehow prevent ourselves from drowning. (Dr 11)</p>", "<p>Complicating this was the fact that when a GP took on one refugee then it was most likely that the rest of the family would then come to see the same GP which could dramatically increase their caseload of patients with high health care needs. Related to this was a fear for one GP clinic of being inundated with refugees if their clinic was promoted as a formalised referral centre for refugees because of difficulties already meeting the needs of their current patient load. A situation where there was no system in place to manage referrals was described by one GP as 'a recipe for burnout'.</p>", "<p>Not having a formal referral pathway to GPs also led to problems with the transfer of health information including results from pre-departure health checks and any health services refugees had accessed in Australia. It was also noted that poor transfer of health information could also result in duplication of services such as immunisation.</p>", "<title>Remuneration</title>", "<p>Half of the GPs identified remuneration as a challenge when working with refugees. This was because refugees were mostly bulk billed and many needed longer consultations which were felt to be inadequately remunerated under the current Medicare billing system:</p>", "<p>...it's just not financially viable because, as we know, long complex consultations are not a way which assists you to run your practice in a way that is financially viable... (Dr 11)</p>", "<p>Remuneration was also a challenge because of the fact that refugees often missed appointments, which meant no remuneration, and work with refugees often involved time consuming extra-consultation activities that could not be charged to Medicare:</p>", "<p>Missed appointments are fairly common...So you miss the remuneration if they don't come. The time factor; the complexity of the consult is more than what is remunerated for the time involved, because your follow-up is often phone calls to various agencies or organising things, writing letters, becoming an advocate, coordinating allied health. Very little of that is remunerated... (Dr 10)</p>", "<p>Despite the strong indication that remuneration was a challenge for GPs, the majority of GPs said that they were either not going to use or were unlikely to use the new Medicare item number for performing an initial health assessment on a refugee although there was moderate support for it in principle. This was either because of a lack of familiarity with already existing Enhanced Primary Care (EPC) item numbers or because of the high administrative burden associated with their use:</p>", "<p>It is not something I am likely to use personally. I think it is a great idea ... but I am not very good at using the specific numbers ... and the EPC items and so on. I just don't have time to sort out all the paperwork for that sort of thing. (Dr 6)</p>", "<p>Another GP was disappointed that the Divisions of General Practice had not produced a template to assist using the item number in the same way that they had with previous EPC item numbers. Finally, a number of GPs were critical that the item number did not go far enough in that it failed to recognise that the greater health care needs of refugees were ongoing.</p>", "<title>Infrastructure supports to perform initial assessments</title>", "<p>There was strong evidence provided by the majority of GPs in this study that they did not have the necessary infrastructure support, i.e. the systems and support staff, to perform initial refugee health assessments:</p>", "<p>We are not well enough equipped. We are not resourced, we do not have the supporting background structure. (Dr 9)</p>", "<p>It was particularly overwhelming for GPs when groups or families all came at once and they had not had a previous health assessment:</p>", "<p>...we were having whole families of recent arrivals come to the surgery and need all their history taken; immunisations brought up-to-date and that was just overwhelming... We do have a practice nurse, but she is usually quite busy doing other things. That was too much for us to handle. (Dr 6)</p>", "<p>The Divisions also believed that a lack of infrastructure support was a reason it would be difficult for GPs to perform initial health assessments and was a reason uptake of the new item number would be limited:</p>", "<p>If you just basically say \"Here is a new item number doctors\" it won't be taken up, because it is going to be all too hard. From an infrastructure perspective most practices lack the infrastructure to really make this work. (Div 3)</p>", "<title>Challenges for Divisions assisting GPs</title>", "<p>The Divisions identified a number of challenges in assisting GPs to provide care to refugees. They expressed concerns that they did not know how many refugees were being resettled as well as precisely where these resettlements were occurring within their Divisions. The Divisions reported also having limited awareness of which GPs in their Divisions were providing care to refugees and, as a result, were not able to determine which GPs might need extra support to do this work. One Division had surveyed GPs to assess this but most had been too busy to respond. Although a number of GPs had been recruited by the IHSS provider to offer care to newly arrived refugees, the Divisions expressed frustration that they did not know who these GPs were. They were also concerned that there was no collaboration with the IHSS provider which might avoid resettling refugees in areas where they might have difficulty accessing GP services:</p>", "<p>... there is no point putting refugees in to an area where there are no GPs. There might be GPs there but they might not want to see refugees or they might have closed their books. We would have that intelligence; they would have no idea about that... (Div 1)</p>", "<p>All Divisions mentioned a lack of funding as a major reason their ability to help GPs was limited. Because refugee health was not a priority area for the Commonwealth, Divisions received no direct funding for refugee health initiatives:</p>", "<p>We are funded by the Department of Health and Ageing; we have got a whole lot of quality indicators that we have got to actually achieve in. Refugee health does not even appear in there. (Div 3)</p>", "<p>The Divisions explained that it was also a lack of funding that limited their ability to assist GPs to utilise the new item number. Despite this, two Divisions had diverted core funding to better support GPs in the area of refugee health but no Divisions had funding for specific services or programs. One Division felt their case to argue for increased funding was limited given the lack of data related to refugee numbers settling in the Division and that there was no empirical evidence that GPs weren't coping even though it was recognised that GPs were too busy to answer surveys that might provide this data.</p>", "<title>Ways GPs could be better supported</title>", "<title>Providing GPs with more resources</title>", "<p>Despite GPs questioning how realistic it was for them to manage many of the exotic health conditions in newly refugees, there was some support for the provision of screening guidelines for use by GPs in private practice provided they were in a simple and readily accessible format – such as linked to the general practice software program Medical Director (MD).</p>", "<p>GPs reported they could be assisted to overcome some of the challenges related to culture if they were provided with more background information about refugee groups either through the provision of information sheets or talks from different community members regarding different cultural practices. It was also suggested that these challenges could be reduced through better provision by settlement services of health information to refugees on arrival including information about common conditions in refugees such as hepatitis B, early intervention and illness prevention activities and better initial orientation to the Australian health care system. Many GPs also believed that settlement services could provide more assistance to refugees to attend appointments. Other ways of improving refugee navigation of the health system included a greater role for voluntary organisations, practice nurses or community health care workers who could be health advocates for refugees:</p>", "<p>At times you feel like you're running around doing a lot of the work that could be partly done through either a practice nurse or another allied health worker or somebody who can, on the ground, advocate for that refugee individual. (Dr 10)</p>", "<p>In relation to referral pathways of refugees to general practice, it was strongly stated by a number of GPs that this should involve a consent process which assessed the ability of a GP to take on the care of a new refugee or refugee family:</p>", "<p>It is not a kind of fair system to plonk someone onto a practice. I think agencies should actually liaise with either one of the senior doctors or, if there is a nurse... so a referral can actually be properly organised and assessed as to whether the practice can take someone on. I think the ways it has happened in the past have been really unsatisfactory. (Dr 11)</p>", "<p>Despite limited enthusiasm for the new Medicare item number, a small number of GPs were still interested in learning more about how to use it through their Division and there was also some support for the provision of a template by the Divisions which could also be incorporated into MD.</p>", "<p>The Divisions suggested a number of ways they could assist GPs to perform initial assessments if they were provided with funding. This included improving the utilisation of the item number by educating practice nurses and developing a template as well as Division funded 'clinical attachments' at the state funded specialist refugee service to provide GPs with greater expertise in managing health conditions unique to refuges. It was also suggested that Division funded refugee health infrastructure grants could provide assistance to GPs to better set up their clinics to care for refugees via IT support and the provision of business cases for employing practice nurses. One Division, however, expressed uncertainty about the likelihood that GPs would be willing to build such systems into their practices if they were only seeing small numbers of refugees, particularly given that there was limited enthusiasm to build similar systems for high prevalence chronic diseases in the general population.</p>", "<title>Providing initial refugee health care via a specialist service</title>", "<p>Despite indicating ways they could be assisted, a number of GPs believed that the responsibility of providing initial care to refugees should not lie with GPs in private practice:</p>", "<p>There should be a front line somewhere. I don't think general practice should be the front line. (Dr 8)</p>", "<p>Instead, a system where refugees received an initial assessment via a specialist refugee or community health service was strongly supported by both GPs and the Divisions. One GP described the advantages of a community health service where a range of services could be provided to refugees in one location:</p>", "<p>I think the community health service is the best stop for an initial assessment because of the complexity of the presentations, usually, and the need for accessing a lot of different services that is really beyond most private centre GPs to be able to do that adequately. If you have got access within one building, for example, to workers who can do some of the chasing up, some of the phoning and some of the coordination, then you are much more likely to give people a good service. (Dr 10)</p>", "<p>A number of GPs indicated that they would be much happier to accept referrals of refugees if they had had an initial assessment where they could then focus on their more day-to-day health needs.</p>" ]

[ "<title>Discussion</title>", "<p>This study highlights the many and diverse challenges faced by GPs in private practice when providing health care to refugees in their initial resettlement period. These challenges and their policy implications are discussed below.</p>", "<title>Challenges performing initial assessments</title>", "<p>The extent of the challenges faced by GPs providing initial care to refugees in this study is not surprising given that it is during their early resettlement period that refugees are most likely to experience multiple medical problems, many of an exotic nature, when language and cultural barriers are likely to be greatest and when refugee knowledge of the Australian health care system and general health literacy are likely to be most limited.</p>", "<p>Whilst many of the challenges identified support previous research in this area, the most striking feature of this study is the strong evidence that GPs in private practice are not sufficiently resourced to provide initial care effectively to newly arrived refugees with multiple and complex health needs. For GPs in this study, the lack of resources existed both at an individual GP level, with GPs lacking comprehensive knowledge of the health conditions unique to refugees, as well as at a more structural health system level, where GPs lacked both the time and the infrastructure support to do this work effectively. Further, the lack of resources was not related to the length of time GPs had been providing care to refugees or the intensity of their involvement other than that GPs with less experience were less familiar with the use of interpreter services.</p>", "<p>Given that the first point of contact with the Australian healthcare system for refugees currently arriving in SA is with a GP in private practice, there are a number of health consequences for refugees if GPs do not have the necessary resources to provide them with effective care in their initial resettlement period. These include GPs missing or inadequately treating physical and mental health problems unique to refugees, refugees not following through with treatments or referrals and refugees under engaging with illness prevention activities because of poor health literacy [##REF##16805158##30##]. This can result in refugees experiencing a reduced health status compared with the non-refugee population as well as potentially greater costs to the health system because of later and more expensive treatments.</p>", "<title>Implications for policy</title>", "<title>Barriers to building capacity of GPs to perform initial assessments</title>", "<p>Although GPs mentioned a number of ways they could be assisted to provide more effective initial care to refugees, they also indicated that there would be major limitations attempting to build this capacity.</p>", "<p>Firstly, GPs questioned the expectation that they develop the specific 'refugee health' expertise needed for performing initial assessments which competed with their role as 'generalists'. It is likely that, at best, developing the required expertise to perform initial assessments will only appeal to a small number of GPs.</p>", "<p>Secondly, even those GPs who have an interest in doing this work may not want to identify themselves as a 'refugee doctor' for fears, as stated by one GP, that they will become inundated with referrals. GPs in this study indicated that there were limits to the amount of work they could do with refugee patients given the often multiple and complex needs on initial presentation. GPs, however, operate in a primary health care (PHC) system where they have little control over how patients are referred to them. Further, GPs performing initial health assessments are most likely to be the GPs who provide the ongoing care (more so now given that refugees are initially settled in more permanent accommodation). As a result, GPs providing initial health care can quickly end up with very high numbers of patients with multiple and complex health needs. A number of GPs in this study indicated that this had contributed to them closing their books to new patients. Refugee health service providers in Adelaide as well as interstate[##UREF##1##2##] have also experienced the difficulties sustaining GP involvement under these circumstances. To avoid overburdening a small number of GPs would mean, however, offering more general training to a large number of GPs. This is unlikely to be a cost effective approach and also, as evidenced by this study, developing the necessary expertise and building the practice systems required to provide effective initial health care to refugees will, at best, appeal only to a small number of GPs.</p>", "<p>Thirdly, GPs indicated that providing initial care to refugees was time consuming but the fee-for-service structure of general practice combined with GP workforce shortages limited the time GPs could offer to refugees to manage their multiple and complex health needs – a problem shared with other groups who have greater health care needs [##UREF##18##31##]. Under these circumstances, GPs are unlikely to take on a role that will require them to offer a greater number of longer consultations. This could be one reason why the new Medicare item number received limited support from GPs in this study despite the fact that a lack of remuneration was an issue for a number of them. As suggested by the Divisions, this could also be an indication that the new item number, in its current form, does very little to address the resource problems described above. It is interesting to note that the initial uptake of the item number was greatest in Victoria [##UREF##19##32##] where a large number of refugees receive initial GP care in community health centres with the support of refugee health nurses which highlights the importance of providing adequate time and infrastructure support when doing this work [##UREF##20##33##]. A further limitation of the item number, also mentioned by GPs in this study, is that it does not take into account the fact that the greater initial health care needs often persist beyond the first visit with a GP.</p>", "<title>The role of a specialist health service</title>", "<p>An alternative to providing initial care to refugees in private general practice is for this to be provided in a specialist refugee service or community health setting. Such a service delivery model received strong support from participants in this study. Previous studies have similarly highlighted the central importance of community health services providing initial health care to patients with complex health needs, including refugees, as a result of better access to resources and infrastructure support [##UREF##1##2##,##REF##10232324##26##,##UREF##21##34##].</p>", "<p>It is acknowledged that there is not a one size fits all approach when determining which model, specialist or mainstream, best meets the special service needs of refugees in their initial resettlement period [##UREF##22##35##] and that receiving PHC via a specialist service may delay refugee engagement with local mainstream PHC services [##UREF##1##2##]. Where there is limited capacity, for mainstream services to provide for these special needs, and this study provides strong evidence that this is the case in private general practice in SA, there is a role for a specialist service to fill this service gap [##UREF##22##35##,##REF##16876836##36##]. In SA, such a state funded service already exists, although it is not currently being utilised as the current settlement service provider has adopted a policy of connecting refugees directly with mainstream health services immediately on arrival to Adelaide. It makes sense to utilise the current refugee health expertise and resources of this state funded service to provide initial health care services to refugees, especially those with complex health needs and significant resettlement challenges. Whilst this is a centralised service, the highly centralised population distribution of SA in Adelaide, combined with the relatively small number of refugee arrivals, means that it is accessible for the majority of refugees in SA. Further, the ability to deliver refugee services in multiple community health locations, such as in Victoria, is limited in Adelaide because of a lack of medical presence at these sites. It is recognised, however, that a centralised specialist service is not well suited to larger Australian cities such as Sydney and Melbourne. Finally, if initial health assessments are provided by a specialist service, it is important that a clear, transparent and effective referral system to a nominated general practice is part of this process when initial health care needs have been met. Ongoing links between general practices and the specialist provider would also address a number of the other challenges identified by GPs and Divisions in this research.</p>", "<title>Study strengths and limitations</title>", "<p>This study provided a unique and detailed insight into the experience of GPs providing health care to refugees. However, given the small number of participants in this study, these results cannot be generalised to all GPs in Adelaide or GPs in other locations. To do this, a larger quantitative study would be required. The low response rate from GPs could have meant that those GPs involved were more motivated to participate because of dissatisfaction with the current system of provision of initial health care to refugees. This low response rate, however, mirrors the experience of Divisions in this study and their difficulties getting GPs to participate in research and respond to surveys. Further, it is generally believed that at the time data was collected for this study there were a limited number of GPs (although the exact number is not available) providing initial care to refugees in SA. It is likely, therefore, that the views of GPs in this study not only provide a reasonably comprehensive summary of the challenges of providing initial care but are also the experience of most GPs doing this work in SA at the time. Whilst GPs interstate are likely to face many similar challenges providing initial care to refugees, it is beyond the scope of this paper to comment on how well resourced they are to provide effective care.</p>" ]

[ "<title>Conclusion</title>", "<p>This study provides evidence that, due to a range of challenges, GPs in private practice in SA are insufficiently resourced to provide initial health care effectively to refugees and that attempting to overcome these challenges would face a number of obstacles. Whilst further evidence is required to document the extent of these challenges in SA and how they might be best addressed, it makes sense for the existing state funded refugee health service to be involved in the delivery of initial PHC services to refugees, especially those with complex health needs and significant resettlement challenges.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Introduction</title>", "<p>Many refugees arrive in Australia with complex health needs. In South Australia (SA), providing initial health care to refugees is the responsibility of General Practitioners (GPs) in private practice. Their capacity to perform this work effectively for current newly arrived refugees is uncertain. The aim of this study was to document the challenges faced by GPs in private practice in SA when providing initial care to refugees and to discuss the implications of this for policy relating to optimising health care services for refugees.</p>", "<title>Methods</title>", "<p>Semi-structured interviews with twelve GPs in private practice and three Medical Directors of Divisions of General Practice. Using a template analysis approach the interviews were coded and analysed thematically.</p>", "<title>Results</title>", "<p>Multiple challenges providing care to refugees were found including those related to: (1) refugee health issues; (2) the GP-refugee interaction; and (3) the structure of general practice. The Divisions also reported challenges assisting GPs to provide effective care related to a lack of funding and awareness of which GPs required support. Although respondents suggested a number of ways that GPs could be assisted to provide better initial care to refugees, strong support was voiced for the initial care of refugees to be provided via a specialist refugee health service.</p>", "<title>Conclusion</title>", "<p>GPs in this study were under-resourced, at both an individual GP level as well as a structural level, to provide effective initial care for refugees. In SA, there are likely to be a number of challenges attempting to increase the capacity of GPs in private practice to provide initial care. An alternative model is for refugees with multiple and complex health care needs as well as those with significant resettlement challenges to receive initial health care via the existing specialist refugee health service in Adelaide.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>DJ conceptualised the study, conducted the literature review, undertook data collection and analysis, and drafted the paper. AZ and TB advised on all stages of the work including analysis of the data as well as reviewing and contributing to drafts of the paper. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The authors thank the participants of this study for generously devoting their time to make this research possible. This report was funded through the Commonwealth Primary Health Care Research, Evaluation and Development Program (PHC RED), the Central Northern Adelaide Health Service and the Survivors of Torture and Trauma Assistance and Rehabilitation Service. The study was conducted independently of the funding bodies and the views expressed in this paper are solely those of the authors. Anna Ziersch's involvement was supported by the Flinders University Department of Public Health. The PHC RED funding was provided through the Discipline of General Practice at the University of Adelaide. Teresa Burgess was involved in her capacity as Senior Lecturer in this discipline.</p>" ]

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[ "<title>Background</title>", "<p>The department of psychiatry in a general hospital setting has a multidimensional role, providing inpatient care, maintaining strong interaction with community psychiatric services and offering specialist services to the general hospital wards either as part of the multidisciplinary approach to patient management or by offering specialist inpatient care to patients already hospitalised in other departments by transferring certain patients to the psychiatry department [##REF##4597302##1##,##REF##2669009##2##].</p>", "<p>The consultation-liaison psychiatry service is the link between any general hospital ward and the department of psychiatry [##REF##3406340##3##]. But what are the reasons for transferring a patient from a non-psychiatric bed to an inpatient psychiatric unit?</p>", "<p>To the best of our knowledge, there are only a few reports on patient transfer issues although it is a common practice. In this context, in the present work we put forward our experience and thoughts on the factors that drive the patients transfer from general medicine to psychiatry.</p>", "<p>We examined demographic and clinical backgrounds of a group of patients transferred from internal medicine or surgery to the psychiatric ward. A comparison was made of this data with data obtained from a group of non-transferred patients, also seen by the same consultation-liaison psychiatry service.</p>" ]

[ "<title>Patients and methods</title>", "<p>The present study was carried out at the Peripheral General Hospital of Athens 'G. Gennimatas', an approximately 650 bed community-based hospital with a 18 bed psychiatric unit that covers the greater Athens area. During the study period the psychiatric ward at 'G. Gennimatas' only received voluntary admissions, and operated as an open, short-term unit (the first author of this study worked at the above department during the study period).</p>", "<p>The files of the patients transferred to the psychiatric unit by the consultation-liaison service between 1 March 1989 (opening of the inpatient psychiatric unit) and 31 December 1999 were reviewed. In the year 2000 the law for compulsory hospitalisation of the mentally ill in Greece changed, therefore, all the psychiatric units housed in general hospitals were obligated to also receive compulsory admissions. This change of status has influenced not only the atmosphere in the psychiatric unit but also the admissions by the consultation-liaison service.</p>", "<p>The data collected from the review of the transferred patients' charts included: age, sex, marital status, ward from which the patient was transferred, current psychiatric complaint, medical diagnosis, length of hospital stay, prior psychiatric history, psychiatric inpatient treatment, psychiatric diagnosis and use of psychotropic medication; socioeconomic status was also deduced using the patients' files. This data was compared with data from non-transferred patients' files (control group, corrected for age and sex) during the year 1994–1995 (during this year the first author of this study was responsible for the consultation-liaison service).</p>", "<p>The psychiatric diagnoses are according to the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IIIR [##UREF##0##4##] and DSM-IV [##UREF##1##5##] categories. For quantitative comparisons a t test was employed, whereas for qualitative comparisons we used a two-tailed Fisher's exact test.</p>" ]

[ "<title>Results</title>", "<p>In total, 294 patients (139 men and 155 women) were transferred to the psychiatric ward during the 11-year period of the study (1989 to 1999). The mean number of transfers per year was 26.7, ranging from 18 (1989) to 35 (1991 and 1998). During the above time period the psychiatric unit offered inpatient treatment to 2,974 patients; thus, the admissions by the consultation-liaison service accounted for 9.9% of the total admissions. In the same period, the overall number of referrals for psychiatric assessment was 5,567; thus, 5.2% of the patients seen by the consultation-liaison service were eventually transferred to the psychiatric ward.</p>", "<p>The control group consisted of 225 patients (110 men and 115 women). The majority of the control group came from medicine (156, corresponding to 69.3%), and the remainder (69, 30.7%) came from surgery; the majority of the transferred patients also came from medicine (215, 73.1%) and the remainder (79, 26.8%) from surgery.</p>", "<p>Table ##TAB##0##1## shows demographic data from the transferred and the control groups. There were no significant differences regarding age and sex between the two groups. The transferred group patients were more likely to be single, divorced, or widowed compared to the non-transferred group patients, who were more likely to be married. In all, 44 (15.6%) patients of the transferred group had serious social, family and financial problems versus 11 (4.8%) of the non-transferred group (Fisher's exact test, p &lt; 0.001).</p>", "<p>Among the 294 transferred patients, 223 (75.8%) had a prior psychiatric history whereas 71 (24.1%) did not. Of the non-transferred group, 142 (63.1%) patients had a previous psychiatric history whereas 83 (36.9%) did not. This difference between the two groups is statistically significant (Fisher's exact test, p &lt; 0.01). Of the transferred patients, 124 (42.1%) had prior psychiatric inpatient treatment, whereas 170 (57.8%) did not have any psychiatric treatment in their history, versus 21 (9.3%) and 204 (90.6%) of the control group (Fisher's exact test, p &lt; 0.001).</p>", "<p>Table ##TAB##1##2## shows the main psychiatric complaints of both groups. Suicide attempts and disruptive behaviour/non-compliance were the most often encountered psychiatric complaints in the transferred group. Suicide attempts (146) represent 49.6% of transfers, 103 (70.5%) of them being related to drug overdose (self-poisoning), whereas 43 were not drug related.</p>", "<p>The mean hospital stay for the transferred patients was 26.31 ± 21.15 days (the hospital stay of 23 patients who left against medical advice is not included). During the 11-year period of the study, the longest mean hospital stay for the patients admitted through the outpatient psychiatric clinic and the emergency department (20.9 ± 22.4 days) was observed in 1995; nevertheless, the mean hospital stay for the transferred patients was significantly greater than the above number (t = 2.88, p &lt; 0.01). We noticed that the patients with suicide attempts that were not drug-related (43) together with the patients with serious social problems (46) had the longest hospital stays (table ##TAB##2##3##).</p>", "<p>Table ##TAB##3##4## shows the diagnoses and comparison of the two groups. The transferees were more likely to have been diagnosed with a mood disorder (including bipolar disorder types I and II, unipolar depression, dysthymic disorder) or a personality disorder, whereas the non-transferred were more likely to have been diagnosed with adjustment disorder as well as having 'no psychopathology'. In the other diagnostic categories there are no significant differences. In the transferred group, 23 patients had diagnoses on both axes I and II of the five axis system used for mental health diagnosis, compared with 9 non-transferred patients with the same pattern. Thus, overall, 56 of the transferees (19.0%) compared to 19 (8.4%) patients of the control group had a diagnosis on axis II (Fisher's exact test, p &lt; 0.001). Of the transferred patients, 21 had a second diagnosis on axis I related to addictions, versus 13 patients of the control group. No diagnosis was made for 23 of the transferees and 16 of the non-transferred patients.</p>", "<p>Table ##TAB##4##5## shows the medical diagnoses for the two groups. We note that the number of injured/poisoned patients in the transferred group was significantly greater than the number of the corresponding non-transferred patients.</p>" ]

[ "<title>Discussion</title>", "<p>During the study period (1989 to 1999), the transfers to the psychiatric unit handled by the consultation-liaison service accounted for approximately 9.9% of total admissions, with transfers representing the third source of admissions to the unit after the psychiatric emergency service (59.6%) and the psychiatric outpatient clinic (27.3%). The mean number of admissions per year was 26.7, or 5.3% of the referrals for psychiatric assessment during the 11-year period of the study. Similar percentages in the literature range from 8 to 14.9% [##REF##2721941##6##, ####REF##7141212##7##, ##REF##6628985##8##, ##REF##8435691##9####8435691##9##].</p>", "<p>In fact, the above numbers and percentages of transfers to psychiatric wards may seem relatively small given that psychiatrists reportedly believe that psychopathology in the hospitalised population at any moment, even with conservative estimations, exceeds 30% and ranges from 30 to 50% [##REF##7304795##10##]. Psychiatric units have been said to be reluctant to receive patients transferred from the general hospital and this has been an important issue. Their 'preference' to patients with psychiatric diagnoses only is based not only on the pressure from the community for such admissions, but also on the argument that patients with somatic illnesses may exert a 'negative' influence on the therapeutic environment or are difficult to take care of [##REF##367914##11##,##REF##3926608##12##].</p>", "<p>At this point, we should perhaps clarify that the term 'difficult to take care of' usually refers to those patients who present with a variety of, mainly behavioural, problems in addition to their somatic illness, which actually makes them 'not wanted' in any ward [##REF##634331##13##, ####REF##16342837##14##, ##UREF##2##15####2##15##]. Some of these problems may have been the reason that led their physicians to refer them for a psychiatric consultation or even discuss a transfer to psychiatry in the first place.</p>", "<p>Marital status seems to be a basic discriminating factor between the two groups. Transferred patients were more likely to be single, divorced or widowed compared to controls that were more likely to be married. The same conclusion was reported by Leibenluft <italic>et al</italic>. [##REF##2721941##6##]. The patient spouse and/or family seem to play an important role in the compliance to inpatient treatment in any general hospital ward and make the need for a transfer to psychiatry less likely [##REF##11766968##16##, ####REF##12667162##17##, ##REF##14706941##18##, ##REF##16905722##19####16905722##19##].</p>", "<p>Serious social (unemployment, extreme poverty, homelessness, lack of health insurance, etc) and family problems also seem to prevail in the transferred group. The absence of social support systems makes psychiatric inpatient treatment and the transfer to psychiatry more likely [##REF##14706941##18##, ####REF##16905722##19##, ##REF##2508882##20##, ##REF##2511981##21##, ##REF##1621137##22##, ##REF##17227704##23####17227704##23##].</p>", "<p>The transferred patients were significantly more likely to have a prior psychiatric history and a prior inpatient psychiatric treatment compared to the non-transferred group. It has been reported that the best predictors of hospitalisation are previous rehospitalisations, more severe symptoms and dissatisfaction with family relations [##REF##8564506##24##]. However, a significant number of transferees (58.6%) had their first inpatient psychiatric treatment after their admission to the general hospital for the treatment of a physical illness, and this happens in the majority of the transferred patients. How can we explain this number? This is probably due to the relatively poor psychiatric care system in Greece. Some of the inpatients with suicide attempts (especially the ones with a first suicide attempt without prior psychiatric history), who would have been referred to an outpatient psychiatric service after leaving the hospital ward, need to remain for a few days in the psychiatric unit to ensure they have regained adequate control of their life. Another reason would be that psychiatric services are not readily available in general or not friendly enough to people who may need them at their time of need. Thus, a long-standing psychiatric problem is often revealed, or it is seen how important it is, or it is aggravated, when a patient is hospitalised for a medical problem. We would even go as far as to say that it seems the presence of a psychiatric unit in a general hospital setting makes psychiatry more available, or more 'justifiable', at least to people with coexisting medical problems.</p>", "<p>The mean hospital stay of the transferees was longer than the mean hospital stay of the direct psychiatric admissions. The co-existence of medical or surgical problems together with psychiatric problems, for instance, a serious trauma after a suicide attempt, sometimes requires a long hospital stay and makes a longer hospital stay more likely in the transferees [##REF##3111277##25##, ####REF##1992834##26##, ##REF##16145188##27####16145188##27##]. By contrast, medical co-morbidity was present in a substantial number of psychiatric inpatients in the general hospital units and this was associated with a prolonging of the length of their hospital stay as well [##REF##11927754##28##]. The interaction of depression, which is the most common diagnosis amongst the transferred inpatients, and physical illness, has been reported to increase the length of stay in psychiatric units [##REF##10024064##29##]. Nevertheless, in our study there were no important differences in the presence of physical illness between the two groups, excepting traumatic injuries and self-poisoning after attempted suicide, more often found in the transferred group.</p>", "<p>The social conditions (marital status, unemployment, extreme poverty, homelessness, lack of health insurance etc) that some of the transferees were experiencing can give an additional explanation for the longer stay in the psychiatric ward [##REF##3131674##30##,##REF##9299923##31##].</p>", "<p>The majority of the transferees had a recent suicide attempt (46.6%). This percentage proved to be higher compared to the 19–40% reported by similar international studies [##REF##2721941##6##,##REF##7141212##7##]. Suicide attempts are reported to be increasing in many countries. Consequently, attempted suicide is a regular reason for admission to a general hospital for both sexes [##REF##9549454##32##, ####REF##9519096##33##, ##REF##12831086##34####12831086##34##].</p>", "<p>Undoubtedly, a suicide attempt is among the conditions that alarm and sensitise physicians on medical and surgical wards. A recent suicide attempt, or a suicide attempt that takes place within a hospital ward, alerts the physicians and makes them very sensitive to any thought or action that could be considered self-destructive, even months after the attempt. What is more, it is not unusual for patients with a recent suicide attempt or suicidal ideation or major depression to be treated in overcrowded wards or on high floors near windows that cannot be safely locked, or in rooms that cannot be easily inspected by the nursing station [##REF##7141212##7##]. The transfer of such patients to the psychiatric unit is dictated not only by the above-described lack of rehabilitation psychiatric services but also by pressure from physicians and, of course, by the understanding on the psychiatrist's side of the stress the physicians and the staff involved in treating such patients go through.</p>", "<p>Behavioural problems and non-compliance are often encountered in the transferred patients (12%). Although psychiatrists usually try to keep such patients in the medical and surgical wards, when the efforts of the physicians are aimed rather at controlling the patients' impulsivity and disruptive behaviour than on the treatment of their somatic illness, their transfer to psychiatry often appears the only way to deal with them. In addition, the negative feelings of the doctors, the staff, and the rest of the patients treated in the same wards towards such 'difficult' patients create a burden carried not only by the people around them but also by the patient [##REF##2721941##6##,##REF##7141212##7##,##REF##634331##13##, ####REF##16342837##14##, ##UREF##2##15####2##15##].</p>", "<p>As for the psychiatric diagnoses, the mood disorders (mainly depressive and dysthymic disorder) and the disorders on axis II seem to discriminate the two groups. Specifically, the transferred group was significantly more likely to have a mood disorder or a disorder on axis II. Depression is the most common diagnosis in patients suffering from a physical illness, and it was evaluated either by self-rated depression scales or by structured psychiatric interview [##REF##3521339##35##, ####REF##2189874##36##, ##REF##8521939##37##, ##UREF##3##38####3##38##]. However, at this point we would like to stress again that in the present study the difference in the diagnoses between the two groups is mainly attributed to the increased number of transferees with suicide attempts.</p>" ]

[ "<title>Conclusion</title>", "<p>According to our findings, the typical transferred patient, either female or male, is single, divorced or widowed, lives alone, belongs to a lower socioeconomic class, presents with a disturbed and disruptive behaviour, has a recent suicide attempt with persistent suicidal ideas, suffers from a mood disorder, has a prior psychiatric history and a diagnosis on axis II. Psychiatric diagnosis on axis I (except mood disorders) does not seem to play an important role in the decision of transferring a patient to the psychiatric ward. It is also worth mentioning that the medical diagnosis does not seem to play a major role in the transfer to the psychiatric ward. As for the future, it might be of help if our efforts aim at considering and testing in the long run, by prospective studies, reliable criteria and factors, that should be acknowledged every time a transfer to psychiatry is decided.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>There are only a few reports on issues related to patient transfer from medical and surgical departments to the psychiatric ward by the consultation-liaison psychiatry service, although it is a common practice. Here, we present a study assessing the factors that influence such transfers.</p>", "<title>Method</title>", "<p>We examined the demographic and clinical backgrounds of a group of patients transferred from internal medicine and surgery to the psychiatric ward over an 11-year period. A comparison was made of this data with data obtained from a group of non-transferred patients, also seen by the same consultation-liaison psychiatry service.</p>", "<title>Results</title>", "<p>According to our findings, the typical transferred patient, either female or male, is single, divorced or widowed, lives alone, belongs to a lower socioeconomic class, presents initially with (on the whole) a disturbed and disruptive behaviour, has had a recent suicide attempt with persistent suicidal ideas, suffers from a mood disorder (mainly depressive and dysthymic disorders), has a prior psychiatric history as well as a prior psychiatric inpatient treatment, and a positive diagnosis on axis II of the five axis system used for mental health diagnosis.</p>", "<title>Conclusion</title>", "<p>The transfer of a patient to the psychiatric ward is a decision depending on multiple factors. Medical diagnoses do not seem to play a major role in the transfer to the psychiatric ward. From the psychiatric diagnosis, depressive and dysthymic disorders are the most common in the transferred population, whilst the transfer is influenced by social factors regarding the patient, the patient's behaviour, the conditions in the ward she/he is treated in and any recent occurrence(s) that increase the anxiety of the staff.</p>" ]

[ "<title>Limitations</title>", "<p>The above study of the factors that influence the transfer of inpatients from the medical and surgical wards to psychiatry has the limitations of any retrospective study. The socioeconomic status of the transferees was deduced from information from the patients' social history; such information included: lack of health insurance, lack of permanent residence or homelessness, prolonged unemployment, lack of any income. The severity of the psychiatric and the physical illness are not precisely assessed. During the study period the psychiatrists who were responsible for the patients' transfer presented here were not the same person.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>" ]

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[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Demographic data</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>Transfers</bold></td><td align=\"center\" colspan=\"2\"><bold>Control group non-transfers</bold></td><td align=\"right\"><bold>Two-tailed Fisher's exact test (p value)</bold></td></tr><tr><td/><td align=\"center\"><bold>(n = 294)</bold></td><td align=\"center\"><bold>%</bold></td><td align=\"center\"><bold>(n = 225)</bold></td><td align=\"center\"><bold>%</bold></td><td/></tr></thead><tbody><tr><td align=\"left\"><bold>Marital status:</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">Married</td><td align=\"center\">92</td><td align=\"center\">31.3</td><td align=\"center\">130</td><td align=\"center\">57.8</td><td align=\"right\">&lt; 0.001</td></tr><tr><td align=\"left\">Single</td><td align=\"center\">122</td><td align=\"center\">41.5</td><td align=\"center\">68</td><td align=\"center\">30.2</td><td align=\"right\">&lt; 0.01</td></tr><tr><td align=\"left\">Divorced</td><td align=\"center\">35</td><td align=\"center\">11.9</td><td align=\"center\">9</td><td align=\"center\">4.0</td><td align=\"right\">&lt; 0.01</td></tr><tr><td align=\"left\">Widowed</td><td align=\"center\">45</td><td align=\"center\">15.7</td><td align=\"center\">18</td><td align=\"center\">8.0</td><td align=\"right\">&lt; 0.01</td></tr><tr><td align=\"left\"><bold>Sex:</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">Male</td><td align=\"center\">139</td><td align=\"center\">47.2</td><td align=\"center\">110</td><td align=\"center\">48.9</td><td align=\"right\">NS</td></tr><tr><td align=\"left\">Female</td><td align=\"center\">155</td><td align=\"center\">52.8</td><td align=\"center\">115</td><td align=\"center\">51.1</td><td align=\"right\">NS</td></tr><tr><td/><td/><td/><td/><td/><td/></tr><tr><td align=\"left\"><bold>Age </bold>(t-test)</td><td align=\"center\" colspan=\"2\">46.5 ± 17.3 (16–87)</td><td align=\"center\" colspan=\"2\">49.2 ± 19. (14–85)</td><td align=\"right\">t = -1.65, NS</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Psychiatric complaint</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>Transfers</bold></td><td align=\"center\" colspan=\"2\"><bold>Control group non-transfers</bold></td><td align=\"right\"><bold>Two-tailed Fisher's exact test (p value)</bold></td></tr><tr><td/><td align=\"center\"><bold>(n = 294)</bold></td><td align=\"center\"><bold>%</bold></td><td align=\"center\"><bold>(n = 225)</bold></td><td align=\"center\"><bold>%</bold></td><td/></tr></thead><tbody><tr><td align=\"left\">Attempted suicide</td><td align=\"center\">146</td><td align=\"center\">49.6</td><td align=\"center\">50</td><td align=\"center\">22.2</td><td align=\"right\">&lt; 0.001</td></tr><tr><td align=\"left\">Psychiatric history/medication</td><td align=\"center\">23</td><td align=\"center\">7.8</td><td align=\"center\">41</td><td align=\"center\">18.2</td><td align=\"right\">&lt; 0.001</td></tr><tr><td align=\"left\">Psychiatric symptomatology*</td><td align=\"center\">94</td><td align=\"center\">32.0</td><td align=\"center\">93</td><td align=\"center\">41.3</td><td align=\"right\">&lt; 0.05</td></tr><tr><td align=\"left\">Disruptive behaviour/non-compliance</td><td align=\"center\">31</td><td align=\"center\">10.5</td><td align=\"center\">11</td><td align=\"center\">4.9</td><td align=\"right\">&lt; 0.05</td></tr><tr><td align=\"left\">Subjective complaints without objective findings</td><td align=\"center\">0</td><td align=\"center\">0.0</td><td align=\"center\">12</td><td align=\"center\">5.3</td><td align=\"right\">&lt; 0.001</td></tr><tr><td align=\"left\">Not described</td><td align=\"center\">0</td><td align=\"center\">0.0</td><td align=\"center\">18</td><td align=\"center\">8.0</td><td align=\"right\">&lt; 0.001</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Average hospital stay (in psychiatric ward) in days</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\" colspan=\"2\"><bold>Transfers to psychiatry ward by consultation-liaison (C-L) service</bold></td><td align=\"center\"><bold>t Test</bold></td><td align=\"center\"><bold>p Value</bold></td></tr></thead><tbody><tr><td align=\"left\">Suicide attempts, not drug related (n = 43)</td><td align=\"left\">Remainders of the transferees (n = 228*)</td><td/><td/></tr><tr><td align=\"left\">38.8 ± 26.3</td><td align=\"left\">25.2 ± 20.6</td><td align=\"center\">3.21</td><td align=\"center\">&lt; 0.01</td></tr><tr><td align=\"left\">Serious socioeconomic problems (n = 46)</td><td align=\"left\">Remainders of the transferees (n = 225)</td><td/><td/></tr><tr><td align=\"left\">40.2 ± 31.0</td><td align=\"left\">25.3 ± 21.3</td><td align=\"center\">3.11</td><td align=\"center\">&lt; 0.01</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Psychiatric diagnoses</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>Transfers</bold></td><td align=\"center\" colspan=\"2\"><bold>Control group non-transfers</bold></td></tr><tr><td/><td align=\"center\"><bold>(n = 294)</bold></td><td align=\"center\"><bold>%</bold></td><td align=\"center\"><bold>(n = 225)</bold></td><td align=\"center\"><bold>%</bold></td></tr></thead><tbody><tr><td align=\"left\">Delirium</td><td align=\"center\">33</td><td align=\"right\">11.2</td><td align=\"center\">36</td><td align=\"right\">16.0</td></tr><tr><td align=\"left\">Addictions</td><td align=\"center\">27</td><td align=\"right\">9.2</td><td align=\"center\">22</td><td align=\"right\">9.8</td></tr><tr><td align=\"left\">Schizophrenia</td><td align=\"center\">32</td><td align=\"right\">10.9</td><td align=\"center\">22</td><td align=\"right\">9.8</td></tr><tr><td align=\"left\">Other psychotic disorders</td><td align=\"center\">24</td><td align=\"right\">8.2</td><td align=\"center\">12</td><td align=\"right\">5.3</td></tr><tr><td align=\"left\">Mood disorders</td><td align=\"center\">78^a</td><td align=\"right\">26.5</td><td align=\"center\">38^a</td><td align=\"right\">16.9</td></tr><tr><td align=\"left\">Anxiety disorders</td><td align=\"center\">13</td><td align=\"right\">4.4</td><td align=\"center\">15</td><td align=\"right\">6.7</td></tr><tr><td align=\"left\">Somatoform disorder</td><td align=\"center\">7</td><td align=\"right\">2.3</td><td align=\"center\">11</td><td align=\"right\">4.9</td></tr><tr><td align=\"left\">Personality disorders</td><td align=\"center\">33^b</td><td align=\"right\">11.2</td><td align=\"center\">10^b</td><td align=\"right\">4.4</td></tr><tr><td align=\"left\">Adjustment disorders</td><td align=\"center\">18^b</td><td align=\"right\">6.1</td><td align=\"center\">32^b</td><td align=\"right\">14.2</td></tr><tr><td align=\"left\">Eating disorders</td><td align=\"center\">3</td><td align=\"right\">1.0</td><td align=\"center\">1</td><td align=\"right\">0.4</td></tr><tr><td align=\"left\">No psychopathology</td><td align=\"center\">3^a</td><td align=\"right\">1.0</td><td align=\"center\">10^a</td><td align=\"right\">4.4</td></tr><tr><td align=\"left\">Undiagnosed</td><td align=\"center\">23</td><td align=\"right\">7.8</td><td align=\"center\">16</td><td align=\"right\">7.1</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T5\"><label>Table 5</label><caption><p>Physical problems</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\"><bold>Transfers (n = 294)</bold></td><td align=\"center\"><bold>Control group (non-transfers; n = 225)</bold></td></tr></thead><tbody><tr><td align=\"left\">Injuries/poisoning</td><td align=\"center\">146^a</td><td align=\"center\">82^a</td></tr><tr><td align=\"left\">Central nervous system diseases</td><td align=\"center\">35</td><td align=\"center\">19</td></tr><tr><td align=\"left\">Vascular diseases</td><td align=\"center\">17</td><td align=\"center\">16</td></tr><tr><td align=\"left\">Gastrointestinal tract diseases</td><td align=\"center\">18</td><td align=\"center\">25</td></tr><tr><td align=\"left\">Cancer</td><td align=\"center\">8</td><td align=\"center\">11</td></tr><tr><td align=\"left\">Endocrine diseases</td><td align=\"center\">16</td><td align=\"center\">10</td></tr><tr><td align=\"left\">Musculoskeletal diseases</td><td align=\"center\">10^b</td><td align=\"center\">17^b</td></tr><tr><td align=\"left\">Urinary tract diseases</td><td align=\"center\">5</td><td align=\"center\">1</td></tr><tr><td align=\"left\">Kidney diseases</td><td align=\"center\">6</td><td align=\"center\">2</td></tr><tr><td align=\"left\">Hematological diseases</td><td align=\"center\">5</td><td align=\"center\">1</td></tr><tr><td align=\"left\">Infectious diseases</td><td align=\"center\">8</td><td align=\"center\">6</td></tr><tr><td align=\"left\">Other diseases</td><td align=\"center\">11</td><td align=\"center\">15</td></tr><tr><td align=\"left\">Not clarified</td><td align=\"center\">9^a</td><td align=\"center\">20^a</td></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>NS, not significant.</p></table-wrap-foot>", "<table-wrap-foot><p>*Including: confusion, agitation, depression, anxiety and delusions.</p></table-wrap-foot>", "<table-wrap-foot><p>*A total of 23 patients who left against medical advice are not included.</p></table-wrap-foot>", "<table-wrap-foot><p>Two-tailed Fisher's exact test:^ap &lt; 0.05, ^bp &lt; 0.01.</p></table-wrap-foot>", "<table-wrap-foot><p>Two-tailed Fisher's exact test: ^ap &lt; 0.01, ^bp &lt; 0.05.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Gender plays an important, but not necessarily appropriate part, in medical decision-making. Research has shown that differences between men and women regarding biological processes, socioeconomic conditions, risk behaviour and environmental risk factors may all contribute to differences in health [##UREF##0##1##,##REF##10741575##2##]. Thus it is sometimes appropriate to investigate and treat male and female patients differently. On the other hand, there is also evidence that women, for no apparent medical reasons, are not offered the same treatment as men, which raises the possibility of gender bias. For example, many studies show that women are less likely than men to receive more advanced diagnostic and therapeutic interventions [##UREF##1##3##, ####REF##16002197##4##, ##REF##16449728##5##, ##UREF##2##6##, ##UREF##3##7####3##7##]. In the clinical situation it is often difficult to know the extent to which gender differences in management reflect physicians' gender bias or are due to other physician, patient or communication characteristics related to gender [##UREF##4##8##, ####UREF##5##9##, ##REF##9196634##10####9196634##10##].</p>", "<p>Patients' wishes and communication behaviour contribute to gender differences in health care [##UREF##6##11##,##REF##8643986##12##]. It is argued, for instance, that men describe their symptoms in a straightforward and demanding way while women often act in a submissive way during consultations, give vague descriptions of their symptoms and are hesitant to accept potentially dangerous measures such as surgery [##UREF##3##7##,##REF##9023524##13##]. In studies of psychosocial adaptation to cancer a recurring result is that men, more than women, prefer to share information while women tend to adapt to the stressful situation by expressing and sharing emotions [##REF##10194883##14##,##REF##14606973##15##]. However, even if there is some tendency for men to be information-oriented and women emotion-oriented, the research results are more complex. There are reports showing that men experience distress when there are social constraints to express emotions [##REF##14606973##15##], that the need for information is more pronounced among women [##REF##10217026##16##], and that women are more knowledgeable about their disease than men [##REF##1286656##17##].</p>", "<title>Perceptions about gender differences</title>", "<p>Assumptions and beliefs about differences in men and women regarding behaviour, skills, emotions and needs are widespread in society. Assumed gender differences are often polarized as opposites, for example: males are associated with order, control and individualism while females are considered incapable of controlling their feelings and as having a natural sense for the family [##UREF##7##18##,##UREF##8##19##]. The man is seen as strong and active, and the woman as weak and passive; the man is symbolised by work, while the woman is associated with the home. In research, generalisations and stereotypes about men and women and other social groups are mainly treated as problematic since they bias interpretations of human activities and are sources of discrimination [##UREF##9##20##]. However, there is also an ongoing discussion about how much truth there might be in a given stereotype [##UREF##10##21##].</p>", "<p>It is debatable whether there is a male and a female \"language\", but research shows that there are often gender differences in the way language is used [##UREF##11##22##,##UREF##12##23##]. Analyses of conversations show fairly clearly that men and women talk differently. Issues such as turn-taking, politeness, interruption, use of swear words etc. are distinctly gender marked and may be read as signifying power relationships [##UREF##12##23##, ####UREF##13##24##, ##UREF##14##25####14##25##]. Studies also provide evidence of differences in the way men and women write [##UREF##15##26##,##UREF##16##27##]. In her research on autobiographies, Mary Gergen found substantial differences in the narratives written by men and women. Men focused on the career and achievements of the subject [##UREF##15##26##]. Emotional ties were mentioned only as 'facts', i.e., male authors did not try to recreate in the reader emphatic emotional responses. On the other hand, in narratives written by women the career line was important but was mingled with other issues that had great personal impact. Furthermore, women's autobiographies dealt extensively with relationships with others.</p>", "<p>Discourse processes have developed out of what and how things are told, who speaks (characterised for example by gender, age, ethnicity, education and social position), and who listens (characterized according to the same factors). Similarly, a gender perspective on written language includes not only the author and the narrative itself but also the reader's interpretation. Since readers of a text are usually aware of whether the author is a man or woman, their expectations and interpretations might be affected by their preconceptions about gender [##UREF##15##26##]. In an experiment involving an authentic text in two versions, identical in all but the simulated male or female author, differences were shown in how readers viewed the author. Based on the same text, the male writer was considered more trustworthy and intelligent and the female writer more humane [##UREF##17##28##]. This shows that preconceptions have a great influence on how a text is judged and gendered expectations of manstories and womanstories might be more important than content and facts [##UREF##15##26##].</p>", "<p>In a previous study we investigated gender differences in 83 patients' letters concerning their experiences when being diagnosed with cancer [##REF##15669017##29##]. It was found that more women than men wrote long, personal and emotional narratives, thus confirming the earlier results about gender differences in communication behaviour described above. However, the majority of letters, about 60%, were neither long, personal or emotional, nor short, impersonal or unemotional, and thus they were hard to categorize. When discussing these results we asked if the gender differences were significant enough to be detectable if all obvious hints about the patient's sex were removed (i.e., pronouns and expressions like \"my wife\" and \"my husband\")? If so, this would confirm that there are genuine differences in male and female patients' descriptions, differences that are not just creations of the reader knowing the sex of the patient-writer on beforehand.</p>", "<p>The aim of the present paper was, therefore, to investigate the extent to which it was possible to identify the patients' sex by reading the same letters, once all information regarding the sex of the patient-writer had been removed. Students of psychology and medicine were invited to be participants. In order to provide nuances to the results, and hints about how gender was created, the students' explanations of their choice of sex were scrutinized in a few of the letters where the students had varying rates of success in determining the authors' sex.</p>" ]

[ "<title>Method</title>", "<title>Letters from patients</title>", "<p>The study was based on letters written by patients with a recent diagnosis of cancer. The letters were collected at an oncology department in Sweden during a five-month period in the late 90s, in order to analyse the manner in which the patients had received their diagnosis. All patients aged 18–70 who had received their cancer diagnosis 2–8 months previously, were asked to \"...write a page or two describing how you received your diagnosis... including what the physician told you, how you reacted and how you felt afterwards. In addition, please describe both what you perceived as beneficial and what was detrimental...\" Out of 187 consecutive patients invited, 138 (74%) submitted a written narrative [##REF##12190266##30##].</p>", "<p>In the present study, all the letters about breast cancer (n = 53) were excluded since this group of patients are subjected to a mammography-screening programme that was considered difficult to blind. Two letters were removed because they were illegible. The remaining letters (n = 83) were typed and all names, places and dates were systematically changed to prevent identification of patients, doctors and others concerned. To blind the letters all information that revealed the patient's sex was removed. The words \"husband\" and \"wife\" were consistently changed to \"co-habiter\", \"mother\" and \"father\" were changed to \"parent\". Detailed descriptions with references to specific clinics or surgical procedures were made less specific if they provided clues to the patient's sex. For example, any references to prostate or gynaecological symptoms or statements about women's clinics or urology departments were changed or removed. Abbreviations and spelling mistakes were retained as in the original letter. During this process two letters were excluded since it was considered too difficult to change them without distortion. Eighty-one letters remained, 42 written by men and 39 by women. The ethics committee of Umeå University approved the study.</p>", "<title>Participants</title>", "<p>A total of 130 participants, 87 medical students and 43 psychology students at Umeå University, volunteered to take part in the study that was carried out on five occasions during the fall of 2005. The participants were aged 18 to 42 years (M = 24.4); 45 of them were men and 85 women.</p>", "<p>In a pilot study we found that it was too demanding and time consuming for participants to read 81 letters. Therefore, two test-groups of participants where formed, groups A and B, each reading one half of the letters. The goal was to achieve an equal distribution of participants with respect to the number and sex in the two test groups (Table ##TAB##0##1##).</p>", "<title>Data collection</title>", "<p>The participants were informed that the study was based on patients' authentic stories. Each participant was first asked to answer questions about their own sex, age and social background. They then read the letters belonging to their test group. For each letter they were asked to make a decision about the patient's sex (man or woman) and to explain their choice of sex in an open-ended question. The participants were instructed to read through the letters rapidly and make decisions based on their first impressions.</p>", "<p>The current paper focuses on the decision about the author's sex and uses the explanations for the choice of sex in four letters to illuminate the complexity in the findings.</p>", "<title>Missing data</title>", "<p>In total there were 5263 decisions about sex to be made, but data were missing for 79 of these decisions spread over letters and male and female participants. There is no reason to believe that this gap had any systematic influence on the results.</p>", "<title>Analysis</title>", "<p>The relationships between participant characteristics (sex and discipline [medicine or psychology]) and participant success rate for the sex-decision task were analysed using the statistical program SPSS 11.0 for Windows. Unpaired t-tests and ANOVAs where used to compare differences between means. The level of significance was set at p &lt; 0.05.</p>", "<p>In order to shed more light on the statistical findings four letters were analyzed further. The letters that were selected were those with the highest and lowest frequencies of correct decisions about sex, together with two letters where about 50% of the participants' hade made a correct decision (see* in Table ##TAB##2##3##). For these letters the open-ended answers about the motives for the decision about sex were read and coded by three of the researchers (JA, MB-H, EK). In a joint session with all five researchers, the codes were compared, discussed and sorted into the broad categories: length, language and content. In a few cases of disagreement about how to categorize, the researchers discussed to find a solution. In this paper the motives for choosing a male or female patient are presented in typical examples, to illustrate the reasoning connected with each letter.</p>" ]

[ "<title>Results</title>", "<title>Quantitative analysis</title>", "<p>An independent t-test showed that there were no significant differences between the results of the medical and psychology students (p = 0.377) nor between the average numbers of correct decisions about sex in the test groups A and B (p = 0.250). There were also no significant differences between the success rates of male and female participants (p = 0.628).</p>", "<p>Table ##TAB##1##2## shows the percentage of correct decisions for all letters and male and female letters, made by all participants, and by male and female participants separately. The mean value for correct decisions among all participants for all letters was 61.7% with a variation from 26.8% to 82.5%. Comparing these results to chance, i.e. 50% correct decisions, independent t-tests showed that the participants were significantly better than chance in their judgements of male as well as female letters (p-values not shown in the table). The participants also chose 'male patient' significantly more often than 'female patient' (p &lt; 0.000).</p>", "<p>Table ##TAB##2##3## shows the distribution of letters according to the proportions of correct decisions made. For six of the 81 letters (7.4%) the proportion of correct decisions was lower than 30%, for 48 letters (59.3%) it was between 30 and 70 %, and for the remaining 27 letters (33.3%) the proportion of correct decisions was higher than 70%. Both male and female participants had a higher success rate on male than on female letters (p &lt; 0.000).</p>", "<p>All six letters with a success rate below 30% were written by women. A majority of the letters (17/27) with a success rate above 70% were written by men.</p>", "<title>Qualitative examination</title>", "<p>The students' explanations as to why they believed the author was a male or female patient varied from just a few words, e.g. \"short letter\" or \"lot of emotions\", to three or four sentences where they expressed several reasons and reflections. In the following, the letter on which the participants had the lowest proportion of correct decisions about sex is labelled 'the most difficult letter', the one with the highest proportion of correct answers is 'the easiest letter', and the two letters where the participants were quite divided on whether the author was a male or female patient are labelled the 'in-between letters'.</p>", "<title>The most difficult letter</title>", "<p>Letter 32, written by a woman, was very short and consisted of only two sentences:</p>", "<p>\"My cancer was detected in the following way: I had a severe cough and cold and blood started to come from the rectum.\"</p>", "<p>Sixty participants read this letter. Five made a correct decision about sex and two of them explained their choice. They referred to \"the sentence structure\" and the disclosure of medical facts strongly linked to personal integrity as reasons for believing it was written by a woman patient.</p>", "<p>Fifty-five participants thought that a man had written the letter and 46 of them explained their choice. Their reasons for a male author could be summarized as follows: The narrative was short and contained factual information and no emotions; the patient seemed dissociated from feelings and unwilling to share experiences and thoughts; several participants claimed that a female writer would have given a more balanced description and put some more effort into the assignment.</p>", "<title>The easiest letter</title>", "<p>The letter where the sex of the author was correctly identified most often (number 24), was written by a woman. It was longer than most letters, at 408 words, and was written in a format that described events in temporal order as well as the patient's reactions to these events. The patient mentioned family and friends, described medical staff and openly shared personal thoughts and feelings.</p>", "<p>All 61 participants who read this letter correctly judged that the patient was a woman and 58 explained their choice. The length of the letter was often mentioned, the language was described as soft and vivid and the use of descriptions such as \"smooth and gentle\" about the male oncologist were frequently given as the motive for choosing a female writer. That the patient shared emotions, mentioned weakness and tears, described family members, friends and medical staff, and emphasized the meaning of support and network, were all seen as clues to the sex of the author. The attitudes of those around the patient were also seen as indicating a female author, e.g. when the patient was informed about the diagnosis, the doctor embraced and held the patient's hands.</p>", "<title>The two 'in-between' letters</title>", "<p>Letter 45 was written by a woman. The letter was 376 words long and started with the following sentence: <italic>\"You have asked for my experiences concerning the manner in which I received the information that I had malignant cell changes\"</italic>.</p>", "<p>Following this the patient gave a detailed description of the course of events and the feelings involved. The letter also contained observations concerning positive and negative experiences of her treatment and how she was met by the staff.</p>", "<p>Sixty-seven participants read this letter, and 35 of them (52%) correctly decided it was written by a woman. Sixty explained their choice. Participants who believed the author was a woman found the letter long and detailed. The language was described as proper, with an introduction ensuring that the reader did not forget why the letter was written. Some participants referred to the patient's use of the Swedish word \"gräsligt\", (\"horrible\" in English), as hinting that this was a female patient. The narrative was seen as emotional with disclosures of feelings of weakness and fear, seen as strong hints of a 'female writer'. Other reasons related to content concerned \"the extensive description of the course of events\" and the way the patient reflected on and analyzed the course of events.</p>", "<p>Participants who thought that the writer was a man found the introduction formal. The sentences were described as short and the language academic. The use of the Swedish word \"pallade\" (\"managed\" in English) was associated with a man. The narrative was described as carefully prepared, based on factual information and focused on events rather than emotions. It gave the participants the impression of an evaluation, written on order. The patient seemed energetic and put more trust in himself than in those around him.</p>", "<p>Letter 74 was written by a man. It consisted of 110 words and started with a description of the patient's shock when the black spot on the arm was diagnosed as a malignant tumour. The writer mentioned the doctor by title and full name and gave him credit for how the bad news was delivered. The patient described feelings of depression following the diagnosis. However, after receiving psychoactive drugs the patient felt a lot better and had returned to work.</p>", "<p>Thirty-tree of the 67 participants (49%) who read the letter thought that the writer was a man while the other half thought it was a woman. Fifty participants explained their decision. Those who thought it was a man described the narrative as short and concise, distant and formal. They stated that the patient focused on the disease as a diagnosis rather than on the emotions involved. Other explanations focused on the patient's comment that it was important to get back to work, and the long waiting time before seeing a doctor.</p>", "<p>The participants who believed it was written by a female patient thought, on the other hand, that a long waiting time to see a doctor indicated that the patient was a woman. They also found hints about gender in \"the style\" and \"the choice of words\". Other clues mentioned were how the patient referred to the doctor by title and full name and the comments about reception. But the most common motives for choosing 'female patient' were that the patient described anxiety and weakness and was not afraid to ask for help or medical treatment for depression.</p>" ]

[ "<title>Discussion</title>", "<title>Summary</title>", "<p>The results showed that in 62% of the cases the university student participants succeeded in identifying the sex of the patient who had written the letter, which was significantly more accurate than a chance allocation. Male and female students did not differ in this regard. The success rate varied between letters and for one third of the letters more than 70% of the participants made a correct decision. For the remaining two thirds of the letters many participants thus had problems identifying the patient's sex. There were significant differences between the students' success rate for male and female letters, with more correct decisions being made for male letters. In four letters the explanations for the choice of sex were analysed and the participants based their choices on three factors – length, language and content. The participants were more likely to say that a particular letter was written by a man if the letter was short, the language was more formal and academic, and the content focussed on the factual info. If the letter was long, written in more expressive manner, and described emotions and relationships the participants were more likely to decide that the author was a woman. However, depending on whether the participants believed the patient was a man or a woman, the same utterances and expressions were interpreted in different ways.</p>", "<title>On method</title>", "<p>That the narratives were authentic and written by \"real patients\" with cancer, as opposed to constructed paper cases, increased the credibility of the study. Further, the letters were not initially collected with a gender study in mind but to study the communication of bad news. This fact, presumably, limited the risk that the patients consciously adjusted their narratives according to societal norms about male and female patients' behaviour.</p>", "<p>In research comparing men and women there is always a risk of circular arguments. Men's and women's behaviours, thoughts or narratives are compared, and differences and similarities are noted. This raises the question of whether the interpretations are true differences or biased by the observers' preconceptions and expectations of a gendered pattern. A strength in this study was the use of the written narrative form, making it possible to create \"neutral\" patients in the sense that there was no obvious information within the text that revealed the patient's sex. On the other hand, differences between men and women in writing about illness might not be the same as differences when talking about illness, for example when seeing a physician. The interpretation of gender may be different in reading compared to listening. Thus the design with neutral patients and written narratives inherited weaknesses along with the strengths.</p>", "<p>The students' participation in the study was voluntary and more women than men took part. However, comparisons of the results showed no significant differences in the responses of men and women students. The gender topic of the study may have contributed to a preponderance of male and female students with a special interest in gender issues. Whether this influenced the results is beyond our knowledge, but even participants aware of gender issues had to rely on their preconceptions and beliefs when sorting the letters.</p>", "<p>The instructions to read through the letters rapidly and make judgements based on their first impressions forced the participants to be categorical. Many found this unpleasant, indicating an aversion towards using categorical generalisations. In studies of stereotypes and attitudes it is regularly found that people are inclined to express themselves in politically correct terms and try to distance themselves from gender stereotyping [##UREF##18##31##]. Yet, on an unconscious level they nevertheless rely on the stereotypes they are trying to avoid. Thus, if our students had felt less pressure it is likely that the same stereotypes would have emerged, but probably in more guarded terms.</p>", "<title>On results</title>", "<title>Better than chance allocation</title>", "<p>The participants made accurate decisions about the sex of the author in approximately 62% of the cases and in one third of the letters the success rate was even higher. These results indicate, hardly surprisingly, that there were gender differences in the illness narratives and confirmed our findings from a previous study that differences between male and female letters are detectable on a group level [##REF##15669017##29##]. This finding is consistent with other studies that show fairly stable gender differences in conversations and use of language [##UREF##12##23##, ####UREF##13##24##, ##UREF##14##25##, ##UREF##15##26####15##26##]. Reliable gender differences have also been found in meta-analyses of behaviours and traits in areas such as cognitive performance, cognitive attitudes, personality and group behaviour [##UREF##10##21##]. However, the fact that the students in our study were able to recognize gender differences, i.e. to make accurate decisions about the patient's sex in a majority of the blinded narratives, shows that knowledge and awareness of gender differences is common and widespread among people.</p>", "<p>It is hard to know whether he success rate would have been different had the participants been qualified psychologists or physicians with more clinical experience. In a study with a similar design, English professors in fact did worse than college students when they were asked to identify whether a man or a woman had written different 100-word passages of American fiction [##UREF##16##27##]. Experience in reading or analysing texts did not increase their ability to categorize the author by sex.</p>", "<p>In our study the success rate varied greatly across the letters showing that although preconceptions about gender differences rest on a basis of 'reality' on a group level, there are many exceptions and variations on the individual level. This fact illustrates the risk of making prejudiced assessments and biased interpretations in everyday communication.</p>", "<p>The lack of a sex difference in students' ability to identify the patients' sex, i.e. to recognize gendered patterns in the narratives, is in line with previous research showing that on the whole men and women are aware of gender differences in behaviour to the same extent [##UREF##10##21##] and have similar associations and preconceptions about gender [##UREF##18##31##,##UREF##19##32##].</p>", "<p>The participants were more successful at identifying which letters were written by male than female patients. One possible explanation for this might be related to 'the male norm', i.e., that men and behaviours associated with men are seen as the norm and the point of reference while women, and their needs and behaviour, are seen as exceptions in many situations [##UREF##20##33##]. One consequence of this might be that the participants were more inclined to see a man in the narratives and more prone to guess on a male writer when they were unsure. This would also explain why the participants chose 'male patient' more often. Another possible explanation is that women tend to show a greater capacity to vary their text than men, and to a greater extent adjust their language to existing circumstances [##UREF##15##26##]. In a social system where the man is the norm and women run the risk of being disregarded or not taken seriously, there might be a purpose behind women adjusting their language to a male language norm [##UREF##13##24##]. Some female writers may employ a discourse that seems like male writing, or at least seems to be in line with how readers are used to seeing male writing [##UREF##16##27##].</p>", "<title>The decision process</title>", "<p>When comparing the reasons for choosing a male or female writer in letters with the highest and lowest success rates, pros and cons of applying common generalizations about men and women in individual cases are illustrated. Letter 24 was written by a woman and fulfilled preconceptions about women's writing regarding length, personal and emotional content, and inclusion of family members and other people in the text. Subsequently, all participants succeeded in correctly identifying the author as a woman. Letter 32 was difficult since it was written by a woman whose letter did not fit into the stereotypes about women's way of communicating. On the contrary, she wrote 'like a man'. The participants commented that the author was 'writing briefly' and 'with factual information and no emotions', characteristics they associated with a male author. This indicates that the letters with very high rates of successful allocation corresponded with generalizations about gender while those with very low success rates clearly deviated from gender stereotypes.</p>", "<p>The reasons underlying the allocation of the 'in-between' letters suggest that the participants were sifting and weighting a variety of factors when categorizing the author by sex. It appears that they created an image of the narrator's sex from some information they initially gleaned, and then they looked for clues to confirm their belief about the writer's sex. We do not know, however, to what extent these initial clues concerned 'length', 'language', 'content', or even something else, and we do not know the hierarchy governing these clues, i.e., was a 'long narrative' more likely to supersede the use of 'academic language' or 'emotional expressions' as cueing the initial impression that formed the basis for the decision. These are interesting questions that remain to be considered in forthcoming research. It was, nevertheless, striking that the same phenomenon or expressions were interpreted in quite different ways depending on whether the participant decided that the patient was a man or a woman. For example: the content in letter 45 was described as \"emotional\" or \"reflective and analysing\" by the participants who thought the author was a woman. The same content, on the other hand, was commented on as \"based on facts and events\" or \"written on order like an evaluation\" by the participants who thought the writer was a man. In the process, interpretations of identical utterances were thus biased by the participants' gendered preconceptions. These findings correspond with the results from earlier experiments, where identical articles gave the readers quite different views of the author depending on whether the simulated author was man or woman [##UREF##17##28##].</p>", "<p>Our results were gathered in a study with an experimental design and the participants were students. It is reasonable to believe that similar interpretative processes take place in a clinical situation. In the clinic there are seldom doubts about a patient's sex and, depending on whether the patient is a man or a woman, the clinician has different preconceptions and expectations about the story that will be told. Thus, when a male patient describes his symptoms, needs and experiences, it is likely that the doctor, psychologist, or other health care staff member, will interpret and remember the narrative differently compared to when a female patient tells the same story [##UREF##21##34##,##REF##16719596##35##]. This might be one clue to the mental processes causing the gender bias in diagnoses and treatment identified in many studies in various fields of health care [##UREF##1##3##, ####REF##16002197##4##, ##REF##16449728##5##, ##UREF##2##6##, ##UREF##3##7####3##7##,##UREF##22##36##,##REF##18211484##37##].</p>", "<p>The participants' explanations for their decisions contained many stereotypes, preconceptions and ideas about men and women. In the black-and-white generalizations that the participants uses the categories man and woman stood out as complete opposites defined by their difference. For example, participants explained their choice by comments such as \"a woman wouldn't express herself this way\". However, if stereotypical assumptions and generalizations were completely false and there were no differences between male and female writers, the participants would not have done significantly better than chance allocations in their judgements. Generalizations about men and women are based on individual experiences as well as abstractions and conceptions in society, and stereotypical ideas often contain some truth when compared to results from observational studies and other research [##UREF##10##21##]. In fact, people use generalizations and preconceptions as an aid in understanding the world. Without generalisations we would be lost in a fragmented social world that was difficult to understand. However, generalizations and stereotypes are also problematic as they bias what we see and hear; they imply a risk of neglecting variation, less well-known aspects, and of making skewed assessments.</p>", "<p>To ensure that students in the health care field are aware of the risk of interpreting patients' behaviour in ways that reflect gender bias it seems essential to include reflections about the impact of preconceptions and generalisations in medical education. In healthcare, it is also vital to safeguard a working climate where generalisations and stereotypical attitudes towards men and women (and also other social groups, e.g. immigrants, unemployed, elderly and disabled) are constantly questioned and reflected on in clinical discussions among doctors, psychologists and other healthcare staff. The results from this article could contribute to such discussions, and the different interpretations of identical expressions depending on whether the author was taken for a man or a woman might be used as examples.</p>", "<p>More knowledge about the cognitive, behavioural and communication processes leading to gender bias in medical work is needed. Observations of authentic consultations in different clinics would be valuable [##UREF##23##38##]. In a future study, we will focus on the explanations for the participants' choice of sex in more of the 'in-between' letters.</p>" ]

[ "<title>Conclusion</title>", "<p>It was possible for participants to detect gender differences in patients' illness narratives although the narratives were blinded. The reasons the participants gave for their sex-categorisation reflected common gender stereotypes suggesting that such stereotypes are more than problematic clichés; at least on a group level they correspond to differences in male and female patients' illness descriptions. However, it was also obvious that preconceptions about gender obstructed and biased the participants' interpretations of the letters. Depending on whether the participants believed the author was a male or a female patient they interpreted the same utterances in different ways. Translated into the clinical situation our results suggest that on the one hand there are gender differences that are recognizable and useful in clinical work consistent with common gender stereotypes. On the other hand, stereotyped preconceptions and generalisations about gender imply that there is a risk that health care staff might interpret a story told by a male patient differently than the identical story told by a woman, and this could result in gender biased investigations and treatments. These findings are important for further understanding of and research about gender bias processes in clinical work.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>In many diseases men and women, for no apparent medical reason, are not offered the same investigations and treatment in health care. This may be due to staff's stereotypical preconceptions about men and women, i.e., gender bias. In the clinical situation it is difficult to know whether gender differences in management reflect physicians' gender bias or male and female patients' different needs or different ways of expressing their needs. To shed some light on these possibilities this study investigated to what extent it was possible to identify patients' sex when reading their blinded illness narratives, i.e., do male and female patients express themselves differently enough to be recognised as men and women without being categorised on beforehand?</p>", "<title>Methods</title>", "<p>Eighty-one authentic letters about being diseased by cancer were blinded regarding sex and read by 130 students of medicine and psychology. For each letter the participants were asked to give the author's sex and to explain their choice. The success rates were analysed statistically. To illuminate the participants' reasoning the explanations of four letters were analysed qualitatively.</p>", "<title>Results</title>", "<p>The patient's sex was correctly identified in 62% of the cases, with significantly higher rates in male narratives. There were no differences between male and female participants. In the qualitative analysis the choice of a male writer was explained by: a short letter; formal language; a focus on facts and a lack of emotions. In contrast the reasons for the choice of a woman were: a long letter; vivid language; mention of emotions and interpersonal relationships. Furthermore, the same expressions were interpreted differently depending on whether the participant believed the writer to be male or female.</p>", "<title>Conclusion</title>", "<p>It was possible to detect gender differences in the blinded illness narratives. The students' explanations for their choice of sex agreed with common gender stereotypes implying that such stereotypes correspond, at least on a group level, to differences in male and female patients' illness descriptions. However, it was also obvious that preconceptions about gender obstructed and biased the interpretations, a finding with implications for the understanding of gender bias in clinical practice.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>All five authors contributed to the design, data collection and drafting of this article. JA, PS and KH were mainly responsible for the final drafting. All five authors read and approved the manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The research was supported by The Swedish Research Council and The Swedish Society of Medicine.</p>" ]

[]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Distribution of letters and characteristics of participants in the test groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td/><td align=\"center\" colspan=\"2\">Letters (N = 81)</td><td/><td align=\"center\" colspan=\"3\">Participants (N = 130)</td></tr><tr><td/><td align=\"center\">Number</td><td align=\"center\">Written by<break/> men/women</td><td/><td align=\"center\">Number</td><td align=\"center\">Men/Women</td><td align=\"center\">Medicine/<break/>Psychology</td></tr><tr><td colspan=\"3\"><hr/></td><td/><td colspan=\"3\"><hr/></td></tr><tr><td align=\"center\">Group A</td><td align=\"center\">41</td><td align=\"center\">20/21</td><td/><td align=\"center\">63</td><td align=\"center\">22/41</td><td align=\"center\">43/20</td></tr><tr><td align=\"center\">Group B</td><td align=\"center\">40</td><td align=\"center\">22/18</td><td/><td align=\"center\">67</td><td align=\"center\">23/44</td><td align=\"center\">44/23</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Percentage of correct decisions about patient's sex made by male and female participants.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">All letters<break/> (N = 81)</td><td align=\"left\">Male letters<break/> (n = 42)</td><td align=\"left\">Female letters<break/> (n = 39)</td><td align=\"left\">p-values*</td></tr></thead><tbody><tr><td align=\"left\">Male participants<break/> (n = 45)</td><td align=\"left\">61.2</td><td align=\"left\">65.7</td><td align=\"left\">55.9</td><td align=\"left\">&lt; 0.000</td></tr><tr><td align=\"left\">Female participants<break/> (n = 85)</td><td align=\"left\">62.0</td><td align=\"left\">64.8</td><td align=\"left\">58.5</td><td align=\"left\">&lt; 0.000</td></tr><tr><td align=\"left\">All participants<break/> (N = 130)</td><td align=\"left\">61.7</td><td align=\"left\">65.2</td><td align=\"left\">58.0</td><td align=\"left\">&lt; 0.000</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>The letters sorted according to the percentage of correct decisions made about the patient's sex.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Correct decision<break/> interval (%)</td><td align=\"left\">Letter label</td><td align=\"center\">Number of<break/> letters (%)</td><td align=\"left\">Patient's sex^†</td></tr></thead><tbody><tr><td align=\"left\">0 – 5.0</td><td/><td/><td/></tr><tr><td align=\"left\">5.1 – 10.0</td><td align=\"left\">32*</td><td align=\"center\">1 (1.2)</td><td align=\"left\">2</td></tr><tr><td align=\"left\">10.1 – 15.0</td><td align=\"left\">59</td><td align=\"center\">1 (1.2)</td><td align=\"left\">2</td></tr><tr><td align=\"left\">15.1 – 20.0</td><td/><td/><td/></tr><tr><td align=\"left\">20.1 – 25.0</td><td/><td/><td/></tr><tr><td align=\"left\">25.1 – 30.0</td><td align=\"left\">4, 18, 25, 66</td><td align=\"center\">4 (4.9)</td><td align=\"left\">2, 2, 2, 2</td></tr><tr><td align=\"left\">30.1 – 35.0</td><td align=\"left\">44, 79, 36, 37,</td><td align=\"center\">4 (4.9)</td><td align=\"left\">1, 1, 1, 2</td></tr><tr><td align=\"left\">35.1 – 40.0</td><td align=\"left\">68</td><td align=\"center\">1 (1.2)</td><td align=\"left\">1</td></tr><tr><td align=\"left\">40.1 – 45.0</td><td align=\"left\">58, 55, 57</td><td align=\"center\">3 (3.7)</td><td align=\"left\">2, 1, 1</td></tr><tr><td align=\"left\">45.1 – 50.0</td><td align=\"left\">33, 81, 6, 23, 75, 2, 22, 74*</td><td align=\"center\">8 (9.9)</td><td align=\"left\">2, 2, 1, 1, 2, 1, 2, 1</td></tr><tr><td align=\"left\">50.1 – 55.0</td><td align=\"left\">45*, 50, 53, 54, 30, 39</td><td align=\"center\">6 (7.4)</td><td align=\"left\">2, 1, 2, 1, 2, 1</td></tr><tr><td align=\"left\">55.1 – 60.0</td><td align=\"left\">34, 13, 19</td><td align=\"center\">3 (3.7)</td><td align=\"left\">1, 2, 2</td></tr><tr><td align=\"left\">60.1 – 65.0</td><td align=\"left\">15, 29, 38, 48, 63, 5, 20, 46, 43, 62, 65, 73, 80</td><td align=\"center\">13 (16.0)</td><td align=\"left\">2, 2, 1, 2, 2, 2, 1, 2, 1, 1, 2, 2, 1</td></tr><tr><td align=\"left\">65.1 – 70.0</td><td align=\"left\">10, 14, 72, 56, 60, 69, 1, 11, 17, 35</td><td align=\"center\">10 (12.3)</td><td align=\"left\">2, 2, 1, 1, 1, 2, 1, 2, 1, 1</td></tr><tr><td align=\"left\">70.1 – 75.0</td><td align=\"left\">76, 7, 21, 52, 77, 31</td><td align=\"center\">6 (7.4)</td><td align=\"left\">1, 1, 1, 2, 1, 1</td></tr><tr><td align=\"left\">75.1 – 80.0</td><td align=\"left\">64, 78, 3, 9, 26, 41, 51</td><td align=\"center\">7 (8.6)</td><td align=\"left\">2, 1, 2, 1, 1, 2, 1</td></tr><tr><td align=\"left\">80.1 – 85.0</td><td align=\"left\">61, 71, 8, 40, 49</td><td align=\"center\">5 (6.2)</td><td align=\"left\">2, 2, 2, 1, 2</td></tr><tr><td align=\"left\">85.1 – 90.0</td><td align=\"left\">27, 12, 47, 67, 28, 42, 70</td><td align=\"center\">7 (8.6)</td><td align=\"left\">1, 1, 1, 1, 2, 1, 1</td></tr><tr><td align=\"left\">90.1 – 95.0</td><td align=\"left\">16</td><td align=\"center\">1 (1.2)</td><td align=\"left\">1</td></tr><tr><td align=\"left\">95.1 – 100</td><td align=\"left\">24*</td><td align=\"center\">1 (1.2)</td><td align=\"left\">2</td></tr><tr><td colspan=\"4\"><hr/></td></tr><tr><td align=\"left\">Total</td><td/><td align=\"center\">81 (100)</td><td/></tr></tbody></table></table-wrap>" ]

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[ "<table-wrap-foot><p>* p-values comparing the success for male and female letters.</p></table-wrap-foot>", "<table-wrap-foot><p>^†1 = man, 2 = woman</p><p>*Letters further examined in the qualitative analysis.</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Recently, a cutaneous operator independent force-frequency relation recording system as been validated in the stress echo lab, based on first heart sound amplitude variations at increasing heart rates [##REF##18031588##1##,##UREF##0##2##]. Contractility quantification and systolic/diastolic time measurement through the system has been previously demonstrated [##REF##18031588##1##,##REF##18426559##3##]. A further application could be the assessment of Second Heart Sound (S2) amplitude variations at increasing heart rates. In fact, the maximum amplitude of vibrations measured by the sensor following the ECG T wave originates from the physical phenomenon of the abrupt deceleration of the moving aortic blood mass. The audible components of this deceleration give rise to the Second Heart Sound (S2) [##REF##1880906##4##, ####REF##5912766##5##, ##REF##1000782##6####1000782##6##]. The aim of this study was to assess the relationship between second heart sound amplitude variations at increasing heart rates and hemodynamic changes.</p>" ]

[ "<title>Methods</title>", "<title>Patient selection</title>", "<p>We enrolled 146 consecutive patients (99 males, 60 ± 14 years) referred for stress echocardiography. Patients' characteristics are summarized in Table ##TAB##0##1##. The type of stressor was chosen by the attending cardiologist/echocardiographist at time of testing in relation to relative contraindications of one stressor over the other [##REF##17765820##7##,##REF##18579481##8##]. Ninety-nine subjects underwent exercise stress (13 non competitive athletes were the controls). Twenty-four patients unable to exercise and 17 patients scheduled for coronary flow reserve evaluation underwent dipyridamole stress echo. Six patients with permanent pace maker (DDD in 5, BIV in 1) underwent pacing stress. Coronary artery disease was defined by the presence of angiographically assessed coronary stenosis (with quantitatively assessed diameter reduction in major coronary vessels) or previous myocardial infarction. The local Ethical Committee approved the study protocol. All patients gave their written informed consent before entering the study. All patients met the following inclusion criteria: 1) referred to stress echo for clinically-driven testing. 2) acoustic window of acceptable quality 3) willingness to enter the study. From the initially considered population of 152 patients, 4 were excluded for poor acoustic window (n = 4), or refusal to give written informed consent (n = 2).</p>", "<title>Semi-supine bicycle exercise</title>", "<p>Graded bicycle semi-supine exercise echo was performed starting at an initial workload of 25 watts lasting for 2 minutes; thereafter the workload was increased stepwise by 25 watts at 2 minutes interval. A 12-lead electrocardiogram and blood pressure determination were performed at baseline and every minute thereafter [##REF##17765820##7##]. Two-dimensional echocardiographic monitoring was performed throughout and up to 5 min after the end of peak stress. Two-dimensional images were recorded at baseline and at the end of each step.</p>", "<title>Dipyridamole stress echo</title>", "<p>Two-dimensional echocardiography and 12-lead electrocardiographic (ECG) monitoring were performed in combination with high dose dipyridamole (up to 0.84 mg over 6 min) in accordance to well established protocols [##REF##17765820##7##,##REF##18579481##8##]. Contraindications to using dipyridamole were asthma, hypotension, and bradyarrhythmias.</p>", "<title>Pacing stress echo</title>", "<p>The pacing protocol was accelerated (with a 10-beat increment every 60 s) until one of the following criteria was reached: 1 – 85% of maximal heart rate (age-corrected: 220 – age for men, 200 – age for women); or 2 – PM maximal programmable heart rate (which varied widely, according to the model of PM, up to 170 bpm during stress). Stimulation was performed, wherever possible, in atrial stimulation mode, or dual-chamber (DDD) pacing to have normal contraction sequence [##UREF##1##9##].</p>", "<title>Regional wall motion analysis</title>", "<p>Regional wall motion analysis was evaluated at baseline and at peak stress with a semiquantitative assessment of a wall motion score index (WMSI), with the 17 segment model of the left ventricle, each segment ranging from 1 = normal/hyperkinetic to 4 = dyskinetic, according to the recommendations of the American Heart Association and American Society of Echocardiography. WMSI was derived by dividing the sum of individual segment scores by the number of interpretable segments [##REF##18579481##8##,##REF##11815441##10##]. Test positivity was defined as the occurrence of at least one of the following conditions: 1) new dyssynergy in a region with normal rest function (i.e., normokinesia becoming hypokinesia, akinesia or dyskinesia) in at least two adjacent segments.</p>", "<title>Diagnostic end points and interruption criteria</title>", "<p>The diagnostic end-points for all types of stress were: the development of obvious echocardiography positivity. Non-echocardiographic test end-points were the following: peak dipyridamole dose; 85% of target heart rate; achievement of conventional end-points (such as severe chest pain and/or diagnostic ST segment changes). The test was also stopped, in the absence of diagnostic endpoints, for one of the following reasons of constituting a submaximal, non-diagnostic test: intolerable symptoms; limiting asymptomatic side effects, consisting of: a) hypertension (systolic blood pressure &gt;220 mmHg; diastolic blood pressure &gt;120 mmHg); b) hypotension (relative or absolute): &gt;30 mmHg fall of blood pressure; c) supraventricular arrhythmias: supraventricular tachycardia or atrial fibrillation; d) ventricular arrhythmias: ventricular tachycardia; frequent, polymorphous premature ventricular beats [##REF##18579481##8##].</p>", "<title>Blood pressure analysis</title>", "<p>One nurse recorded blood pressures at rest and during each individual study. The blood pressure recording was made using a sphygmomanometer and the diaphragm of a standard stethoscope. Systolic and diastolic blood pressure was obtained in the right arm. During exercise test, blood pressure recording was obtained with patient lying in a left rotated semi supine position and instructed to hand grip to the left support with their left hand. Patients have been told to let their right hand go limp when blood pressure was measured.</p>", "<p>By selection, 75 out of the 99 patients of the exercise group had simultaneous S2 amplitude and systemic blood pressure measurement at the first, third and fifth post exercise minute time.</p>", "<title>Volume analysis</title>", "<p>All patients underwent transthoracic echocardiography at baseline and during stress. Left ventricular end-diastolic and end-systolic volumes were measured from apical four- and two-chamber view, by an experienced observer using the biplane Simpson-method. Only representative cycles with optimal endocardial visualization were measured and the average of three measurements was taken. The endocardial border was traced, excluding the papillary muscles. The frame captured at the R wave of the ECG was considered to be the end-diastolic frame, and the frame with the smallest left ventricular cavity the end systolic frame. Images were acquired at baseline and at each increase in heart rate of 10 beats during stress.</p>", "<title>Systemic Vascular Resistance (SVR)</title>", "<p>SVR were calculated according to the traditional formula:</p>", "<p></p>", "<p>where 5 is an approximation of the right atrial pressure and MAP is mean arterial pressure.</p>", "<title>Systemic arterial compliance</title>", "<p>Systemic arterial compliance (C) was calculated as stroke volume index/systemic arterial pulse pressure; were pulse pressure = systolic blood pressure – diastolic blood pressure [##UREF##2##11##].</p>", "<title>Arterial elastance and ventricular-arterial coupling</title>", "<p>In all, ventricular arterial coupling was indexed by the ratio of left ventricular systolic elastance index (systolic pressure/end-systolic volume index) to arterial elastance (Ea, ratio of end-systolic pressure by stroke volume). Echocardiography (for ESV and stroke volume) and cuff sphygmomanometer (systolic pressure, multiplied × 0.90 to obtain end-systolic pressure) provided the raw measurements.</p>", "<p>Because stroke volume (and input impedance) varies directly with body size, arterial elastance was adjusted for body surface area (EaI) to better reflect differences in arterial properties with age and between the genders adjusted for differences in body size [##REF##1638719##12##]. Of note ventricular-arterial coupling is ventricular elastance/arterial elastance, which can further be described as: end-systolic pressure/end-systolic LV volume divided by end-systolic pressure/stroke volume: the pressure terms in the numerator and the denominator cancel out, and ventricular-arterial coupling equals to stroke volume/end-systolic volume.</p>", "<title>Operator-independent second heart sound quantification</title>", "<p>The transcutaneous force sensor is based on a linear accelerometer from STMicroelectronics (LIS3). The device includes in one single package a MEMS sensor that measures a capacitance variation in response to movement or inclination and a factory trimmed interface chip that converts the capacitance variations into analog signal proportional to the motion. The device has a full scale of ± 2·<italic>g </italic>(<italic>g </italic>= 9.8 m/s²) with a resolution of 0.0005·<italic>g</italic>. We housed the device in a small case (Figure ##FIG##0##1##) which was positioned in the mid-sternal precordial region and was fastened by a solid gel ECG electrode. The acceleration signal was converted to digital and recorded by a laptop PC, together with an ECG signal. The system is also provided with a user interface that shows both the acceleration and the ECG signals while the acquisition is in progress[##REF##18031588##1##]. The data were analyzed by using software developed in Matlab (The MathWorks, Inc). A peak detection algorithm, synchronized with the ECG, scans the first 150 ms following the R wave to locate the first heart sound vibration. Subsequently, the interval between the first heart sound and the following R wave is analyzed to record the amplitude (nadir to peak) of second heart sound vibration for each cardiac beat [##REF##18426559##3##]. The accelerometer simply records naturally generated heart vibrations, which audible components give rise to the second heart sound 'See additional file ##SUPPL##0##1##: Appendix'.</p>", "<p>The curve of S2 peak amplitude variation as a function of heart rate was finally computed as the increment with respect to the resting amplitude value [##UREF##3##13##]. All the parameters were acquired as instantaneous values at baseline and during stress; mobile mean was utilized to assess baseline value (1 minute recording), at each incremental stress test, at peak test, and during recovery (Figure ##FIG##1##2##). Baseline, peak stress, peak-rest difference as absolute value, and delta % rest-peak stress values were computed.</p>", "<p>Non myocardial noising vibrations (skeletal muscles, body movements, breathing) were eliminated by frequency filtering.</p>" ]

[ "<title>Results</title>", "<title>Resting and stress echocardiographic findings</title>", "<p>Technically adequate images were obtained in all patients at baseline (by selection) and during stress.</p>", "<title>At Peak Exercise</title>", "<p>Heart rate was lower in the dipyridamole than in the exercise and pacing groups. The mean ejection fraction increased in the exercise and Dip groups, while decreased in the pacing group. Regional wall motion abnormalities occurred in 5 patients of the exercise, 1 patient of Dip and 2 patients of the pacing groups (Table ##TAB##1##2##).</p>", "<title>Peripheral pressures, load and coupling</title>", "<p>Arterial elastance increased in the exercise and the Pacing groups, while decreased in the dipyridamole, mainly due to a greater dipyridamole induced arterial compliance (Table ##TAB##1##2##).</p>", "<p>Despite similar baseline values, diastolic blood pressure increased in the exercise, decreased in the dipyridamole, while unchanged in the pacing group, although the response was heterogeneous at the individual level (Table ##TAB##1##2##).</p>", "<title>Sensor built second heart sound amplitude variations</title>", "<p>A consistent second heart sound signal was obtained in all patients at rest and during stress (Figure ##FIG##1##2##). In the patients as a whole, baseline S2 was 7.2 ± 3.3 m<italic>g</italic>, increasing to 12.7 ± 7.7 m<italic>g </italic>at peak stress. S2 trends during exercise or dipyridamole are shown in Figure ##FIG##2##3##.</p>", "<p>Mean S2 percentage increase was + 133 ± 104% in the 99 exercise patients, + 2 ± 22% in the 41 dipyridamole patients and + 31 ± 27% in the 6 pacing patients (p &lt; 0.05 between groups) (Table ##TAB##1##2##).</p>", "<p>In the exercise group the S2 amplitude percentage increase was similar in the 13 control and in the 86 patients (+ 140 ± 123% vs. 132 ± 102%, p = ns)</p>", "<p>At linear regression analysis significant positive determinants of the S2 amplitude changes during stress were the systemic blood pressure, the heart rate, and cardiac index rest-peak changes (Table ##TAB##2##3##). Scatter plots demonstrating correlations between S2 changes and arterial pressure rest-peak changes are displayed in Figure ##FIG##3##4##.</p>", "<title>Second heart sound undershoot and the post exercise hypotension</title>", "<p>A significant correlation was found between post exercise hypotension and recovery S2 undershoot: 44 (80%) of the 55 patients with post-exercise hypotension had S2 undershoot in the recovery, while 19 (96%) of the 20 patients without post-exercise hypotension had stable rate-S2 curve at recovery (Table ##TAB##3##4##) (Figure ##FIG##2##3##).</p>" ]

[ "<title>Discussion</title>", "<p>A stable, reproducible, and consistent S2 force signal was recorded in all patients at rest and during stress. Baseline force value had an ample range (from 2 to 23 <italic>g </italic>* 10^-3). The most widely accepted theory for the genesis of the second heart sound is the \"cardiohemic model,\" which states that the sounds are produced by the vibration of the entire heart and its contents [##REF##3329595##14##]. This vibration is triggered by valve closure (the aortic and pulmonary valves for the second heart sound). The amplitude of these sounds depends on the force with which the valves close, which, in turn, depends on the pressure gradient across the valve at the time of closure. We previously demonstrated that in adult patients undergoing stress testing, the first heart sound amplitude was directly related to myocardial contractility [##REF##18031588##1##]. In this investigation, blood pressure (systolic, diastolic and mean) correlated closely with S2 amplitude. This may be explained by the fact that amplitude is primarily determined by one factor, the force of valve closure [##REF##8329628##15##].</p>", "<title>Biophysics of the second heart sound</title>", "<p>Early studies of the hemodynamic determinants of the amplitude of the S2 have related the aortic component amplitude of the S2 vibration to the aortic pressure, in agreement with clinical findings that hypertensive patients frequently have loud second heart sounds [##REF##1880906##4##,##REF##3966382##16##,##REF##696634##17##]. In their proposed mechanism for the origin of the second heart sound, Sabbah and Stein [##REF##1000782##6##] showed a relation between the amplitude of S2 and the driving pressure. Driving pressure, in the heart, refers to the instantaneous difference between arterial and ventricular pressure shortly after semilunar closure. Kusukawa and associates [##REF##5912766##5##] previously found a good correlation of the amplitude of the second heart sound with the difference of pressure between the aorta and the left ventricle coincident with the dicrotic notch. But patients suffering from myocardial infarction and/or heart failure, often exhibit reduced S2 amplitude, even when the aortic pressure is normal [##REF##624169##18##]. They showed that the amplitude of S2 was linearly related to the rate of change of the pressure gradient that develops across the aortic valve during diastole (r = .82). The latter is also correlated with negative dP/dt (r = .62).</p>", "<p>In normotensive patients with poor ventricular performance, the rate of isovolumic relaxation may be compromised and this would cause a reduction in negative dP/dt which in turn causes a reduction of the rate of change of the pressure gradient that develops across the valve during diastole. A diminished S2, therefore, would result due to the more slowly developing driving pressure, which directly affects the characteristics of valvular vibration. Tanigawa et al [##REF##1880906##4##] demonstrated in instrumented dogs, that when the time constant of left ventricular pressure fall \"T\" was normal, the aortic systolic pressure and diastolic pressure were good predictors of S2 intensity. When LV relaxation was impaired, increasing T greater than 135% of control, the S2 intensity for any given aortic pressure was reduced. When relaxation was hyperactive, decreasing T less than 65% of control, S2 intensity was increased. Aortic pressure/T which assessed both aortic pressure and relaxation ability, is a better determinant of A2 intensity than aortic systolic pressure or aortic diastolic pressure alone.</p>", "<title>Second heart sound frequencies or amplitude to get clinical information?</title>", "<p>Previous studies have shown that it is possible to estimate systemic blood pressure using the spectral information of the second heart sound. A mathematical model for the vibration of the closed aortic valve was proposed by Zhang et al [##REF##17946534##19##], showing that the increasing aortic pressure results in an increase both in frequency and amplitude of produced sound. The results of this study also suggest that it is the increasing resonant frequency and amplitude of the blood column induced by elevated distending pressure that plays significant role in the process.</p>", "<p>Various mathematical methods have been used to describe heart sounds, including the frequency domain (FFT) and the time domain (RMS) amplitude.</p>", "<title>The frequency domain (FFT)</title>", "<p>The frequencies present in heart sounds are determined by the volume of the vibrating mass (smaller volume has a higher resonance frequency) and the tension generated in the walls of the heart and great vessels. This explains the fact that S2 is normally of higher frequency than S1 (the aorta is of lower volume than the heart) and that younger children exhibited higher heart sound frequencies than older children [##REF##9972738##20##]. Other Authors [##REF##6708485##21##] stated that the major concentration of energy, for both first heart sound (M1) and second heart sound (S2), is below 150 Hertz (Hz) which may indicate that both sounds are caused by vibrations within the same structure, possibly the entire heart. However S2 spectra have greater amplitude than S1 spectra above 150 Hz, which may be due to vibrations within the aorta and pulmonary artery. Because peak frequency is a descriptor of only a single point, it is therefore not a useful factor in describing heart sound changes resulting from variations in myocardial contractility or systemic pressure changes 'See additional file ##SUPPL##0##1##: Appendix'.</p>", "<title>The amplitude domain (RMS)</title>", "<p>In previous investigation [##REF##9972738##20##], hemodynamic variables (heart rate and blood pressure) correlated more closely with amplitude than with frequency. This may be explained by the fact that amplitude is primarily determined by one factor – force of valve closure – whereas frequency depends on the force of closure, heart volume, and the resonance frequencies of the heart and great vessels. Thus, differences in heart size and intravascular volume status could explain the greater variability (and, thus, weaker statistical correlation) in frequency than amplitude characteristics.</p>", "<p>This is the motive for we used a peak amplitude (nadir to peak) signal analysis system for both the first and the second heart sounds vibrations [##REF##18031588##1##,##REF##18426559##3##].</p>", "<title>The properties of the chest wall in the transmission of sound from inside the thorax to the surface of the chest</title>", "<p>The chest wall is a low-pass filter. Cardiac vibrations propagate as mechanical shear waves, and the intervening viscoelastic thoracic tissue attenuates the higher frequencies and introduces a variable propagation delay [##REF##14003144##22##,##UREF##4##23##].</p>", "<p>In contrast to the dynamics observed epicardially, Wood [##REF##7967843##24##] demonstrated that heart sound frequency law was dominated by quasi-stationary and impulse-like components implying that the instantaneous power and the power spectrum contain most of the diagnostic information in heart sound.</p>", "<p>Modelling the heart/thorax acoustic system in dogs, based on the simultaneous recording of the intracardiac and thoracic phonocardiograms, Durand and co-workers [##REF##2246923##25##] showed that the heart/thorax acoustic system acts like a band pass filter having a higher attenuation for A2 than for M1. Between 20 and 100 Hz, the mean attenuation of M1 is 30 dB while that of A2 is 46 dB. Above 100 Hz, the attenuation slope is -12 dB per octave for M1 and -6 dB per octave for A2. Again, the frequency domain is influenced by the heart/thorax acoustic system, and the frequency based heart sound information is jeopardized by a further variable. Using heart sound amplitude to get clinical information, the absolute force value in the single patient is certainly related to the transthoracic propagation of cardiac vibrations. In fact, when measured epicardially or on the aortic root, S2 vibrations are up to 10 times more powerful than when measured on the chest, and cannot be used as absolute value for interpatient comparison. However the amplitude (force) % changes (i.e. contractility for M1 and systemic pressure for S2) are not influenced by the heart/thorax acoustic system and the data can be used for intrapatient changes as for contractility or systemic pressure changes.</p>", "<title>Second heart sound and stress changes</title>", "<p>Previous phonocardiography research has been focused on the determination of heart sound production at rest, but relatively little work has been done to investigate heart sounds under stress testing. Luisada et al. [##REF##3485870##26##] stated that heart sound changes during stress may be more rapid and sensitive than changes in heart rate and blood pressure. Of the 146 study patients, 39 unchanged or decreased diastolic blood pressure at peak stress vs. rest (mainly dipyridamole group, 28 out of 41 pts) while 107 increased diastolic blood pressure (mainly exercise group, 91 out of 99 pts). Patients with increased pressure had + 116 ± 106% second heart sound amplitude increase vs. + 26 ± 67% in patients with unchanged or decreased diastolic blood pressure at peak stress. In our study, a mismatch between increased diastolic pressure, but blunted S2 amplitude, occurred in 7 patients out of the 107 with stress increased diastolic pressure. According to the physiological basis, in these case the blunted S2 increase should be related to a diminished driving pressure between the aorta and the left ventricle, with delayed or altered active LV relaxation. These 7 patients had coronary artery disease. Obviously, sensor measured S2 amplitude, without ventricular relaxation data, blind us to the quantification of the time constant of left ventricular pressure fall, and/or to negative LV dP/dt. However, this totally noninvasive sensor demonstrated capability to monitor beat to beat systemic pressure changes, at rest and during exercise. Further studies with simultaneous hemodynamic in humans should be done to address this issue.</p>", "<title>Second heart sound and post exercise hypotension</title>", "<p>Post exercise hypotension has been demonstrated both in hypertensive and healthy subjects [##REF##15076798##27##] . In normotensive subjects, it has been attributed to a decrease in cardiac output and/or systemic vascular resistance [##REF##15181391##28##,##REF##8866370##29##]. Moreover, it has been accompanied by a decrease in peripheral sympathetic activity [##REF##8866370##29##,##REF##15542577##30##] and an increase in cardiac sympathetic activity [##REF##8282635##31##]. Other studies demonstrated that the acute post-exercise reduction in blood pressure was clinically similar following high intensity short duration exercise and moderate intensity longer duration exercise [##REF##16896732##32##]. Acute exercise may serve as a non-pharmacological aid in the treatment of hypertension. S2 amplitude monitoring could be a method to assess efficacy of the acute post-exercise blood pressure reduction. In the selected patients of our study, a significant correlation was found between post exercise hypotension and recovery second heart sound lower amplitude, to confirm the capability of the sensor to mirror diastolic pressure trend.</p>" ]

[ "<title>Conclusion</title>", "<p>Continuous and non-invasive monitoring of blood pressure (BP) is important to prevent hypertensive patients from stroke and heart attack. However, most of the prevalent BP devices can provide solely intermittent measurements. S2 recording quantitatively documents systemic pressure changes: S2 amplitude trend is up-sloping when pressure increases as may occur during physical exercise or is flat for a flat pressure trend as may occur during dipyridamole induced vasodilatation. A new concept of non-invasive blood pressure measurement by heart sound pattern analysis is described. The known diagnostic criterion of the 'accentuated' second heart sound of a hypertensive patient is here converted into a computer-aided pattern-recognition process for the second heart sound, applicable over the entire range of blood pressure. The method is in principle suited for automatically repeated blood pressure measurements, but further development is still needed for conversion into a widely practicable procedure. Integrating first heart sound [##REF##18031588##1##], second heart sound amplitude and first-second heart sound time delay [##REF##18426559##3##], a cutaneous operator-independent force sensor describes in real time systolic elastance, diastolic time, and systemic pressure trend, offering a new chance to monitor failing hearts.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Recently, a cutaneous force-frequency relation recording system based on first heart sound amplitude vibrations has been validated. A further application is the assessment of Second Heart Sound (S2) amplitude variations at increasing heart rates. The aim of this study was to assess the relationship between second heart sound amplitude variations at increasing heart rates and hemodynamic changes.</p>", "<title>Methods</title>", "<p>The transcutaneous force sensor was positioned in the precordial region in 146 consecutive patients referred for exercise (n = 99), dipyridamole (n = 41), or pacing stress (n = 6). The curve of S2 peak amplitude variation as a function of heart rate was computed as the increment with respect to the resting value.</p>", "<title>Results</title>", "<p>A consistent S2 signal was obtained in all patients. Baseline S2 was 7.2 ± 3.3 m<italic>g</italic>, increasing to 12.7 ± 7.7 m<italic>g </italic>at peak stress. S2 percentage increase was + 133 ± 104% in the 99 exercise, + 2 ± 22% in the 41 dipyridamole, and + 31 ± 27% in the 6 pacing patients (p &lt; 0.05). Significant determinants of S2 amplitude were blood pressure, heart rate, and cardiac index with best correlation (R = .57) for mean pressure.</p>", "<title>Conclusion</title>", "<p>S2 recording quantitatively documents systemic pressure changes.</p>" ]

[ "<title>Statistical analysis</title>", "<p>SPSS 11 for Windows was utilized for statistical analysis. The statistical analyses included descriptive statistics (frequency and percentage of categorical variables and mean and standard deviation of continuous variables).</p>", "<p>The one-way ANOVA was used to compare continuous variables between groups; when homogeneity of variance was not present, the Kruskal-Wallis test for nonparametric independent samples was used. Intergroup comparison was performed with Scheffe and Tamhane post hoc tests, respectively.</p>", "<p>Relations between variables were assessed using linear regression analysis and Pearson's correlation coefficient. Cardiac or vascular properties significantly related to the S2 amplitude changes were graphically displayed with simple scatter plots. Crosstabs' statistics and measures of association for post exercise hypotension vs. post exercise S2 amplitude undershoot were performed in 75 selected patients.</p>", "<title>Limitations of the study</title>", "<p>We used intermittent auscultatory methods to determine exercise and post-exercise blood pressure. These auscultatory methods are prone to sampling error and may provide inaccurate results. Since diastolic isovolumic relaxation occurs simultaneously with the physical phenomenon (the abrupt deceleration of the moving aortic blood mass), that gives rise to the S2 amplitude, the S2 amplitude is an algebraic sum of the myocardial and of the aortic blood mass effects. Several scenarios can occur for S2 amplitude. 1 – With constant ventricular relaxation rate, S2 amplitude is directly related to the diastolic aortic pressure: 2 – With constant aortic diastolic pressure, S2 amplitude is directly related to the ventricular relaxation rate. Obviously, sensor measured S2 amplitude, without ventricular relaxation hemodynamics, cannot sense the ventricular component of the S2. Further studies in humans with simultaneous hemodynamic assessment should be done to address this issue. Another limitation of the study could arise from the fact that we didn't measure the split in the second cardiac sound [##REF##16574092##33##,##REF##16705255##34##]. The continuous wavelet transforms (CWTs) method is capable of detecting its two components, A2 and P2, allowing therefore the measurement of the delay between them. This delay, called the split, is very important in the diagnosis of many pathological cases, but it was not the aim of this study</p>", "<title>Characteristics of the population and inducible ischemia</title>", "<p>Eight (5%) of the 146 patients had stress induced ischemia. The low rate of test positivity depends on many factors. The test indication class was not always I or IIa: low appropriateness in a high volume laboratory setting mainly depends on too often repeated tests in the absence clinical changes [##REF##17509703##35##]. Second, stress test was often performed in young patients with low pre-test probability of CAD (13 controls and 39 patients with atypical chest pain and/or systemic hypertension). Third, valvular heart disease patients (moderate aortic stenosis in 9, moderate mitral regurgitation in 10) were referred for Doppler stress echo. Fourth, 17 CAD patients underwent dipyridamole stress for coronary flow reserve evaluation of left anterior descending coronary artery [##REF##18579481##8##].</p>", "<title>Abbreviations</title>", "<p>A2: aortic component of the second heart sound; BSA: body surface area; C: systemic arterial compliance; DBP: diastolic blood pressure; CAD: coronary artery disease; CO: cardiac output; DCM: idiopathic dilated cardiomyopathy; EaI: effective arterial elastance index; EDV: end-diastolic volume; EF: ejection fraction; ESV: end-systolic volume; FFR: force-frequency relation; <italic>g</italic>: acceleration unit (9.8 m/sec²); HR: heart rate; LV: left ventricle/ventricular; LVMI: left ventricular mass index; M1: mitral component of the first heart sound; P2: pulmonary component of the second heart sound; S1: first heart sound; S2: second heart sound; SBP: systolic blood pressure; SVR: systemic vascular resistance; WMSI: wall motion score index.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>TB conceived this study, performed the data analysis, and drafted the manuscript; LV, CP, EPa, LP and MP were responsible for data collection and revised the manuscript; VG, EB, FF and MG were responsible for technology development and digital signal processing; GA gave a contribution to data discussion; EPi gave a contribution to preparation of study design, data discussion, and critical revision of the manuscript.</p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>Publication cost has been funded by: Prof. Giorgio Arpesella, Director, Heart Transplant Unit, Bologna University, Department of Cardiac Surgery, Heart and Lung Transplantation Program, Policlinico S. Orsola, Via Massarenti, 9, 40138 Bologna, Italy.</p>", "<p>Phone: ++39-051-6364733</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Isovolumic contraction force and second heart sound (S2) amplitude</bold>. A Micro-Electro-Mechanical Systems (MEMS) accelerometer is temporarily positioned in the mid-sternal precordial region before starting the scheduled stress test in all patients. A peak detection algorithm, synchronized with the ECG, scans the first 150 ms following the R wave to record the isovolumic contraction force vibration and then the interval before the following R wave to record the second heart sound amplitude (S2, pink symbol). All the parameters are acquired as instantaneous values at baseline and during stress. The data can be also read remotely by a wireless bluetooth sensor network, with reliable continuous remote monitoring 'See additional file ##SUPPL##0##1##: Appendix'.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Computing the second heart sound amplitude variation as a function of heart rate</bold>. All the parameters are acquired as instantaneous values at baseline and during stress; mobile mean is utilized to assess baseline value (1 minute recording), at each incremental stress test, at peak test, and during recovery. Left panel: instantaneous S2 amplitude scattering (blue points exercise, red points recovery) depends on the respiratory cycle and thorax expansion; blue and red curves = S2 amplitude mobile mean. Right panel: blue curve = exercise in progress; red curve = recovery.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Second heart sound (S2) amplitude recording simultaneously with diastolic blood pressure during stress</bold>. Left panel: similar S2-frequency trend during stress (blue symbols) and recovery (red symbols) in a patient with normal exercise pressure changes and without post exercise hypotension. Middle panel: S2-frequency trend during stress (blue symbols) and recovery (red symbols) in a patient with exercise induced diastolic hypertension and post exercise hypotension. Right panel: flat-negative S2-frequency trend during dipyridamole stress induced hypotension.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Second Heart sound recording quantitatively documents systemic pressure changes</bold>. Scatter plots demonstrating relationship between sensor Second Heart Sound amplitude % changes (y axis) and systemic pressure rest-peak changes values (x axis) in the whole group of 146 patients. Red symbols: exercise stress; green symbols: dipyridamole stress; blue symbols: pacing stress. Left panel: systolic pressure (SBP) changes. Middle panel: diastolic pressure (DBP) changes. Right panel: mean pressure (MP) changes.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Characteristics of the study patients</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">EXERCISE</td><td align=\"left\">DIP</td><td align=\"left\">PACING</td></tr></thead><tbody><tr><td align=\"left\">Pt n°</td><td align=\"left\">99</td><td align=\"left\">41</td><td align=\"left\">6</td></tr><tr><td align=\"left\">Age (years)</td><td align=\"left\">56 ± 14</td><td align=\"left\">68 ± 11</td><td align=\"left\">68 ± 10</td></tr><tr><td align=\"left\">Males</td><td align=\"left\">68</td><td align=\"left\">27</td><td align=\"left\">4</td></tr><tr><td align=\"left\">Controls</td><td align=\"left\">13</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">CAD</td><td align=\"left\">36</td><td align=\"left\">29</td><td align=\"left\">3</td></tr><tr><td align=\"left\">Previous PTCA/By pass</td><td align=\"left\">27</td><td align=\"left\">19</td><td align=\"left\">1</td></tr><tr><td align=\"left\">Previous myocardial infarction</td><td align=\"left\">25</td><td align=\"left\">13</td><td align=\"left\">2</td></tr><tr><td align=\"left\">Arterial hypertension</td><td align=\"left\">18</td><td align=\"left\">5</td><td align=\"left\">-</td></tr><tr><td align=\"left\">Valvular disease</td><td align=\"left\">19</td><td align=\"left\">2</td><td align=\"left\">1</td></tr><tr><td align=\"left\">Atipical chest pain</td><td align=\"left\">12</td><td align=\"left\">3</td><td align=\"left\">1</td></tr><tr><td align=\"left\">DCM</td><td align=\"left\">1</td><td align=\"left\">2</td><td align=\"left\">1</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Rest and stress data</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\">EXERCISE</td><td/><td align=\"center\">DIP</td><td/><td align=\"center\">PACING</td></tr></thead><tbody><tr><td align=\"left\">N of pts</td><td align=\"center\">99</td><td/><td align=\"center\">41</td><td/><td align=\"center\">6</td></tr><tr><td align=\"left\">Age (yrs)</td><td align=\"center\">56 ± 14</td><td align=\"center\">§</td><td align=\"center\">68 ± 11</td><td/><td align=\"center\">68 ± 10</td></tr><tr><td align=\"left\">Gender (M/F)</td><td align=\"center\">68/31</td><td/><td align=\"center\">27/14</td><td/><td align=\"center\">4/2</td></tr><tr><td align=\"left\">BSA (m²)</td><td align=\"center\">1.88 ± .19</td><td/><td align=\"center\">1.83 ± .16</td><td/><td align=\"center\">1.87 ± .28</td></tr><tr><td align=\"left\"><bold>Standard echo measurements</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">LVMI (g/m²)</td><td align=\"center\">104 ± 28</td><td/><td align=\"center\">104 ± 20</td><td/><td align=\"center\">138 ± 34</td></tr><tr><td align=\"left\">HR rest (bpm)</td><td align=\"center\">73 ± 16</td><td/><td align=\"center\">66 ± 13</td><td/><td align=\"center\">71 ± 10</td></tr><tr><td align=\"left\">HR peak (bpm)</td><td align=\"center\">131 ± 24</td><td align=\"center\">Δ</td><td align=\"center\">84 ± 13</td><td align=\"center\">*</td><td align=\"center\">132 ± 13</td></tr><tr><td align=\"left\">LV EF % rest</td><td align=\"center\">59 ± 11</td><td/><td align=\"center\">58 ± 13</td><td/><td align=\"center\">51 ± 11</td></tr><tr><td align=\"left\">LV EF % peak</td><td align=\"center\">67 ± 14</td><td align=\"center\">‡</td><td align=\"center\">62 ± 13</td><td align=\"center\">*</td><td align=\"center\">45 ± 16</td></tr><tr><td align=\"left\">WMSI rest</td><td align=\"center\">1.11 ± .29</td><td/><td align=\"center\">1.17 ± .32</td><td/><td align=\"center\">1.28 ± .46</td></tr><tr><td align=\"left\">WMSI peak</td><td align=\"center\">1.13 ± .31</td><td/><td align=\"center\">1.19 ± .32</td><td/><td align=\"center\">1.4 ± .46</td></tr><tr><td align=\"left\">Δ WMSI (rest-peak)</td><td align=\"center\">.02 ± .10</td><td/><td align=\"center\">.01 ± .07</td><td/><td align=\"center\">.15 ± .24</td></tr><tr><td align=\"left\"><bold>Sensor built second heart sound (S2) amplitude changes</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">S2 rest (m<italic>g</italic>)</td><td align=\"center\">7.7 ± 4.9</td><td/><td align=\"center\">7.1 ± 2.8</td><td/><td align=\"center\">5.8 ± 1.4</td></tr><tr><td align=\"left\">S2 peak (m<italic>g</italic>)</td><td align=\"center\">15.9 ± 8.7</td><td align=\"center\">§</td><td align=\"center\">7.2 ± 3</td><td/><td align=\"center\">7.7 ± 2.4</td></tr><tr><td align=\"left\">S2 Δ rest-peak (m<italic>g</italic>)</td><td align=\"center\">8.2 ± 6.1</td><td align=\"center\">§</td><td align=\"center\">.1 ± 1.5</td><td/><td align=\"center\">1.8 ± 1.9</td></tr><tr><td align=\"left\">S2 Δ % (rest-peak)</td><td align=\"center\">133 ± 104</td><td align=\"center\">§</td><td align=\"center\">2 ± 22</td><td/><td align=\"center\">31 ± 27</td></tr><tr><td align=\"left\"><bold>Perpheral pressures, load and coupling</bold></td><td/><td/><td/><td/><td/></tr><tr><td align=\"left\">SBP rest (mmHg)</td><td align=\"center\">134 ± 21</td><td/><td align=\"center\">137 ± 20</td><td/><td align=\"center\">131 ± 25</td></tr><tr><td align=\"left\">SBP peak (mmHg)</td><td align=\"center\">189 ± 26</td><td align=\"center\">§</td><td align=\"center\">127 ± 26</td><td/><td align=\"center\">137 ± 37</td></tr><tr><td align=\"left\">Δ SBP (rest-peak, mmHg)</td><td align=\"center\">55 ± 25</td><td align=\"center\">§</td><td align=\"center\">-8 ± 17</td><td/><td align=\"center\">6 ± 17</td></tr><tr><td align=\"left\">DBP rest (mmHg)</td><td align=\"center\">74 ± 12</td><td/><td align=\"center\">71 ± 12</td><td/><td align=\"center\">74 ± 11</td></tr><tr><td align=\"left\">DBP peak (mmHg)</td><td align=\"center\">94 ± 13</td><td align=\"center\">§</td><td align=\"center\">67 ± 13</td><td/><td align=\"center\">75 ± 15</td></tr><tr><td align=\"left\">Δ DBP (rest-peak, mmHg)</td><td align=\"center\">20 ± 13</td><td align=\"center\">§</td><td align=\"center\">-4 ± 10</td><td/><td align=\"center\">1 ± 15</td></tr><tr><td align=\"left\">Mean pressure rest (mmHg)</td><td align=\"center\">94 ± 13</td><td/><td align=\"center\">93 ± 12</td><td/><td align=\"center\">93 ± 14</td></tr><tr><td align=\"left\">Mean pressure peak (mmHg)</td><td align=\"center\">126 ± 15</td><td align=\"center\">§</td><td align=\"center\">88 ± 17</td><td/><td align=\"center\">96 ± 20</td></tr><tr><td align=\"left\">Δ mean pressure (rest-peak, mmHg)</td><td align=\"center\">32 ± 14</td><td align=\"center\">§</td><td align=\"center\">-5 ± 12</td><td/><td align=\"center\">2 ± 15</td></tr><tr><td align=\"left\">SVR rest (dyne * sec * cm^-5)</td><td align=\"center\">2134 ± 802</td><td/><td align=\"center\">2118 ± 702</td><td/><td align=\"center\">1652 ± 533</td></tr><tr><td align=\"left\">SVR peak (dyne * sec * cm^-5)</td><td align=\"center\">1501 ± 547</td><td/><td align=\"center\">1551 ± 747</td><td/><td align=\"center\">1546 ± 620</td></tr><tr><td align=\"left\">Δ SVR (rest-peak, dyne * sec * cm^-5)</td><td align=\"center\">-632 ± 669</td><td align=\"center\">‡</td><td align=\"center\">-567 ± 613</td><td/><td align=\"center\">-106 ± 382</td></tr><tr><td align=\"left\">Arterial compliance rest (mL *m^-2/mmHg)</td><td align=\"center\">0.49 ± 0.18</td><td/><td align=\"center\">0.48 ± 0.2</td><td/><td align=\"center\">0.7 ± 0.38</td></tr><tr><td align=\"left\">Arterial compliance peak (mL *m^-2/mmHg)</td><td align=\"center\">0.33 ± 0.11</td><td align=\"center\">Δ</td><td align=\"center\">0.55 ± 0.22</td><td/><td align=\"center\">0.4 ± 0.2</td></tr><tr><td align=\"left\">Δ Arterial compliance (rest-peak, mL *m^-2/mmHg)</td><td align=\"center\">-0.17 ± 0.17</td><td align=\"center\">Δ</td><td align=\"center\">0.07 ± 0.15</td><td align=\"center\">*</td><td align=\"center\">-0.3 ± 0.24</td></tr><tr><td align=\"left\">Arterial elastance index rest (mmHg/mL/m²)</td><td align=\"center\">4.7 ± 1.5</td><td/><td align=\"center\">4.5 ± 1.5</td><td/><td align=\"center\">3.6 ± 1.1</td></tr><tr><td align=\"left\">Arterial elastance index peak (mmHg/mL/m²)</td><td align=\"center\">6.2 ± 1.8</td><td align=\"center\">Δ</td><td align=\"center\">4.1 ± 1.1</td><td align=\"center\">*</td><td align=\"center\">6.3 ± 2.5</td></tr><tr><td align=\"left\">Δ Arterial elastance index (rest-peak, mmHg/mL/m²)</td><td align=\"center\">1.5 ± 1.6</td><td align=\"center\">Δ</td><td align=\"center\">-.4 ± 1.4</td><td align=\"center\">*</td><td align=\"center\">2.7 ± 1.7</td></tr><tr><td align=\"left\">Ventricular/arterial coupling rest (SP/ESV/EaI ratio)</td><td align=\"center\">1.8 ± .9</td><td/><td align=\"center\">1.8 ± .9</td><td/><td align=\"center\">1.3 ± .7</td></tr><tr><td align=\"left\">Ventricular/arterial coupling peak (SP/ESV/EaI ratio)</td><td align=\"center\">2.9 ± 1.9</td><td align=\"center\">§</td><td align=\"center\">2.1 ± 1.1</td><td/><td align=\"center\">1.1 ± .8</td></tr><tr><td align=\"left\">Δ Ventricular/arterial coupling (rest-peak)</td><td align=\"center\">1.1 ± 1.6</td><td align=\"center\">§</td><td align=\"center\">.4 ± .6</td><td/><td align=\"center\">-0.2 ± .4</td></tr><tr><td align=\"left\">Cardiac index rest (L/min/m²)</td><td align=\"center\">2 ± 0.7</td><td/><td align=\"center\">1.9 ± 0.5</td><td/><td align=\"center\">2.5 ± 0.7</td></tr><tr><td align=\"left\">Cardiac index peak (L/min/m²)</td><td align=\"center\">3.9 ± 1.3</td><td align=\"center\">§</td><td align=\"center\">2.7 ± 0.9</td><td/><td align=\"center\">2.7 ± 0.8</td></tr><tr><td align=\"left\">Δ Cardiac index (rest-peak, L/min/m²)</td><td align=\"center\">1.9 ± 1.2</td><td align=\"center\">§</td><td align=\"center\">0.7 ± 0.6</td><td/><td align=\"center\">0.3 ± 0.5</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Significant determinants of the sensor second heart sound (S2) amplitude values</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">Rest S2</td><td align=\"left\">Peak S2</td><td align=\"left\">S2 Δ % rest-peak</td></tr></thead><tbody><tr><td align=\"left\">Age (yrs)</td><td align=\"left\">-.359 (&lt;.01)</td><td align=\"left\">-.476 (&lt;.01)</td><td align=\"left\">-.153 (&lt;.05)</td></tr><tr><td align=\"left\">BSA (m2)</td><td/><td/><td/></tr><tr><td align=\"left\">LVMI (g/m2)</td><td align=\"left\">-.194 (&lt;.05)</td><td/><td/></tr><tr><td align=\"left\">LV EF %</td><td/><td align=\"left\">.215 (&lt;.01)</td><td/></tr><tr><td align=\"left\">WMSI</td><td/><td/><td/></tr><tr><td align=\"left\">HR (bpm)</td><td align=\"left\">.206 (&lt;.01)</td><td align=\"left\">.516 (&lt;.01)</td><td align=\"left\">.453 (&lt;.01)</td></tr><tr><td align=\"left\">Diastolic Blood Pressure (mmHg)</td><td align=\"left\">.183 (&lt;.05)</td><td align=\"left\">.319 (&lt;.01)</td><td align=\"left\">.502 (&lt;.01)</td></tr><tr><td align=\"left\">Systolic Blood Pressure (mmHg)</td><td/><td align=\"left\">.338 (&lt;.01)</td><td align=\"left\">.544 (&lt;.01)</td></tr><tr><td align=\"left\">Mean Blood Pressure (mmHg)</td><td/><td align=\"left\">.345 (&lt;.01)</td><td align=\"left\">.567 (&lt;.01)</td></tr><tr><td align=\"left\">Ventricular elastance (mmHg/mL/m2)</td><td/><td align=\"left\">.144 (&lt;.05)</td><td align=\"left\">.218 (&lt;.01)</td></tr><tr><td align=\"left\">Arterial elastance</td><td/><td align=\"left\">.307 (&lt;.01)</td><td align=\"left\">.281 (&lt;.01)</td></tr><tr><td align=\"left\">SVR (dyne * sec * cm^-5)</td><td/><td/><td/></tr><tr><td align=\"left\">Arterial compliance (mL *m^-2/mmHg)</td><td/><td align=\"left\">-.340 (&lt;.01)</td><td align=\"left\">-.300 (&lt;.01)</td></tr><tr><td align=\"left\">Ventricular/arterial coupling</td><td/><td/><td/></tr><tr><td align=\"left\">Cardiac index</td><td align=\"left\">.153 (&lt;.05)</td><td align=\"left\">.432 (&lt;.01)</td><td align=\"left\">.388 (&lt;.01)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Crosstabs' statistics and measures of association for post exercise hypotension vs. post exercise second heart sound amplitude undershoot in 75 selected patients</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">Exercise recovery hypotension</td><td align=\"left\">Exercise recovery isopressure</td><td align=\"left\">Total</td></tr></thead><tbody><tr><td align=\"left\">SHS Recovery Under shot</td><td align=\"left\"><bold>44</bold></td><td align=\"left\"><bold>1</bold></td><td align=\"left\">45</td></tr><tr><td align=\"left\">SHS Recovery same shot</td><td align=\"left\"><bold>11</bold></td><td align=\"left\"><bold>19</bold></td><td align=\"left\">30</td></tr><tr><td align=\"left\">Total</td><td align=\"left\">55</td><td align=\"left\">20</td><td align=\"left\">75</td></tr></tbody></table></table-wrap>" ]

[ "<disp-formula>SVR = 80 * (MAP-5)/CO,</disp-formula>" ]

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[]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Appendix. Sound – Heart sounds – Accelerometer to measure peak heart sounds vibration amplitude – Wireless – Wireless telemedicine – Telemedicine is healthcare's new frontier.</p></caption></supplementary-material>" ]

[ "<table-wrap-foot><p>§= significant differences between exercise and both dipyridamole and pacing stress pts; ‡ = significant differences between exercise and pacing stress pts; * = significant differences between dipyridamole and pacing stress pts; Δ = significant differences between exercise and dipyridamole stress pts</p></table-wrap-foot>", "<table-wrap-foot><p>Linear regression analysis to identify significant relationship between predictor variables (first column) and the sensor second heart sound (S2) amplitude was performed for baseline (second column) peak stress (third column) and rest-peak delta values (fourth column).</p><p>Pearson's correlation coefficients (and significance value within brackets) are reported in cells with significant (p &lt; 0.05) relationships.</p></table-wrap-foot>", "<table-wrap-foot><p>Kendall's tau-c = 0.591 P &lt; 0.001</p></table-wrap-foot>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Atopic asthma is a common inflammatory disorder of the airway epithelium characterized by tissue obstruction and remodeling, bronchial smooth muscle cell hyperreactivity to allergens and chronic bronchial inflammation. It classically involves allergen-driven T helper 2 (Th2) lymphocyte polarisation with coordinate production of interleukin (IL)-4, IL-5, IL-13 and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are encoded in one gene cluster on chromosome 5q31-34 [##REF##17083345##1##]. IL-4 and IL-13 are critically involved in the pathogenesis of allergic asthma by regulating IgE-production by B cells, inducing airway hyperreactivity and triggering key features of airway remodeling, whereas IL-5 is a key factor for eosinophilia [##REF##11477111##2##,##REF##18266877##3##]. Activation of IgE receptors on mast cells triggers the release of preformed vasoactive mediators such as histamine, the synthesis of prostaglandins and leukotrienes, and, via a positive feedback loop, expression IL-4 and IL-13 [##REF##11477111##2##].</p>", "<p>Its apparent association with airway diseases has recently focussed interest on the novel IL-7-like cytokine thymic stromal lymphopoietin (TSLP). TSLP expression is increased in asthmatic airways and correlates with both the expression of Th2-attracting chemokines and with disease severity [##REF##15944327##4##, ####REF##16432252##5##, ##REF##16785889##6####16785889##6##], indicating a link between TSLP and human asthma. Furthermore it was shown that experimental lung-specific expression of TSLP leads to transgene-induced allergic airway inflammation characterized by a massive infiltration of leukocytes, goblet cell hyperplasia, and subepithelial fibrosis, as well as by increased serum IgE levels [##REF##16142237##7##].</p>", "<p>TSLP is a typical four-helix-bundle cytokine 140 amino acid residues in length and was first cloned in humans in 2001 [##REF##11480573##8##, ####REF##11418668##9##, ##REF##17129180##10####17129180##10##]. The human TSLP gene is localized on chromosome 5q22, interestingly close to the gene cluster encoding several Th2-related cytokines such as IL-4, IL-5, IL-9, and IL-13 [##REF##16142237##7##,##REF##16172260##11##]. Human TSLP is produced by different cell types in atopic asthma, mainly by epithelial and smooth muscle cells and induces an inflammatory Th2 response. The TSLP receptor (TSLPR) is a heterodimeric cytokine receptor consisting of the IL-7 receptor alpha chain (IL-7Rα) and a TSLP-specific receptor chain with similarity to the common gamma receptor chain (γc). The TSLPR, also known as CRLF2, is expressed in heart, skeletal muscle, kidney and liver, but also on asthma-relevant dentritic cells [##REF##11418668##9##,##REF##11474172##12##]. In this review, the signal transduction around human TSLP in the cascade of events in the development of atopic asthma is discussed. We first describe the regulation of TSLP production in airway epithelial and other cells, then cover the TSLPR-mediated effects on TSLP target cells such as DCs and mast cells, and finally treat the DC-triggered onset of a specific Th2 response.</p>", "<title>Regulation of TSLP expression</title>", "<p>In the human airway system, fibroblasts, smooth muscle cells, epithelial cells and mast cells all have the potential to produce TSLP [##REF##12055625##14##, ####REF##17213320##15##, ##REF##17242164##16##, ##REF##17617600##17##, ##REF##17513456##18####17513456##18##]. Airway epithelial cells (AECs) were found to have increased TSLP mRNA levels in human asthmatics [##REF##15944327##4##]. Importantly, overexpression of TSLP in AECs induces experimental asthma in mice [##REF##16142237##7##].</p>", "<p>TSLP expression is enhanced by different stimuli with relevance in asthma. Primary small airway epithelial cells (SAECs) produce biologically active TSLP in response to bacterial peptidoglycan, and lipoteichoic acid as well as to poly I:C (mimicking viral double-stranded RNA) [##REF##17242164##16##]. IL-1β and TNF-α, two cytokines associated with pulmonary inflammation and strongly upregulated in the asthmatic lung [##REF##10586089##19##,##REF##17034639##20##] can, under appropriate conditions, induce human TSLP expression in normal human bronchial epithelial cells (NHBECs) [##REF##17213320##15##,##REF##17617600##17##], SAECs [##REF##17242164##16##] and human airway smooth muscle cells (HASMCs) [##REF##17513456##18##]. Similarly, TGF-β, IFN-β, IL-4, IL-13, and, in particular, a combination of TNF-α and IL-4 or IL-13 upregulate TSLP expression in NHBEs [##REF##17617600##17##].</p>", "<p>It is established that rhinovirus and respiratory syncytial virus (RSV) can trigger exacerbations of asthma [##REF##11080702##21##]. TSLP expression in human bronchial epithelial cells is stimulated by both viruses and an involvement of signal transduction through p38 and Jun kinase (JNK) has been demonstrated [##REF##17959543##22##]. Stimulation of TSLP expression evoked by rhinoviral dsRNA and RSV proteins via toll-like receptors (TLRs) is synergistically enhanced by IL-4, indicating a contribution of JAK/STAT signalling [##REF##17617600##17##]. The notion of cooperative signalling to TSLP gene transcription by cytokine and toll-like receptors is supported by the observation that tumor necrosis factor- (TNF-) α and IL-4 or IL-13 jointly drive TSLP expression in NHBECs, but none of the factors has a respective effect on its own. The induction of TSLP by combination of TNF-α and Th2 cytokines but not by the individual cytokines suggests that NFκB and STATs cooperate in transcriptional regulation of the TSLP gene [##REF##17617600##17##].</p>", "<p>HASMCs which also act as effector cells in initiating or perpetuating airway inflammation [##REF##15294996##23##,##REF##16951370##24##], respond by TSLP release to stimulation with TNF-α and IL-1β both <italic>in vitro </italic>and <italic>in vivo </italic>[##REF##17513456##18##]. Pharmacological inhibitors of ERK1/2 and p38, but not blockers of phosphatidylinositid-3 kinase (PI-3K) specifically suppress TSLP secretion induced by both factors individually or combination, suggesting that TSLP expression in HASMC is controlled via MAPK pathways [##REF##17513456##18##]. Crosstalk of NFκB- and MAPK pathways is suggested by experiments in cells with mutated mediators NFκB and Ras which show a strong decrease in transcriptional activity of the human TSLP promoter [##REF##15492243##25##].</p>", "<p>Studies employing deletion constructs of the TSLP gene promoter indicated that a DNA fragment extending from 3.74 to 3.86 kb upstream of the transcriptional start site contains a cis element required for transcriptional induction by IL-1β. Inspection of this ~120-bp sequence revealed consensus cognate elements for NFκB and IRF-1 as well as a putative AP-1 binding site [##REF##17213320##15##]. Mutations in these motifs indicated that the induction of TSLP gene expression seen in cells stimulated with IL-1β is likely to be mediated through NFκB, whose subunits p65/p50 bind to the NFκB cognate motif in the human TSLP promoter. One of the major pathways for NFκB activation involves the phosphorylation of the inhibitor IκBα, which is followed by IκBα degradation and the subsequent migration of NFκB dimers (each monomer consisting of a p50 and a p65 subunit) from cytoplasm to the nucleus [##REF##3129195##26##]. A dominant-negative mutant of IKKβ (IκB) inhibits IL-1β-mediated transcription of the TSLP gene [##REF##17213320##15##].</p>", "<p>Since TLSP induction in the airway epithelium of asthmatics appears to be a associated with allergen contact, it is important to note that engagement of TLRs by allergene provocation activates NFκB [##REF##15229469##27##]. TLR2, TLR3, TLR8, and TLR9 can all induce human TSLP expression in airway epithelial cells [##REF##17213320##15##,##REF##17617600##17##], suggesting that TSLP may become upregulated in the lung epithelium upon allergen challenge. In line with this hypothesis, we have recently observed that direct stimulation of lung epithelial cells with different allergens induces the expression of TSLP mRNA (Borowski et al., manuscript in preparation).</p>", "<p>Viral dsRNA is sensed, apart from TLR3, also by the recently identified cytosolic RNA helicases RIG-I and MDA5 [##REF##16027039##28##,##REF##16762830##29##]. Activation of TLR3, RIG-I, and MDA5 by dsRNA is transmitted to transcription factors NFκB and IRF-3, leading to transcriptional upregulation of pro-inflammatory genes and expression of type I interferons including IFN-β. siRNA experiments suggested that TSLP is directly induced by dsRNA in airway epithelial cells, and that the response is mediated by a pathway involving TLR3, NFκB and and IRF-3, but is independent of interferon signalling. Enhancement of dsRNA-dependent TSLP expression by IL-4 is significantly inhibited by siRNA targeting STAT6, supporting the notion of STAT6 as an important transcription factor in the control of TSLP expression [##REF##17617600##17##].</p>", "<p>Very recent work showed that TSLP expression in the murine lung is influenced by peptidyl-propyl isomerase (PIN1), an important regulator of survival-promoting and proinflammatory cytokines in T-cells. Active PIN1 inactivates adenosine-uridine binding factor 1 (AUF1), whose function is to destabilize mRNA by interaction with adenosine-uridine rich elements. Since TSLP expression is blocked by a PIN1 inhibitor after challenging lung with allerges and the 3'-untranslated region of TSLP mRNA contain an AUF1 binding site, PIN1 is likely to be a modulator of TSLP expression in asthma at the posttranscriptional level [##REF##18374404##30##].</p>", "<title>Activation of the TSLP receptor and intracellular signal transduction</title>", "<p>The specific, low affinity TSLP receptor α chain (TSLPRα) is a member of the hematopoietic (type 1) cytokine receptor family. In combination with the IL-7Rα chain it forms the heterodimeric TSLP receptor (TSLPR) which, upon TSLP binding, transmits signals towards STAT activation and proliferation into the cell interior [##REF##11480573##8##,##REF##11418668##9##,##REF##10881176##31##,##REF##10974032##32##]. The TSLPRα chain has some atypical features for a type 1 cytokine receptor, both in its extracellular and intracellular region. The exodomain, for instance, lacks one of the four cysteine residues conserved within the receptor family, perhaps indicating a unique tertiary structure. Intracellularly, the TSLPRα lacks one of the two conserved sequence boxes present in other cytokine receptors that govern the interaction with Janus kinases (JAKs).</p>", "<p>Signal transduction emanating from the dimerized TSLPR is similar to signalling from the IL-7R, reflecting the overlapping utilization of the IL-7Rα chain by the two systems. The IL-7 receptor utilizes the γc chain as a dimerization partner for IL-7Rα, which recruits JAK3 via its box1 element. Ligand-induced crosslinking of both the TSLP and IL-7 receptors results in the activation of STAT5 and STAT3 and concomitant specific gene regulation [##REF##11480573##8##,##REF##10570284##33##,##REF##11907084##34##]. However, unlike the IL-7R, the TSLPR appears to drive STAT activity via an uncommon mechanism without an involvement of Janus kinases. Evidence for this interpretation comes from experiments showing that no JAK phosphorylation was evoked by the activated TSLPR and that dominant-negative forms of JAK1 and JAK2 did not block TSLP-mediated STAT5 activation [##REF##9916685##35##]. It was puzzling, however, that fusions of TSLPR cytoplasmic domain with the exodomains of the erythropoietin receptor or CD8 did activate JAK2 upon ligand-dependent homo-dimerization [##REF##10733486##36##, ####REF##10872831##37##, ##REF##14993294##38####14993294##38##]. From results obtained with dominant-negative versions of Tec, a role of this cytoplasmic Src-related tyrosine kinase in the TSLPR-mediated activation of STAT5 was inferred [##REF##10570284##33##]. Src-type mediators are also involved in proliferative signaling, TSLP-induced cell proliferation is blocked by the Src family inhibitor PP1 [##REF##11907084##34##]. While both STAT5 activation and cell proliferation require the box1 domain of TSLPRα and IL-7Rα, the single intracellular tyrosine residue of TSLPRα receptor is critical only for proliferative signaling, but not for TSLP-dependent STAT5 activation [##REF##11907084##34##]. Apart from STAT5, TSLP initiates STAT3 phosphorylation in murine DCs without the induction of any of the four Janus kinases (JAKs) [##REF##11418668##9##,##REF##10881176##31##,##REF##10974032##32##] and activates STAT1 in murine pro B cells (A. Wohlmann and K. Friedrich, unpublished results). TSLP does not stimulate the activation of ERK1/2 and p70S6K [##REF##11480573##8##]. Thus, neither the MAPK pathway nor the p70S6K pathway appear to be involved in the signal transduction pathway elicited by TSLP. Details of TSLPR signaling are far from being settled, but the present view is that Src type kinases are mediating the proliferative response and unknown (non-JAK) kinases are critical for STAT activation and, ultimately, regulation of target genes (Figure ##FIG##0##1##).</p>", "<title>Activation of effector cells of the innate and adaptive immune system through TSLP</title>", "<p>Compelling evidence has been acumulated for a determinative role of TSLP in the initiation and maintenance of the allergic response in the context of atopic asthma [##REF##16432252##5##,##REF##16785889##6##]. Human TSLP is able to directly activate effector cells of the innate immune system like mast cells (MCs), which are known to play an important role in the pathogenesis of atopic diseases [##REF##7491066##39##,##REF##16750987##40##]. Functional receptors for TSLP are expressed <italic>in vivo </italic>on MCs infiltrating the bronchial mucosa of asthmatic patients as revealed by immunostaining of biopsy specimen [##REF##17242164##16##]. In inflammatory conditions mimicked by the presence of IL-1 and TNF α, TSLP is a potent activator of MCs leading to the production of high levels of proinflammatory Th2 cytokines and chemokines such as IL-5, IL-13, IL-6, GM-CSF, CXCL8, and CCL1 [##REF##17242164##16##] (Figure ##FIG##1##2##). Signaling pathways underlying this complex gene regulation have not been characterized yet.</p>", "<p>Apart from mast cells, the second main sentinel of the innate immune system is represented by DCs localized at the epithelial surface. DCs are also operative in the creation of a microenvironment that directs T cells towards Th1 or Th2 differentiation. A strong Th2 type response is typical in the context of the allergic syndrome. Human TSLP strongly activates immature CD11c⁺DCs while it does not appear to have any direct biological effects on B cells, T cells, NK-cells or neutrophils [##REF##11418668##9##,##REF##12055625##14##,##REF##15741223##41##]. TSLP induces DCs to up-regulate the expression of major histocompatibility class I and II and costimulatory molecules, including CD40, CD80, CD86. Importantly, it also strongly upregulates expression of the mRNA for OX40L, a member of the TNF superfamily that has been implicated in the initiation of Th2 cell responses [##REF##9787171##42##, ####REF##10637281##43##, ##REF##11157058##44####11157058##44##]. TSLP also stimulates DCs to produce the Th2-attracting chemokines TARC (thymus and activation regulated chemokine, CCL17) and MDC (macrophage derived chemokine, CCL22) [##REF##12055625##14##], as well as IL-8, IL-15 and eotaxin-2, clearly suggesting that TSLP-activated DCs may represent an initial key step in the development of allergic inflammation [##REF##11907084##34##,##REF##16275760##45##]. In the asthmatic bronchial mucosa, elevated expression of TSLP was also accompanied by and correlated with elevated expression of the CCR4 ligands TARC and MDC at the mRNA level [##REF##15944327##4##]. As revealed by a comparative global transcriptome analysis of naive and TSLP treated DCs, TSLP does not stimulate DCs to produce the Th2-polarizing cytokine IL-4 and, at the same time, suppresses the anti-inflammatory cytokine IL-10 as well as interferon- (IFN-) γ [##REF##16275760##45##].</p>", "<p>When TSLP treated DCs are stimulated with CD40 ligand, they induce the differentiation of CD8⁺T cells into cytolytic effector cells which produce IFN-γ as well IL-4 and IL-13. Interestingly, expression of these ctokines has as before been considered mutually exclusive [##REF##12707303##46##] (Figure ##FIG##1##2##).</p>", "<title>Indirect effects of TSLP on Th2 differentiation via OX40L</title>", "<p>OX40L, a member of the TNF superfamily has been identified as crucial mediator of Th2 cell responses driven by DCs [##REF##10637281##43##,##REF##12672051##47##]. In mice, blocking of OX40L by inhibitory antibodies inhibited the immune response induced by TSLP, indicated by reduction of cytokine secretion, Th2 inflammatory cell infiltration and IgE production [##REF##18060034##48##]. It appears that OX40L and IL-4 act synergistically and sequentially in driving Th2 cell responses in cocultures of T cell and DCs activated by TSLP [##REF##17129180##10##,##REF##16275760##45##]. Interestingly, in the presence of IL-12, OX40L is unable to induce a Th2 cell response but rather directs T cell differentiation towards the Th1 phenotype, indicating a functional dominance of IL-12 over TSLP [##REF##16275760##45##].</p>", "<p>Historically, CD4⁺Th2 cells are defined as effector T cells with the capacity to produce IL-4, -5, -10, and -13 [##REF##2523712##49##,##REF##9529145##50##]. IL-4 and IL-13 are typical pro-inflammatory cytokines, but IL-10 does not appear to contribute to allergic inflammation in either humans or mice [##REF##8648025##51##,##REF##11422123##52##], but even suppresses allergic inflammation [##REF##10536118##53##, ####REF##11477409##54##, ##REF##12209095##55####12209095##55##]. Importantly, dendritic cells activated by TSLP prime naive CD4⁺T cells to differentiate into a particular subtype of Th2 cells that produce the classical Th2 cytokines IL-4, IL-5 and IL-13, but no IL-10. It is also remarkable that these special Th2 cells secrete very high levels of TNF-α [##REF##12055625##14##]. TNF-α is prominent in asthmatic airways and genotypes that correlate with increased TNF-α secretion are associated with an increased risk of asthma [##REF##9097957##56##]. Because of their unique profile of cytokine production, Th2 cells induced by TSLP-activated DCs have been designated inflammatory Th2 cells to discriminate them from the classical regulatory Th2 cells [##REF##17129180##10##] (Figure ##FIG##1##2##).</p>", "<p>Th1 and Th2 cell differentiation is regulated by the key transcription factors T-bet for Th1 and GATA-3 and c-Maf for Th2. Th1 cells express high levels of T-bet but low GATA-3 and c-Maf, while Th2 cells show a reverse expression pattern, hence these transcription factors can be used as molecular markers for Th1 or Th2 cells [##REF##12461566##57##]. CD4⁺T cells primed by TSLP-activated DCs show the typical Th2 pattern high GATA-3 and c-Maf and low T-bet. However, IL-12 can override this Th2-specific gene regulation by inhibiting GATA-3 and c-Maf and strongly up-regulating T-bet [Ito <italic>et al. </italic>2005]. Regulation processes behind this phenomenon may involve temporal IL-12-induced upregulation of the IL-12R signaling subunit (IL-12Rβ2) and concomitant signal transduction via STAT4, which has been observed in CD4+ cells upon activation of OX40 [##REF##18250420##58##].</p>", "<p>TSLP has also been discussed as a player in the maintenance and regulation of Th2 memory cells. This interpretation comes from the finding that DCs activated by TSLP can induce an expansion of a CD4⁺T cell subset expressing the prostaglandin D2 receptor (CRTH2), a property of human Th2 central memory T cells [##REF##16782037##59##]. Interestingly, TSLP-activated DCs enhance the allergy-inducing properties of Th2 memory cells by up-regulating their expression of pro-allergic genes, particularly IL-17RB, the receptor for IL-25. IL-25, in turn, was shown to trigger the proliferation of Th2 memory cells and increase to the production of IL-4, IL-5, and IL-13, but not TNF-α or IFN-γ. These results suggest that IL-25 may costimulate the proliferation and further polarization of Th2 memory cells induced by TSLP-activated DCs [##REF##17635955##60##].</p>", "<p>Importantly for the development of asthmatic symptoms, the activation of inflammatory Th2 cells through TSLP and their production of the inflammatory cytokines IL-4, IL-5, IL-13 and TNF-α triggers IgE production, eosinophilia, mucus production and fibroblast proliferation [##REF##8893001##61##,##REF##9143690##62##]. The effector mechanisms of atopic asthma ultimately involve IgE-coated mast cells that undergo degranulation upon contact with the allergen and induce an immediate response, leading to the symptoms of local inflammation and bronchospasm [##REF##17466594##63##].</p>" ]

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[ "<title>Conclusion</title>", "<p>In atopic asthma, many different agents such as viruses, bacteria and allergens can induce a TSLP-dependent inflammatory response, leading to an inappropriate activation of both the innate and the adaptive immune system. With regard to the innate branch of the response, TSLP acts on mast cells and dendritic cells as well as, according to recent results, natural killer cells. Mast cells play a prominent role in the development of asthmatic symptoms, because they secrete inflammatory cytokines in response to TSLP. The fact that mast cells also produce TSLP indicates a potential amplification loop by the action of mast cell-derived TSLP on epithelial DCs. A critical switch governed by DCs is the TSLP-induced expression of OX40L, a Th2 cell polarizing signalling molecule. A further emerging role of TSLP is the generation of Th2 memory T cells. The IL-25-mediated collaborative interactions between eosinophils/basophils and Th2 memory cells perhaps propagate a positive feedback loop between innate effector and adaptive immunity, leading to the amplification of allergic inflammation (Figure ##FIG##1##2##).</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<p>Thymic stromal lymphopoietin (TSLP), a novel interleukin-7-like cytokine, triggers dendritic cell-mediated inflammatory responses ultimately executed by T helper cells of the Th2 subtype. TSLP emerged as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells (DCs). DCs activated by epithelium-derived TSLP can promote naïve CD4+ T cells to adopt a Th2 phenotype, which in turn recruite eosinophilic and basophilic granulocytes as well as mast cells into the airway mucosa. These different cells secrete inflammatory cytokines and chemokines operative in inducing an allergic inflammation and atopic asthma. TSLP is, thus, involved in the control of both an innate and an adaptive immune response. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understandimg of the disease and for the development of new therapeutics.</p>" ]

[ "<title>List of abbreviations used</title>", "<p>AEC: Airway Epithelial Cells; DCs: Dendritic Cells; GM-CSF: Granulocyte-Macrophage Colony Stimulating Factor; HASMCs: Human Airway Smooth Muscle Cells; IgE: Immunoglobulin E; IL: Interleukin; IFN: Interferon; JAK: Janus Kinase; JNK: Jun Kinase; MAPK: Mitogen Activated Protein Kinase; MCs: Mast Cells; NFκB: Nuclear Factor κB; NHBECs: Normal Human Bronchial Epithelial Cells; NK cells: Natural Killer Cells; PI-3K: Phosphatidylinositid 3-Kinase; SAECs: Small Airway Epithelial Cells; TLR: Toll-like Receptor; TNF: Tumor Necrosis Factor; TSLP: Thymic Stromal Lymphopoietin; TSLPR: Thymic Stromal Lymphopoietin Receptor; STAT: Signal Transducer and Activator of Transcription.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>KS and AB collected information from the literature and prepared iniatial versions of large parts of the manuscript. MK provided additional information from an immunological and clinical point of view and edited major parts of the manuscript. KHF conceived the overall organization of the manuscript, added extended sections of text and did the final editing. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>This work was supported by the Deutsche Forschungsgemeinschaft through grants FR 854/2-4 and LU 623/2-3.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Structure and signal transduction of the heterodimeric TSLP receptor complex.</bold> For details see text.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Central role of TSLP in the orchestration of an asthmatic response upon contact of the airway epithelium with allergens or other challenging agents.</bold> Intercellular communication among different cell types via cytokines evokes activity of the native (bottom part) as well as the adaptive (upper part) of the immune system. For details see text.</p></caption></fig>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Pain is a personal and subjective experience. The psychological factors therefore play important roles in shaping pain perception. One of these factors is expectation. In clinical situations, when pain is anticipated, patients often report the worsening of pain [##REF##7880221##1##, ####REF##11460803##2##, ##REF##15829386##3####15829386##3##]. Conversely, expectation of pain relief is considered to be effective means for producing placebo analgesia [##REF##14976306##4##, ####REF##16385107##5##, ##REF##16186029##6####16186029##6##]. Recently, Keltner <italic>et al. </italic>demonstrated that the level of expected pain intensity significantly altered perceived pain when the comparison was made between two noxious thermal stimuli of almost equal intensity [##REF##16624963##7##].</p>", "<p>Interests in pain anticipation-related brain activity has increased in recent years. EEG recording revealed significantly enhanced signals during anticipation of painful stimuli or priming with pain-related adjectives [##UREF##0##8##,##REF##10771160##9##]. Functional imaging studies suggested that expectation of pain could cause alteration in both pain perception and forebrain pain transmission [##REF##10373114##10##, ####REF##11943821##11##, ##REF##11839418##12####11839418##12##], even amplify brain responses to nonpainful somatosensory stimulation [##REF##11007903##13##]. More interestingly, the pain anticipation-related areas are largely overlapped with pain-related areas, such as the primary somatosensory cortex (SI), anterior cingulate cortex (ACC), periaqueductal grey (PAG), insular cortex (IC), prefrontal cortex (PFC), and cerebellum [##REF##10373114##10##, ####REF##11943821##11##, ##REF##11839418##12####11839418##12##,##REF##10076873##14##,##REF##12757820##15##].</p>", "<p>Although expectation of pain has been extensively studied in humans using neuroimaging techniques, neural mechanisms underlying the modulating effect of expectation are far from clear. Functional imaging studies are able to detect expectation-related signal changes, but can not directly measure neuronal spike activity. In fact, the correlation between imaging signals and action potential firing of neurons is still unclear [##REF##14499413##16##]. Furthermore, activation of brain sites revealed by imaging studies can not resolve issues as to how the information is transferred among the brain regions. The issues can only be addressed in animal experiments [##UREF##1##17##].</p>", "<p>We reported previously that the medial and lateral pain pathways were activated in parallel manner by cutaneous noxious radiant heat in awake rats, using a multiple-channel single-unit recording method [##REF##14625065##18##]. In that study, we incidentally observed anticipatory responses in the medial pain system, including the ACC and medial dorsal thalamus (MD). To further explore this issue, we investigated the effect of pain anticipation on nociceptive behavior and neural activity in awake rats. To establish anticipation in rats, we employed a Pavlovian conditioning paradigm, in which a neutral conditioned stimulus (tone) was paired with a noxious unconditioned stimulus (laser). The establishment of pain expectation was determined by the acquisition of conditioned responses (tone-induced avoidance). The aim of this study was to determine whether and how pain expectation could alter nociceptive processes and functional connectivity in the nociceptive neural networks.</p>" ]

[ "<title>Methods</title>", "<title>Animals</title>", "<p>All experiments were performed on nine male Sprague-Dawley rats (250–300 g) individually housed in a room maintained with a 12-h light/dark cycle. Food and water were available <italic>ad libitum</italic>. The experimental protocols were approved by the Animal Care and Use Committee at the Chinese Academy of Sciences and in accordance with the IASP guidelines for animal study. Every effort was made to minimize both animal suffering and the number of animals used.</p>", "<title>Surgery for microelectrode implantation</title>", "<p>After a 7-day period of habituation, animals received the surgery of microelectrode implantation. As in the previously described procedure [##REF##14625065##18##], rats were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg i.m.) and mounted on a stereotaxic frame. Following the retraction of skin and soft tissue, small holes corresponding to the recording sites were drilled on the skull. Then arrays of eight stainless steel Teflon-coated microwires (50 μm diameter, Biographics, Inc. Winston-Salem, NC, USA) were implanted unilaterally into the target brain areas. The coordinates were as follows: 3.2 mm anterior (A) to bregma, 0.8 mm lateral (L) to midline, 2.5 mm ventral (V) from the skull surface for the ACC; 2.3 mm posterior (P), 0.8 L, 5.5 V for the MD; and 1.0 P, 2.0 L, 2.0 V for the SI cortex. The microarrays were fixed in place with dental cement. Animals were housed individually and allowed to recover from surgery for at least 7 days before being subjected to the experiment.</p>", "<title>Apparatus and laser stimulation</title>", "<p>The experimental chamber was 44 × 22 × 44 cm in dimension and made of transparent acrylic plastics. The floor of the chamber contained an array of holes (diameter 6 mm) spaced 10 mm apart (centre to centre). A computer-controlled CO₂laser stimulator (Model DM-300, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Science) was used to deliver pain stimuli. The laser radiation was 10.6 μm in wavelength and 2.5 mm spot diameter. The output power and duration of the stimulation were set at 8 W and 20 ms, which was designed to activate the primary nociceptive afferents without damaging the skin or the subcutaneous tissue [##REF##7652034##52##]. The CO₂laser was equipped with a He-Ne aiming beam, which was visualized prior to firing the laser. The laser beam was emitted through the holes at the floor of the chamber to the plantar surface of rat hindpaw contralateral to the recording brain regions. A tone generator (Coulbourn Inc. USA) was mounted on the side wall of the chamber for audio stimuli (80 dB, 800 Hz, 100 ms). In tone-laser pairing trials, a laser pulse was delivered 900 msec after the tone. The onsets and durations of the tone and laser were controlled by a PC running program <italic>Magnet </italic>(Biographics, Inc.). A video camera was positioned in front of the chamber to record behavioral activity.</p>", "<title>Experimental protocol</title>", "<p>The experiments were conducted under normal lighting. In the first 3 days, rats were placed individually in the experimental chamber each day for 1 h. The experimental sessions started from day 4. All rats underwent 3 experimental sessions in 3 successive days (Fig. ##FIG##5##6##). The behavior of rats was continuously videotaped throughout the sessions for later analysis.</p>", "<p>In Session 1, two blocks of stimuli were delivered. One consisted of 40 painful laser stimuli and the other consisted of 30 tone stimuli, with inter-trial interval of no less than 60 sec. The order of the stimuli was balanced across animals. This session serves as a normal control for the following two sessions.</p>", "<p>In the second session, animals were subjected to 60 tone-laser pairing stimuli trials. This conditioning training formed a stable linkage between auditory cues and animals' nociceptive behaviors, i.e. the auditory cue will forecast the impending painful stimulus. This learning phase was followed by a testing phase (30 trials), in which the tone was administered without paired with laser, to determine the acquisition and extinction of the learned responses. The Session 2 was used to assess the effects of anticipation on neural activity and functional connectivity within central pain networks.</p>", "<p>In Session 3, animals received another 40 trials of laser stimuli. This session tests the possibility of sensitization or tolerance for noxious stimuli.</p>", "<title>Unit recording</title>", "<p>The simultaneous extracellular recording of the three selected brain areas was performed throughout the experimental sessions. Neural electric signals were obtained from the stainless steel microwires and passed from the headset assemblies to a preamplifier via two lightweight cables and a commutator. The commutator was free to turn as necessary, permitting unrestricted movement of the rat. The signals were band-pass filtered between 0.5 and 5 kHz (6 dB cutoff) before being sent to a spike-sorting device (Biographics, Inc.). Valid spikes were selected using amplitude and duration thresholds and recorded into a database file with PC-based software (<italic>Magnet</italic>, Biographics, Inc.). The identity of clearly sorted single neurons was verified by graphical capture of waveforms. Onset of tone and stimulation events was recorded into the data file with a resolution of 1 ms.</p>", "<title>Histology</title>", "<p>Animals were sacrificed with overdosed pentobarbital at the end of experiment. Recording sites were marked by electrophoretically deposited iron (20 μA, 10 – 20 s) at the tips of selected wires. Animals were then perfused with 4% paraformaldehyde and the brains were post-fixed, frozen, and cut coronally into 40-μm sections. The iron deposits could be visualized as blue dots under light microscope. Data obtained from the microwires outside the target regions were not included in the analysis.</p>", "<title>Behavioral assessment and analysis</title>", "<p>Behavioral responses to nociceptive stimuli were assessed by off line video analysis. According to the method of Fan <italic>et al.</italic>, the laser-induced nociceptive responses in rats can be classified as eight categories: head movement (Hm), body movement (Bm), foot jumping (Fj), foot elevation (Fe), foot movement (Fm), licking (Li), rearing (Re), and grooming (Gr) [##REF##7652034##52##]. The frequency and duration of each response were then used to quantitatively evaluate nociceptive behaviors. Here we modified the method by focusing on five of the eight categories listed above, i.e., Hm, Bm, Fj, Fe, and Fm. A score of 0 was assigned if the rat stayed quietly; a score of 1 if the rat displayed Hm (exploring); a score of 2 if Bm or Fm (motivation of avoidance) was observed; a score of 3 if Fj or Fe (successful escape) occurred. The behavioral response was measured with cumulative scores every 5 successive trials. One-way ANOVA followed by the <italic>Dunnett </italic>test for multiple comparisons was used to compare behavior scores between sessions. The behavioral assessment was used to examine whether and when the tone-laser association was steadily formed.</p>", "<title>Data analysis</title>", "<p>The neuronal firing rate was quantified for each neuron using peri-event time histograms (PSTHs). The bin size was 0.1 s for the computation of PSTHs. Bin counts for each trial were calculated using the analysis program NeuroExplorer (Plexon, Dallas, TX) and the results were exported to Matlab (The MathWorks, Inc.) in spreadsheet form. Neural responses to auditory or noxious stimulation were evaluated using a sliding window averaging technique, in which a 1-s time window was slid through the entire period of a trial at 0.1-s step. The bin counts of each window were compared with those of a preset 3-s control window 10 s before the stimulation event by Student's <italic>t</italic>-test. The differences were considered significant only when it reached a significance level of <italic>p </italic>&lt; 0.005 in three consecutive steps, thus to achieve a global significance of <italic>p </italic>&lt; 0.05. Units that significantly increased their activities after tone or laser stimuli were defined as excitatory; those that decreased their activities were considered inhibitory. To compare the neural responses between different sessions, the neuronal firing rates were transferred into Z scores using MatLab program: Z = (X-M)/S, where X is the actual firing rate obtained from PSTH, M and S are mean and standard deviation of the baseline discharging (-5 – -2 s), respectively.</p>", "<p>To identify the functional interactions between the recorded areas, cross-correlation and partial directed coherence (PDC) analyses were performed. In the cross-correlation analysis, one neuron was selected as the reference neuron and all other neurons were defined as partner neurons. The peri-spike histogram of a partner neuron within -0.5~0.5 sec around the reference neuron were calculated with a 5-ms bin size and a 3-bin <italic>Gaussian </italic>smooth. The significance level of the cross-correlograms was defined by 95% confidence level in the <italic>Nex </italic>program. Data falling into the 10-s period after laser stimulation were calculated.</p>", "<p>PDC is a frequency domain representation of the key concept of <italic>Granger </italic>causality. Briefly, if knowledge of <italic>x</italic>(n)'s past significantly improves prediction of <italic>y</italic>(n), we could then states that an observed time-series <italic>x</italic>(n) <italic>Granger</italic>-causes series <italic>y</italic>(n). This relation between time-series is by no means reciprocal. Absence of PDC between two structures at a given frequency means the lack of a direct link between them. Thus, PDC allows the detection of coactivations among simultaneous neuronal activities by highlighting one neuronal group that possibly drives another. The detailed methodology of PDC has been described elsewhere [##REF##10638818##53##, ####REF##11417058##54##, ##REF##11752471##55##, ##REF##17318884##56##, ##REF##18339481##57####18339481##57##]. In the present study, principal component analysis (PCA) for neurons in each brain area was first performed in <italic>Nex</italic>. Then the first principal component (PC1) of a given brain area that had the largest response were exported into <italic>MatLab</italic>, and the value of PDC across 1 – 50 Hz for each 2.5-s analysis time window were calculated. These values were then averaged around the laser stimulation events (0–10 s post-stimulus) and normalized to Z scores relative to the baseline (before stimulation) data.</p>", "<p>ANOVAs and non-parametric <italic>Chi</italic>-square tests were performed to determine differences in <italic>Z </italic>scores and percentage of correlated neurons between sessions, respectively. <italic>Bonferroni</italic>'s test was used for <italic>post hoc </italic>test and <italic>p </italic>&lt; 0.05 was considered to be level of significance for all the statistics.</p>" ]

[ "<title>Results</title>", "<title>Behavioral response</title>", "<p>Rats responded to auditory stimuli with high level exploratory activity in the early stage of Session 1 as evidenced by rearing, head movement, and short-lasting freezing. After repeated tone presentation, these exploring behaviors substantially subsided, as Fig. ##FIG##0##1A## (Session 1) illustrated. By contrast, noxious laser stimulation caused marked escaping responses such as foot jumping, lifting and licking. Such nociceptive behaviors always occurred throughout the first session (Fig. ##FIG##0##1B##).</p>", "<p>In the second session, rats received tone-laser conditioning training. As can be seen in Fig. ##FIG##0##1A## (Session 2), rats gradually learned to escape after tone but prior to the delivery of noxious stimulation. Following 25 tone-laser pairing trials, the auditory cue became a reliable predictor for the forthcoming painful stimuli. In the testing phase, it was observed that the tone alone was able to elicited escaping behavior (Fig. ##FIG##0##1C##), indicating the acquisition of the conditioned response. Interestingly, at the same time when rats are sensitive to the warning signal, the nociceptive behavior induced by actual pain stimulation was significantly reduced compared to Session 1 (9.18 ± 1.05 <italic>vs. </italic>12.92 ± 0.16, <italic>p </italic>&lt; 0.05).</p>", "<title>Laser- and tone-induced neuronal activity</title>", "<p>A total of 216 – 224 single units were simultaneously recorded (72 – 73 from the ACC, 61 – 64 MD, and 83 – 87 SI, varied between sessions due to neuron drifting). Noxious laser induced predominantly excitatory responses, displayed in sharp or sustained manner, as shown in Fig. ##FIG##1##2A##. The neurons exhibiting excitatory responses accounted for 32%, 51%, and 60% in ACC, MD thalamus, and SI cortex, respectively. Inhibitory neuronal responses were occasionally encountered and less than 5%. Auditory stimuli also elicited discharge of a small proportion of neuron within the recorded areas, with 7% in ACC, 16% in MD, and 11% in SI, as shown in Fig. ##FIG##1##2B##. Fig. ##FIG##1##2C## illustrated the typical neuronal response produced by tone-laser pairing.</p>", "<p>We compared the laser-induced neuronal responses during post-stimulus time across the three sessions. As shown in Fig. ##FIG##2##3A##, a significant difference was detected across sessions (ACC, <italic>F</italic>(2,2130) = 59.74, <italic>p </italic>&lt; 0.0001; MD, <italic>F</italic>(2,1850) = 63.57, <italic>p </italic>&lt; 0.0001; SI, <italic>F</italic>(2,2520) = 109.2, <italic>p </italic>&lt; 0.0001). A post hoc <italic>Bonferroni </italic>test for multiple comparisons showed that the pain-related responses in Session 2 were significantly higher than in Session 1 (<italic>p </italic>&lt; 0.05) for all the recorded areas, suggesting that anticipation of pain may enhance the nociceptive transmission in the brain. No significant difference was found between Sessions 1 and 3, indicating that no sensitization or tolerance was developed throughout Session 1–3. Tone-related responses were relatively weak with respect to the laser-induced responses. As illustrated in Fig. ##FIG##2##3B##, comparing with tone presentation alone, paring the tone cue with nociceptive stimulation significantly increased tone-related neural activity in the ACC but not in the MD and SI. These results suggest that the ACC may play a significant role in neural processing involved in pain anticipation.</p>", "<title>Functional connectivity within and between the recorded areas during noxious stimulation</title>", "<p>Correlations between the neurons within the same region were observed more often than those between different regions. For the within-area cross-correlations, <italic>Chi</italic>-square tests showed that the correlated activity in Session 2 was significantly higher than that in Session 1 (<italic>p </italic>&lt; 0.05) for all the recorded areas (Fig. ##FIG##3##4A##). For the between-area cross-correlations, the significantly enhanced correlated activity was observed between the MD and the other two regions (ACC and SI) in session 2 in comparison with session 1 (Fig. ##FIG##3##4B##).</p>", "<title>Information flow between the recorded areas during noxious stimulation</title>", "<p>PDC analysis was used to determine the direction of information flow from one region to others under different experimental conditions. A two-way ANOVA was performed to measure the difference in the normalized PDC between the Session 1 and 2. There was no significant Session × Direction interaction for all the regional pairs. However, significant effect was found in directions for ACC-SI (<italic>F</italic>(1,31) = 13.86, <italic>p </italic>= 0.0008) and MD-SI (<italic>F</italic>(1,30) = 5.184, <italic>p </italic>= 0.0301), which indicated that the information flow from the ACC to SI, and the MD to SI was significantly larger than that in the opposite direction in both sessions (Fig. ##FIG##4##5A## and ##FIG##4##5B##).</p>" ]

[ "<title>Discussion</title>", "<p>In the present study, simultaneous single unit recording was performed in the ACC, MD and SI to study the neural mechanism underlying the effect of pain expectation using tone-laser conditioning model in rats. There were three main findings. First, under anticipation condition, neuronal responses to the auditory cue were significantly increased only in the ACC whereas those to nociceptive stimuli were enhanced in all the recorded areas. Second, expectation of impending pain enhanced correlated neural activity within and between recording areas following noxious stimuli and third, there were larger amounts of information flow from the medial (ACC and MD) to the lateral (SI cortex) pathway during pain processing.</p>", "<p>Neuroimaging studies have identified anticipation-related activation in many cortical areas including the SI, ACC, IC, and the PFC [##REF##10373114##10##, ####REF##11943821##11##, ##REF##11839418##12####11839418##12##,##REF##10076873##14##,##REF##12757820##15##]. In the present study, we found significant increase in the neuronal response in the ACC but not in the SI during pain expectation. There are two possible explanations for the inconsistency between our and others' results. First, the animal model used in the present study is the type of 'certain' expectation, in which the neutral conditioned stimulus (tone) reliably predict the noxious unconditioned stimulus (laser). In contrast, most imaging studies on human expectation employed an uncertain paradigm. Certain and uncertain expectations have been demonstrated to be mediated by different neural pathways; the former is associated with activity in the ACC [##REF##10373114##10##,##REF##12757820##15##,##REF##9252330##19##, ####REF##9620699##20##, ##REF##10334900##21####10334900##21##], whereas the latter involves changes in the SI [##REF##11943821##11##,##REF##10076873##14##,##REF##7816140##22##]. Thus, our results provide evidence that the ACC, rather than the SI, is a structure critically involved in the neural process underlying certain expectation of pain. Another possible explanation is derived from the attention-related focusing mechanism. Previous studies in rats have found that the ACC is involved in tasks required visual or audio attention and preferentially activated during presentation of the conditional stimulus [##REF##8670672##23##, ####REF##9383513##24##, ##REF##10848573##25##, ##REF##14555761##26##, ##REF##15621378##27####15621378##27##]. The increased activity in the ACC observed in the present study could also be due to the conditioning experiment employed.</p>", "<p>As previously described, expectation of an aversive event (painful stimulation) can modify subsequent behavior (pain reactivity). Conditioned expectation (certain expectation) is associated with the emotional state of fear, which produces hypoalgesia [##REF##3524387##28##, ####REF##9276841##29##, ##REF##9364083##30####9364083##30##]. In contrast, unconditioned expectation (uncertain expectation) is related to anxiety, which has the opposite effect on pain, i.e., hyperalgesia [##REF##10212051##31##, ####REF##10883806##32##, ##REF##10601674##33####10601674##33##]. In our study, we found that the nociceptive behaviors (paw lifting and licking) induced by actual pain stimulation were reduced during conditioning, which is consistent with prior studies that the conditioned fear leads to decreased behavioral reactivity [##REF##10601674##33##]. Unexpectedly, the analysis of neuronal activity suggested that the laser-elicited responses in all recorded areas were enhanced under the expectation conditions. This seemed in contradiction with the behavioral findings that pain was decreased. It should be noted that noxious stimulation-elicited fear itself is a negative emotion. The emotional component of pain has been known to involve pathways through the medial thalamus to the ACC [##REF##10737061##34##,##REF##12015237##35##]. In addition, emotional states are found to be closely related to attentional states [##REF##11839418##12##]. There is evidence in humans and animals for the involvement of the ACC as well as the primary somatosensory cortices in the attentional modulation of pain [##REF##2599050##36##, ####REF##10393884##37##, ##REF##10692599##38####10692599##38##]. Thus, the neuronal responses in the recorded areas may reflect a mixed effect exerted by expectation. On the other hand, the increased neuronal activity and cross-correlations in ACC and MD during noxious stimulation may represent an endogenous antinociceptive activation instead of signalling nociceptive information. Early studies indicate that both the medial thalamus and ACC are involved in the activation of descending pain suppression mechanisms. Projections from the midline thalamic nuclei and ACC to the PAG have been described [##REF##7085925##39##,##REF##10888743##40##]. A high density of opioid receptors and activation induced by fentanyl within ACC support the participation of it in the down-regulation of pain perception [##REF##8989012##41##]. Therefore, the inconsistence between the behavioral findings and neural activity change suggest a more complex role of medial system in pain processing, including both mediation and suppression.</p>", "<p>Converging evidence indicates that pain is a multi-dimensional experience that involves distributed brain regions comprising lateral and medial systems [##REF##10068155##42##, ####REF##11126640##43##, ##REF##14993415##44##, ##REF##16011543##45####16011543##45##]. The medial pain system consists of the ACC and the medial thalamic nuclei and is believed to process the emotional-motivational component of pain [##REF##9252330##19##,##REF##12015237##35##]. The functional relationship and anatomical connection between the ACC and the medial thalamus have been demonstrated by numerous studies [##REF##64477##46##, ####REF##762282##47##, ##REF##6633977##48##, ##REF##1282403##49##, ##REF##14672812##50####14672812##50##]. The present study simultaneously recorded neurons in these areas, and found a significant increase in the number of correlated neuronal pairs within the same and between different brain areas under anticipation conditions, compared to those under non-anticipation conditions. An increased synchronized activity observed in the present study suggests temporal coding may play a significant role in processing pain perception under the conditioning state. Together with previous discussion, these results indicated an enhanced network processing in the pain neuromatrix under the expectation of pain. Further studies will be required to elucidate the implication of this finding.</p>", "<p>Another interesting finding of this study is larger amount of information flow from the medial (ACC and MD) to the lateral (SI cortex) pathway as compared to those in the opposite direction. PDC analysis reveals causality of coherent neural activity of two regions. In this view, our result indicates that the emotion-related neural circuits may modulate the neuronal activity in the somatosensory pathway during nociceptive transmission. Our previous study on tonic pain has demonstrated an increase in the information flow from the medial to the lateral pain pathway during the first hour after formalin injection [##REF##16603156##51##]. Although little available evidence supports direct linkage between the medial and the lateral pain systems [##REF##10737061##34##], our prior and current results both suggest the medial system may modulate lateral system during nociceptive processing, Based on the fact that the medial system is composed of the medial and intralaminar thalamic nuclei and limbic cortical areas which have descending projections to nociception regulating centres such as PAG, it is possible that the medial system modulates somatosensory nociceptive transmission through the brainstem structures that control both spinal and trigeminal dorsal horn pain transmission neurons. Thus, clarifying the anatomical and functional interaction between the parallel systems can provide deeper insight into the neural mechanism of expectation related pain modulation and may help us to improve the treatment of clinical pain.</p>" ]

[ "<title>Conclusion</title>", "<p>The present study demonstrated that anticipation of pain enhanced the neuronal discharges and correlated neural activity within and between brain regions in the medial and lateral pain pathways induced by the following noxious stimuli, indicating that the nociceptive processing in both medial and lateral pain systems is modulated by the expectation of pain.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Expectation is a very potent pain modulator in both humans and animals. There is evidence that pain transmission neurons are modulated by expectation preceding painful stimuli. Nonetheless, few studies have examined the influence of pain expectation on the pain-related neuronal activity and the functional connectivity within the central nociceptive network.</p>", "<title>Results</title>", "<p>This study used a tone-laser conditioning paradigm to establish the pain expectation in rats, and simultaneously recorded the anterior cingulate cortex (ACC), the medial dorsal thalamus (MD), and the primary somatosensory cortex (SI) to investigate the effect of pain expectation on laser-induced neuronal responses. Cross-correlation and partial directed coherence analysis were used to determine the functional interactions within and between the recorded areas during nociceptive transmission. The results showed that under anticipation condition, the neuronal activity to the auditory cue was significantly increased in the ACC area, whereas those to actual noxious stimuli were enhanced in all the recorded areas. Furthermore, neuronal correlations within and between these areas were significantly increased under conditions of expectation compared to those under non-expectation conditions, indicating an enhanced synchronization of neural activity within the pain network. In addition, information flow from the medial (ACC and MD) to the lateral (SI cortex) pain pathway increased, suggesting that the emotion-related neural circuits may modulate the neuronal activity in the somatosensory pathway during nociceptive transmission.</p>", "<title>Conclusion</title>", "<p>These results demonstrate that the nociceptive processing in both medial and lateral pain systems is modulated by the expectation of pain.</p>" ]

[ "<title>Abbreviations</title>", "<p>ACC: Anterior cingulate cortex; MD: Medial dorsal thalamus; SI: primary somatosensory cortex; PAG: Periaqueductal grey; IC: Insular cortex; PFC: Prefrontal cortex; PSTH: Peri-event time histogram; PDC: Partial directed coherence; PCA: Principal component analysis.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>JYW participated in the design of the study and drafted the manuscript. HTZ carried out all the experiment and performed the statistical analysis. JYC and DJW assisted with the electrophysiological recordings and data analysis. LAB contributed to the data analysis and interpretation. FL conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>This work was funded by a NNSF grant (30700223), and a grant for young scientist (07CX051005) from the Chinese Academy of Sciences to JYW, NNSF grants (30370461, 30570577, and 30770688), the 100 Talented Plan of the Chinese Academy of Sciences, and a grant from the 863 project (2006AA02Z431) to FL, NIH grants NS-43441 and NS 40628, TW-006144 to JYC, and NS-19608 to DJW.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Tone or laser-elicited behavior in the first two sessions</bold>. (A) The learning effect demonstrated by tone alone-elicited behavior. The behavioral score was accumulated every 5 successive trials. One-way ANOVA followed by the <italic>Dunnett </italic>test for multiple comparisons were used to compare the difference of behavioral scores between Session 2 and 1 (* <italic>p </italic>&lt; 0.05). As can be seen, rats learned to escape immediately after the tone after about 25 tone-laser pairing trials. (B) Laser-induced nociceptive behavior in the first session. (C) The acquisition and extinction of the conditioned response demonstrated by tone alone-elicited behavior. *, *** <italic>p </italic>&lt; 0.05, <italic>p </italic>&lt; 0.001, respectively, compared with \"Baseline\"; ###, <italic>p </italic>&lt; 0.001, compared with \"Trained\", one-way ANOVA followed by the Newman-Keuls Multiple Comparison Test. \"Baseline\" and \"Trained\" are the averaged behavioral scores in the first and second sessions (trials 1–30 in Session 1 and trials 51–55 in Session 2, see above Fig. 1A), respectively.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>The typical neuronal response elicited by simple laser (A), simple tone (B), and paired tone and laser (C)</bold>. PSTHs illustrated the average firing rate of a neuron around a stimulus. Time = 0 on the x-axis corresponded to the time of noxious (A, C) or tone (B) stimulus onset.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Laser (A) and tone-induced (B) responses in each session</bold>. The magnitude of neuronal discharge was assessed by <italic>Z</italic>-scores. The laser-induced response was presented as a time course post laser stimulus and averaged every 1 sec. The tone-elicited response was calculated 0 – 1 s following the onset of the tone. *, # <italic>p </italic>&lt; 0.05 indicate significantly different from Session 1 and Session 3, respectively.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>The percent of significantly correlated neuronal pairs within (A) and between (B) recorded brain areas during noxious stimulation</bold>. * <italic>p </italic>&lt; 0.05 indicates significantly different from Session 1.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Result of partial directed coherence analysis during noxious stimulation</bold>. (A) Two-way ANOVA showed that the information flow from ACC to SI, and MD to SI was significantly larger than that in the opposite direction in both Session 1 and 2. 'ACC-MD' indicates directed coherence from ACC to MD, and the same as the other regional pairs. Values are normalized to the pre-stimulation baseline level. * <italic>p </italic>&lt; 0.05, *** <italic>p </italic>&lt; 0.001, compared with PDC in the opposite direction. (B) An example of the amount of partial directed coherence observed between recorded areas. These PDC values were normalized to z-scores relative to the mean and variance of baseline (pre-stimulation) PDC. The normalized PDCs exceeding 95% confident interval of the baseline were displayed in pseudo colour. Warm and cool colours indicate the increase and decrease in PDC, respectively. The direction of information flow are from the column area to the row area.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>Experimental procedure</bold>. Session 1 contains two blocks of stimuli, one consisted of 40 laser stimuli and the other consisted of 30 tone stimuli. Session 2 involves 60 tone-laser pairing stimuli trials. Session 3 comprises another 40 trials of laser stimuli. The inter-trial interval was no less than 60 sec.</p></caption></fig>" ]

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[ "<title>Background</title>", "<p>The APOBEC family of cytosine deaminases includes numerous members that can deaminate cytosine to uracil within DNA and/or RNA molecules. Among these enzymes, the APOBEC3 sub-family has been discovered when human APOBEC3G (hA3G) was reported to restrict HIV replication ([##REF##12167863##1##]; reviewed in [##REF##18304004##2##]). Human hA3G has been shown to trigger extensive deamination of cytosine in the negative viral DNA strand during reverse transcription and to lead to deleterious G-to-A mutations considered as the hallmark of APOBEC3-editing activity. Subsequently, several other human APOBEC3 proteins – including APOBEC3A (hA3A) [##REF##16527742##3##], APOBEC3B (hA3B) [##REF##15956565##4##,##REF##15466872##5##], APOBEC3C (hA3C) [##REF##15466872##5##], APOBEC3DE (hA3DE) [##REF##16920826##6##], APOBEC3F (hA3F) [##REF##15296757##7##, ####REF##15152192##8##, ##REF##15141007##9####15141007##9##] and APOBEC3H (hA3H) [##REF##16571802##10##] – have been shown to exhibit antiviral effects against a variety of viruses, including numerous retroviruses – i.e. HIV, SIV, MLV, HTLV and foamy viruses –, hepatitis B virus and adeno-associated virus (AAV) (for review [##REF##17303427##11##]). In contrast to humans, the mouse genome encodes only one APOBEC3 (mA3) protein, which, like human APOBEC3 proteins, displays antiviral effects [##REF##12859895##12##]. Aside from the antiviral function of APOBEC3 proteins against exogenous viruses, some inhibitory effects have been reported on intracellular targets (for review [##REF##18304004##2##]) and several studies support the notion that the primary function of APOBEC3 proteins could be to prevent the propagation of mobile elements. Indeed, mammalian genomes have accumulated numerous transposable elements which account for &gt; 45% of the genomic DNA [##REF##11237011##13##,##REF##12466850##14##]. These elements can be grouped into two main classes: the strictly intracellular non-LTR (Long Terminal Repeat) retrotransposons, namely long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), which account for ~30% of each mammalian genome, and the LTR-containing retroelements (including the endogenous retroviruses, ERVs), accounting for ~10% of the genomes and closely related to retroviruses. The life cycle of ERVs includes the formation of virus-like particles (VLPs) that, in several instances – but not systematically – can remain strictly intracellular as observed for the well-characterized murine intracisternal A-particle (IAP) and MusD elements (the so-called \"intracellularized\" ERVs, [##REF##15479948##15##, ####REF##17151128##16##, ##REF##15107856##17##, ##REF##18256233##18####18256233##18##]), or that can bud at the cell membrane for an extracellular cycle as observed for the recently identified murine intracisternal A-particle-related envelope-encoding (IAPE; [##REF##18256233##18##]) and the human endogenous retrovirus HERV-K(HML2) elements [##REF##17077319##19##,##REF##17257061##20##]. Although most of these elements are no longer active due to the accumulation of inactivating mutations, some of them are still functional and have been cloned, thus allowing direct <italic>ex vivo </italic>assay of the effect of APOBEC proteins on their mobility. Accordingly, several APOBEC3 proteins, including hA3A, hA3B, hA3C and hA3F have been demonstrated to restrict the retrotransposition of the human LINE-1 (L1) elements [##REF##16527742##3##,##REF##16735504##21##,##REF##16648136##22##], as well as the L1-dependent transposition [##REF##12897783##23##] of the human <italic>Alu </italic>SINE elements [##REF##16728505##24##]. Moreover, although no effect on the retrotransposition of L1 elements was observed in the presence of hA3G [##REF##16735504##21##,##REF##15322092##25##, ####REF##15674295##26##, ##REF##17079095##27####17079095##27##], reports have shown that hA3G can prevent the retrotransposition of <italic>Alu </italic>elements [##REF##17079095##27##,##REF##17030807##28##] by sequestering <italic>Alu </italic>RNAs in cytoplasmic high-molecular-mass (HMM) ribonucleoprotein complexes [##REF##17030807##28##]. Similarly, the cloning of active copies for the intracellular murine IAP and MusD elements [##REF##15479948##15##,##REF##15107856##17##] made possible to demonstrate susceptibility of these retroelements to murine APOBEC3 and to most of the human APOBEC3 proteins [##REF##16728505##24##,##REF##15674295##26##,##REF##16407327##29##]. In addition, <italic>in silico </italic>analyses of the naturally present genomic copies of these elements in the murine genome have revealed \"traces\" of APOBEC3 editing on these elements ([##REF##15674295##26##]; see also [##REF##17967065##30##]), thus supporting the physiological relevance of the observed <italic>ex vivo </italic>assays, and the genomic impact of APOBEC3 protein activity.</p>", "<p>Here we take advantage of the recent identification of the infectious progenitor of the intracellularized IAP retrotransposon, namely IAPE, to analyze the possible restriction of a <italic>bona fide </italic>murine ERV, in a state close to that at the time of its initial endogenization step when the element still behaved as an infectious retrovirus, having not yet reached its highly adapted \"intracellularized\" state [##REF##18256233##18##]. In parallel, we performed a similar analysis on the human progenitor of the HERV-K(HML2) family members that we had \"reconstituted\", resulting in the <italic>Phoenix </italic>element which proved to be a <italic>bona fide </italic>endogenous retrovirus, the element being able to enter cells by infection and integrate with all the characteristic features of the genomic copies presently found in the human genome [##REF##17077319##19##]. These two functional human and murine \"extracellular\" ERVs were used to assess the effects of APOBEC3 proteins on mammalian endogenous retroviruses in appropriate <italic>ex vivo </italic>assays, and refined <italic>in silico </italic>analyses of the naturally present copies of these elements in their target host genomes finally unambiguously demonstrated \"traces\" of APOBEC3 editing, with identifiable signatures. Altogether, the data show that APOBEC3 proteins play a role not only on the intracellular retrotransposons found in humans and mice, but also on their retroviral \"progenitors\" endowed with an extracellular life style, thus <italic>de facto </italic>filling the gap between the described effects of APOBEC3 proteins on <italic>bona fide </italic>exogenous retroviruses on the one hand and intracellular retroelements on the other.</p>" ]

[ "<title>Methods</title>", "<title>Plasmids</title>", "<p>The human (HERV-K) and murine (IAPE-D) <italic>neo</italic>-marked ERV copies (pBS CMV-Kcons Stop Env neoAS and pCMV RU5 IAPE neoAS, respectively), the VSV-G and IAPE-D <italic>env </italic>expression vectors, and the <italic>neo</italic>-marked autonomous murine IAP retrotransposon (pGL3-IAP92L23 neo^TNF) have been previously described [##REF##15107856##17##, ####REF##18256233##18##, ##REF##17077319##19####17077319##19##]. The APOBEC3 expression plasmids were obtained from M. Malim (hA3A), the NIH AIDS Research and Reference Reagent program (hA3B, hA3C and hA3F), Open Biosystems (hA3DE), A. Hance (hA3G), and N. Landau (mA3). A plasmid expressing a defective hA3G gene (with a premature stop codon) was used as a negative control. All the APOBEC3 ORF-containing fragments were re-cloned into the pcDNA6 expression plasmid (Invitrogen).</p>", "<title>Retrotransposition and infection assays</title>", "<p>Retrotransposition assays with the neo-marked IAP were as described previously [##REF##12034850##38##]. For the infection assays, 293T cells seeded in 60-mm-diameter plates were transfected using the Lipofectamine Plus kit (Invitrogen) with 4.5 μg of the <italic>neo</italic>-marked <italic>env</italic>-defective murine or human ERV, 0.5 μg of the IAPE or VSV-G <italic>env </italic>expression vector, and 5 μg of the APOBEC3 expression vector to be tested. Supernatants were harvested 48 h post-transfection, filtered through 0.45-μm pore-size PVDF membranes, supplemented with Polybrene (4 μg/ml), and used to infect HeLa target cells by spinoculation (1.200 g for 2.5 h at 25°C). Infection events were detected upon G418 selection of target cells and viral titers quantified as the number of G418^Rclones per mL of supernatant [##REF##18256233##18##,##REF##17077319##19##].</p>", "<title>Analysis of integrated proviral DNAs</title>", "<p>Cellular DNA from 20–25 individual G418^Rclones was used to PCR-amplify a 996 bp fragment encompassing the <italic>env </italic>to <italic>neo </italic>gene region (nt 6783–7779) of the IAPE-D element and a 2049 bp fragment spanning the <italic>neo </italic>to <italic>gag </italic>gene region (nt 1093–3142) of the HERV-K element (initial 3 min denaturation step at 94°C; 40 cycles: 94°C, 50 sec; 60°C, 50 sec; 68°C, 150 sec). PCR reactions were performed with sets of appropriate primers in 50 μl containing 0.5 μg of cellular DNA, 1× Buffer II and 1.5 U AccuPrime <italic>Taq </italic>DNA polymerase (Invitrogen). The PCR products were electrophoresed on agarose gels, purified with the Nucleospin Extract II kit (Macherey-Nagel) and a ~800 bp or a ~1600 bp fragment was sequenced (Applied Biosystem sequencing kit) for IAPE-D and HERV-K, respectively.</p>", "<title>Human and Mouse genome analyses</title>", "<p>IAPE-D, IAPE-A and HERV-K endogenous retroviruses were extracted from the mouse and human genome sequence databases (Mouse GoldenPath mm8, February 2006 assembly and Human GoldenPath hg18, March 2006 assembly; <ext-link ext-link-type=\"uri\" xlink:href=\"http://genome.ucsc.edu/\"/>) by using as a querying probe the sequence of the previously described functional IAPE-D1 copy [##REF##18256233##18##], the sequence of the IAPE-A copy with intact <italic>gag</italic>-<italic>pol </italic>open reading frames (chr14-0436, [##REF##18256233##18##]), and the sequence of the HERV-K element (Phoenix-derived; [##REF##17077319##19##]) used in the cell-based infection assay. Twenty sequences displaying the highest homology to their cognate probe were selected for the IAPE-D and HERV-K elements. Twenty sequences with the highest homology to the IAPE-A sequence and localized on the Y chromosome were also selected to be used as a control (see Results). Alignments were performed using the ClustalW and Editsequence softwares and consensus sequences generated. Quantitative analysis of the nucleotide substitutions within the IAPE-A, IAPE-D and HERV-K elements was performed using Excel and Hypermut 2.0 (available at the <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.hiv.lanl.gov/\"/> website) softwares, on the full-length retroviruses. The localization of the analyzed sequences within the mouse and human genomes are given in additional file ##SUPPL##0##1##.</p>", "<title>Statistical analyses</title>", "<p>Significance levels for the data in Figures ##FIG##1##2## and ##FIG##2##3## were calculated using the Kruskal Wallis test (GrapPrism software package). More refined analyses for the occurrence of the G-to-A versus C-to-T mutations were performed using a Poisson regression in a log-linear model. The genmod procedure of the SAS software was used (version 9.1, SAS Institute Inc, Cary, NC). The observed distributions of the G-to-A mutations among the GA, GC, GG and GT contexts for HERV-K or the GXA, GXC, GXG and GXT contexts for IAPE were compared to the distribution of these di- or trinucleotides by the chi square test.</p>" ]

[ "<title>Results and discussion</title>", "<title>Restriction of murine and human infectious ERVs by APOBEC3 proteins</title>", "<p>To assay whether the mouse IAPE element is restricted by APOBEC3 proteins, we used the previously described functional copy of IAPE-D (<ext-link ext-link-type=\"uri\" xlink:href=\"http://genome.ucsc.edu/\"/>; mm9 July 2007 Assembly: chr12: 24,282,555–24,290,874) [##REF##18256233##18##] that was cloned under the control of the CMV promoter, and in which a <italic>neo </italic>resistance gene was inserted in reverse orientation into the <italic>env </italic>gene (Figure ##FIG##0##1A##). The effect of APOBEC3 proteins on HERV-K was analyzed by using the \"reconstituted\" <italic>Phoenix </italic>element cloned under the control of the CMV promoter, in which the <italic>env </italic>gene is stopped and an anti-sense-oriented <italic>neo </italic>resistance gene is inserted into its 3'-LTR (Figure ##FIG##0##1##). Proviral clones of IAPE-D or HERV-K (4.5 μg), complemented with an expression vector for a functional IAPE or VSV-G Env (0.5 μg) respectively, and the murine (mA3) or human (hA3A-G) APOBEC3 proteins or a control plasmid (5 μg), were transfected in 293T cells. Supernatants were harvested 48 h post-transfection, filtered through 0.45-μm pore-size PVDF membranes, supplemented with Polybrene (4 μg/ml), and transferred onto HeLa target cells. To increase sensitivity, target cells were subjected to spinoculation at 1.200 × g for 2.5 h at 25°C. Infection events were detected after G418 selection of target cells and viral titers expressed as the number of G418^Rclones per mL of supernatant. As illustrated in Figure ##FIG##0##1##, mA3 and hA3G protein expression leads to a dramatic decrease in both the IAPE-D and HERV-K viral titers (Figure ##FIG##0##1B##). In the case of the murine IAPE-D element, only a limited effect – if any – was observed with the human APOBEC3 proteins other than hA3G, with for instance no effect of hA3A which otherwise has a strong effect on the rate of retrotransposition of its intracellular counterpart, <italic>i.e</italic>. the IAP element (Figure ##FIG##0##1##). In the case of the human HERV-K, at variance with what is observed for the murine IAPE-D element, almost all the APOBEC3 proteins (with the exception of hA3C) have an effect, the highest activity being observed with hA3B and hA3F.</p>", "<p>We further assessed whether the observed decrease in viral titers was associated with editing of the viral DNA by sequencing a 800 or 1600 bp fragment of the <italic>de novo </italic>integrated IAPE-D or HERV-K proviral DNA copies, respectively, in 20–25 individual G418^Rclones. As illustrated in Figure ##FIG##1##2## numerous G-to-A transitions were observed in the presence of mA3 or hA3G in both ERVs, as expected for an APOBEC3-mediated editing. For HERV-K, G-to-A editing was also observed with hA3B, hA3DE and hA3F, but not with hA3A, as expected from previous characterization of this enzyme ([##REF##16527742##3##,##REF##16735504##21##,##REF##16728505##24##,##REF##16407327##29##]; reviewed in [##REF##17303427##11##]). Furthermore, mA3 and hA3G editing leads to G-to-A mutations in a <underline>G</underline>XA or <underline>G</underline>G context, respectively, which are the hallmarks previously described for each enzyme [##REF##15674295##26##,##REF##15098018##31##,##REF##15296758##32##]. For hA3B and hA3F, G-to-A editing was observed in the <underline>G</underline>A context [##REF##18304004##2##,##REF##16537839##33##]. In addition, in spite of a low number of G-to-A mutations, hA3DE editing seems to preferentially take place in the <underline>G</underline>A/T context as expected [##REF##16920826##6##]. It has to be stressed that the editing rate is probably underestimated because too heavily mutated <italic>neo </italic>genes present in these ERV DNAs can no longer confer G418 resistance after integration.</p>", "<title>Traces of APOBEC3 past activity on resident IAPE and HERV-K elements in the murine and human genome</title>", "<p>Since the murine IAPE-D and the human HERV-K elements are found to be restricted by APOBEC3 proteins in the <italic>ex vivo </italic>assay above, we asked whether APOBEC3 proteins might have actually impaired the <italic>in vivo </italic>amplification of these elements in the past, by searching for evidence of APOBEC3-editing on the endogenous copies residing in the murine and human genome, respectively. Accordingly, an <italic>in silico </italic>analysis was performed to assess the levels of G-to-A mutations in two sets of full-length genomic IAPE elements, originating from two different subfamilies, namely IAPE-A and IAPE-D, and on full-length HERV-K elements. Both the murine IAPE-D subfamily and the human HERV-K elements have most probably been amplified by reinfection of the germline and therefore could have been subjected to APOBEC3 editing. Conversely, the IAPE-A subfamily has most probably been amplified via gene duplication, with several elements – essentially on the Y chromosome – disclosing identical flanking sequences [##REF##8903725##34##,##REF##8709280##35##], and therefore should not have undergone APOBEC3 editing: this family of elements – closely related to IAPE-D – can therefore be used as an internal control for the <italic>in silico </italic>genomic analyses. For all three families of elements, we selected by BLAST analysis a set of twenty copies displaying the closest sequence similarity to their cognate \"master\" copy: to the functional \"Phoenix\" element for HERV-K, to the functional copy used in the <italic>ex vivo </italic>assay for IAPE-D, and to the unique full-length copy with preserved open reading frames for IAPE-A. A consensus sequence was then derived for each family of elements, and each family member was analyzed for mutations to the consensus. As illustrated in Figure ##FIG##2##3A##, numerous mutations can be found for the three families of elements, consistent with the million years of genome evolution that have elapsed since the initial infection and/or amplification events. However, a specific increase in G-base mutations can be observed for both the IAPE-D and the HERV-K copies, not observed for the IAPE-A copies. These mutations are essentially G-to-A substitutions, with the effect being most probably \"strand-specific\", since the number of such mutations is almost twice that of the C-to-T substitutions. In addition, this bias is not observed for the IAPE-A elements, as expected for a duplicated element which has amplified by chromosomal DNA duplication, without a reverse transcription step prone to APOBEC3 mutagenesis. Interestingly, as illustrated in Figure ##FIG##2##3B##, the observed G-to-A changes are not randomly distributed but seem to be influenced by the neighbouring nucleotides: the <underline>G</underline>XA triplet is the most frequent \"target\" for the G-to-A substitutions in the IAPE-D elements (see arrow in Figure ##FIG##2##3B##), in agreement with previous reports – and data in Figure ##FIG##1##2## – indicating that mA3 preferentially targets <underline>G</underline>XA trinucleotide motifs [##REF##15674295##26##,##REF##15098018##31##,##REF##16537839##33##]. On the other hand, the G-to-A substitutions in the HERV-K copies are most frequently observed in the <underline>G</underline>G context (see arrow in Figure ##FIG##2##3B##), which corresponds to the footprint of hA3G editing [##REF##15098018##31##] and data in Figure ##FIG##1##2##. There is no clear-cut evidence for G-to-A substitutions in the <underline>G</underline>A and <underline>G</underline>T context, excluding any significant contribution of hA3B, hA3DE or hA3F. Noteworthily, a \"non-specific\" bias can be observed for the endogenous IAPE-A, -D and HERV-K elements, which favors G-to-A mutations in C<underline>G</underline> dinucleotides (Figure ##FIG##2##3B##), most probably reflecting an APOBEC3-independent (since it is also observed for the duplicated IAPE-A elements) deamination of methylated-CpG islands. Finally, examples of sub-genomic regions of IAPE-D and HERV-K elements enriched in G-to-A substitutions, are shown in Figure ##FIG##2##3C##, where the di- or tri-nucleotide sequences specific for the hA3G and mA3 APOBEC proteins, respectively, are underlined. Altogether, these <italic>in silico </italic>data strongly suggest that the IAPE and HERV-K elements have been subjected to editing by specific APOBEC3 proteins during their retroviral cycle of amplification and insertion into their target host genome.</p>", "<p>We further explored APOBEC3 editing by analyzing more specifically the G-to-A substitutions at the mA3 and hA3G target sites for each of the twenty IAPE-D and HERV-K proviruses, respectively. As shown in Figure ##FIG##3##4A–B##, for each proviral element, both the total number of G-to-A mutations (grey plus hatched grey) and the number of G-to-A mutations at the mA3- and hA3G-specific sites (hatched grey) were measured, together with the number of C-to-T \"non-strand-specific\" mutations as an internal control (dark bars; also used to order the copies in the Figure). Figure ##FIG##3##4A–B## then clearly shows that i) the total number of G-to-A mutations is for most proviruses higher than that of the \"control\" C-to-T mutations, ii) this increase is essentially due to \"specific\" mutations at the respective APOBEC3 sites, and iii) the extent of the observed mutations is highly variable depending on the proviral copy. Actually, for both the IAPE-D and HERV-K proviruses, more G-to-A mutations than C-to-T mutations can be observed, consistent with a strand specificity that can only have occurred prior to integration; in addition, this excess of G-to-A mutations is in general observed at <underline>G</underline>XA triplet positions for the murine, mA3-sensitive IAPE-D (&gt; 40% of the G-to-A mutations for the majority of the proviruses, namely thirteen out of twenty), and at GG doublet positions for the human, hA3G-sensitive HERV-K elements. For the latter, it should be noted that the extent of specific G-to-A mutations is rather limited (seventeen out of the twenty HERV-K proviruses display &lt; 30% of their G-to-A mutations at <underline>G</underline>G positions), except for two proviruses (ch3-1271 and ch21-0189) which are specifically hypermutated (Figure ##FIG##3##4B–C##), with &gt; 70% of their G-to-A mutations in the <underline>G</underline>G context, without any evidence for a clear-cut gradient along the proviral sequence (Figure ##FIG##3##4C##). These results indicate that HERV-K can indeed be severely edited by hA3G, and that APOBEC3G protein expression at different times of HERV-K amplification in the human genome must have been quite variable.</p>" ]

[ "<title>Results and discussion</title>", "<title>Restriction of murine and human infectious ERVs by APOBEC3 proteins</title>", "<p>To assay whether the mouse IAPE element is restricted by APOBEC3 proteins, we used the previously described functional copy of IAPE-D (<ext-link ext-link-type=\"uri\" xlink:href=\"http://genome.ucsc.edu/\"/>; mm9 July 2007 Assembly: chr12: 24,282,555–24,290,874) [##REF##18256233##18##] that was cloned under the control of the CMV promoter, and in which a <italic>neo </italic>resistance gene was inserted in reverse orientation into the <italic>env </italic>gene (Figure ##FIG##0##1A##). The effect of APOBEC3 proteins on HERV-K was analyzed by using the \"reconstituted\" <italic>Phoenix </italic>element cloned under the control of the CMV promoter, in which the <italic>env </italic>gene is stopped and an anti-sense-oriented <italic>neo </italic>resistance gene is inserted into its 3'-LTR (Figure ##FIG##0##1##). Proviral clones of IAPE-D or HERV-K (4.5 μg), complemented with an expression vector for a functional IAPE or VSV-G Env (0.5 μg) respectively, and the murine (mA3) or human (hA3A-G) APOBEC3 proteins or a control plasmid (5 μg), were transfected in 293T cells. Supernatants were harvested 48 h post-transfection, filtered through 0.45-μm pore-size PVDF membranes, supplemented with Polybrene (4 μg/ml), and transferred onto HeLa target cells. To increase sensitivity, target cells were subjected to spinoculation at 1.200 × g for 2.5 h at 25°C. Infection events were detected after G418 selection of target cells and viral titers expressed as the number of G418^Rclones per mL of supernatant. As illustrated in Figure ##FIG##0##1##, mA3 and hA3G protein expression leads to a dramatic decrease in both the IAPE-D and HERV-K viral titers (Figure ##FIG##0##1B##). In the case of the murine IAPE-D element, only a limited effect – if any – was observed with the human APOBEC3 proteins other than hA3G, with for instance no effect of hA3A which otherwise has a strong effect on the rate of retrotransposition of its intracellular counterpart, <italic>i.e</italic>. the IAP element (Figure ##FIG##0##1##). In the case of the human HERV-K, at variance with what is observed for the murine IAPE-D element, almost all the APOBEC3 proteins (with the exception of hA3C) have an effect, the highest activity being observed with hA3B and hA3F.</p>", "<p>We further assessed whether the observed decrease in viral titers was associated with editing of the viral DNA by sequencing a 800 or 1600 bp fragment of the <italic>de novo </italic>integrated IAPE-D or HERV-K proviral DNA copies, respectively, in 20–25 individual G418^Rclones. As illustrated in Figure ##FIG##1##2## numerous G-to-A transitions were observed in the presence of mA3 or hA3G in both ERVs, as expected for an APOBEC3-mediated editing. For HERV-K, G-to-A editing was also observed with hA3B, hA3DE and hA3F, but not with hA3A, as expected from previous characterization of this enzyme ([##REF##16527742##3##,##REF##16735504##21##,##REF##16728505##24##,##REF##16407327##29##]; reviewed in [##REF##17303427##11##]). Furthermore, mA3 and hA3G editing leads to G-to-A mutations in a <underline>G</underline>XA or <underline>G</underline>G context, respectively, which are the hallmarks previously described for each enzyme [##REF##15674295##26##,##REF##15098018##31##,##REF##15296758##32##]. For hA3B and hA3F, G-to-A editing was observed in the <underline>G</underline>A context [##REF##18304004##2##,##REF##16537839##33##]. In addition, in spite of a low number of G-to-A mutations, hA3DE editing seems to preferentially take place in the <underline>G</underline>A/T context as expected [##REF##16920826##6##]. It has to be stressed that the editing rate is probably underestimated because too heavily mutated <italic>neo </italic>genes present in these ERV DNAs can no longer confer G418 resistance after integration.</p>", "<title>Traces of APOBEC3 past activity on resident IAPE and HERV-K elements in the murine and human genome</title>", "<p>Since the murine IAPE-D and the human HERV-K elements are found to be restricted by APOBEC3 proteins in the <italic>ex vivo </italic>assay above, we asked whether APOBEC3 proteins might have actually impaired the <italic>in vivo </italic>amplification of these elements in the past, by searching for evidence of APOBEC3-editing on the endogenous copies residing in the murine and human genome, respectively. Accordingly, an <italic>in silico </italic>analysis was performed to assess the levels of G-to-A mutations in two sets of full-length genomic IAPE elements, originating from two different subfamilies, namely IAPE-A and IAPE-D, and on full-length HERV-K elements. Both the murine IAPE-D subfamily and the human HERV-K elements have most probably been amplified by reinfection of the germline and therefore could have been subjected to APOBEC3 editing. Conversely, the IAPE-A subfamily has most probably been amplified via gene duplication, with several elements – essentially on the Y chromosome – disclosing identical flanking sequences [##REF##8903725##34##,##REF##8709280##35##], and therefore should not have undergone APOBEC3 editing: this family of elements – closely related to IAPE-D – can therefore be used as an internal control for the <italic>in silico </italic>genomic analyses. For all three families of elements, we selected by BLAST analysis a set of twenty copies displaying the closest sequence similarity to their cognate \"master\" copy: to the functional \"Phoenix\" element for HERV-K, to the functional copy used in the <italic>ex vivo </italic>assay for IAPE-D, and to the unique full-length copy with preserved open reading frames for IAPE-A. A consensus sequence was then derived for each family of elements, and each family member was analyzed for mutations to the consensus. As illustrated in Figure ##FIG##2##3A##, numerous mutations can be found for the three families of elements, consistent with the million years of genome evolution that have elapsed since the initial infection and/or amplification events. However, a specific increase in G-base mutations can be observed for both the IAPE-D and the HERV-K copies, not observed for the IAPE-A copies. These mutations are essentially G-to-A substitutions, with the effect being most probably \"strand-specific\", since the number of such mutations is almost twice that of the C-to-T substitutions. In addition, this bias is not observed for the IAPE-A elements, as expected for a duplicated element which has amplified by chromosomal DNA duplication, without a reverse transcription step prone to APOBEC3 mutagenesis. Interestingly, as illustrated in Figure ##FIG##2##3B##, the observed G-to-A changes are not randomly distributed but seem to be influenced by the neighbouring nucleotides: the <underline>G</underline>XA triplet is the most frequent \"target\" for the G-to-A substitutions in the IAPE-D elements (see arrow in Figure ##FIG##2##3B##), in agreement with previous reports – and data in Figure ##FIG##1##2## – indicating that mA3 preferentially targets <underline>G</underline>XA trinucleotide motifs [##REF##15674295##26##,##REF##15098018##31##,##REF##16537839##33##]. On the other hand, the G-to-A substitutions in the HERV-K copies are most frequently observed in the <underline>G</underline>G context (see arrow in Figure ##FIG##2##3B##), which corresponds to the footprint of hA3G editing [##REF##15098018##31##] and data in Figure ##FIG##1##2##. There is no clear-cut evidence for G-to-A substitutions in the <underline>G</underline>A and <underline>G</underline>T context, excluding any significant contribution of hA3B, hA3DE or hA3F. Noteworthily, a \"non-specific\" bias can be observed for the endogenous IAPE-A, -D and HERV-K elements, which favors G-to-A mutations in C<underline>G</underline> dinucleotides (Figure ##FIG##2##3B##), most probably reflecting an APOBEC3-independent (since it is also observed for the duplicated IAPE-A elements) deamination of methylated-CpG islands. Finally, examples of sub-genomic regions of IAPE-D and HERV-K elements enriched in G-to-A substitutions, are shown in Figure ##FIG##2##3C##, where the di- or tri-nucleotide sequences specific for the hA3G and mA3 APOBEC proteins, respectively, are underlined. Altogether, these <italic>in silico </italic>data strongly suggest that the IAPE and HERV-K elements have been subjected to editing by specific APOBEC3 proteins during their retroviral cycle of amplification and insertion into their target host genome.</p>", "<p>We further explored APOBEC3 editing by analyzing more specifically the G-to-A substitutions at the mA3 and hA3G target sites for each of the twenty IAPE-D and HERV-K proviruses, respectively. As shown in Figure ##FIG##3##4A–B##, for each proviral element, both the total number of G-to-A mutations (grey plus hatched grey) and the number of G-to-A mutations at the mA3- and hA3G-specific sites (hatched grey) were measured, together with the number of C-to-T \"non-strand-specific\" mutations as an internal control (dark bars; also used to order the copies in the Figure). Figure ##FIG##3##4A–B## then clearly shows that i) the total number of G-to-A mutations is for most proviruses higher than that of the \"control\" C-to-T mutations, ii) this increase is essentially due to \"specific\" mutations at the respective APOBEC3 sites, and iii) the extent of the observed mutations is highly variable depending on the proviral copy. Actually, for both the IAPE-D and HERV-K proviruses, more G-to-A mutations than C-to-T mutations can be observed, consistent with a strand specificity that can only have occurred prior to integration; in addition, this excess of G-to-A mutations is in general observed at <underline>G</underline>XA triplet positions for the murine, mA3-sensitive IAPE-D (&gt; 40% of the G-to-A mutations for the majority of the proviruses, namely thirteen out of twenty), and at GG doublet positions for the human, hA3G-sensitive HERV-K elements. For the latter, it should be noted that the extent of specific G-to-A mutations is rather limited (seventeen out of the twenty HERV-K proviruses display &lt; 30% of their G-to-A mutations at <underline>G</underline>G positions), except for two proviruses (ch3-1271 and ch21-0189) which are specifically hypermutated (Figure ##FIG##3##4B–C##), with &gt; 70% of their G-to-A mutations in the <underline>G</underline>G context, without any evidence for a clear-cut gradient along the proviral sequence (Figure ##FIG##3##4C##). These results indicate that HERV-K can indeed be severely edited by hA3G, and that APOBEC3G protein expression at different times of HERV-K amplification in the human genome must have been quite variable.</p>" ]

[ "<title>Conclusion</title>", "<p>The restriction effects of APOBEC proteins on endogenous retroelements have essentially concerned retrotransposons with a strictly intracellular life cycle, namely the LINE/SINE non-LTR retrotransposons, and LTR-retrotransposons including the yeast Ty1 element [##REF##15823539##36##,##REF##16000409##37##], and the IAP and MusD murine elements [##REF##16527742##3##,##REF##16728505##24##,##REF##15674295##26##,##REF##16537839##33##]. In these cases severe restriction has been observed, both in <italic>ex vivo </italic>assays and by <italic>in silico </italic>analysis of the traces that APOBEC proteins have left through DNA edition in the course of reverse transcription of the retroelements [##REF##15674295##26##]. Here we show that similar effects take place at the level of endogenous retroviruses with an extracellular life cycle, with an unambiguous restriction of the murine IAPE by a murine APOBEC3 protein, and of the human HERV-K element by a human APOBEC3 protein. Taking into account that an infectious IAPE retrovirus with an extracellular life cycle has been the progenitor of the IAP element, the restriction observed for IAP by mA3 appears simply to be the consequence of the restriction that initially controlled the progenitor infectious IAPE invading the rodent ancestor, with the effect being maintained in the evolution of the endogenized IAP retroelements. Although it concerns a heterologous – and therefore not necessarily very relevant-situation, it is noteworthy that the human hA3A protein can control the murine intracellular IAP retroelement, a property not observed for the IAPE infectious progenitor. This is most probably relevant to the localization of the hA3A protein – in the nucleus – and to its rather atypical mode of action – not involving editing of the reverse transcribed DNA – which identifies this restriction factor as more specifically devoted to intracellular retrotransposons, consistent with the absence of reported effects of this factor on – most – infectious retroviruses (reviewed in [##REF##18304004##2##]). Finally, <italic>in silico </italic>analysis of the genomic copies of the elements demonstrates that APOBEC3 editing has taken place in evolution for these amplified elements, with clear-cut evidence for a severe heterogeneity in the extent of the editing process.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>APOBEC3 cytosine deaminases have been demonstrated to restrict infectivity of a series of retroviruses, with different efficiencies depending on the retrovirus. In addition, APOBEC3 proteins can severely restrict the intracellular transposition of a series of retroelements with a strictly intracellular life cycle, including the murine IAP and MusD LTR-retrotransposons.</p>", "<title>Results</title>", "<p>Here we show that the IAPE element, which is the infectious progenitor of the strictly intracellular IAP elements, and the infectious human endogenous retrovirus HERV-K are restricted by both murine and human APOBEC3 proteins in an <italic>ex vivo </italic>assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. <italic>In silico </italic>analysis of the naturally occurring genomic copies of the corresponding endogenous elements performed on the mouse and human genomes discloses \"traces\" of APOBEC3-editing, with the specific signature of the murine APOBEC3 and human APOBEC3G enzymes, respectively, and to a variable extent depending on the family member.</p>", "<title>Conclusion</title>", "<p>These results indicate that the IAPE and HERV-K elements, which can only replicate via an extracellular infection cycle, have been restricted at the time of their entry, amplification and integration into their target host genomes by definite APOBEC3 proteins, most probably acting in evolution to limit the mutagenic effect of these endogenized extracellular parasites.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>CE, SP and DR carried out the experimental work and drafted the manuscript. OH performed the <italic>in silico </italic>analyses and drafted the manuscript. TH conceived the study and drafted the manuscript. All authors read and approved the final manuscript.</p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>The authors wish to thank Anne Aupérin for help in the statistical analyses and Christian Lavialle for critical reading of the manuscript. This work was supported by the CNRS, a grant from the Ligue Nationale contre le Cancer (Equipe labellisée) and fellowships from the CNRS to SP and the Association pour la Recherche sur le Cancer (ARC) to DR.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Murine and human APOBEC3 proteins inhibit endogenous retroviruses. (A) Rationale of the assay for detection of infection events by endogenous retroviruses in the presence of APOBEC3 proteins. The IAPE-D and HERV-K elements used in the assay are marked with the <italic>neo </italic>reporter gene – inserted in reverse orientation – and carry their own functional genes, except for the <italic>env </italic>gene which is supplied in <italic>trans</italic>, thus allowing only for single rounds of infection. Human 293T cells are co-transfected with the indicated expression vectors for APOBEC3 family members, the supernatants collected 2-days post-transfection to infect HeLa target cells, and infection events detected upon G418 selection. (B) Analysis of the activity of murine and human APOBEC3 proteins on the indicated endogenous retroviruses. Viral titers are given as percentages relative to a control (no apobec: expression vector with a nonfunctional hA3G; 622 and 549 G418^Rclones/ml for IAPE-D and HERV-K, respectively). Data are the means ± standard deviations (s.d.) for at least three independent experiments. Bottom: retrotransposition frequency of an active autonomous IAP element marked with a <italic>neo </italic>indicator gene for retrotransposition [##REF##15107856##17##] in the presence of the corresponding APOBEC3 proteins; the assay was performed by cotransfection of HeLa cells with the marked IAP and APOBEC expression vector as previously described [##REF##15674295##26##]; values are the means ± standard deviations (s.d.) for at least three independent experiments and are given as percentages relative to the control (no apobec; 1.3 × 10^-3G418^Rclones/cell).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>APOBEC3 proteins induce specific G-to-A hypermutations. Two-entry tables showing nucleotide substitution preferences in the presence of the indicated APOBEC3 proteins for the IAPE-D and HERV-K integrated proviruses. n, total number of bases sequenced. The adjacent graphs represent the relative frequencies of observed G-to-A mutations as a function of the G neighboring nucleotides (+2 position for the expected mA3 footprint, +1 position for the other APOBEC3s); for the two-entry tables, p-values calculated by a Poisson regression in a log-linear model for the occurrence of the G-to-A versus C-to-T mutations yielded p &lt; 0.03 in all cases (except for hA3DE (p = 0.18) due to the low number of mutations); for the adjacent graphs, p-values calculated by a chi square test were p &lt; 0.01 in all cases (except again for hA3DE, p = 0.7); similar levels of significance (or even higher) were obtained using the Kruskal Wallis test.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p>Distribution of the nucleotide substitutions in the IAPE-D and HERV-K genomic copies residing in the mouse and human genomes. Endogenous sequences were extracted from the mouse and the human genome databases, aligned and compared to the derived consensus. (A) Upper panels: percentage of substitutions for each nucleotide, for the endogenous IAPE-D and HERV-K elements (with the IAPE-A elements used as a control). Lower panels: two-entry tables showing nucleotide substitutions preferences, with the G-to-A values in bold (higher that the \"non-specific\" C-to-T value for IAPE-D and HERV-K, and identical in the case of the IAPE-A control). n, total number of nucleotides analyzed. (B) Influence of nucleotides at position -2, -1, +1 and +2 on G-to-A mutations (the mutated G is at position 0). Data represent the percentage of indicated target di- or trinucleotide sequences bearing G-to-A mutations. X represents any nucleotide. P-values calculated for the two-entry tables (in A) by a Poisson regression in a log-linear model for the occurrence of the G-to-A versus C-to-T mutations yielded p &lt; 0.003 for IAPE-D and HERV-K; in B, p-values calculated by a chi square test were p &lt; 0.001; similar levels of significance were obtained using the Kruskal Wallis test. (C) Example of G-to-A mutations present in twenty IAPE-D (upper panel) and HERV-K (lower panel) sequences. GXA trinucleotides and GG dinucleotides are underlined in the consensus sequence of IAPE-D and HERV-K, respectively.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p>Variability of the number of G-to-A mutations within the endogenous IAPE-D and HERV-K proviruses depending on the element. The total numbers of G-to-A mutations (plain + hatched grey bars) for each IAPE-D (A) and HERV-K (B) proviruses are represented, together with that of the C-to-T (black bars) \"none-strand-specific\" mutations, given as an internal control (also used for ordering the elements) indicative of the genetic drift-associated age-dependent amount of mutations for each copy (same rank order as the sum of all non-G-to-A base substitutions). The number of G-to-A mutations specifically associated with the mA3 or hA3G APOBEC footprints is indicated with hatched grey. (C) Positioned G-to-A mutations (red bars) specifically associated with the hA3G APOBEC footprint (\"GG\") for the individual HERV-K elements in (B); yellow bars correspond to deletions in the proviruses (relative to the Phoenix consensus sequence). The data and the image shown in figure 4C were generated using the Hypermut 2.0 software available at the <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.hiv.lanl.gov/\"/> website.</p></caption></fig>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>table 1. localization of the analyzed sequences within the mouse and human genomes.</p></caption></supplementary-material>" ]

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[ "<title>Background</title>", "<p>Chronic obstructive pulmonary disease (COPD) is a growing burden to patients and the National Health Service. Disease progression is preventable by early detection combined with smoking cessation [##REF##7966841##1##,##REF##871704##2##]. The annual costs are estimated at £1,639 per patient, 54% of this is for unscheduled care relating to exacerbations [##REF##12647945##3##]. In primary care, earlier diagnosis and the use of interventions aimed at preventing exacerbations and delaying the progression of the disease may be the best way to tackle these costs [##REF##16953994##4##].</p>", "<p>The guidelines of the National Institute for Health and Clinical Excellence (NICE) on COPD diagnosis and management [##REF##15041752##5##] are clear and comprehensive. However, major problems regarding the practical implementation of these guidelines have been identified that may affect the accuracy of COPD diagnosis and treatment.</p>", "<p>Spirometry is a fundamental component of COPD diagnosis [##REF##15041752##5##,##UREF##0##6##], and can be performed accurately in primary care [##REF##14514938##7##]. However, some studies have found major problems with existing spirometry [##REF##10453871##8##, ####UREF##1##9##, ##REF##16701691##10##, ##REF##17761057##11####17761057##11##]. Up to one third of practices had no spirometer, in addition to which practice nurses lacked training and support in performing spirometry and interpreting the results [##UREF##1##9##].</p>", "<p>At present, neither the NICE nor the GOLD guidelines recommend reversibility testing in the diagnosis of COPD. Post-bronchodilator spirometry is necessary for diagnosis of COPD according to the GOLD guidelines [##UREF##0##6##], but not the NICE guidelines [##REF##15041752##5##].</p>", "<p>Reversibility testing using bronchodilators affects the frequency of diagnoses of COPD [##REF##16085729##12##,##UREF##2##13##]. Prevalence of COPD was 27% lower using post-bronchodilator spirometry compared to spirometry without bronchodilator [##REF##16085729##12##].</p>", "<p>Reversibility testing may also influence treatment decisions. For example, based on a study that used post-bronchodilator readings [##REF##10807619##14##], inhaled steroids are recommended for patients who have a forced expiratory volume in one second (FEV1) ≤ 50% predicted and two or more exacerbations per year [##REF##15041752##5##]. Thus, failure to use bronchodilators may cause some patients to be given treatment they do not need.</p>", "<p>There is widespread evidence of poor adherence to guidelines in the management of COPD [##REF##14533285##15##, ####REF##16953997##16##, ##REF##17240616##17##, ##REF##15863417##18##, ##REF##15481268##19##, ##REF##16882107##20####16882107##20##]. Studies in primary care have demonstrated problems with provision of most treatments including medications, vaccination, smoking cessation advice and referral to pulmonary rehabilitation. For example, 25% of general practitioners (GPs) systematically prescribed inhaled corticosteroids to patients with severe COPD, but nearly half of them were unaware of the guideline-based indication for steroids [##REF##15481268##19##]. In the United Kingdom, pulmonary rehabilitation is available to only 2% of those who need it [##UREF##3##21##]. Provision of advice on smoking cessation is also poor. Hyland et al. [##REF##16524709##22##] reported that more than one third of current smokers with COPD had not been offered help with smoking cessation such as referral to a smoking cessation clinic or pharmacological therapy.</p>", "<p>At present, the quality of diagnosis, assessment and management of COPD is variable and does not always relate to the available knowledge about the disease and the burden it represents. The aims of this study were to assess the diagnostic accuracy of COPD registers in general practice and to evaluate guideline adherence in the treatment of stable COPD.</p>" ]

[ "<title>Methods</title>", "<title>Recruitment procedure</title>", "<p>All practices in the Plymouth area were invited to take part. The first 13 practices to respond were included. Three North Devon practices agreed to participate.</p>", "<p>Project nurses (Respiratory Specialist Nurses trained and experienced in primary care management of COPD including spirometry) collected data from February 2005 to March 2006. Practice registers were electronically searched using diagnostic codes for COPD. Exclusion criteria were: serious co-morbidity affecting the patient's ability to take part or to perform spirometry, or inability to attend the surgery. Records of all patients on the COPD register were examined and those with coding errors or normal spirometry were excluded. The remaining suitable patients were invited to an appointment with the project nurse.</p>", "<title>Audit procedure</title>", "<p>The South West Multicentre Research Ethics Committee confirmed that as a service evaluation, formal research ethics approval was not required for the audit. Patients were informed about the study and confidentiality issues. Patient consent was obtained to collect and analyse the data using an electronic consent form approved by the NHS information security and registration authority.</p>", "<p>Patient assessment was based on a custom written software package. Demographic and clinical data were entered by the project nurse (Table ##TAB##0##1##) and questionnaire data were entered by the patients. Spirometry was performed without bronchodilator therapy in all patients according to European Respiratory Society and American Thoracic Society standards [##REF##15219010##23##]. The spirometers used were a MicroLab ML3500 (Plymouth) and a MicroLab ML3300 (North Devon). Reversibility testing was performed using 400–800 mcg salbutamol via a large volume spacer, with spirometry repeated after 15 minutes. In Plymouth, reversibility testing was carried out only if clinically indicated to separate asthma from COPD; in North Devon, reversibility testing was performed on all patients.</p>", "<p>After clinical assessments, patients completed on-screen questionnaires. One question was seen at a time, and each possible response was numbered. Patients selected their response and pressed the appropriate number on the keyboard.</p>", "<p>Questionnaires included the Medical Research Council (MRC) Dyspnoea Scale [##REF##13823475##24##], the Clinical COPD Questionnaire (CCQ) [##REF##12773199##25##] and the Lung Information Needs Questionnaire (LINQ) [##REF##16524709##22##].</p>", "<p>Patients were also asked about their sputum production, (in order to assess the need for mucolytic therapy), and whether their symptoms were relieved by short-acting bronchodilators, (to help assess whether they were receiving adequate bronchodilator treatment).</p>", "<p>Following completion of clinical assessments and questionnaires, the program automatically determined the patient's recommended treatment according to the NICE guidelines. For example, inhaled steroids were recommended if FEV1 was less than 50% predicted and if the patient had had two or more exacerbations during the previous year. Recommendations for bronchodilator treatment were based on the nurse's clinical judgement and whether their current therapy relieved their symptoms rather than computer logic. Mucolytic therapy was recommended to be considered if the patient had a chronic productive cough.</p>", "<p>At the end of the assessment, the nurse reviewed the collected data and confirmed the diagnosis. The computer then produced two separate reports summarising the results of the assessments; a clinical report for the GP or practice nurse and a report in layman's terms for the patient.</p>", "<title>The COPD assessment software</title>", "<p>The software was revised and improved during the process of the audit in accordance with feedback from patients, project nurses and primary care clinicians. Revisions included requesting data on treatment according to guidelines, shortening GP reports, and simplifying patient reports. As a result of this, there are varying numbers of patients in the statistical analyses.</p>", "<title>Diagnostic criteria</title>", "<p>Spirometric results were interpreted according to the NICE recommendations [##REF##15041752##5##]. If reversibility testing was applied, diagnosis was based on post-bronchodilator values. A diagnosis of restriction was given if the FEV1 (% predicted) was less than 80% and the ratio of FEV1 to forced vital capacity (FVC) was greater or equal to 0.7.</p>", "<p>Asthma was diagnosed on the basis of both spirometric and clinical features such as the patient's history and family history which were obtained from the patient and examination of their primary care records. Asthma was confirmed if a patient with airflow obstruction returned to normal spirometric values or if a large change in FEV1 (&gt; 400 ml) was observed in response to bronchodilators. Some patients had clinical features of both asthma and COPD as described in the NICE guidelines.</p>", "<title>Data analysis</title>", "<p>Data was transferred into SPSS (Version 14.0, SPSS Inc., Chicago IL). Descriptive statistics and cross-tables were used to evaluate the accuracy of diagnostic registers and guideline adherence in treatment. If applicable, t tests or chi square tests were used to analyse between-group differences (α = 0.05).</p>" ]

[ "<title>Results</title>", "<title>Characteristics of invited and excluded patients</title>", "<p>Of the 841 patients invited for assessment, 619 were seen. Thirty-nine patients (6%) were unable to perform spirometry.</p>", "<p>To examine the possibility of selection bias, a sub-sample of 288 patients on the COPD register were examined in more detail. Of these patients, 47 (16%) were excluded as they were unable to attend the practice; the remaining 241 patients were invited. Of these, 43 (18%) patients declined. No differences in age and gender were seen between those who attended and those who declined (Table ##TAB##1##2##).</p>", "<title>Accuracy of diagnostic registers</title>", "<p>Of the 580 patients who underwent spirometry, 88 (15%) patients had normal values, 456 (79%) showed obstruction, and 36 (6%) showed restriction.</p>", "<p>Final diagnoses based on spirometric and clinical features are shown in Figure ##FIG##0##1##. Of 580 patients who had spirometry, 158 (27%) were found not to have COPD, as per the NICE guidelines. Of all 422 patients who received the final diagnosis of COPD, 25 patients (6%) had both asthma and COPD.</p>", "<title>Reversibility testing in patients with airflow obstruction</title>", "<p>Airway obstruction was found in 456 patients and reversibility testing was undertaken in 232 (51%). In 140 patients reversibility testing was performed routinely (North Devon patients) and in 92 patients as clinically indicated to exclude asthma from COPD (Plymouth patients).</p>", "<p>Reversibility testing led to a change in diagnosis for 25/232 (11%) patients. Where reversibility testing was performed routinely, the diagnosis changed in 7/140 (5%), with five being changed from a diagnosis of obstruction to normal, and two changing from a diagnosis of obstruction to one of restriction. Where reversibility testing was performed as clinically indicated, diagnostic changes occurred in 18/92 (20%) patients, with 14 changing from a diagnosis of obstruction to normal, and four changing from a diagnosis of obstruction to restriction.</p>", "<p>As regards the magnitude of change in FEV1, 162 patients (70%) changed by up to 200 ml, 58 patients (25%) changed by 200–400 ml, and in 12 patients (5%) the change was larger than 400 ml.</p>", "<p>In patients who showed a small change of FEV1 (up to 200 ml), diagnosis was changed in 8/162 (5%). In patients with a change up to 400 ml, diagnosis was changed in 12/58 (21%), and in those with a change of more than 400 ml, diagnosis was changed in 5/12 (42%). In the large majority of cases the diagnosis was changed from COPD to asthma. Five of the 12 patients with more than 400 ml changes in FEV1 through reversibility testing met the criteria for asthma based on post bronchodilator readings alone.</p>", "<p>The severity grading of airflow obstruction based on pre-bronchodilator readings changed after bronchodilator in 41/232 (18%) patients. Thirty-one patients changed from moderate to mild disease, ten from severe to moderate. Three patients had lower spirometry readings after bronchodilators and in these cases their pre-bronchodilator readings were used to classify severity grading.</p>", "<title>Characteristics of patients with confirmed COPD</title>", "<p>The cohort of 422 patients with confirmed COPD was examined further. The mean FEV1 was 1.25 (SD 0.44) litres and mean FEV1% predicted was 50.3% (SD 14.3). Based on the NICE guidelines, 227 (54%) patients had mild COPD, 159 (38%) patients had moderate COPD, and 36 (9%) patients had severe COPD.</p>", "<p>The mean (SD) age was 69.2 (8.7) years. Body mass index was low (&lt; 18.5) in 26/420 (6%); 32% were normal and 62% were overweight (BMI &gt; 25).</p>", "<p>One hundred and thirty-seven patients (33%) were current smokers with a mean consumption of 20 cigarettes per day and an exposure of 48 pack years; 276 (65%) were ex-smokers with a mean exposure of 43 pack years and ten patients (2%) had never smoked. Of the current smokers, 76% had been offered help to quit smoking, either with nicotine gum or patches, or by referral to a smoking cessation clinic.</p>", "<p>Exacerbations occurred in all grades of airflow obstruction. Patients with severe airflow obstruction had received more steroid courses in the previous year than patients with milder obstruction. Interestingly, healthcare consumption (out of hours visits, accident and emergency attendances, bed days spent in hospital) was not particularly skewed towards patients with severe airflow obstruction (Table ##TAB##2##3##). Respiratory failure or cor pulmonale was noted in five patients.</p>", "<p>Figure ##FIG##1##2## shows counts of MRC dyspnoea scale scores in the COPD sample, for different degrees of severity. Results for the LINQ and the CCQ will be reported elsewhere.</p>", "<p>In the previous two years, 93/422 (22%) patients had attended a consultant chest physician; 36/422 (9%) patients had seen a COPD specialist nurse and 3/422 (0.7%) had seen a respiratory specialist physiotherapist. In the previous five years, 187/384 (49%) of patients had had a chest x-ray, at time of diagnosis. Patients who had attended a consultant chest physician were similar to those who did not attend in respect of diagnosis and spirometry: mean FEV1% of predicted was 45.7% (SD 14.8) and 48.5% (SD 13.8) respectively (<italic>t </italic>= 1.68, <italic>p </italic>= 0.09), but had higher MRC dyspnoea scores: median 3.1 (IQR 1.3) and 2.7 (IQR 1.50), <italic>p </italic>= 0.002. No differences were noted between those attending and those not attending secondary care were noted with respect of age, smoking status, pack years or in the proportions that were on the recommended treatment with short and long acting anti-cholinergic, long acting beta-2-agonists inhaled steroids or mucolytics therapies.</p>", "<title>Management of COPD and compliance with guidelines</title>", "<p>Data on current or recommended treatment were available for 278 of the 422 patients diagnosed with COPD (Table ##TAB##3##4##).</p>", "<p>Long acting bronchodilators were recommended for more patients than had received them and were therefore under prescribed. The NICE guidelines recommend that mucolytic therapy should be considered in patients with a chronic cough productive of sputum. Although 53% had a chronic productive cough, only 4% of these were receiving mucolytics. By contrast, 60% of patients were receiving inhaled steroids, of which only 23% met the indication of FEV1, less than 50% of predicted and two or more exacerbations per year [##REF##15041752##5##]. Nine patients for whom inhaled steroids would be recommended had not received them, again highlighting the problem of inappropriate prescribing.</p>", "<p>Practices varied substantially in prescribing long acting bronchodilators. The proportion of patients prescribed long acting beta-2-agonists ranged between 23–56% in different practices, and between 9–25% for long acting anticholinergics.</p>", "<p>Prescribing outside of licensed indications was noted in six patients who were taking short acting anticholinergics and long acting anticholinergics therapy at the same time. Although long acting anticholinergics are only licensed for use in COPD, six patients who were found not to have COPD were on long acting anticholinergics.</p>", "<p>Vaccination status was recorded for immunisation against pneumococcus and influenza, both of which are recommended in COPD. Influenza vaccine was up to date in 346/398 (87%) and pneumococcal vaccination was up to date in 227/380 (60%).</p>", "<p>Data on attendance or recommendation to attend pulmonary rehabilitation were available for 372 COPD patients. Recommendations were based on the NICE guidelines which state that \"Pulmonary rehabilitation should be offered to all those who consider themselves functionally disabled by COPD (usually MRC dyspnoea scale score of three and above)\" [##REF##15041752##5##].</p>", "<p>Pulmonary rehabilitation was not available in North Devon, but 54/134 (40%) North Devon patients had an MRC dyspnoea scale score of three or more. None of these patients had attended pulmonary rehabilitation. In Plymouth, 121/238 (51%) had MRC dyspnoea scale score of three or more and 80/238 patients (34%) were willing, suitable and able to take part. Of these, five had an MRCDS score of less than three and 7/80 (9%) had attended rehabilitation.</p>" ]

[ "<title>Discussion</title>", "<p>COPD is an important disease that compromises patients' quality of life and creates a huge financial burden to the National Health Service. Our study confirms that disease management takes place largely in primary care. Guidelines suggest that an accurate diagnosis should be followed by effective treatments of stable disease and exacerbations of COPD.</p>", "<p>Diagnostic registers have inherent problems. According to the present findings, registers include large numbers without COPD. In this study, 27% of individuals listed as having COPD were eligible for reclassification of their disease following structured clinical assessment by a trained nurse. These findings confirm the problems previously observed with application and interpretation of spirometry in primary care.</p>", "<p>The application of reversibility testing is a controversial issue in the diagnosis of COPD in primary care [##REF##16375199##26##]. Recent guidelines have suggested that reversibility testing should be used where clinically indicated to separate asthma from COPD [##REF##15041752##5##,##UREF##0##6##], but there is a lack of good evidence to underpin these recommendations [##REF##16375199##26##]. Reversibility testing records the change in FEV1 before and after bronchodilators and the magnitude of this change has been used to differentiate asthma from COPD [##REF##15041752##5##,##UREF##4##27##].</p>", "<p>Reversibility testing was found to change the diagnosis from that made on pre-bronchodilator spirometry in 11% of cases. This finding demonstrates the benefits of judicious use of reversibility testing based on clinical need to exclude asthma as opposed to performing reversibility testing in all cases. This study confirms that performing pre-bronchodilator spirometry alone leads to overestimation of the prevalence and severity of COPD, with the potential to cause errors in treatment. The use of pre-bronchodilator spirometry alone cannot be recommended for diagnosis and severity assessment-spirometry in primary care should be done only after administration of bronchodilators. This approach is in keeping with the current GOLD guidelines, whereas the NICE guidelines do not specify the importance of post-bronchodilator readings.</p>", "<p>This study examined current treatment against treatment as recommended by the NICE guidelines. Under-prescribing with bronchodilators, particularly long acting agents was apparent. Long acting bronchodilators are known to improve lung function, exercise tolerance, symptoms, and quality of life, and to reduce exacerbations [##UREF##0##6##]. Furthermore, it is known that bronchodilators interact with other treatments to improve outcomes. The combination of fluticasone and salmeterol reduced decline in lung function over 3 years, [##REF##17314337##28##] and outcomes in pulmonary rehabilitation were improved by tiotropium [##REF##15764761##29##]. The reason why these drugs are not prescribed may be due to concerns over price, a lack of knowledge about the benefits of these medicines [##REF##15481268##19##] or from an unjustified nihilistic approach to COPD management [##REF##16493152##30##]. There was no evidence that this phenomenon was limited to primary care, those attending a consultant chest physician in the previous five years were no more likely to be treated according to guidelines.</p>", "<p>This study highlights an apparent over-treatment with inhaled steroids. Only a minority of those who received inhaled steroids met the NICE criteria. In some cases, treatment with steroids may once have been not only appropriate, but also effective in reducing exacerbations until they were no longer appeared necessary. Over-treatment suggests a waste of resources and puts patients at risk of adverse effects. These findings concur with Decramer et al. [##REF##14533285##15##], who revealed that 49% of GPs prescribed inhaled steroids to all of their COPD patients. The role of mucolytics is debated [##REF##15866309##31##], but despite the evidence-based recommendations in NICE that mucolytics should be considered in patients with a chronic productive cough, mucolytics are seldom prescribed to such patients.</p>", "<p>Quitting smoking improves patients' prognoses in COPD, and offering support to stop smoking reduces mortality and morbidity from COPD [##REF##7966841##1##]. It is disappointing that a quarter of current smokers with COPD had not been offered practical help in terms of referral to smoking cessation service or drug therapy. While patients may not accurately report the help they have been offered, Rutschmann et al. [##REF##15481268##19##] found that many physicians reported feeling uncomfortable giving smoking cessation advice, and a fifth of physicians were unaware that smoking cessation had a positive impact on life expectancy and disease progression.</p>", "<p>Pulmonary rehabilitation is highly effective and is a cost effective intervention recommended by national and international COPD guidelines. The finding in this study was striking-in certain geographic regions pulmonary rehabilitation was not available at all and where it was available, only 9% of eligible patients had attended. Identification of patients suitable for rehabilitation is an important component of COPD assessment, as currently the proven benefits of pulmonary rehabilitation are being denied to many patients.</p>", "<p>This study used a novel information technology system that provides a systematic and comprehensive assessment package for COPD, and facilitates the delivery of high-quality care according to guidelines. The system assesses clinical data and examines patient-centred outcomes such as quality of life and patients' perceived information needs. By providing reports to both GPs and patients it offers a new approach to COPD management in primary care. Management according to the guidelines is promoted with the potential to improve care. The system also has an educational function as GPs and nurses are given the opportunity to learn about COPD management. Given these advantages, use of the system has the potential to enable long-term improvement of COPD management. However, using a different IT-system for guideline-based management of COPD and asthma, Tierney et al. [##REF##15762903##32##] reported no enhanced adherence to guidelines and no beneficial effects on patient-centred and clinical outcomes over a year. Research into the benefits of our system in terms of long-term improvement of clinical care and patient-centred outcomes is ongoing.</p>", "<p>This study has some limitations. Although the participating practices provided a spectrum of COPD services and varied widely in their size and socio-demographic features, they may not representative as they were all in Devon and were included on the basis of their interest in the project. Furthermore, as only patients who were able to attend primary care clinics were included, those most severely affected by their COPD and those with important co-morbidities may have been under-represented.</p>", "<p>The software system made recommendations for specific treatments based on guidelines and some recommendations were not definite statements that the treatment was required, but that the treatment should be considered, (for example mucolytics therapy). Thus the apparent under-prescribing in these situations is more difficult to interpret and may be appropriate use of therapy. A further limitation was that this was a cross-sectional study and did not assess the impact of recommendations on changing treatment or other outcomes. The actual decision by the GP to prescribe, the patients' compliance, response to treatment and duration were outside the scope of the study. The software system had not been formally validated in other studies.</p>" ]

[ "<title>Conclusion</title>", "<p>The present study represents one of the first studies to report the true levels of severity of COPD and the provision of appropriate treatments in primary care. The study demonstrates that compared to diagnoses made by expert nurses with the help of a standardised, guideline-based computer program, the diagnostic registers in primary care are inaccurate in 27% of cases. The role of reversibility testing was examined and it was found that pre-bronchodilator readings alone overestimated both prevalence and the severity of COPD. Reversibility testing was useful in detecting some cases where a diagnosis of asthma was considered. On this basis it is suggested that only post bronchodilator readings should be used routinely in primary care, but reversibility testing is appropriate in specific cases where asthma is suspected. The management of patients with COPD seldom followed guideline based recommendations in terms of drug treatment and pulmonary rehabilitation.</p>", "<p>In the future, computer-based assessment systems which provide management recommendations may facilitate optimal diagnosis and treatment for patients with COPD in primary care.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Guidelines on COPD diagnosis and management encourage primary care physicians to detect the disease at an early stage and to treat patients according to their condition and needs. Problems in guideline implementation include difficulties in diagnosis, using spirometry and the disputed role of reversibility testing. These lead to inaccurate diagnostic registers and inadequacy of administered treatments. This study represents an audit of COPD diagnosis and management in primary care practices in Devon.</p>", "<title>Methods</title>", "<p>Six hundred and thirty two patients on COPD registers in primary care practices were seen by a visiting Respiratory Specialist Nurse. Diagnoses were made according to the NICE guidelines. Reversibility testing was carried out either routinely or based on clinical indication in two sub-samples. Dyspnoea was assessed. Data were entered into a novel IT-based software which computed guideline-based treatment recommendations. Current and recommended treatments were compared.</p>", "<title>Results</title>", "<p>Five hundred and eighty patients had spirometry. Diagnoses of COPD were confirmed in 422 patients (73%). Thirty nine patients were identified as asthma only, 94 had normal spirometry, 23 were restrictive and 2 had a cardiac disorder. Reversibility testing changed diagnosis of 11% of patients with airflow obstruction, and severity grading in 18%. Three quarters of patients with COPD had been offered practical help with smoking cessation. Short and long-acting anticholinergics and long acting beta-2 agonists had been under-prescribed; in 15–18% of patients they were indicated but not received. Inhaled steroids had been over-prescribed (recommended in 17%; taken by 60%), whereas only 4% of patients with a chronic productive cough were receiving mucolytics. Pulmonary rehabilitation was not available in some areas and was under-used in other areas.</p>", "<title>Conclusion</title>", "<p>Diagnostic registers of COPD in primary care contain mistakes leading to inaccurate prevalence estimates and inappropriate treatment decisions. Use of pre-bronchodilator readings for diagnosis overestimates the prevalence and severity in a significant minority, thus post bronchodilator readings should be used. Management of stable COPD does often not correspond to guidelines. The IT system used in this study has the potential to improve diagnosis and management of COPD in primary care.</p>" ]

[ "<title>Competing interests</title>", "<p>Dr Jones has received educational, research or travel grants from Glaxo Smith Kline; Astra Zeneca; Boehringer-Ingelheim; Pfizer; Ivax; Novartis and Altana. Dr Jones was a director of Patient Centred Software Ltd., the provider of the software used in this project.</p>", "<title>Authors' contributions</title>", "<p>RJ and BS designed and supervised the study and contributed to the manuscript. MS analysed data and drafted the manuscript. MM recruited North Devon practices and carried out assessments in North Devon. DM acted as the project nurse and contributed to data preparation. All authors have read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>The authors wish to express their gratitude to David Wade and colleagues who designed the software, Rowan Russell who contributed to data preparation, Janet Comyn for her help in collating the data, and Professor John Campbell for his advice on the manuscript. We also thank the patients and staff of participating practices.</p>", "<p>The study was funded by contributions from Boehringer Ingelheim, Glaxo Smith Kline and Astra Zeneca.</p>", "<p>Dr Jones is in receipt of a Department of Health Primary Care Researcher Development Award.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Final diagnoses in the Plymouth COPD audit sample.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p>Distribution of MRC dyspnoea score for different degrees of severity of airflow obstruction.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Data gathered by the COPD assessment software (questionnaires not shown)</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\">Demographic data</td></tr><tr><td align=\"left\">Weight, height and body mass index (BMI)</td></tr><tr><td align=\"left\">Spirometry results: FEV1 and FVC (litres and % of predicted) pre and post bronchodilator</td></tr><tr><td align=\"left\">Number of exacerbations in previous year</td></tr><tr><td align=\"left\">Number of antibiotics for respiratory tract infections in the previous 12 months</td></tr><tr><td align=\"left\">Number of oral steroid courses in the previous 12 months</td></tr><tr><td align=\"left\">Number of out of hours visits in the previous 12 months</td></tr><tr><td align=\"left\">Number of attendances of Accident and Emergency (A &amp; E) in the previous 12 months</td></tr><tr><td align=\"left\">Number of hospital admissions in the previous 12 months</td></tr><tr><td align=\"left\">Number of bed days in the previous 12 months</td></tr><tr><td align=\"left\">Whether patient had an x-ray at the time of diagnosis or in the previous 5 years</td></tr><tr><td align=\"left\">Vaccination status (pneumococcus and influenza)</td></tr><tr><td align=\"left\">Current COPD medication: short and long acting bronchodilators, inhaled corticosteroids, mucolytics, other prescribed medications</td></tr><tr><td align=\"left\">Inhaler technique: good, moderate, poor</td></tr><tr><td align=\"left\">Use of nebuliser</td></tr><tr><td align=\"left\">Smoking history: age smoking started, date of cessation, average number of cigarettes per day</td></tr><tr><td align=\"left\">Whether patient had undergone smoking cessation treatment</td></tr><tr><td align=\"left\">Oxygen assessment and therapy, cor pulmonale, cyanosis</td></tr><tr><td align=\"left\">Pulse oximetry value</td></tr><tr><td align=\"left\">Attendance of specialist services: respiratory specialist nurse/physiotherapy in the previous 2 years, chest clinic in the previous 5 years, pulmonary rehabilitation ever</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Age and gender of North Devon patients who declined and who were seen</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">Declined</td><td align=\"left\">Attended</td><td align=\"left\">Significance</td></tr></thead><tbody><tr><td align=\"left\">Age</td><td align=\"left\">66.4 (11.3)</td><td align=\"left\">68.1 (10.0)</td><td align=\"left\">t(241) = -0.997, p = 0.320</td></tr><tr><td align=\"left\">Gender (Males)</td><td align=\"left\">18/43 (42%)</td><td align=\"left\">127/198 (64%)</td><td align=\"left\">X²(1) = 0.27, p = 0.603</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Frequency of exacerbations, antibiotic and steroid courses and healthcare consumption in patients with confirmed COPD</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\">Number in previous year of:</td><td align=\"left\">Mild (N = 226)</td><td align=\"left\">Moderate (N = 158)</td><td align=\"left\">Severe (N = 36)</td><td align=\"left\">All (N = 420)</td></tr></thead><tbody><tr><td align=\"left\">Exacerbations</td><td align=\"left\">1.3 (1.7)</td><td align=\"left\">1.3 (1.6)</td><td align=\"left\">1.7 (2.3)</td><td align=\"left\">1.4 (1.8)</td></tr><tr><td align=\"left\">Antibiotics courses</td><td align=\"left\">1.2 (1.6)</td><td align=\"left\">1.3 (1.6)</td><td align=\"left\">1.7 (2.3)</td><td align=\"left\">1.3 (1.6)</td></tr><tr><td align=\"left\">Steroid courses</td><td align=\"left\">0.7 (1.3)</td><td align=\"left\">0.7 (1.4)</td><td align=\"left\">1.4 (2.2)</td><td align=\"left\">0.8 (1.4)</td></tr><tr><td align=\"left\">Out of hours visits</td><td align=\"left\">0.1 (0.4)</td><td align=\"left\">0.2 (0.5)</td><td align=\"left\">0.2 (0.8)</td><td align=\"left\">0.1 (0.5)</td></tr><tr><td align=\"left\">Attended A&amp;E</td><td align=\"left\">0.1 (1.1)</td><td align=\"left\">0.1 (0.5)</td><td align=\"left\">0.2 (0.5)</td><td align=\"left\">0.1 (0.9)</td></tr><tr><td align=\"left\">Bed days</td><td align=\"left\">0.5 (3.0)</td><td align=\"left\">0.8 (4.0)</td><td align=\"left\">0.7 (2.4)</td><td align=\"left\">0.6 (3.3)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Current and recommended treatment in patients with confirmed COPD and proportion of patients in whom a treatment was recommended who were receiving that treatment</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"left\">No. currently receiving <break/>the treatment</td><td align=\"left\">No. for whom the treatment <break/>was recommended</td><td align=\"left\">No. receiving the treatment <break/>recommended for them</td></tr><tr><td/><td align=\"left\">(N = 278)</td><td align=\"left\">(N = 278)</td><td align=\"left\">(N = 278)</td></tr></thead><tbody><tr><td align=\"left\">SAAC</td><td align=\"left\">124 (44.6%)</td><td align=\"left\">109 (39.2%)</td><td align=\"left\">59/109 (54%)</td></tr><tr><td align=\"left\">LAB2 agonists</td><td align=\"left\">124 (44.6%)</td><td align=\"left\">80 (28.8%)</td><td align=\"left\">37/80 (46%)</td></tr><tr><td align=\"left\">LAAC</td><td align=\"left\">50 (18.0%)</td><td align=\"left\">53 (19.1%)</td><td align=\"left\">12/53 (23%)</td></tr><tr><td align=\"left\">Inhaled steroids</td><td align=\"left\">167 (60.0%)</td><td align=\"left\">48 (17.0%)</td><td align=\"left\">39/48 (81%)</td></tr><tr><td align=\"left\">Mucolytics</td><td align=\"left\">9 (3%)</td><td align=\"left\">144 (53.0%)</td><td align=\"left\">6/144 (4%)</td></tr></tbody></table></table-wrap>" ]

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[ "<graphic xlink:href=\"1465-9921-9-62-1\"/>", "<graphic xlink:href=\"1465-9921-9-62-2\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>Pax2 is a member of the Pax family of transcription factors [##REF##1977574##1##,##REF##1311084##2##], characterised by the presence of a paired-type homeodomain [##REF##8939674##3##,##REF##7893124##4##]. <italic>Pax2 </italic>is expressed in a number of different organs in the developing mouse embryo, including the ureteric bud, kidneys [##REF##1977574##1##,##REF##1311084##2##] and otic vesicle [##REF##1977575##5##]. In the developing nervous system, <italic>Pax2 </italic>is first detected at embryonic day (E) 7.5 in the neural plate, in the area of the presumptive midbrain-hindbrain region [##REF##7577673##6##]. At E8.0, <italic>Pax2 </italic>displays a broad expression domain in this region, which by E9.5 is restricted to the isthmus at the midbrain-hindbrain boundary [##REF##7577673##6##]. Pax2 expression in the isthmus ceases after E11 [##REF##1457381##7##]. In the cerebellum, Pax2 is specifically expressed by a subset of cerebellar GABAergic interneurons and their precursors, from E12 until the end of cerebellar development (postnatal day 15) [##REF##10512984##8##]. In the spinal cord, Pax2 expression is found in the intermediate zone as early as E10.5 [##REF##1977575##5##,##REF##1457381##7##]. In the developing eye,<italic>Pax2 </italic>expression is initiated at E9 in the ventral half of the optic vesicle. By E11, after invagination of the optic vesicle, both Pax2 transcript and protein are detected at high levels in the ventral opening of the optic cup, the optic fissure, and the optic stalk, ending at the border with the diencephalon [##REF##1977575##5##,##REF##1457381##7##,##REF##9473325##9##,##REF##8951055##10##]. After E12.5, Pax2 protein expression is still present in the ventral optic cup, although the level is decreased, and it is no longer detected after E16.5 [##REF##9473325##9##]. Pax2 is also expressed in glial cells in the optic nerve [##REF##1977575##5##,##REF##9473325##9##,##REF##8951055##10##].</p>", "<p>Pax6, another member of the Pax family, is expressed in the dorsal telencephalon, diencephalon, hindbrain regions and spinal cord [##REF##8126546##11##] and throughout the developing optic cup, but is absent from the optic stalk and optic nerve [##REF##7789273##12##]. Mutant mice lacking functional Pax6 protein (<italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mice), display a large range of nervous system defects, including absence of eyes [##REF##1684639##13##], disruption of dorsoventral telencephalic patterning [##REF##8951061##14##,##REF##11050125##15##] and the diencephalic-mesencephalic boundary [##REF##9169845##16##,##REF##9108373##17##]. Pax6 and Pax2 are expressed in neighbouring, but mutually exclusive domains in the developing eye (with the exception of the ventral optic cup), [##REF##12100888##18##], and in the diencephalic-mesencephalic region [##REF##10354469##19##]. In the developing spinal cord, Pax2 is expressed by many types of early differentiated neurons, located in the mantle zone and surrounding Pax6-positive neural precursors in the ventricular zone [##REF##9409667##20##]. Fewer Pax2-positive interneurons are found in the <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutant, indicating that Pax6 is required for their development [##REF##9409667##20##].</p>", "<p>We have recently described a group of diencephalic cells at the border region between the diencephalon and the telencephalon that expresses Pax2 protein at E10.5 [##REF##16957084##21##]. At E12.5, these Pax2-immunopositive cells form a distinct cell population at the most dorso-lateral tip of the eminentia thalami [##REF##16957084##21##], a diencephalic structure that joins the ventral diencephalon to the dorsal and ventral telencephalon [##REF##4540788##22##, ####REF##7939711##23##, ##REF##10906711##24####10906711##24##]. Here, we describe previously unidentified areas of Pax2 expression in the developing mouse forebrain. Further, we show that Pax2 expression is maintained in the septum of <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants, which are known to have an enlarged septum. This study highlights the value of Pax2 as a novel marker of forebrain development.</p>" ]

[ "<title>Methods</title>", "<title>Mice</title>", "<p>Animal care was in accordance with institutional guidelines and UK Home Office regulations. The day the vaginal plug was detected was considered E0.5. Wild type mice on a CBA genetic background were used for the Pax2 expression pattern analysis. <italic>Pax6</italic>^{<italic>Sey</italic>/+}heterozygotes, kept on a mixed CD1-Swiss genetic background, were intercrossed to generate homozygous null embryos. These were identified by the absence of eyes, as previously described [##REF##1684639##13##]. Wild type mice from the same genetic background were collected for comparison by intercrossing wild types.</p>", "<title>Histology</title>", "<p>Embryos were harvested between E10.5 and E16.5 and pups between P1 and P15. Whole embryos or heads of P1 were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4°C. P7 and P15 pups were anesthetized with Avertin and perfused through the heart with fixative, followed by tissue dissection and overnight incubation in fresh fix. Embryonic and postnatal tissue were all processed following standard conditions [##REF##16957084##21##], embedded in paraffin and cut in serial 10 μm sections (embryonic tissue) or 12.5 μm (postnatal brains) at a coronal or sagittal plane. At least two wild type embryonic heads or postnatal brains for each age were used, and six <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants with the corresponding wild types were examined with each marker.</p>", "<title>Immunohistochemistry and Immunofluorescence</title>", "<p>These were performed according to standard protocols. Antigen retrieval was achieved by microwaving sections at full power in 10 mM citrate buffer, pH 6.0 for four times, 5 min each. Primary antibodies were rabbit anti-Pax2 (Covance, 1:200), rabbit anti-calbindin (Swant, 1:500), monoclonal mouse anti-Pax6, Lim1/2 (DSHB, both 1:100) and nestin (DSHB, 1:50), and goat anti-choline acetyltransferase (Millipore – AB144P, 1:50). Secondary non-fluorescence antibodies were biotylinated anti-rabbit IgG (Dako, 1:200), biotylinated anti-mouse IgG (Dako, 1:200), biotylinated anti-goat IgG (Dako, 1:100),</p>", "<p>For single immunohistochemistry experiments the dark brown signal was revealed after incubation with the ABC kit (Vector), followed by standard diaminobenzidin (DAB, Sigma) and hydrogen peroxide incubation. For the double immunohistochemistry experiments, nuclear staining was first detected using a DAB-nickel detection kit (Vector) resulting in a grey/black staining. Sections were then incubated with the second antibody and appropriate secondary and the dark brown filamentous signal was revealed after incubation with the ABC kit (Vector), followed by a DAB reaction using the DAB detection kit (Vector). For the double immunofluorescence experiments, Pax6 or Lim1/2 signal were amplified with a biotinylated anti-mouse IgG antibody and signal was revealed after incubating with streptavidin conjugated to Alexa fluor 488 dye (Invitrogen, 1:200). Monoclonal anti-β tubulin isotype III (Sigma – clone SDL.3D10, 1:400) was detected using as secondary antibody an anti-mouse IgG conjugated to Alexa fluor 488 dye (Invitrogen, 1:200). For double immunohistochemistry followed by immunofluorescence, after detection of the first antibody with DAB or DAB-Nickel immunohistochemistry as described above, sections were incubated with a second antibody which was detected by means of immunofluorescence. Polyclonal Pax2 and calbindin were detected using as secondary antibodies an anti-rabbit IgG conjugated to Alexa fluor 488 dye and an anti-rabbit IgG conjugated to Alexa fluor 568 dye, respectively (Invitrogen, 1:200). Appropriate controls were used in all cases by incubating some sections with all but the primary antibodies. No immunostaining occurred under these conditions.</p>", "<title>Microscopy</title>", "<p>A Leica microscope connected to a Leica DFC 480 digital camera was used to capture images of DAB and immunofluorescent labelled sections. Confocal images were captured with a Leica TCS NT confocal microscope.</p>" ]

[ "<title>Results</title>", "<title>Domains of Pax2 expression in the developing mouse forebrain</title>", "<p>We examined Pax2 expression in the early forebrain using immunohistochemistry on sagittal sections of E10.5 and E11.5 embryos (Fig. ##FIG##0##1##). At E10.5, a few Pax2-immunopositive cells were detected within the ventral telencephalon in lateral sagittal sections (Fig. ##FIG##0##1A##, a-arrowheads), with the staining becoming more intense in mid-sagittal sections (Fig. ##FIG##0##1a'##). In addition, a small population of Pax2 immunopositive cells was detected in the neuroepithelium of the anterior hypothalamus, adjacent to the optic recess area (Fig. ##FIG##0##1B,b##). At E11.5, strong Pax2 expression was found in the developing septum (Fig. ##FIG##0##1C,c##) with no Pax2-immunopositive cells detected in the neighbouring lamina terminalis (arrow in Fig. ##FIG##0##1c##). As in E10.5 embryos, a number of Pax2-positive cells were found close to the base of the hypothalamus but also in more dorsal areas of the anterior hypothalamus (Fig. ##FIG##0##1C,c##). At both E10.5 and E11.5, Pax2 expression was found in previously described regions such as the ventral neuroepithelium of the optic recess (Fig. ##FIG##0##1B,b,C,c##) [##REF##1977575##5##,##REF##1457381##7##,##REF##9473325##9##,##REF##8951055##10##], the optic cup (arrowhead in Fig. ##FIG##0##1A## and not shown) [##REF##1977575##5##,##REF##1457381##7##,##REF##9473325##9##,##REF##8951055##10##] and near the diencephalic-telencephalic boundary (asterisk in Fig. ##FIG##0##1A## and not shown), which will give rise to the eminentia thalami [##REF##16957084##21##].</p>", "<p>Pax2 protein expression was then examined at E12.5, on coronal and sagittal sections along the caudo-rostral axis of the developing mouse forebrain (Fig. ##FIG##1##2##). The strongest expression domain of Pax2 was detected in the telencephalon. The Pax2 antibody labelled most cells of the septal neuroepithelium, located in close proximity to the dorso-medial telencephalon (Fig. ##FIG##1##2A,a##). This strong and characteristic Pax2 expression in the septum was also observed in sagittal sections, in the region where the lamina terminalis joins the septum (Fig. ##FIG##1##2D,d##). Only a few Pax2-immunopositive cells were detected within the neighbouring lamina terminalis (arrows in Fig. ##FIG##1##2d##).</p>", "<p>Specific Pax2 expression was also detected in small clusters of cells in regions of the hypothalamus. The Pax2 antibody labelled a group of cells located at the lateral hypothalamic area (Fig. ##FIG##1##2B##, arrowheads in b), and a narrow strip of cells parallel to the anterior hypothalamic ventricular zone (Fig. ##FIG##1##2B##, arrows in b'). A few Pax2-immunopositive cells were also detected along the base of the hypothalamus, excluding the midline region (Fig. ##FIG##1##2b##, arrows). These groups of Pax2-positive cells can also be distinguished in a sagittal plane (Fig. ##FIG##1##2D,d'##). The cell populations indicated by the arrowheads and small arrows (Fig. ##FIG##1##2d'##), located just above the optic recess area, correspond to the respective populations indicated in Fig. ##FIG##1##2b##. The Pax2 positive cells located close to the third ventricle, indicated with large arrows in Fig. ##FIG##1##2d'##, correspond to those shown in Fig. ##FIG##1##2b'##. In coronal sections of the caudal forebrain, specific Pax2 expression was also detected in a small cluster of cells resembling a nucleus in the neuroepithelium of the anterior hypothalamus (Fig. ##FIG##1##2C,c##). In accordance with previous reports, Pax2 expression was also detected in the ventral neuroepithelium of the optic recess area at the base of the hypothalamus (Fig. ##FIG##1##2d'##, asterisk), retina (not shown) [##REF##1977575##5##,##REF##1457381##7##,##REF##9473325##9##,##REF##8951055##10##] and eminentia thalami at the level depicted in Fig. ##FIG##1##2B## (not shown) [##REF##16957084##21##].</p>", "<p>Double immunofluorescence with Pax2 and β-tubulin III (Tuj1), a marker of early neural differentiation found in neurites, revealed that a very low proportion of septal cells labelled with Pax2 co-expressed β-tubulin III (Fig ##FIG##2##3A##), suggesting that the majority of the Pax2-positive cells in this region are neural precursors. This was confirmed by double immunohistochemistry with Pax2 (black, nuclear staining) and nestin (brown, filament staining), an intermediate filament protein found in radial glia, which showed that the β-tubulin III-negative/Pax2-positive cells in the septum express nestin (Fig. ##FIG##2##3B##). In the hypothalamus, most of the Pax2-positive cells found close to the ventricle (panels 2b' and 2c) are also positive for β-tubulin III, showing that these cells are newly formed neurons (Fig. ##FIG##2##3C##). The Pax2-positive population indicated by arrowheads in panels 2b and 2d' also expressed β-tubulin III, even more extensively than the other hypothalamic Pax2-positive cells (Fig. ##FIG##2##3D##). Finally, the Pax2-positive cells located close to the hypothalamic ventral midline (small arrows in panels 2b and 2d') did not express β-tubulin III, but were positive for nestin, indicating that they correspond to neural progenitors (data not shown).</p>", "<p>At E13.5, Pax2 was detected in cells located at the septal midline (Fig. ##FIG##1##2E##). Expression was strong and specifically confined to the septal neuroepithelium, at the point where the septum joins with the future hippocampus via the lamina terminalis (Fig. ##FIG##1##2F##). At more rostral telencephalic levels, a small number of Pax2-immunopositive cells were found in the differentiating field of the septum (Fig. ##FIG##1##2G##).</p>", "<p>Pax2 expression in the hypothalamus at this age was similar to that seen at E12.5, with the exception that the immunopositive cells in the lateral hypothalamic area and the base of the hypothalamus shown in Fig. ##FIG##1##2b##, were no longer detectable (data not shown). No Pax2-immunopositive cells were found in the eminentia thalami after E13.5 (not shown).</p>", "<p>At E14.5, coronal telencephalic sections revealed similar Pax2 expression to that described at E13.5 (data not shown). As for the earlier ages examined, Pax2 expression was mainly confined to the septum, in the neuroepithelium adjacent to the lamina terminalis, as depicted in the mid-sagittal section in Fig. ##FIG##3##4A## and ##FIG##3##4a##. In the E14.5 hypothalamus, Pax2 expression was limited to a very narrow band of cells, localized in the differentiating field of the anterior hypothalamus and reaching the medial horn of the lateral ventricle (Fig. ##FIG##3##4B##, arrows in b). We could not detect these immunopositive cells in the coronal plane, probably because of their narrow field of expression. Expression was also found at the optic recess area as previously described [##REF##9473325##9##], (arrowhead in Fig. ##FIG##3##4A,B##).</p>", "<p>By E16.5, the main Pax2 expression domain in the developing septum was located in the differentiating field of the medial septum. It was mainly found in a stripe-like cell arrangement, parallel to the midline and surrounded by the fibre bundles of the fornix (Fig. ##FIG##3##4D##). At this developmental stage, the only Pax2 expression detected in the hypothalamus was in the optic stalk epithelium, just above the optic chiasm region (Fig. ##FIG##3##4C##), in a few cells of the hypothalamic neuroepithelium, adjacent to the suprachiasmatic nucleus (arrowheads in Fig. ##FIG##3##4C##) and in a few scattered cells in the medial preoptic nucleus.</p>", "<p>During early postnatal development only a few Pax2-positive cells were detected in the forebrain. At postnatal day (P) 1, Pax2 immunopositive cells were detected scattered in the most caudal sections of the septal area, at the level where the fornix is found in proximity to the anterior commissure (Fig. ##FIG##4##5A##). As at E16.5, a few cells were found close to the fornix, in the medial septal area, as well as in the border zone between the medial and lateral septum (Fig. ##FIG##4##5A,B##). Pax2 also labelled the medial preoptic nucleus of the hypothalamus (Fig. ##FIG##4##5B##). At P8, a similar expression pattern was observed (Fig. ##FIG##4##5C, D##). In addition, Pax2 was detected in the subfornical organ (Fig. ##FIG##4##5E##), one of the circumventricular organs of the brain involved in fluid balance [##REF##16394195##25##]. Pax2 expression was no longer detected at P15 (not shown).</p>", "<title>Expression of Pax2 in the septum in <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants</title>", "<p>To validate Pax2 as a marker of the septal neuroepithelium, we used the <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutant, which has been shown to have an enlarged septum [##REF##11050125##15##]. Using double immunofluoresence, we first examined expression of Pax2 and Pax6 in E12.5 wild type embryos. In rostral telencephalic sections, Pax6 was strongly expressed in the dorsal, lateral and ventral pallium, with its most ventral expression domain expanding into the lateral ganglionic eminence, just below the pallial-subpallial boundary (Fig. ##FIG##5##6A##), as previously described [##REF##8126546##11##,##REF##8951061##14##,##REF##10906711##24##]. Pax6 was expressed at lower levels in the medial pallium but did not overlap with the expression domain of Pax2 in the septal neuroepithelium (Fig. ##FIG##5##6A##, arrows).</p>", "<p>Using <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutant embryos, we examined whether loss of Pax6 affects Pax2 expression in the septum. No gross changes in the extent of the main Pax2 expression domain were observed, although the intensity of Pax2 staining appeared reduced in <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants compared to the wild type (area between arrows in Fig. ##FIG##5##6B,C##). In addition, Pax2 expression was observed in a more dorsal area compared to wild type, revealing a larger septum in the mutant (compare areas between dotted lines in Fig. ##FIG##5##6B## and ##FIG##5##6C##), in accordance with previous published data [##REF##11050125##15##]. This result was consistent in all mutants examined (n = 6). Double immunofluorescence with antibodies for Pax2 and the septal marker Lim1 (also known as Lhx1), using an antibody that detects both Lim1 and Lim2 (Lim1/2) [##REF##9880598##26##,##REF##9022063##27##], showed that in both wild types and <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants, Pax2 expression was confined within the Lim1/2 positive domain (Fig. ##FIG##5##6D,E##). This shows that the shifted area of Pax2 expression observed in the <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutant most likely corresponds to ventral telencephalic tissue, and not to ectopic Pax2 expression in the dorsal telencephalon.</p>", "<title>Pax2-positive cells in the early postnatal septum do not express markers of GABA-ergic or cholinergic neurons</title>", "<p>To gain further insight into the neurochemical properties of the differentiated Pax2 cells detected in the septum, we examined co-expression of Pax2 and markers of GABA-ergic and cholinergic neurons, the principal neuronal types of this structure [##REF##8863126##28##, ####REF##3022187##29##, ##REF##3607436##30####3607436##30##].</p>", "<p>To examine whether the Pax2-positive cells detected postnatally might be GABA-ergic we performed double immunostaining experiments with Pax2 and calbindin, a calcium-binding protein that has been shown to label a large population of GABA-ergic somatospiny neurons in the adult septum [##REF##2199841##31##,##REF##1981215##32##]. A large number of neurons were labelled with an antibody for calbindin in the postnatal septum (Fig. ##FIG##6##7B##). However, Pax2-positive cells did not colocalize with calbindin, neither at P1 (not shown) nor at P8 (Fig. ##FIG##6##7A–C##). To examine whether the Pax2 immunolabelled neurons might be cholinergic, we performed double immunostaining experiments with appropriate markers. At P1, we examined co-expression of Pax2 and Islet1, a protein that has been shown to label some populations of cholinergic septal neurons [##REF##18367596##33##,##REF##11301207##34##]. No co-localisation of these two proteins was detected (not shown). Choline acetyltransferase (Chat), the acetylcholine-synthesizing enzyme in cholinergic neurons, can not be detected clearly in septal neurons by means of immunohistochemistry before P8 [##REF##8863126##28##,##REF##3607436##30##]. We examined co-localization of Chat and Pax2 at P8 by means of double immunostaining (immunohistochemistry followed by immunofluorescence). As shown in Fig. ##FIG##6##7##, at P8 a few cells in the septum start expressing Chat but do not express Pax2 (##FIG##6##7D–F##).</p>" ]

[ "<title>Discussion</title>", "<p>Specific markers expressed in different regions of the developing nervous system are widely used as tools to study neural development. Here, the expression of Pax2, a well-studied marker of the isthmus, spinal cord and developing eye, has been re-examined, using a polyclonal antibody, and novel areas of Pax2 protein expression have been identified in the ventral telencephalic septum and the developing hypothalamus. The polyclonal antibody used in the present study recognizes the same epitope described by Dressler and Douglass (1992) and has been previously used by several groups to characterize Pax2 protein distribution [##REF##1311084##2##,##REF##1457381##7##,##REF##9473325##9##]. In this study, the use of paraffin sections subjected to microwaving for antigen retrieval, in contrast to the cryostat sections used previously, may have allowed the identification of the previously undescribed domains of Pax2 expression.</p>", "<p>Pax2 staining in the hypothalamus is first detected at E10.5 and comprises a few cells in the ventricular zone, dorsal to the optic recess. By E11.5, Pax2 positive cells are found in a region that extends from the area of the optic recess to the lateral ventricle that will give rise to the anterior hypothalamus. Comparison of the distribution of Pax2 positive cells in this region at E12.5 and E10.5 suggests that they might follow a migratory path towards dorsal regions of the anterior hypothalamus. By E14.5, fewer Pax2-positive cells are present than at E12.5, and these are restricted to the dorsal anterior hypothalamus. Pax2 is expressed in the optic stalk, a structure that joins the optic cup to the brain, and that ends at the base of the hypothalamus [##REF##1977575##5##,##REF##1457381##7##,##REF##9473325##9##,##REF##8951055##10##]. It is therefore possible that the small population of Pax2/β tubulin III-positive cells in the developing anterior hypothalamus described here, may arise from the optic stalk and collaborate with other cellular hypothalamic cues in guiding the trajectory of the optic nerve.</p>", "<p>Pax2 is also expressed in the eminentia thalami, a transient developmental structure of unknown function [##REF##10479068##35##] that joins the ventral diencephalon to the telencephalon [##REF##4540788##22##, ####REF##7939711##23##, ##REF##10906711##24####10906711##24##]. Pax2 expression in the eminentia thalami is first detected at E10.5 at the dorsal border between the diencephalon and telencephalon [##REF##16957084##21##], and this staining can not be detected after E13.5. The Pax2-positive cells in the eminentia thalami do not express β-tubulin III, indicating that they are most likely to be neuronal precursors. The early appearance of these cells at the diencephalic-telencephalic boundary (this study and [##REF##16957084##21##]) suggests that they might be important for the formation of this boundary.</p>", "<p>Consistent, high levels of Pax2 expression were also observed in the septum of the basal forebrain. Pax2 is first expressed at E10.5 by a small number of cells located in the ventral telencephalon. It seems likely that these cells give rise to the Pax2-positive population observed in the septal area one day later. By E12.5, septal Pax2 expression although strong, is confined to the septal neuroepithelium, mainly at levels proximal to the lamina terminalis. Only a few Pax2-positive cells are observed in the differentiating layer of the septum. Septal expression is also observed at later developmental stages, but by E16.5 it is downregulated and becomes restricted to a small population of differentiated cells in the medial septum. During septal development in rodents, cells migrate from the lateral ventricle towards the midline. The medial nucleus is one of the first nuclei formed in the septum [##REF##758337##36##, ####REF##4416651##37##, ##REF##3354843##38####3354843##38##]. Therefore, it is possible that the sparse Pax2-positive cells observed at E16.5 correspond to the Pax2-positive cells observed in the differentiating field of the septum between E12.5 and E14.5 (Fig. ##FIG##2##3A##, ##FIG##1##2G## and not shown).</p>", "<p>Pax2 is still expressed by a few cells located in the medial and lateral septal areas during postnatal development and it is not detected after P15. The majority of neurons in the medial and lateral septum are cholinergic or GABA-ergic [##REF##8863126##28##,##REF##3607436##30##,##REF##2199841##31##]. Mature cholinergic neurons express the enzyme choline acetyltransferase (Chat) [##REF##3523620##39##,##UREF##0##40##], whose expression in the septum becomes detectable at around P8 [##REF##8863126##28##,##REF##3607436##30##]. At this age, Pax2 expressing neurons are still found in the septal region but do not co-express Chat, suggesting that these cells might not be of the cholinergic type. However, it is also possible that the Pax2 immunopositive cells might develop into cholinergic neurons at later time points when Chat expression has increased and Pax2 expression has been turned off. Similarly, calbindin is expressed by a large number of somatospiny GABA-ergic neurons in the adult septum [##REF##2199841##31##,##REF##1981215##32##] and it is present in the postnatal septal area, but it does not co-localize with Pax2, indicating that the Pax2 septal neurons are not of this particular GABA-ergic type. As there many different types of GABA-ergic neurons in the septum [##UREF##1##41##], it is possible that the Pax2-positive cells might develop into a different type, such as the septohippocampal projection neurons, a prominent GABA-ergic population comprised of parvalbumin-positive cells [##REF##8863126##28##,##REF##2924136##42##,##REF##2215923##43##]. Although a few of these cells are first detected at around P8 [##REF##8863126##28##,##REF##3607436##30##], when Pax2 expression is still detectable in the septum, they become clearly visible after P15, when Pax2 expressing cells are no longer present in the septum. Again, this expression pattern precludes us from being able to draw conclusions about the specific neuronal type of these cells based solely on immunostaining techniques. Cell fate experiments using a Pax2-cre mouse strain and an appropriate cre reporter strain would allow us to address which type of neurotransmitter fate and properties the Pax2-positive septal neurons adopt.</p>", "<p>In the developing eye, there is a sharp boundary between the domains of Pax2 expression in the optic stalk and Pax6 expression in the optic cup, [##REF##12100888##18##,##UREF##2##44##,##REF##11003833##45##]. Mutual cross-repressive interactions between Pax2 and Pax6 are essential for formation of this boundary [##REF##11003833##45##]. Here we show that Pax2 and Pax6 are expressed in neighbouring, non-overlapping domains in the rostral telencephalon, reminiscent of the pattern observed in the developing eye [##REF##12100888##18##,##REF##11003833##45##]. Given this expression pattern, and the fact that the septum is enlarged in <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants [##REF##11050125##15##], we hypothesised that the Pax2 septal expression domain might be expanded in this mutant. However, we found no increase in the extent or intensity of Pax2 expression in this region in <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants. Nevertheless, the Pax2 expression domain was found at a more dorsal position than in the wild type, consistent with the previously described size increase of the septum in the <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutant [##REF##11050125##15##]. The Pax2 expression domain still lies within the ventral telencephalon, as shown by co-expression of the septal marker Lim1 [##REF##9880598##26##,##REF##9022063##27##].</p>", "<p>There are several mouse models with different types of mutations in the <italic>Pax2 </italic>locus, including the Krd mice (Krd/+), a mutant with a chromosomal deletion that includes this locus [##REF##9473325##9##,##REF##9112988##46##,##REF##7835879##47##], <italic>Pax2</italic>^-/-null mutants [##REF##8951055##10##,##REF##8575306##48##], and <italic>Pax2</italic>^{1<italic>Neu </italic>}mice with a frameshift mutation in <italic>Pax2 </italic>[##REF##8943028##49##]. All of these mutants display defects in kidney formation, optic nerve trajectory, and inner ear patterning, consistent with previously identified expression domains of <italic>Pax2 </italic>transcript [##REF##8951055##10##,##REF##8575306##48##, ####REF##8943028##49##, ##REF##15242798##50####15242798##50##]. However, defects in the midbrain-hindbrain region range from complete loss of the posterior mesencephalon and cerebellum in the <italic>Pax2</italic>^{1<italic>Neu </italic>}mouse [##REF##8943028##49##], to no phenotypic alteration in the <italic>Pax2</italic>^-/-mutant [##REF##8951055##10##,##REF##9405645##51##], possibly as a consequence of differences in genetic background [##REF##8951055##10##]. It would be of interest to study the telencephalic septum in these different mutants, to identify any possible alterations due to loss of Pax2 expression in this region.</p>" ]

[ "<title>Conclusion</title>", "<p>Pax2 is expressed in the anterior hypothalamus, eminentia thalami and telencephalic septum in the developing mouse forebrain, in neuronal progenitors and early born neurons. Between E11.5 and E14.5, it is strongly expressed in the septal neuroepithelium, at a level close to the lamina terminalis and it is no longer detectable by P15. Further, the absence of functional Pax6 does not cause gross alterations of Pax2 expression in the septum. Thus Pax2 represents an ideal marker for the study of the developing septum.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The availability of specific markers expressed in different regions of the developing nervous system provides a useful tool for the study of mouse mutants. One such marker, the transcription factor Pax2, is expressed at the midbrain-hindbrain boundary and in the cerebellum, spinal cord, retina, optic stalk, and optic chiasm. We recently described a group of diencephalic cells that express Pax2 as early as embryonic day (E) 10.5, and become part of the eminentia thalami by E11.5. The discovery of this previously undescribed cell population prompted us to examine Pax2 protein expression in the developing mouse forebrain in more detail.</p>", "<title>Results</title>", "<p>We determined the expression pattern of Pax2 in the forebrain of wild type mouse embryos between E10.5 and postnatal day (P) 15. Pax2 expression was detected in the septum of the basal forebrain, hypothalamus, eminentia thalami and in the subfornical organ. To evaluate Pax2 as a marker for septal cells, we examined Pax2 expression in <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants, which have an enlarged septum. We found that Pax2 clearly marks a population of septal cells equivalent to that seen in wild types, indicating its utility as a marker of septal identity. These cells did not express the GABAergic marker calbindin nor the cholinergic marker choline acetyltransferase and were not detectable after P15.</p>", "<title>Conclusion</title>", "<p>Pax2 is expressed in populations of cells within the developing septum, hypothalamus, and eminentia thalami. It seems especially useful as a marker of the telencephalic septum, because of its early, strong and characteristic expression in this structure. Further, its expression is maintained in the enlarged septum of <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutants.</p>" ]

[ "<title>Abbreviations</title>", "<p>E: embryonic day; P: postnatal day; Chat: choline acetyltransferase.</p>", "<title>Authors' contributions</title>", "<p>VF designed and carried out the experiments, analysed the results and wrote the manuscript. DJP and JOM participated in the analysis and writing of the manuscript. All authors read and approved the final manuscript.</p>" ]

[ "<title>Acknowledgements</title>", "<p>Work in the authors' laboratory is funded by the BBSRC, Wellcome Trust and the MRC. We thank Christine Morrison and Tamsin Lannagan for excellent technical assistance, Trudi Gillespie for confocal imaging, Catherine Carr and Tian Yu for providing embryos, Dario Magnani and Petrina Georgala for postnatal brains and staff of the University of Edinburgh Biological Research Resource facility at Little France for animal care. The Lim1/2 antibody was generated by T. Jessell and S. Brenner-Morton, the nestin antibody by S. Hockfield and the Pax6 antibody by A. Kawakami. They were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa (Department of Biological Sciences, Iowa City, IA).</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Pax2 protein expression in the mouse forebrain at E10.5 and E11.5</bold>. <bold>A-B</bold>, <bold>a-b</bold>: E10.5 sagittal sections showing Pax2 expression in the developing forebrain. In the ventral telencephalon, a few Pax2-positive cells can be detected in lateral sections (arrowheads in <bold>a</bold>) and become more abundant in more medial sections (<bold>a'</bold>). A few Pax2-positive cells are detected in the hypothalamus, close to the strongly Pax2-positive optic recess (or) area (<bold>B, b</bold>). The previously described staining in the future eminentia thalami of the diencephalon (asterisk in <bold>A</bold>) and the optic cup (arrowhead in <bold>A</bold>) are also shown. <bold>C</bold>, <bold>c</bold>: E11.5 sagittal sections revealing strong Pax2 expression in the developing septum (Se). No Pax2 expression is detected in the neighbouring lamina terminalis (LT) (arrow in <bold>c</bold>) at this developmental stage. Pax2-immunopositive cells are also found in the anterior hypothalamus (arrowheads in <bold>c</bold>) and may originate from the ventral neuroepithelium of the optic recess area (or). Expression in the eminentia thalami is not shown. Note the Pax2 expression in the spinal cord (SC) (arrows in <bold>C</bold>). <bold>a, b </bold>and <bold>c </bold>are high power images of the boxed areas in <bold>A, B </bold>and <bold>C </bold>respectively. <bold>a' </bold>is a high power image of a sagittal section at a more medial level than that depicted in <bold>a</bold>. Scale bars: <bold>C</bold>, 400 μm; <bold>A</bold>, <bold>B</bold>, <bold>c</bold>, 200 μm; <bold>a</bold>, <bold>a'</bold>, <bold>b</bold>, 50 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Pax2 protein expression in the mouse forebrain at E12.5 and E13.5</bold>. <bold>A-C</bold>: Low power views of coronal E12.5 forebrain sections immunoreacted with Pax2; the boxed areas are shown at higher magnification in panels <bold>a-c</bold>. <bold>D</bold>: Low power view of a sagittal E12.5 section immunoreacted with Pax2. Note the strong expression of Pax2 in the isthmic region (Is) and the spinal cord (SC), in accordance with previous reports. <bold>d</bold>, <bold>d' </bold>higher magnification of the boxed areas in <bold>D</bold>. <bold>A</bold>, <bold>a</bold>, <bold>d</bold>: Strong Pax2 expression is detected in the septum (Se) of the basal forebrain, mainly in the septal neuroepithelium. A few Pax2-positive cells are detected in the lamina terminalis (LT) (arrows in <bold>d</bold>). <bold>b-c</bold>, <bold>d'</bold>: Pax2 is detected in groups of cells found in the lateral hypothalamic area (arrowheads in <bold>b </bold>and <bold>d'</bold>) and in the anterior hypothalamic neuroepithelium (arrows in <bold>b' </bold>and <bold>d'</bold>, <bold>c</bold>). These cell populations might originate from cells located at the base of the hypothalamus (small arrows in <bold>b </bold>and <bold>d'</bold>), Pax2 expression can also be seen in the ventral neuroepithelium of the optic recess (asterisk in <bold>d'</bold>), as previously described. <bold>E-G</bold>: Low and high power images of E13.5 coronal sections reveal strong Pax2 expression in the septum, mainly in the neuroepithelium (<bold>F</bold>). A few immunopositive cells are found in the differentiating layer of the septum (<bold>G</bold>). The boxed areas in the inset panels in <bold>E-G </bold>indicate the areas shown in the respective high power images. <bold>A-C </bold>and <bold>E-G </bold>are sections from the same specimens respectively. Scale bars: <bold>D</bold>, <bold>E-G</bold>-insets, 1000 μm; <bold>A-C</bold>, 500 μm; <bold>a</bold>, <bold>d'</bold>, <bold>F</bold>-high power, 200 μm; <bold>b-c</bold>, <bold>d</bold>, <bold>E</bold>, <bold>G</bold>-high power, 50 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Pax2 is primarily expressed in neural progenitors in the septum and in early differentiated neurons in the hypothalamus</bold>. <bold>A</bold>: Double immunofluorescence with Pax2 (red) and β-tubulin III (Tuj1) (green) on a sagittal E12.5 telencephalic section shows that only a minority of Pax2-positive cells in the septum also express Tuj1, an early marker of differentiated neurons. This is further confirmed with double immunohistochemistry with Pax2 (black) and nestin (brown) (<bold>B</bold>), revealing that these Pax2-positive cells have nestin-positive filaments. Panels (<bold>A</bold>) and (<bold>B</bold>) show correspond to high power images taken within the region depicted in <bold>Fig. 2D, d</bold>. In the hypothalamus (<bold>C, D</bold>), most Pax2-positive cells express the early neural marker β-tubulin III (green), showing that they correspond to early generated neurons. Pax2-positive cells in (<bold>C</bold>) correspond to those shown in <bold>Fig. 2b'</bold>, and those in (<bold>D</bold>) correspond to the cells indicated by arrowheads in <bold>Fig. 2b</bold>. The position of the ventricular zone (VZ) is indicated in sections <bold>A, C </bold>and <bold>D</bold>. Scale bars: <bold>A</bold>, 20 μm; <bold>B-D</bold>, 5 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F4\"><label>Figure 4</label><caption><p><bold>Pax2 protein expression in the mouse forebrain at E14.5 and E16.5</bold>. <bold>A</bold>, <bold>a</bold>: Low and high power images of an E14.5 sagittal section showing strong Pax2 staining in the neuroepithelium of the septum (Se) adjacent to the foramen of Monro (FM) in an E14.5 sagittal section. This intense Pax2 staining is observed at the level where the lamina terminalis (LT) joins the septum. <bold>a </bold>shows a higher magnification of the boxed area in panel <bold>A</bold>. <bold>B</bold>, <bold>b</bold>: In more lateral sagittal sections Pax2 expression is found in a cell population (arrows) within the anterior hypothalamus (AH) reaching the medial horn (mh) of the lateral ventricle. <bold>b </bold>shows a higher magnification of the boxed area in panel <bold>B</bold>. <bold>C</bold>: E16.5 coronal section showing Pax2 expression around the optic chiasm (oc) region, in the optic stalk epithelium and in a few cells of the hypothalamic neuroepithelium next to the suprachiasmatic nucleus (SCH) (arrows). <bold>D</bold>: E16.5 coronal section showing Pax2 expression in the differentiating layer of the medial septum surrounded by the axonal bundles of the fornix (fx). Scale bars: <bold>A</bold>, <bold>B</bold>, 1000 μm; <bold>a</bold>, <bold>C</bold>, <bold>D</bold>, 200 μm; <bold>C</bold>-inset, 250 μm; <bold>b</bold>, 100 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F5\"><label>Figure 5</label><caption><p><bold>Pax2 protein expression in the mouse forebrain during early postnatal development</bold>. <bold>A-E</bold>: Coronal forebrain sections immunostained for Pax2 at P1 (<bold>A-B</bold>) and at P8 (<bold>C-E</bold>), showing the presence of few disperse Pax2-positive cells in the septal area (Se) (<bold>A-C</bold>, high power in <bold>D</bold>), the medial preoptic nucleus (po) (asterisk in <bold>A </bold>and <bold>B</bold>), and the subfornical organ (SFO) (arrow in <bold>E</bold>). The fibre tracts are indicated in <bold>A </bold>and <bold>B </bold>for orientation purposes (ac, anterior commissure; cc, corpus callosum; fx, fornix). The boxed areas in <bold>A </bold>and <bold>C </bold>delineate the high power images depicted in B and D respectively. Scale bars: <bold>A</bold>, <bold>C</bold>, 100 μm; <bold>B</bold>, <bold>E</bold>, 25 μm; <bold>D</bold>, 12.5 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F6\"><label>Figure 6</label><caption><p><bold>The Pax2 expression domain in the telencephalic septum is located in a more dorsal position in the <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutant than in wild type</bold>. Pax2 expression in the telencephalic septum in E12.5 wild type (wt) (<bold>A, B, D</bold>) and <italic>Pax6</italic>^{<italic>Sey</italic>/<italic>Sey </italic>}mutant (<italic>Sey</italic>) (<bold>C, E</bold>) coronal sections. <bold>A</bold>: Double immunofluorescence with Pax2 (red) and Pax6 (green) reveals that the two proteins are expressed in non-overlapping, mutually exclusive domains. The arrows in <bold>A </bold>indicate the ventral and dorsal limits of Pax6 and Pax2 expression respectively. <bold>B-C</bold>: Pax2 expression in wt and <italic>Sey </italic>embryos, showing that the Pax2 expression domain is shifted dorsally in the <italic>Sey </italic>mutant (<bold>C</bold>) compared to wt (<bold>B</bold>). The septum and the Pax2 expression domain are indicated by dashed lines and arrows respectively. <bold>D-E</bold>: Double immunofluorescence with Pax2 (red) and Lim1/2 (green) in the septum shows that Pax2 expression is found within the Lim-positive domain in both wild types (<bold>D</bold>) and <italic>Sey </italic>mutants (<bold>E</bold>), suggesting that the shifted Pax2 expression domain in the <italic>Sey </italic>mutant is still within the limits of the ventral telencephalon. Note that panels B and C correspond to slightly more rostral sections than those shown in D and E respectively. Scale bars: <bold>A</bold>, <bold>B</bold>, <bold>C</bold>, 200 μm; <bold>D</bold>, <bold>E</bold>, 100 μm.</p></caption></fig>", "<fig position=\"float\" id=\"F7\"><label>Figure 7</label><caption><p><bold>Pax2 expression in the medial septum does not co-localise with that of calbindin and choline acetyltransferase (Chat)</bold>. <bold>A-C</bold>: Double immunohistochemistry with Pax2 (black) (<bold>A</bold>) and calbindin (magenta) (<bold>B</bold>) on P8 coronal sections reveal the presence of both calbindin-positive and Pax2-positive neurons in the septal area, but not co-expression of these proteins (<bold>C</bold>). Similarly (<bold>D-F</bold>), septal neurons that express Pax2 (green) (<bold>D</bold>) and Chat (brown) (<bold>E</bold>), do not co-express these proteins (<bold>F</bold>). The panels are high powers of the level depicted in <bold>Fig. 5C</bold>. Scale bar for all panels, 10 μm.</p></caption></fig>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Background</title>", "<p>The CD44 family of transmembrane glycoproteins mediates the response of cells to their extracellular microenvironment by regulating growth, survival, differentiation and motility. All human CD44 proteins are encoded by a single, highly conserved gene containing 20 exons, 12 of each undergo alternative splicing [##REF##1465456##1##] (see figure ##FIG##0##1A##). Complex alternative splicing of the central region of the gene is responsible for the incorporation of the variable domains to shape, predominantly, the extracellular, membrane-proximal stem structure of the protein. The heterogeneity of the CD44 protein products can be further increased by post-translational modifications [##REF##1465456##1##, ####REF##2466575##2##, ##REF##10737932##3##, ##REF##12511867##4####12511867##4##]. The sequence encoded in exon v3 contains an optimal Ser-Gly-Ser-Gly (SGSG) consensus motif for modification by heparan sulfate (HS) side chains, to which several heparin-binding proteins attach [##REF##7532176##5##]. This unique HS addition site is critical for CD44v3 isoforms' capacity to bind and present HS-dependent growth factors.</p>", "<p>Human variable exon v3 can follow a specific alternative splicing route different from that affecting other variable exons so it can be included in the mRNA together with other variable exons or independently from them [##REF##9665479##6##,##REF##16530165##7##]. This inclusion is regulated by a multisite bipartite exonic splicing enhancer (ESE) consisting in a tandem nonamer (XX motif) and a heptamer (Y motif) that act cooperatively for the efficient recognition of the splice sites [##REF##17940137##8##]. The XX motif is located centrally in the exon while the Y motif is located within the sequence coding for the glycosaminglycan (GAG) binding site immediately downstream from the SGSG motif in v3 (figure ##FIG##0##1B##).</p>", "<p>In order to address the existence and functional nature of the XXY ESE in non-human species we have evaluated the overall level of conservation of CD44 exon v3, including its splicing regulatory elements-the 3' splice site (3'ss), the XXY splicing enhancer- and the GAG binding site, in 95 mammalian species. We also provide data of CD44v3 inclusion into mRNA from peripheral blood samples, by means of RT-PCR, in some representative mammalian taxa with differing levels of conservation of the sequence elements analyzed.</p>" ]

[ "<title>Methods</title>", "<p>Frozen (-80°C) blood samples were selected from the animal tissue bank of the Department of R+D+I, Laboratorio Dr. Echevarne, Barcelona, Spain. Genomic DNA was isolated from 200 μl of blood using the NucleoSpin Blood kit (Macherey-Nagel) following manufacturer's instructions. PCR amplification of CD44v3 was performed with INT6SF and I7wtR primer set or -49v3F and I7wtR primer set (table ##TAB##0##1##) using PCR Master Mix (Promega). PCR bands of interest were isolated from agarose using the NucleoSpin Extract II kit (Machery-Nagel), sequenced in both directions with the primers used during PCR and the CEQ Dye Terminator Cycle Sequencing Quick Start kit (Beckman Coulter) and analyzed in a CEQ 8800 Genetic Analysis System (Beckman Coulter). All sequences were edited to remove ambiguous base calls and primer sequences and submitted to GenBank.</p>", "<p>CD44 RT-PCR was performed from total RNA extracted from frozen blood samples with a modification of the QIAamp RNA Blood Mini kit protocol (QIAGEN). Briefly, 150 μl of frozen blood were lysed at 70°C for 10 min with RLT/β-mercaptoethanol buffer containing 4 mg/ml Proteinase K and centrifuged at 10,000 × g for 3 min. 450 μl of the lysate supernatant were mixed with 225 μl of absolute ethanol and loaded in a QIAamp spin column following manufacturer's instructions. Eluted RNA was treated with RQ1 RNase-free DNase (Promega) at 37°C for 30 min and purified following the QIAamp RNA Mini protocol for RNA cleanup (QIAGEN). The first-strand reaction was performed with random primers (Promega) and SuperScript II Reverse Transcriptase (Invitrogen). As control of RNA quality, total CD44 isoforms were amplified with degenerate E20F-VI and E20R-QEM primer set (table ##TAB##0##1##) using GC-Rich PCR System (Roche).</p>", "<p>In order to amplify CD44v3 containing isoforms, PCR primers were designed based on a multiple sequence alignment containing the sequences corresponding to the 95 mammalian species. Exon v3 positions that showed full conservation were identified and selected to locate the 3' ends of the primers ensuring perfect matches. According to this, v3 amplification was perfomed with primers 13v3F and 100v3R (table ##TAB##0##1##) and PCR Master Mix (Promega). As control of complete genomic DNA digestion, non reverse-transcribed RNAs were tested amplification negative with primers 13v3F and 100v3R.</p>", "<p>Molecular conservation analyses were conducted using the MEGA version 3.1 software [##REF##15260895##9##] and sequence logos were generated with the WebLogo application [##REF##15173120##10##].</p>" ]

[ "<title>Results</title>", "<title>CD44v3 sequencing</title>", "<p>There is little sequence data available for CD44 variable exons from most animal species. The orthologue prediction for human CD44 in Ensembl <italic>release 48 </italic>provides v3 exon sequence for 16 species of mammals. In order to increase the data available we studied CD44 exon v3 in most of the animal samples stored in our tissue bank.</p>", "<p>A region that enabled amplification of CD44v3, was located by a Blast search against multiple species using a human genomic fragment spanning intron6-v3-intron7. Alignment of sequences corresponding to the 10 nt at the 3' end of the INT6SF primer matched perfectly in <italic>Macaca mulatta</italic>, <italic>Canis familiaris</italic>, <italic>Oryctolagus cuniculus </italic>and <italic>Pan troglodytes </italic>and presented a single nucleotide missmatch in <italic>Mus musculus</italic>, <italic>Rattus norvegicus</italic>, <italic>Loxodonta africana </italic>and <italic>Bos taurus</italic>. Likewise, primer I7wtR matched perfectly with the exception of <italic>Mus musculus </italic>where there was a single nucleotide missmatch. We tested primers INT6SF and I7wtR in <italic>Loxodonta africana</italic>, <italic>Canis familiaris</italic>, <italic>Bos taurus</italic>, <italic>Oryctolagus cuniculus </italic>and in non-mammalian species such as <italic>Spheniscus humboldti</italic>, <italic>Psittacus erithacus </italic>and <italic>Varanus niloticus</italic>. Sequence confirmation of PCR products showed that only mammalian species amplified CD44v3. We failed to amplify v3 (exonic and/or flanking intronic sequences) from <italic>Spheniscus humboldti</italic>, <italic>Psittacus erithacus</italic>, <italic>Cygnus atratus</italic>, <italic>Threskiornis aethiopicus</italic>, <italic>Dacelo novaguineae</italic>, <italic>Ciconia ciconia</italic>, <italic>Amazona aestiva</italic>, <italic>Guaruba guarouba</italic>, <italic>Varanus niloticus</italic>, <italic>Sparus aurata</italic>, <italic>Merluccious merlucius </italic>and <italic>Plesionika edwarsii</italic>. In the absence of v3 specific PCR amplification we cannot evaluate the presence/absence of v3 nor the lack of conservation of primer regions. Lack of v3 amplification from bird, fish or reptile DNA is in agreement with the absence of exon v3 from available genomes of <italic>Gallus gallus </italic>[GenBank: <ext-link ext-link-type=\"gen\" xlink:href=\"NC_006092\">NC_006092</ext-link>], <italic>Xenopus tropicalis </italic>[Ensembl: ENSXETG00000007556] and <italic>Gasterosteus aculeatus </italic>[Ensembl: ENSGACG00000011430].</p>", "<p>In view of this, our sample set has been restricted to 95 mammalian species distributed in 29 families. The region amplified comprises, relative to the known human CD44v3 sequence, a 5' partial fragment of intron 6 and a 3' partial fragment of exon v3 (117 nucleotides out of 126) (see figure ##FIG##0##1B##). The resulting sequences are shown in Additional file ##SUPPL##0##1##.</p>", "<title>CD44v3 splicing regulatory elements conservation</title>", "<p>We have compared sequences of 140 nucleotides long spanning 23 nucleotides of intron 6 and 117 nucleotides of exon v3, in the mammalian species listed in Additional file ##SUPPL##0##1##. The genomic region amplified enables the analysis of the CD44v3 splicing regulatory elements, namely, the 3'ss and the XXY ESE [##REF##17940137##8##]. The level of conservation at each position of the alignment in the 95 species analyzed is shown in figure ##FIG##1##2A##. Sequence alignment of exon v3 in these species reveals 69 (59%) conserved and 48 (41%) variable residues. In this way, the 95 species studied are represented by 28 different nucleotide sequences.</p>", "<p>The 3'ss is fully conserved in 84 out of 95 species. The rest of species (11 out of 95) have single-nucleotide substitutions at positions -5 (n = 1), -6 (n = 2), -7 (n = 6) or -8 (n = 2) (see figure ##FIG##1##2B##). The functional significance of these varying positions in the splice site is addressed below by means of v3 expression analysis in peripheral blood.</p>", "<p>The percentage of conserved residues in the ESE and in exon v3 is 68% and 59%, respectively. These values suggest that the level of conservation of the ESE with respect to the exon is of the same range (figure ##FIG##1##2A##). Upon ESE dissection, XX reveals higher variability relative to Y (figure ##FIG##1##2A## and ##FIG##1##2C##) which remains conserved in all species with the exception of <italic>Elephas maximus</italic>, <italic>Loxodonta africana </italic>and <italic>Ursus arctos </italic>which present synonymous variations (see Additional file ##SUPPL##0##1##). If we consider the XXY ESE as a whole unit of 25 nucleotides long, such unit is represented by 13 different sequences whose relative frequency is shown in table ##TAB##1##2##. The most common sequence is <italic># </italic>10, present in 42% of the species tested and followed by <italic># </italic>2, present in 28% of the species. The latter corresponds to the previously described human sequence [##REF##17940137##8##] and it is also detected in other primate families. The third most common sequence is <italic># </italic>8, present in 11% of the species. The analysis of the XXY sequences reveals 17 (68%) conserved and 8 (32%) variable residues of which most are single-nucleotide substitutions. Within the XX motif, positions 3 (conserved in 58 sequences out of 95), 4 (92 out of 95) and 10 (88 out of 95) are represented by 3 different nucleotides. The positions that have been functionally shown elsewhere to decrease CD44v3 inclusion in mutant expression constructs (X mutant: AAATGggtA and Y mutant: ATGggtA) [##REF##17940137##8##] remain invariant, corroborating their functional importance during splicing.</p>", "<title>CD44v3 GAG binding site conservation</title>", "<p>Considering v3's reading frame (codon start = 3), the 117 nucleotides of CD44 exon v3 translate into a 38 amino acid extracellular protein domain. Amino acid sequence alignment of this domain from our data set shows 12 (32%) conserved and 26 (68%) variable residues classifying the 95 species studied into 28 different amino acid sequence groups. This alignment also reveals full conservation of the SGSG motif (figure ##FIG##2##3A##) with the exception of <italic>Tursiops truncatus </italic>and <italic>Oryctolagus cuniculus </italic>where the sequences have been changed to SGSD and PGSG, respectively (see Additional file ##SUPPL##0##1##). Bourdon et al [##REF##3472204##11##] identified the amino acid sequence homology around the SG dipeptide sites that serves as GAG attachment sites in the core proteins of proteoglycans. The core protein must contain acidic amino acids on the amino-terminal side of the sequence SGXG, where X stands for any amino acid. The S is the most critical of the invariant residues and the relative importance of the residues is S &gt; first G &gt; second G &gt; acidic residues. According to this, only the rabbit's v3 domain would not be expected to bind HS.</p>", "<p>Human exon v3 contains acidic residues both upstream and downstream of the SGSG motif. The eight amino acids located downstream of the SGSG site consist of acidic residues flanked by hydrophobic residues that are necessary for the specific addition of HS at this site [##REF##9891022##12##]. The species analyzed maintain a conserved GAG binding site both at the nucleotide (see figure ##FIG##1##2A##) and the amino acid level (figure ##FIG##2##3B##) implying that the secondary and/or tertiary structure around the SGSG motif is critical to initiate HS attachment, and this may have further contributed to the conservation of the Y ESE motif contained therein in all species tested.</p>", "<title>CD44v3 expression in mammalian species</title>", "<p>CD44v3 has been reported to be constitutively expressed in human peripheral blood cells, irrespective of their activation status [##REF##11180125##13##, ####REF##10914548##14##, ##REF##9558399##15####9558399##15##]. We have used peripheral blood accordingly as a model to evaluate the expression of CD44v3 isoforms in some taxon-representative mammalian species. The RT-PCR results (see Additional files ##SUPPL##0##1## and ##SUPPL##1##2##) show that there is no correlation between sequence variation within the 3'ss or the ESE and lack of v3 expression in peripheral blood. All species tested have revealed v3 expression implying that the conservation observed is sufficient to maintain v3 inclusion. The human CD44 protein contains an unique HS binding site coded by CD44 exon v3 [##REF##7532175##16##], therefore enabling only CD44v3 containing isoforms to carry HS side chains and to bind and present heparin-binding growth factors and cytokines. On this basis, presence of a conserved GAG attachment site in all mammals studied may reflect a similar function [##REF##10037743##17##,##REF##9531542##18##] for CD44v3 in these species.</p>", "<p>In addition to mammals, CD44 constitutive exons are also found in birds, amphibians and fish as described in public databases although their expression in certain tissues in such taxa has not been addressed. In conclusion, we have obtained CD44v3 sequence from 95 mammalian species but have failed to amplify the homologous fragment from bird, reptile or fish species, in agreement with the lack of CD44 variable exons from available genome sequences of model organisms in these taxa. This implies that CD44v3 appears to be an exclusive mammalian gene trait. The sequence conservation observed in our dataset would support a common origin and function for this exon in all mammals. Furthermore, CD44v3 sequence conservation in mammalian species enables maintenance of functional splicing regulatory elements and the GAG binding site. The level of conservation of the sequence encoding the GAG binding site, which in turn contains the Y motif of the ESE analysed, is higher than the overall level found for the rest of the exon. Whether this phenomenon is due to purifying selection pressure contributed by the GAG attachment domain alone or in conjunction with the Y motif of the ESE remains undetermined. Functional inclusion of CD44v3 has also been demonstrated in peripheral blood from mammalian species representative of the different sequence variations observed, implying <italic>in vivo </italic>use of exon v3 in these species. Further work is required to search for the exact evolutionary origin of CD44 exon v3 in mammals.</p>" ]

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[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44.</p>", "<title>Findings</title>", "<p>We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences.</p>", "<title>Conclusion</title>", "<p>CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait.</p>" ]

[ "<title>Abbreviations</title>", "<p>SGSG, Ser-Gly-Ser-Gly; HS, heparan sulfate; ESE, exonic splicing enhancer; GAG, glycosaminglycan; 3'ss, 3' splice site.</p>", "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>EV conceived and designed the study, carried out the molecular genetic studies and sequence analysis and was the principal author of the manuscript. JMH participated in the design of the study, completed analyses, interpreted findings and reviewed the manuscript. MD and HFB participated in taxonomically certified sample acquisition and reviewed the manuscript. MI participated in the design and coordination of the study, interpreted findings and revised critically the manuscript for its final approval.</p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>This work was supported in part by PROFIT-01000-2004-212 from the Spanish Ministry of Industry, Tourism and Trade. EV is registered at the Department of Cellular Biology, Physiology and Immunology at the Autonomous University of Barcelona, Spain, for PhD programme administration purposes.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>CD44 exon v3</bold>. A) Genomic structure of human CD44 gene (gray boxes, constitutive exons; white boxes, alternative exons; black box, variable exon v3; black line, introns). B) Schematic representation of exon v3 and its flanking introns (gray boxes, relative location of the XX and Y splicing enhancer motifs; white box, relative location of the nucleotides coding for the GAG binding site and the SGSG motif). Genomic DNA amplification was performed with PCR primers located in intron 6 and in the v3-intron 7 junction (indicated by arrows).</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Nucleotide sequence conservation</bold>. A) Top, line plot displaying the level of conservation at each position of the alignment of the 95 species analyzed (dark gray boxes, location of 3'ss, XX and Y splicing enhancer motifs; light gray box, GAG binding site). Bottom, schematic representation of the genomic region aligned. B) Sequence logo of the 3'ss. Positions are numbered according to the exon sequence. C) Sequence logo of the XX and Y splicing enhancer motifs. Positions are numbered according to the exon sequence.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Amino acid sequence conservation</bold>. A) Line plot displaying the level of conservation at each position of the alignment of the 95 species analyzed (dark gray box, location of SGSG motif; light gray box, GAG binding site). B) Sequence logo of the GAG binding site. Positions are numbered according to the v3 coded domain.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Primers</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><italic>Primer</italic></td><td align=\"center\"><italic>Region</italic></td><td align=\"left\"><italic>Sequence</italic></td></tr></thead><tbody><tr><td align=\"left\">INT6SF</td><td align=\"center\">Intron 6</td><td align=\"left\">5'-ACCTTCTGTGCCTGATTTTC-3'</td></tr><tr><td align=\"left\">I7wtR</td><td align=\"center\">Exon v3-intron 7</td><td align=\"left\">5'-AATTGATTATTCTTACTGGTGCTGG-3'</td></tr><tr><td align=\"left\">-49v3F</td><td align=\"center\">Intron 6</td><td align=\"left\">5'-GCTTGGCGTCCAGCTCAG-3'</td></tr><tr><td align=\"left\">E20F-VI</td><td align=\"center\">Exon 20</td><td align=\"left\">5'-GGGCAGAAGAAAAAGCTAGTNAT-3'</td></tr><tr><td align=\"left\">E20R-QEM</td><td align=\"center\">Exon 20</td><td align=\"left\">5'-CCAAATGCACCATYTCYTG-3'</td></tr><tr><td align=\"left\">13v3F</td><td align=\"center\">Exon v3</td><td align=\"left\">5'-ATATCATCTCAGCAGGCT-3'</td></tr><tr><td align=\"left\">100v3R</td><td align=\"center\">Exon v3</td><td align=\"left\">5'-TCATCAATGCCTGATCCA-3'</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Sequences representing the XXY ESE in CD44v3</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"right\"><italic>XXY ID</italic></td><td align=\"left\"><italic>Sequence (XX_Y)</italic></td><td align=\"right\"><italic>N Species</italic></td><td align=\"right\"><italic>freq (%)</italic></td><td align=\"right\"><italic>N changes*</italic></td></tr></thead><tbody><tr><td align=\"right\">1</td><td align=\"left\">AAaTGAAGAAAAcGAAGA_ATGAAGA</td><td align=\"right\">2</td><td align=\"right\">2.11</td><td align=\"right\">2</td></tr><tr><td align=\"right\">2</td><td align=\"left\">AAaTGAAGAAAATGAAGA_ATGAAGA</td><td align=\"right\">27</td><td align=\"right\">28.42</td><td align=\"right\">1</td></tr><tr><td align=\"right\">3</td><td align=\"left\">AAaTGAAGAAAATGAAGA_ATGAgGA</td><td align=\"right\">2</td><td align=\"right\">2.11</td><td align=\"right\">2</td></tr><tr><td align=\"right\">4</td><td align=\"left\">AAaTGAAGAcAATGAAGA_ATGAAGA</td><td align=\"right\">3</td><td align=\"right\">3.16</td><td align=\"right\">2</td></tr><tr><td align=\"right\">5</td><td align=\"left\">AAaTGAgGAAAATGAAGA_ATGAAGA</td><td align=\"right\">1</td><td align=\"right\">1.05</td><td align=\"right\">2</td></tr><tr><td align=\"right\">6</td><td align=\"left\">AACaGAAGAAAAcGAAGA_ATGAAGA</td><td align=\"right\">2</td><td align=\"right\">2.11</td><td align=\"right\">2</td></tr><tr><td align=\"right\">7</td><td align=\"left\">AACcGAAGAAAAcGAAGA_ATGAAGA</td><td align=\"right\">1</td><td align=\"right\">1.05</td><td align=\"right\">2</td></tr><tr><td align=\"right\">8</td><td align=\"left\">AACTGAAGAAAAcGAAGA_ATGAAGA</td><td align=\"right\">11</td><td align=\"right\">11.58</td><td align=\"right\">1</td></tr><tr><td align=\"right\">9</td><td align=\"left\">AACTGAAGAAAATGAAGA_AcGAAGA</td><td align=\"right\">1</td><td align=\"right\">1.05</td><td align=\"right\">1</td></tr><tr><td align=\"right\">10</td><td align=\"left\">AACTGAAGAAAATGAAGA_ATGAAGA</td><td align=\"right\">40</td><td align=\"right\">42.11</td><td align=\"right\">0</td></tr><tr><td align=\"right\">11</td><td align=\"left\">AACTGAAGAcAATGAAGA_ATGAAGA</td><td align=\"right\">3</td><td align=\"right\">3.16</td><td align=\"right\">1</td></tr><tr><td align=\"right\">12</td><td align=\"left\">cAgTGAAGAAAATGAAGA_ATGAAGA</td><td align=\"right\">1</td><td align=\"right\">1.05</td><td align=\"right\">2</td></tr><tr><td align=\"right\">13</td><td align=\"left\">cAgTGAAGAtAAcGAAGA_ATGAAGA</td><td align=\"right\">1</td><td align=\"right\">1.05</td><td align=\"right\">4</td></tr></tbody></table></table-wrap>" ]

[]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Summarized results. GenBank accession number, taxonomic information, nucleotide sequence, GAG binding site motif and v3 expression results are listed for each sample. Nucleotide sequence is split into exon v3, intron 6, 3' splice site and XXY ESE. The XXY identification number (XXY ID) and its relative representation among species (XXY freq) are indicated. Results of v3 expression are displayed as positive expression (++) or data not available (n.a.).</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional file 2</title><p>Raw RT-PCR expression data of CD44v3 containing isoforms. CD44v3 containing isoforms were amplified from equivalent amounts of cDNA (RT+) and non-retrotranscribed RNA (RT-) digested with DNase. All samples were amplified in the same experiment with primers 13v3F and 100v3R. Negative control (C-) consisted of amplification in the absence of template cDNA/RNA. Positive control (<italic>H. sapiens</italic>) consisted of a human sample where v3 is reported to be expressed in normal peripheral blood cells [##REF##11180125##13##, ####REF##10914548##14##, ##REF##9558399##15####9558399##15##]. PCR product size (bp) is compared to molecular weight ladder (MW). Interpretation of results: samples were considered positive for CD44v3 expression when the net intensity of the amplification of the RT+ was higher than the RT-counterpart. Amplification in some RT-samples is compatible with residual genomic DNA.</p></caption></supplementary-material>" ]

[ "<table-wrap-foot><p>Each XXY sequence is represented by an identification number (XXY ID). Variable positions relative to most common sequence (#10) are indicated in lower case. Number of species representing each sequence and its relative frequency are displayed.</p><p>*Total number of changes relative to #10.</p></table-wrap-foot>" ]

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[ "<title>Background</title>", "<p>Gene expression profiling has become a powerful approach to the study of molecular pathophysiology and is a potentially useful diagnostic tool in multiple fields [##REF##15010847##1##]. Oncologists have applied gene expression profiling to predict breast cancer aggressiveness [##REF##16899776##2##], and microarray-driven approaches have been used to analyze cardiovascular diseases such as hypertension, heart failure, cardiac rejection, and atherosclerosis [##REF##16843168##3##, ####REF##12748210##4##, ##REF##16179268##5####16179268##5##]. Ideally, gene expression profiling is performed on the specific cell type and tissue of interest, <italic>i.e</italic>. the tumor, myocardium, or atheroma. However, sampling target tissues from humans is often problematic, and data derived from tissues not routinely available to clinicians limits the diagnostic utility of this approach.</p>", "<p>For the study of biological processes that involve an inflammatory response, gene expression profiles can be obtained from circulating leukocytes [##REF##16503242##6##]. Due to the ease of sampling, gene expression profiling of circulating leukocytes has been applied to the study of cancer [##REF##15709187##7##], atherosclerosis [##REF##12527563##8##,##REF##15728378##9##], and systemic lupus erythematosus [##REF##12642603##10##]. These studies demonstrate the utility of transcriptional analysis of peripheral blood in the study of disease states having a systemic, inflammatory component.</p>", "<p>Tobacco use, whether by smoking or chewing, is associated with the development of many diseases. People who smoke more than 20 cigarettes per day have a 3–6 fold increased incidence of myocardial infarction [##REF##8565161##11##] and increased overall rates of cardiovascular mortality compared to those who have never smoked [##REF##10988015##12##]. The risk of developing lung cancer is 20-fold increased in cigarette smokers [##REF##12527563##8##], and smokers are at increased risk of developing chronic obstructive pulmonary disease, multiple cancers (<italic>e.g</italic>. esophageal, bladder, and leukemia), pneumonia, osteoporosis, and periodontal disease [##REF##14739193##13##]. Despite these major adverse health effects, more than 20% of American adults identify themselves as active smokers [##REF##17065979##14##].</p>", "<p>The mechanistic link between tobacco smoking and related diseases remain incompletely understood. To date, there have been numerous reports analyzing the effect that exposure to cigarette smoke has on the gene expression profiles of various cell types [##REF##17149619##15##, ####REF##15829617##16##, ##REF##14678473##17##, ##REF##15006922##18##, ##REF##17115125##19##, ##REF##16520944##20##, ##REF##17602957##21##, ##REF##17220372##22####17220372##22##]. However, despite this detailed analysis, very little consensus amongst findings has been reported, even when the same cell type has been studied [##REF##15829617##16##]. This lack of significant overlap in conclusions may be the result of the considerable heterogeneity in methodology as well as the relatively small (on average 5–10 test subjects) sample populations in each study. Furthermore, many of these reports rely on the <italic>in vitro </italic>exposure of cells to cigarette smoke condensate, raising the obvious issue of physiological relevance amongst these various studies.</p>", "<p>Here we report a novel method for analyzing the <italic>in vivo </italic>effects of tobacco use on gene expression in circulating leukocytes. The purpose of this study is not to identify biomarkers associated with tobacco use; rather, our approach is aimed at identifying changes in genes and gene sets that result from tobacco use and applying this information to identify potential cellular pathways associated with tobacco-dependent pathology. Our results indicate that tobacco use affects pathways that control cell death, response to stress, macromolecular metabolism and the inflammatory cascade, providing new insights into the systemic effects of smoking that may underlie tobacco-related diseases.</p>" ]

[ "<title>Methods</title>", "<title>Subject Population</title>", "<p>Subjects between the ages of 18 and 50 years (inclusive) referred to UNC Hospitals were considered for enrollment in this University of North Carolina Institutional Review Board-approved study (IRB 04-MED-471). Exclusion criteria included current cancer treatment, pregnancy, lymphoma, leukemia, chronic immunosuppressive therapy, infection with HIV or HCV, history of solid organ transplant, and anemia (<italic>i.e</italic>. conditions which might alter peripheral blood counts or patterns of gene expression). After obtaining informed consent for a one-time blood donation, subjects were interviewed for pertinent medical information, including a detailed history of tobacco use, family history of heart disease and diabetes. Blood cell counts including a white blood cell differential analysis was performed to ensure consistency in cell subtype number between study populations.</p>", "<title>Blood Withdrawal and Processing</title>", "<p>Blood (30 ml) was drawn early in the day from subjects fasted for at least 8 hours to minimize the signals associated with nutritional and diurnal cycles from the microarray data. Processing was begun within 15 minutes of the time of blood draw. Eight ml were collected into a tube containing EDTA and proteinase inhibitors (Becton, Dickinson and Co., Cockeysville, MD) to provide a sample of plasma for cotinine assays. The balance of blood was collected into Na-EDTA Vacutainer tubes (Becton, Dickinson and Co., Cockeysville, MD). Whole blood was treated with 10 volumes of carbonate-buffered 150 mM NH₄Cl to lyse red blood cells. The remaining leukocytes were washed and concentrated by centrifugation [##REF##11937761##23##,##REF##15548831##24##]. RNA and DNA were recovered from leukocytes using a modified one-step acid guanidinium isothiocyanate-phenol-chloroform extraction (RNA-STAT60, Tel-Test, TX). RNA was subsequently post-purified using the RNeasy Mini-kit (Qiagen, Valencia, CA). RNA quantity, purity, and integrity were assessed by spectrophotometry and microcapillary electrophoresis on an Agilent BioAnalyzer 2100. Complete processing of samples occurred within 2 hours, exceeding the standards set by the Consortium for Expression Profiles in Sepsis [##REF##16237642##25##]. Plasma cotinine levels were determined by competitive ELISA using the Serum Cotinine Assay Kit (BioQuant; San Diego, CA) essentially as described by the manufacturer.</p>", "<title>Gene Expression Profiling</title>", "<p>We utilized a \"sample × reference\" experimental design strategy in which RNA from each subject was hybridized to the microarray slide in the presence of labeled human reference RNA (UHRR, Stratagene, La Jolla, CA) [##REF##15113400##26##,##REF##15155546##27##]. Briefly, total RNA (500 ng) was used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5 CTP. Labeled subject cRNA was co-hybridized to Agilent G4112A Whole Human Genome 44 K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled UHRR. Slides were hybridized and washed, then scanned on an Axon 4000b microarray scanner. The data were processed using GenePix Pro 6 software and entered into the UNC Microarray Database [##UREF##0##28##].</p>", "<title>Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) analysis</title>", "<p>Three hundred nanograms of total RNA were reverse transcribed using the iScript Synthesis cDNA Kit (Biorad, Hercules, CA). Real-time PCR reactions were performed using either the Roche Universal Probe Library (Roche Diagnostics, Mannheim, Germany) or pre-validated Taqman^®assays (Applied Biosystems, Framingham, MA). Primers and probes for the indicated human transcripts were designed using Probe Finder (version 2.41, Roche Diagnostics, Mannheim, Germany): <italic>CDKN1C </italic>(left primer GAGCGAGCTAGCCAGCAG, right primer GCGACAAGACGCTCCATC, probe #77); <italic>CX3CR1 </italic>(left primer CTCTGGCTTCTGGGTGGAG, right primer AGACCACGATGTCCCCAATA, probe #30); <italic>SASH1 </italic>(left primer CAGATCCGGGTGAAGCAG, right primer GAGTCCACCACTTGGAATCG, probe #38); <italic>RPS29 </italic>(left primer CCAAGAACTGCAAAGCCATC, right primer GGCATTGGTGACTCTGATGA, probe #26); and <italic>18S </italic>(left primer GGAGAGGGAGCCTGAGAAAC, right primer TCGGGAGTGGGTAATTTGC, probe #40). <italic>PTGDR </italic>and <italic>HRASLS3 </italic>were measured using Taqman^®assays Hs00235003_m1 and Hs00272992_m1, respectively. Real-time PCR reactions were performed using the ABI PRISM^®7900 sequence detection system, software, and reagents. Relative changes in gene expression were calculated using the delta Ct method using ribosomal <italic>18S </italic>to normalize RNA input. Statistical significance was determined using the Student's <italic>t </italic>test. <italic>P </italic>values less than 0.05 were considered significant.</p>", "<title>Statistical Methods</title>", "<p>Microarray data were normalized <italic>via </italic>the loess local intensity normalization [##REF##15709187##7##,##REF##14597310##29##], and probes were filtered for features having a normalized intensity of &lt; 30 aFU in either channel. Probes were removed if &lt; 70% of the data were present across all samples. Missing data points were imputed using the k nearest-neighbors algorithm (k = 10). 18,375 probes passed these filters, and were subsequently used for analysis. Scripts written in the R Statistical Language and Environment (\"R\"; Version 2.2.1, build r36812, release date 2005-12-20.) and Perl (ActiveState Perl 5.8.1, build 807, release date 2003-11-6) were used to standardize (μ = 0, σ = 1) each sample in the data set.</p>", "<title>Statistical Analysis of Microarrays (SAM)</title>", "<p>Lists of differentially expressed genes were identified using the statistical analysis of microarray algorithm [##REF##11309499##30##, ####REF##12710672##31##, ##REF##15927423##32####15927423##32##] (SAM, Version 2.21, release date 2005-8-24; typical false discovery rate of approximately 10%). Unsupervised, semi-supervised, and supervised clustering analysis was performed on gene lists essentially as described [##REF##9843981##33##] using Cluster, version 2.11[##UREF##1##34##]. Heat maps of cluster analyses were visualized with JavaTreeView, version 1.0.12 [##REF##15180930##35##,##UREF##2##36##].</p>", "<title>Gene Set Analysis (GSA)</title>", "<p>GSA [##REF##16199517##37##,##UREF##3##38##] was performed using the Molecular Signatures Database (MSigDB) [##REF##17644558##39##] to identify gene set activity associated with cotinine levels. Mapping to gene ontology categories (GO) [##UREF##4##40##] and identification of putative transcription factor binding sites was performed on gene lists using the GATHER web-based analysis environment [##REF##10802651##41##, ####REF##17000751##42##, ##UREF##5##43####5##43##] using the TRANSFAC V7.0 (public) database [##REF##12520026##44##, ####REF##16381825##45##, ##REF##11125113##46##, ##UREF##6##47####6##47##].</p>", "<title>Hyperclustering</title>", "<p>A median-centered gene list was used for cluster analysis to identify relationships between subject samples (arrays). The clustering file was then used as the basis for a new pre-clustering file to incorporate gene annotation data. Genes were assigned to GO and TRANSFAC categories using the GATHER web interface [##REF##17000751##42##]. Categories showing statistical enrichment (p value &lt; 0.01) were identified, and each gene in the pre-clustering file was annotated as to its membership in the appropriate category. The TRANSFAC predictions of transcription factor binding sites were designated in the pre-clustering file by the value representing the median-centered mean fold change expressed as the Log₂of the ratio of each sample to the reference for each gene. This method of indicating membership was chosen to reflect a relationship between expression level (as measured by microarray) and presence or absence of transcription factor binding sites. Gene membership in GO categories was indicated by a binary value of either 1.00 or 0.00 as a placeholder for the expression ratio. Blue color was added after the fact to heat maps indicating Gene Ontology membership to avoid confusion with expression values. The annotated pre-clustering file was then clustered on only the Y axis (genes) to preserve relationships among arrays. This technique, which we have designated \"Hyperclustering,\" allows both the gene expression data and various other forms of annotation to be represented as a single heat map, effectively illustrating functional relationships among genes.</p>" ]

[ "<title>Results and discussion</title>", "<title>Subject Selection for Gene Expression Analysis</title>", "<p>Initial analysis of the gene transcription data from a cohort of 171 individuals revealed strong signals related to the race and gender of the subject (unpublished observations). Similar signals have been described in other microarray experiments. These signals can hinder attempts to identify signals related to the biological effect being studied [##REF##16005921##48##]. For this reason, we selected the largest cohort in our dataset (Caucasian males) to maximize the statistical power of our analysis. We adopted a case-control approach to our study design and data analysis. Selected subject demographics are presented in Table ##TAB##0##1##.</p>", "<title>Tobacco Use Determination</title>", "<p>Self-reported tobacco use history is notoriously inaccurate [##REF##12773728##49##, ####REF##15461199##50##, ##REF##17046624##51####17046624##51##]. For purposes of this study, we defined tobacco use status by the subject's plasma cotinine concentration. Cotinine, the principle metabolite of nicotine, is a reliable surrogate marker of tobacco use [##REF##12944172##52##,##REF##9772852##53##]. It has a plasma half-life of approximately 24 hours (as opposed to nicotine's <italic>in vivo </italic>half-life of 30 minutes) and tends to reach steady state levels that vary by only 15%–20% in people with regular smoking habits [##REF##12944172##52##]. As seen in Figure ##FIG##0##1##, the distribution of plasma cotinine is similar in both the Caucasian male subpopulation under study and a larger cohort of 171 subjects, with strong bimodal peaks near 0 ng/mL and 150 ng/mL. Cutoffs of plasma cotinine for the definition of active tobacco users and non-users were set at &gt; 100 ng/mL and &lt; 50 ng/mL, respectively, based on previously reported values [##REF##12944172##52##,##REF##9772852##53##].</p>", "<p>Using these criteria, 24 subjects were classified as tobacco users and 38 as non-tobacco users, with 5 subjects having cotinine levels that fell between 50 and 100 ng/mL. These 5 intermediate subjects were removed from further consideration. Comparing each subject's plasma cotinine values with their self-reported tobacco use status revealed overall consistent results (<italic>i.e</italic>. a high cotinine value for subjects who self-reported that they were active tobacco users). Nevertheless, there were notable exceptions. Seven subjects reported that they were non-tobacco users, yet had plasma cotinine levels &gt; 100 ng/mL. Errors in this dimension could be explained by subject misrepresentation or failure of the subjects to disclose nicotine replacement therapy as part of a smoking cessation plan (use of nicotine patches or gum). Interestingly, 3 subjects identified themselves as active smokers, yet had very low plasma cotinine levels. Rapid metabolism of nicotine, smoking of a small number of cigarettes daily, or the use of extremely low-nicotine smoking products could all account for this discrepancy. This discrepancy in self-reported tobacco use and plasma cotinine levels did not appreciably alter the results of our studies (data not shown). All subjects were categorized based only on plasma cotinine levels only. The 2 subject groups will henceforth be referred to as \"high cotinine\" (<italic>i.e</italic>. tobacco users) and \"low cotinine\" (<italic>i.e</italic>. non-tobacco users). Using this criterion, those subjects reporting to be \"smokers\" but who had low plasma cotinine levels were included in the low cotinine group while subjects with high cotinine levels who denied smoking were included in the high cotinine group. To ensure that patient co-morbidities did not influence the gene expression profile, we performed principal components analysis (PCA) on the expression values of genes identified in this paper using the combined significant gene list and visualized in the context of COPD, diabetes, CAD class, and smoking status (Additional File ##SUPPL##0##1##). As expected, the top component of variation appears to be associated only with smoking status.</p>", "<title>Transcriptional Signals of Tobacco Use</title>", "<p>The subjects were stratified based upon the results of the cotinine assay, and differential gene expression was determined by SAM. We identified 38 genes as being differentially expressed (8 genes up-regulated, 30 genes down-regulated in the high-cotinine group) at an 11.7% FDR (Table ##TAB##1##2##). Notable among this list were genes involved in apoptosis, cell cycle regulation, and oncogenesis.</p>", "<p>Visual inspection of the SAM-identified genes revealed that a number of differentially expressed genes are involved in the cell cycle control Gene Ontologies. <italic>CTCF </italic>was down regulated in the high cotinine group. Mutations in this gene have been associated with a variety of cancers [##REF##16989720##54##]. Furthermore, <italic>CTCF </italic>plays an important role in the regulation and differentiation of human myeloid leukemia cells, adding another possible underlying mechanism of leukemiagenesis in tobacco users [##REF##15941718##55##]. Conversely, we found that <italic>SASH1 </italic>(which is implicated in tumorogenesis of colorectal and breast cancer) was up regulated [##REF##17088907##56##]. Interestingly, <italic>CX3CR1 </italic>was significantly down regulated in the high cotinine group. As <italic>CX3CR1 </italic>is up-regulated in atherosclerotic lesions [##REF##16908772##57##], we expected it to be up-regulated in circulating leukocytes of tobacco users due to the increased incidence and severity of CAD in this population (reviewed by Njolstad [##REF##8565161##11##]). However, Barlic, <italic>et al</italic>., showed that macrophage up-regulation of <italic>CX3CR1 </italic>leads to retention of those cells in vessel walls [##REF##16908772##57##]. As the kinetics of the up-regulation of this gene are ill-defined, and it is not yet clear whether circulating monocytes differentially express <italic>CX3CR1 </italic>prior to tissue macrophage transformation, considerably more study will be necessary to elucidate what role it may play in the pathogenesis of smoking-related atherosclerotic disease.</p>", "<p>Further analysis identified genes involved in apoptotic pathways. The pro-apoptotic genes <italic>C1D</italic>, <italic>MTCBP-1</italic>, <italic>CTCF</italic>, <italic>IKIP</italic>, <italic>MAF</italic>, and <italic>YWHAQ </italic>were all significantly down regulated in the high cotinine group. <italic>C1D </italic>(also known as <italic>SUNCOR</italic>) is representative of this group. <italic>C1D </italic>is a multi-functional nuclear protein with DNA-binding properties. When <italic>C1D </italic>is experimentally over-expressed it activates <italic>DNA-PK</italic>, inducing apoptosis [##REF##10362552##58##]. On the other hand, the c-terminal modulator protein (<italic>CTMP</italic>, also known as <italic>THEM4</italic>) was significantly over-expressed in the high cotinine population. CTMP protein stimulates the phosphorylation of AKT/PKB, increasing glucose uptake and blocking apoptosis [##REF##17220372##22##]. The relative mean fold change was modest for most of these genes (Table ##TAB##1##2##); nevertheless, in subjects with high plasma cotinine the overall expression pattern of these genes is anti-apoptotic compared to low cotinine subjects. The combination of increased cell cycle activity, resistance to apoptotic triggers, increased expression of oncogenes, and decreased expression of tumor suppressor genes in circulating leukocytes suggests a mechanism responsible for the low-level, systemic, increased risk of oncogenesis in patients who use tobacco products.</p>", "<p>Testing for differential expression of individual genes does not take advantage of our knowledge of the underlying relationships. Therefore, additional power can be gained by testing for differential expression of gene sets that underlie a common biological process [##REF##16199517##37##,##UREF##3##38##,##REF##15647293##59##]. This idea motivated the development of techniques that pair local statistics of individual gene expression with global statistics based on membership in defined pathways and functional groups. One such algorithm, Gene Set Analysis (GSA), was implemented using the Molecular Signatures database (MSigDB). The GSA algorithm identified 16 gene sets at a p-value &lt; 0.0001 and FDR of 0%. The top three MSigDB pathways were \"Death Pathway,\" \"Dac_IFN_Bladder_Up,\" and \"Metastasis_Adenocarcinoma\" (Table ##TAB##2##3##). Although many of the genes comprising these sets did not reach statistical significance individually, taken as a group they were highly significant. Genes related to apoptosis and type I interferon response were common elements in all of these pathways. Among genes involved in the MSigDB \"Death Pathway,\" expression of <italic>BIRC3 </italic>and <italic>TRAF2 </italic>(anti-apoptotic genes) were up regulated while <italic>CASP9</italic>, <italic>FADD</italic>, and <italic>STK17A </italic>(pro-apoptotic genes) were down regulated in the high cotinine group. This overall expression pattern is indicative of an anti-apoptotic phenotype, which characterizes virtually all cancers. These observations suggest that transcriptional profiles associated with tobacco use may indicate pre-cancerous tendencies. The 71 genes present in the top 3 pathways (Table ##TAB##2##3##) were added to the list of 38 SAM-identified genes to enrich the gene list that was used for further analysis. This list of 109-pooled genes is available as Additional file ##SUPPL##1##2##.</p>", "<title>Pattern Identification <italic>via </italic>the Hyperclustering Technique</title>", "<p>Differentially expressed genes were hyperclustered (see Materials and Methods) and visualized (Figure ##FIG##1##2##) using the pooled gene list. The subjects with the highest mean levels of cotinine were clearly separated from the subjects with the lowest mean cotinine levels using this technique. Moreover, genes were clustered into functional groups based on their expression patterns, membership in Gene Ontologies (Table ##TAB##3##4##, labeled A-G), and presence of predicted transcription factor binding sites. This produced 5 physiologically relevant clusters. The '<italic>Stress</italic>' cluster is comprised of stress-responsive genes involved in signal transduction (<italic>CX3CR1 </italic>and <italic>ITGB1</italic>). The '<italic>Macromolecular Metabolism</italic>' cluster is made up of metabolic genes (<italic>HIPK1</italic>, <italic>SUMO2</italic>, <italic>SULF2</italic>, and <italic>FKBP3</italic>). The third cluster, '<italic>Transcription and Signaling'</italic>, contains genes associated primarily with G protein signaling and transcriptional regulation (<italic>RASGEF1A</italic>, <italic>RAB2</italic>, <italic>ARHGAP1</italic>, <italic>PPP1R12B</italic>, <italic>CREBBP</italic>, and <italic>GNG2</italic>). '<italic>Cell Death and Apoptosis</italic>' is a cluster of genes associated with apoptosis and its regulation. The fifth cluster, '<italic>Interferon' </italic>is defined by genes that potentially contain an interferon-stimulated response element-binding site or are responsive to type-1 interferons.</p>", "<p>The utility of the hyperclustering technique is readily apparent: a single image indicates the relationships among the genes, lending physiological relevance to a data set. A case in point is the '<italic>Interferon' </italic>cluster, comprised of genes that are strongly up regulated in approximately half of the subjects with the highest cotinine levels. The genes in this cluster (<italic>IFI44</italic>, <italic>IFIT1</italic>, <italic>USP18</italic>, and <italic>HERC5</italic>. Figure ##FIG##1##2##) are interferon responsive genes, and are members of the gene class forming the early response to type-I interferons, indicative of a cellular response to viral agents or very specific forms of genotoxicity. Our findings are consistent with those of Grumelli, <italic>et al</italic>. who demonstrated that lymphocytes isolated from lung samples of patients with smoking-related lung damage showed an increase in expression of multiple interferon-inducible proteins [##REF##15526056##60##]. These results indicate that induction of interferon-dependent transcription pathways appear systemically in some tobacco users. Only half of the tobacco users have this expression pattern; the mechanisms of which are unknown, but worthy of future investigation. It is tempting to speculate that these patterns of systemic interferon-responsive induction identify a group of tobacco users who may develop early and severe disease. Longitudinal studies designed to track the patterns of gene expression over time in cohorts of tobacco users and non-users will be necessary to unambiguously determine the meaning of these observations.</p>", "<title>Real time PCR verification of differentially expressed genes</title>", "<p>Quantitative real time PCR was used for both <italic>technical </italic>(microarray) and <italic>biological </italic>verification. Four genes selected from SAM and one gene from GSA: <italic>CX3CR1</italic>, <italic>SASH1</italic>, <italic>HRASLS3</italic>, <italic>PTGDR</italic>, and <italic>CDKN1C</italic>, respectively, were used for technical verification (Figure ##FIG##2##3##, left panel) on samples randomly selected from the low and high cotinine subject population (Caucasian males). The up or down regulation of these genes, irrespective of their method of identification (SAM or GSA) was consistent with the microarray analysis. Furthermore, the relative fold changes determined <italic>via </italic>quantitative real time PCR were either equal to or greater than the fold change measured by the microarray analysis, and significantly different between the low and high cotinine subjects (<italic>P </italic>&lt; 0.05). Analysis using subjects excluded from the microarray analysis (Caucasian females) biologically validated the cotinine-dependent change in expression of two genes, <italic>CDKN1C </italic>and <italic>SASH1 </italic>(Figure ##FIG##2##3##, right panel). <italic>RPS29 </italic>was used as a negative control gene and was not found to be differentially expressed either by microarray or real time PCR analysis.</p>" ]

[ "<title>Results and discussion</title>", "<title>Subject Selection for Gene Expression Analysis</title>", "<p>Initial analysis of the gene transcription data from a cohort of 171 individuals revealed strong signals related to the race and gender of the subject (unpublished observations). Similar signals have been described in other microarray experiments. These signals can hinder attempts to identify signals related to the biological effect being studied [##REF##16005921##48##]. For this reason, we selected the largest cohort in our dataset (Caucasian males) to maximize the statistical power of our analysis. We adopted a case-control approach to our study design and data analysis. Selected subject demographics are presented in Table ##TAB##0##1##.</p>", "<title>Tobacco Use Determination</title>", "<p>Self-reported tobacco use history is notoriously inaccurate [##REF##12773728##49##, ####REF##15461199##50##, ##REF##17046624##51####17046624##51##]. For purposes of this study, we defined tobacco use status by the subject's plasma cotinine concentration. Cotinine, the principle metabolite of nicotine, is a reliable surrogate marker of tobacco use [##REF##12944172##52##,##REF##9772852##53##]. It has a plasma half-life of approximately 24 hours (as opposed to nicotine's <italic>in vivo </italic>half-life of 30 minutes) and tends to reach steady state levels that vary by only 15%–20% in people with regular smoking habits [##REF##12944172##52##]. As seen in Figure ##FIG##0##1##, the distribution of plasma cotinine is similar in both the Caucasian male subpopulation under study and a larger cohort of 171 subjects, with strong bimodal peaks near 0 ng/mL and 150 ng/mL. Cutoffs of plasma cotinine for the definition of active tobacco users and non-users were set at &gt; 100 ng/mL and &lt; 50 ng/mL, respectively, based on previously reported values [##REF##12944172##52##,##REF##9772852##53##].</p>", "<p>Using these criteria, 24 subjects were classified as tobacco users and 38 as non-tobacco users, with 5 subjects having cotinine levels that fell between 50 and 100 ng/mL. These 5 intermediate subjects were removed from further consideration. Comparing each subject's plasma cotinine values with their self-reported tobacco use status revealed overall consistent results (<italic>i.e</italic>. a high cotinine value for subjects who self-reported that they were active tobacco users). Nevertheless, there were notable exceptions. Seven subjects reported that they were non-tobacco users, yet had plasma cotinine levels &gt; 100 ng/mL. Errors in this dimension could be explained by subject misrepresentation or failure of the subjects to disclose nicotine replacement therapy as part of a smoking cessation plan (use of nicotine patches or gum). Interestingly, 3 subjects identified themselves as active smokers, yet had very low plasma cotinine levels. Rapid metabolism of nicotine, smoking of a small number of cigarettes daily, or the use of extremely low-nicotine smoking products could all account for this discrepancy. This discrepancy in self-reported tobacco use and plasma cotinine levels did not appreciably alter the results of our studies (data not shown). All subjects were categorized based only on plasma cotinine levels only. The 2 subject groups will henceforth be referred to as \"high cotinine\" (<italic>i.e</italic>. tobacco users) and \"low cotinine\" (<italic>i.e</italic>. non-tobacco users). Using this criterion, those subjects reporting to be \"smokers\" but who had low plasma cotinine levels were included in the low cotinine group while subjects with high cotinine levels who denied smoking were included in the high cotinine group. To ensure that patient co-morbidities did not influence the gene expression profile, we performed principal components analysis (PCA) on the expression values of genes identified in this paper using the combined significant gene list and visualized in the context of COPD, diabetes, CAD class, and smoking status (Additional File ##SUPPL##0##1##). As expected, the top component of variation appears to be associated only with smoking status.</p>", "<title>Transcriptional Signals of Tobacco Use</title>", "<p>The subjects were stratified based upon the results of the cotinine assay, and differential gene expression was determined by SAM. We identified 38 genes as being differentially expressed (8 genes up-regulated, 30 genes down-regulated in the high-cotinine group) at an 11.7% FDR (Table ##TAB##1##2##). Notable among this list were genes involved in apoptosis, cell cycle regulation, and oncogenesis.</p>", "<p>Visual inspection of the SAM-identified genes revealed that a number of differentially expressed genes are involved in the cell cycle control Gene Ontologies. <italic>CTCF </italic>was down regulated in the high cotinine group. Mutations in this gene have been associated with a variety of cancers [##REF##16989720##54##]. Furthermore, <italic>CTCF </italic>plays an important role in the regulation and differentiation of human myeloid leukemia cells, adding another possible underlying mechanism of leukemiagenesis in tobacco users [##REF##15941718##55##]. Conversely, we found that <italic>SASH1 </italic>(which is implicated in tumorogenesis of colorectal and breast cancer) was up regulated [##REF##17088907##56##]. Interestingly, <italic>CX3CR1 </italic>was significantly down regulated in the high cotinine group. As <italic>CX3CR1 </italic>is up-regulated in atherosclerotic lesions [##REF##16908772##57##], we expected it to be up-regulated in circulating leukocytes of tobacco users due to the increased incidence and severity of CAD in this population (reviewed by Njolstad [##REF##8565161##11##]). However, Barlic, <italic>et al</italic>., showed that macrophage up-regulation of <italic>CX3CR1 </italic>leads to retention of those cells in vessel walls [##REF##16908772##57##]. As the kinetics of the up-regulation of this gene are ill-defined, and it is not yet clear whether circulating monocytes differentially express <italic>CX3CR1 </italic>prior to tissue macrophage transformation, considerably more study will be necessary to elucidate what role it may play in the pathogenesis of smoking-related atherosclerotic disease.</p>", "<p>Further analysis identified genes involved in apoptotic pathways. The pro-apoptotic genes <italic>C1D</italic>, <italic>MTCBP-1</italic>, <italic>CTCF</italic>, <italic>IKIP</italic>, <italic>MAF</italic>, and <italic>YWHAQ </italic>were all significantly down regulated in the high cotinine group. <italic>C1D </italic>(also known as <italic>SUNCOR</italic>) is representative of this group. <italic>C1D </italic>is a multi-functional nuclear protein with DNA-binding properties. When <italic>C1D </italic>is experimentally over-expressed it activates <italic>DNA-PK</italic>, inducing apoptosis [##REF##10362552##58##]. On the other hand, the c-terminal modulator protein (<italic>CTMP</italic>, also known as <italic>THEM4</italic>) was significantly over-expressed in the high cotinine population. CTMP protein stimulates the phosphorylation of AKT/PKB, increasing glucose uptake and blocking apoptosis [##REF##17220372##22##]. The relative mean fold change was modest for most of these genes (Table ##TAB##1##2##); nevertheless, in subjects with high plasma cotinine the overall expression pattern of these genes is anti-apoptotic compared to low cotinine subjects. The combination of increased cell cycle activity, resistance to apoptotic triggers, increased expression of oncogenes, and decreased expression of tumor suppressor genes in circulating leukocytes suggests a mechanism responsible for the low-level, systemic, increased risk of oncogenesis in patients who use tobacco products.</p>", "<p>Testing for differential expression of individual genes does not take advantage of our knowledge of the underlying relationships. Therefore, additional power can be gained by testing for differential expression of gene sets that underlie a common biological process [##REF##16199517##37##,##UREF##3##38##,##REF##15647293##59##]. This idea motivated the development of techniques that pair local statistics of individual gene expression with global statistics based on membership in defined pathways and functional groups. One such algorithm, Gene Set Analysis (GSA), was implemented using the Molecular Signatures database (MSigDB). The GSA algorithm identified 16 gene sets at a p-value &lt; 0.0001 and FDR of 0%. The top three MSigDB pathways were \"Death Pathway,\" \"Dac_IFN_Bladder_Up,\" and \"Metastasis_Adenocarcinoma\" (Table ##TAB##2##3##). Although many of the genes comprising these sets did not reach statistical significance individually, taken as a group they were highly significant. Genes related to apoptosis and type I interferon response were common elements in all of these pathways. Among genes involved in the MSigDB \"Death Pathway,\" expression of <italic>BIRC3 </italic>and <italic>TRAF2 </italic>(anti-apoptotic genes) were up regulated while <italic>CASP9</italic>, <italic>FADD</italic>, and <italic>STK17A </italic>(pro-apoptotic genes) were down regulated in the high cotinine group. This overall expression pattern is indicative of an anti-apoptotic phenotype, which characterizes virtually all cancers. These observations suggest that transcriptional profiles associated with tobacco use may indicate pre-cancerous tendencies. The 71 genes present in the top 3 pathways (Table ##TAB##2##3##) were added to the list of 38 SAM-identified genes to enrich the gene list that was used for further analysis. This list of 109-pooled genes is available as Additional file ##SUPPL##1##2##.</p>", "<title>Pattern Identification <italic>via </italic>the Hyperclustering Technique</title>", "<p>Differentially expressed genes were hyperclustered (see Materials and Methods) and visualized (Figure ##FIG##1##2##) using the pooled gene list. The subjects with the highest mean levels of cotinine were clearly separated from the subjects with the lowest mean cotinine levels using this technique. Moreover, genes were clustered into functional groups based on their expression patterns, membership in Gene Ontologies (Table ##TAB##3##4##, labeled A-G), and presence of predicted transcription factor binding sites. This produced 5 physiologically relevant clusters. The '<italic>Stress</italic>' cluster is comprised of stress-responsive genes involved in signal transduction (<italic>CX3CR1 </italic>and <italic>ITGB1</italic>). The '<italic>Macromolecular Metabolism</italic>' cluster is made up of metabolic genes (<italic>HIPK1</italic>, <italic>SUMO2</italic>, <italic>SULF2</italic>, and <italic>FKBP3</italic>). The third cluster, '<italic>Transcription and Signaling'</italic>, contains genes associated primarily with G protein signaling and transcriptional regulation (<italic>RASGEF1A</italic>, <italic>RAB2</italic>, <italic>ARHGAP1</italic>, <italic>PPP1R12B</italic>, <italic>CREBBP</italic>, and <italic>GNG2</italic>). '<italic>Cell Death and Apoptosis</italic>' is a cluster of genes associated with apoptosis and its regulation. The fifth cluster, '<italic>Interferon' </italic>is defined by genes that potentially contain an interferon-stimulated response element-binding site or are responsive to type-1 interferons.</p>", "<p>The utility of the hyperclustering technique is readily apparent: a single image indicates the relationships among the genes, lending physiological relevance to a data set. A case in point is the '<italic>Interferon' </italic>cluster, comprised of genes that are strongly up regulated in approximately half of the subjects with the highest cotinine levels. The genes in this cluster (<italic>IFI44</italic>, <italic>IFIT1</italic>, <italic>USP18</italic>, and <italic>HERC5</italic>. Figure ##FIG##1##2##) are interferon responsive genes, and are members of the gene class forming the early response to type-I interferons, indicative of a cellular response to viral agents or very specific forms of genotoxicity. Our findings are consistent with those of Grumelli, <italic>et al</italic>. who demonstrated that lymphocytes isolated from lung samples of patients with smoking-related lung damage showed an increase in expression of multiple interferon-inducible proteins [##REF##15526056##60##]. These results indicate that induction of interferon-dependent transcription pathways appear systemically in some tobacco users. Only half of the tobacco users have this expression pattern; the mechanisms of which are unknown, but worthy of future investigation. It is tempting to speculate that these patterns of systemic interferon-responsive induction identify a group of tobacco users who may develop early and severe disease. Longitudinal studies designed to track the patterns of gene expression over time in cohorts of tobacco users and non-users will be necessary to unambiguously determine the meaning of these observations.</p>", "<title>Real time PCR verification of differentially expressed genes</title>", "<p>Quantitative real time PCR was used for both <italic>technical </italic>(microarray) and <italic>biological </italic>verification. Four genes selected from SAM and one gene from GSA: <italic>CX3CR1</italic>, <italic>SASH1</italic>, <italic>HRASLS3</italic>, <italic>PTGDR</italic>, and <italic>CDKN1C</italic>, respectively, were used for technical verification (Figure ##FIG##2##3##, left panel) on samples randomly selected from the low and high cotinine subject population (Caucasian males). The up or down regulation of these genes, irrespective of their method of identification (SAM or GSA) was consistent with the microarray analysis. Furthermore, the relative fold changes determined <italic>via </italic>quantitative real time PCR were either equal to or greater than the fold change measured by the microarray analysis, and significantly different between the low and high cotinine subjects (<italic>P </italic>&lt; 0.05). Analysis using subjects excluded from the microarray analysis (Caucasian females) biologically validated the cotinine-dependent change in expression of two genes, <italic>CDKN1C </italic>and <italic>SASH1 </italic>(Figure ##FIG##2##3##, right panel). <italic>RPS29 </italic>was used as a negative control gene and was not found to be differentially expressed either by microarray or real time PCR analysis.</p>" ]

[ "<title>Conclusion</title>", "<p>In this study we demonstrated that groups of genes in circulating human leukocytes are affected by tobacco use <italic>in vivo</italic>. We identified genes and their relationships using a combination of testing individual genes (SAM), testing gene sets (GSA), and high throughput annotation (GATHER). Hyperclustering using Gene Ontologies and transcription factor binding sites associated with these genes illuminated the functional significance of the differentially regulated genes. The resulting gene expression spectra revealed novel and under-recognized molecular pathways in the pathophysiology of diseases commonly associated with tobacco use. Genomic signals in circulating leukocytes characteristic of cellular metabolism, transcription and signaling, apoptosis, response to stress, and the interferon response were all correlated with nicotine exposure. These results strongly suggest that tobacco use promotes a pro-carcinogenic environment, predisposing individuals to develop cancers in a variety of organ systems.</p>", "<p>Interestingly, some genes that have previously been linked to smoking were not differentially expressed in our 2 subject groups [##REF##12520071##61##, ####REF##15297370##62##, ##REF##17056606##63####17056606##63##]. For example, neither <italic>CYP1B1 </italic>(a cytochrome P450 enzyme playing an important role in chemical carcinogenesis) nor <italic>SOD2 </italic>(which destroys toxic radicals normally produced within cells) had an expression profile that differed significantly between high and low cotinine groups. Although several previous reports identified these genes as being affected by smoking, design and subject pool differences used in the present study could explain the absence of these genes from our profile. <italic>CYP1B1 </italic>is expressed to a greater degree in the females than in males and our data set is all male [##REF##12049173##64##]. <italic>SOD2 </italic>gene expression declines with age [##REF##17005595##65##]. The mean age of one of the studies reporting differential regulation of <italic>SOD2 </italic>was 27 years while the mean age of our study subjects is 46.5 years, which may explain why the <italic>SOD2 </italic>gene expression ratios between the groups in our study did not vary significantly.</p>", "<p>A significant link has been established between smoking and the development of blood-borne cancers such as acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) [##REF##8246285##66##,##REF##17519958##67##]. Exposure to compounds derived from tobacco use is typically highest in the oral and nasal cavities, the laryngotracheobronchial tree, and the urinary system, putting these tissues at the greatest risk of developing tumors [##REF##1548964##68##]. Nevertheless, given chronic exposure to carcinogens, blood tissues are likewise at an increased risk of carcinogenesis [##REF##9498902##69##]. Sandler, <italic>et al</italic>., observed a clear dose response to smoking, with heavy smokers at the highest risk of developing leukemia [##REF##8246285##66##]. The causative mechanism for this observed increase in leukemia among smokers is unknown. Our results identify highly relevant, differentially expressed genes that may serve as the basis for future experiments aimed at addressing the molecular etiology of AML and ALL in smokers. Moreover, these gene signals were detected in an easily obtainable sample of peripheral blood.</p>", "<p>We found a correlation between tobacco use and increased expression of interferon-inducible genes in circulating leukocyte populations. Strong induction of interferon-responsive gene expression was seen in only a subset of tobacco-using subjects, arguing that interferon induction is not a direct effect of tobacco use. The mechanism of induction of these genes is not clear from our data. Previous studies have found a strong correlation between the parenchymal destruction associated with end-stage emphysema and the presence of interferon and interferon-inducible genes in the lung [##REF##15526056##60##]. Intriguingly, 5 of the 6 subjects (83%) with a diagnosis of COPD in this study demonstrated the high-interferon response phenotype. Our observation of elevated peripheral interferon response gene expression may reflect a systemic manifestation of a destructive pulmonary inflammatory response. These observations may provide evidence of a systemic immune basis for smoking-related lung parenchymal destruction. Alternatively, the expression of interferon-responsive genes in the periphery may be secondary to the upper and lower respiratory tract infections to which smokers are prone.</p>", "<p>Hyperclustering revealed 5 distinct, physiologically relevant gene groups in peripheral leukocytes affected by tobacco use <italic>in vivo</italic>. Furthermore, these gene groups belong to pathways and regulatory systems important to the etiology of smoking-related diseases. These novel results enhance our understanding of how tobacco use affects patterns of gene expression in leukocytes, and provide a starting point for elucidating the molecular mechanisms of tobacco-related neoplasia, atherosclerosis, and immune dysfunction. The hyperclustering visualization facilitated interpretation of microarray data by fusing the expression data with functional annotation derived through robust statistical methodology (GSA and GATHER) prior to cluster analysis. This technique is a visual representation that combines gene expression data and any form of additional annotation. Gene expression profiling of readily obtainable peripheral blood samples identified genes that regulate response to stress, macromolecular metabolism, transcription and signaling, interferon response, and cell death and resistance to apoptosis. This profile may identify some novel targets for therapeutic intervention for both smoking-related diseases and, potentially, for smoking cessation.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Strong epidemiologic evidence correlates tobacco use with a variety of serious adverse health effects, but the biological mechanisms that produce these effects remain elusive.</p>", "<title>Results</title>", "<p>We analyzed gene transcription data to identify expression spectra related to tobacco use in circulating leukocytes of 67 Caucasian male subjects. Levels of cotinine, a nicotine metabolite, were used as a surrogate marker for tobacco exposure. Significance Analysis of Microarray and Gene Set Analysis identified 109 genes in 16 gene sets whose transcription levels were differentially regulated by nicotine exposure. We subsequently analyzed this gene set by hyperclustering, a technique that allows the data to be clustered by both expression ratio and gene annotation (<italic>e.g</italic>. Gene Ontologies).</p>", "<title>Conclusion</title>", "<p>Our results demonstrate that tobacco use affects transcription of groups of genes that are involved in proliferation and apoptosis in circulating leukocytes. These transcriptional effects include a <italic>repertoire </italic>of transcriptional changes likely to increase the incidence of neoplasia through an altered expression of genes associated with transcription and signaling, interferon responses and repression of apoptotic pathways.</p>" ]

[ "<title>Competing interests</title>", "<p>The authors declare that they have no competing interests.</p>", "<title>Authors' contributions</title>", "<p>PCC participated in design of the study, recruited subjects, processed samples, analyzed data, performed statistical analysis, and participated in manuscript preparation. BA recruited subjects, analyzed data, and participated in manuscript preparation. EGH processed samples, analyzed data, assisted in statistical analysis, and participated in manuscript preparation. JCS assisted in statistical analysis and manuscript preparation. REL participated in study design and coordination, and assisted in manuscript preparation. SM processed samples. JSP assisted in study design and data management. SSW participated in study coordination and recruited patients. AP assisted with manuscript preparation. GAS participated in study design and coordination, and data analysis. CP conceived of the study, participated in study design and coordination, performed data analysis, and participated in manuscript preparation.</p>", "<title>Pre-publication history</title>", "<p>The pre-publication history for this paper can be accessed here:</p>", "<p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biomedcentral.com/1755-8794/1/38/prepub\"/></p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>This study was supported in part by an American Heart Association Scientist Development Grant (0635100N) to PCC, grants from the NIH (HL072347), CDC (H75/CCH424675 and H75/CCH424677), and UNC School of Medicine (\"Investments in the Future\" program) to CP, and a Doris Duke Charitable Foundation Fellowship to BDA. C.P. is an established investigator of the American Heart Association, and a Burroughs Wellcome Fund Clinician Scientist in Translational Research.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p><bold>Histogram of plasma cotinine concentration</bold>. Distribution of plasma cotinine levels in the total population as well as in the Caucasian male sub-population are demonstrated. Vertical lines represent selected cut-offs for definitions of tobacco users and non-users.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Hyperclustering of cotinine responsive genes</bold>. <bold>A</bold>. The 109 genes identified by SAM and GSA analysis in subjects with high versus low plasma cotinine levels were analyzed by hyperclustering. Clusters (top) were created by incorporating gene expression data with their corresponding TRANSFAC and Gene Ontology (GO) categories. Genes are represented in columns. Individual subject expression profiles (which clustered into 2 groups, high and low cotinine) and TRANSFAC categories are represented in rows and the relative expression of the genes is reflected as indicated in the color scale (upper right). Gene membership in GO categories (also represented in rows) is indicated by Carolina blue. <bold>B</bold>. Enlargement of the five functional groups identified by hyperclustering (bottom). The corresponding TRANSFAC and GO categories are indicated by groups A and B-H, respectively (see Table 4 for detailed category information).</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Histogram of relative expression of selected genes using real time PCR</bold>. <italic>Technical </italic>verification (left) of differentially expressed genes identified in the subject population (Caucasian males) by SAM/GSA (n = 20): <italic>CDKN1C</italic>, <italic>HRASLS3</italic>, <italic>PTGDR</italic>, <italic>CX3CR1</italic>, and <italic>SASH1</italic>. <italic>Biological </italic>verification (right) of two selected genes using independent samples not included in our subject population (Caucasian females, n = 10): <italic>CDKN1C </italic>and <italic>SASH1</italic>. Data is represented as the log base 2 relative change in gene expression (± standard error) and all expression normalized to low cotinine from the subject population samples (Caucasian males). The data labels represent the fold change in high versus low cotinine samples, all of which were statistically significant (<italic>P </italic>&lt; 0.05). The fold change in the gene <italic>RPS29 </italic>was used as a negative control and was not significant (<italic>n.s</italic>.) between the high and low cotinine groups.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Selected demographics of study subjects.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td/><td align=\"center\"><bold>Low Cotinine</bold></td><td align=\"center\"><bold>High Cotinine</bold></td></tr></thead><tbody><tr><td align=\"left\">Number of subjects</td><td/><td align=\"center\">38</td><td align=\"center\">24</td></tr><tr><td align=\"left\">Mean Age ± SD</td><td/><td align=\"center\">47 ± 9</td><td align=\"center\">46 ± 5</td></tr><tr><td align=\"left\">COPD</td><td/><td align=\"center\">2 (5.3%)</td><td align=\"center\">4 (16.7%)</td></tr><tr><td align=\"left\">Diagnosis of Diabetes (Number (% of total))</td><td align=\"left\">*Any</td><td align=\"center\">13 (34%)</td><td align=\"center\">2 (8.3%)</td></tr><tr><td/><td align=\"left\">Type 1</td><td align=\"center\">2 (5%)</td><td align=\"center\">1 (4%)</td></tr><tr><td/><td align=\"left\">Type 2</td><td align=\"center\">11 (29%)</td><td align=\"center\">1 (4%)</td></tr><tr><td align=\"left\">CAD Family History</td><td/><td align=\"center\">20 (53%)</td><td align=\"center\">15 (63%)</td></tr><tr><td align=\"left\">Hyperlipidemia</td><td/><td align=\"center\">24 (63%)</td><td align=\"center\">16 (67%)</td></tr><tr><td align=\"left\">Automated Differential Blood Count</td><td align=\"left\">White Blood Cells (× 10⁹/L ± SD)</td><td align=\"center\">8.42 ± 2.67</td><td align=\"center\">9.00 ± 2.41</td></tr><tr><td/><td align=\"left\">Neutrophils (× 10⁹/L ± SD)</td><td align=\"center\">5.67 ± 2.18</td><td align=\"center\">5.76 ± 1.94</td></tr><tr><td/><td align=\"left\">Lymphocytes (× 10⁹/L ± SD)</td><td align=\"center\">1.90 ± 0.68</td><td align=\"center\">2.31 ± 0.74</td></tr><tr><td/><td align=\"left\">Monocytes (× 10⁹/L ± SD)</td><td align=\"center\">0.42 ± 0.18</td><td align=\"center\">0.46 ± 0.21</td></tr><tr><td/><td align=\"left\">Basophils (× 10⁹/L ± SD)</td><td align=\"center\">0.06 ± 0.04</td><td align=\"center\">0.06 ± 0.04</td></tr><tr><td/><td align=\"left\">Eosinophils (× 10⁹/L ± SD)</td><td align=\"center\">0.22 ± 0.18</td><td align=\"center\">0.26 ± 0.14</td></tr><tr><td/><td align=\"left\">Platelets (× 10⁹/L ± SD)</td><td align=\"center\">252.42 ± 73.97</td><td align=\"center\">250.67 ± 56.06</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Differentially expressed genes identified by SAM analysis.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\" colspan=\"2\"><bold>Down-regulated in High Cotinine Subjects</bold></td><td/><td/><td/></tr><tr><td align=\"left\"><bold>Gene Symbol</bold></td><td align=\"left\"><bold>Gene Name</bold></td><td align=\"left\"><bold>Accession Number</bold></td><td align=\"left\"><bold>Agilent Probe ID</bold></td><td align=\"center\"><bold>Mean FC</bold></td></tr></thead><tbody><tr><td align=\"left\">HRASLS3</td><td align=\"left\">HRAS-like suppressor 3</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_007069\">NM_007069</ext-link></td><td align=\"left\">A_23_P116414</td><td align=\"center\">1.5</td></tr><tr><td align=\"left\">CX3CR1</td><td align=\"left\">Chemokine (C-X3-C motif) receptor 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_001337\">NM_001337</ext-link></td><td align=\"left\">A_23_P407565</td><td align=\"center\">1.3</td></tr><tr><td align=\"left\">GPR56</td><td align=\"left\">G protein-coupled receptor 56</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_005682\">NM_005682</ext-link></td><td align=\"left\">A_23_P206280</td><td align=\"center\">1.3</td></tr><tr><td align=\"left\">PTGDS</td><td align=\"left\">Prostaglandin D2 synthase 21kDa (brain)</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_000954\">NM_000954</ext-link></td><td align=\"left\">A_23_P146554</td><td align=\"center\">1.3</td></tr><tr><td align=\"left\">FLJ23262</td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"BC043173\">BC043173</ext-link></td><td align=\"left\">A_24_P20996</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">BRD1</td><td align=\"left\">Bromodomain containing 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_014577\">NM_014577</ext-link></td><td align=\"left\">A_23_P166536</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">BZRAP1</td><td align=\"left\">Benzodiazapine receptor (peripheral) associated protein 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_004758\">NM_004758</ext-link></td><td align=\"left\">A_23_P152559</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">C1D</td><td align=\"left\">Nuclear DNA-binding protein</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_173177\">NM_173177</ext-link></td><td align=\"left\">A_23_P67992</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">FLJ23262</td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"BC043173\">BC043173</ext-link></td><td align=\"left\">A_24_P20996</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">CTCF</td><td align=\"left\">CCCTC-binding factor (zinc finger protein)</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_006565\">NM_006565</ext-link></td><td align=\"left\">A_24_P347704</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">DNAJB6</td><td align=\"left\">DnaJ (Hsp40) homolog, subfamily B, member 6</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_058246\">NM_058246</ext-link></td><td align=\"left\">A_24_P63827</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"ENST00000320343\">ENST00000320343</ext-link></td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"ENSG00000177197\">ENSG00000177197</ext-link></td><td align=\"left\">A_24_P75688</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">FLJ35696</td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_207387\">NM_207387</ext-link></td><td align=\"left\">A_23_P368484</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">GNG2</td><td align=\"left\">Guanine nucleotide binding protein (G protein), gamma</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_053064\">NM_053064</ext-link></td><td align=\"left\">A_32_P208403</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">HS6ST1</td><td align=\"left\">Heparan sulfate 6-O-sulfotransferase 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AL831893\">AL831893</ext-link></td><td align=\"left\">A_24_P8220</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">IKIP</td><td align=\"left\">IKK interacting protein</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_201613\">NM_201613</ext-link></td><td align=\"left\">A_23_P53467</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">KLRK1</td><td align=\"left\">Killer cell lectin-like receptor subfamily K, member 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_007360\">NM_007360</ext-link></td><td align=\"left\">A_23_P218058</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">MAF</td><td align=\"left\">V-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian)</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AF055376\">AF055376</ext-link></td><td align=\"left\">A_24_P256219</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">MGC61571</td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_182523\">NM_182523</ext-link></td><td align=\"left\">A_24_P408740</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">MTCBP-1</td><td align=\"left\">Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_018269\">NM_018269</ext-link></td><td align=\"left\">A_23_P148194</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AL137798\">AL137798</ext-link></td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_032723\">NM_032723</ext-link></td><td align=\"left\">A_23_P126486</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">OSBPL5</td><td align=\"left\">Oxysterol binding protein-like 5</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_145638\">NM_145638</ext-link></td><td align=\"left\">A_23_P53081</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">PPP1CB</td><td align=\"left\">Protein phosphatase 1, catalytic subunit, beta isoform</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_206877\">NM_206877</ext-link></td><td align=\"left\">A_23_P83414</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">PPP1R12B</td><td align=\"left\">Protein phosphatase 1, regulatory (inhibitor) subunit 12B</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_002481\">NM_002481</ext-link></td><td align=\"left\">A_23_P201790</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">PPP2R2B</td><td align=\"left\">Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_181678\">NM_181678</ext-link></td><td align=\"left\">A_23_P213620</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">SLC25A20</td><td align=\"left\">Solute carrier family 25 (carnitine/acylcarnitine translocase), member 20</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_000387\">NM_000387</ext-link></td><td align=\"left\">A_23_P72025</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">SLC9A3R1</td><td align=\"left\">Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_004252\">NM_004252</ext-link></td><td align=\"left\">A_23_P308519</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">SULF2</td><td align=\"left\">Sulfatase 2</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_198596\">NM_198596</ext-link></td><td align=\"left\">A_23_P154605</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">YWHAQ</td><td align=\"left\">Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_006826\">NM_006826</ext-link></td><td align=\"left\">A_24_P199905</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">PTGDR</td><td align=\"left\">Prostaglandin D2 receptor (DP)</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_000953\">NM_000953</ext-link></td><td align=\"left\">A_23_P393777</td><td align=\"center\">1.1</td></tr><tr><td/><td/><td/><td/><td/></tr><tr><td align=\"left\" colspan=\"2\"><bold>Up-regulated in High Cotinine Subjects</bold></td><td/><td/><td/></tr><tr><td align=\"left\"><bold>Gene Symbol</bold></td><td align=\"left\"><bold>Gene Name</bold></td><td align=\"left\"><bold>Accesion Number</bold></td><td align=\"left\"><bold>Agilent Probe ID</bold></td><td align=\"center\"><bold>Mean FC</bold></td></tr><tr><td align=\"left\">SASH1</td><td align=\"left\">SAM and SH3 domain containing 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_015278\">NM_015278</ext-link></td><td align=\"left\">A_23_P93442</td><td align=\"center\">1.4</td></tr><tr><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"BC107798\">BC107798</ext-link></td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_003283\">NM_003283</ext-link></td><td align=\"left\">A_23_P56050</td><td align=\"center\">1.4</td></tr><tr><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AL442066\">AL442066</ext-link></td><td/><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"AL442066\">AL442066</ext-link></td><td align=\"left\">A_23_P123645</td><td align=\"center\">1.3</td></tr><tr><td align=\"left\">DNAPTP6</td><td align=\"left\">DNA polymerase-transactivated protein 6</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_015535\">NM_015535</ext-link></td><td align=\"left\">A_23_P131255</td><td align=\"center\">1.3</td></tr><tr><td align=\"left\">C1GALT1</td><td align=\"left\">Core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 1,3-galactosyltransferase</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_020156\">NM_020156</ext-link></td><td align=\"left\">A_23_P252145</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">RGL1</td><td align=\"left\">Ral guanine nucleotide dissociation stimulator-like 1</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_015149\">NM_015149</ext-link></td><td align=\"left\">A_23_P115417</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">CTMP</td><td align=\"left\">C-terminal modulator protein</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_176853\">NM_176853</ext-link></td><td align=\"left\">A_23_P149375</td><td align=\"center\">1.2</td></tr><tr><td align=\"left\">LOC283174</td><td align=\"left\">Hypothetical protein LOC283174</td><td align=\"left\"><ext-link ext-link-type=\"gen\" xlink:href=\"NM_001001873\">NM_001001873</ext-link></td><td align=\"left\">A_24_P904484</td><td align=\"center\">1.2</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Summary of GSA.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\"><bold>Gene Set Pathway</bold></td><td align=\"left\"><bold>Description</bold></td><td align=\"center\"><bold>P-value</bold></td><td align=\"center\"><bold>FDR</bold></td></tr></thead><tbody><tr><td align=\"center\">DEATHPATHWAY (c2:161)[##UREF##8##71##]</td><td align=\"left\">Genes involved in signaling via Fas and DR3, 4, and 5.</td><td align=\"center\">&lt; 0.0001</td><td align=\"center\">0</td></tr><tr><td align=\"center\">METASTASIS_ADENOCARC_DN (c2:1553)[##UREF##9##72##]</td><td align=\"left\">Genes involved in metastasis of solid tumors.</td><td align=\"center\">&lt; 0.0001</td><td align=\"center\">0</td></tr><tr><td align=\"center\">DAC_IFN_BLADDER_UP (c2:1304)[##UREF##10##73##]</td><td align=\"left\">Interferon responsive genes upregulated by DAC treatment.</td><td align=\"center\">&lt; 0.0001</td><td align=\"center\">0</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T4\"><label>Table 4</label><caption><p>Hyperclustered TRANSFAC and GO Category Annotations</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"center\">Cluster</td><td align=\"left\">TRANSFAC Annotations</td><td/><td/><td/></tr></thead><tbody><tr><td align=\"center\">A</td><td align=\"left\">V$POU3F2_02</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$ISRE_01: interferon-stimulated response element</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$DEAF1_01</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$E2F1_Q3_01</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$MAZR_01: MAZ related factor</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$KROX_Q6</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$E2F1DP1_01: E2F-1:DP-1 heterodimer</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$HNF1_Q6</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$E2F_Q3_01</td><td/><td/><td/></tr><tr><td/><td align=\"left\">V$E2F1_Q6: E2F-1</td><td/><td/><td/></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"center\">Cluster</td><td align=\"center\">Common GO Parent Node</td><td align=\"center\">Gene Ontology</td><td align=\"center\">GO Level</td><td align=\"center\">GO Name</td></tr><tr><td align=\"center\">B</td><td align=\"center\">signal transduction [##REF##12748210##4##] GO:0007165</td><td align=\"left\">GO:0007264</td><td align=\"left\">6</td><td align=\"left\">small GTPase mediated signal transduction</td></tr><tr><td/><td/><td align=\"left\">GO:0007165</td><td align=\"left\">4</td><td align=\"left\">signal transduction</td></tr><tr><td/><td/><td align=\"left\">GO:0007242</td><td align=\"left\">5</td><td align=\"left\">intracellular signaling cascade</td></tr><tr><td align=\"center\">C</td><td align=\"center\">programmed cell death [##REF##16179268##5##] GO:0012501</td><td align=\"left\">GO:0006917</td><td align=\"left\">8</td><td align=\"left\">induction of apoptosis</td></tr><tr><td/><td/><td align=\"left\">GO:0012502</td><td align=\"left\">7</td><td align=\"left\">induction of programmed cell death</td></tr><tr><td/><td/><td align=\"left\">GO:0043068</td><td align=\"left\">6</td><td align=\"left\">positive regulation of programmed cell death</td></tr><tr><td/><td/><td align=\"left\">GO:0043065</td><td align=\"left\">7</td><td align=\"left\">positive regulation of apoptosis</td></tr><tr><td/><td/><td align=\"left\">GO:0050794</td><td align=\"left\">3*</td><td align=\"left\">regulation of cellular process</td></tr><tr><td/><td/><td align=\"left\">GO:0016265</td><td align=\"left\">3*</td><td align=\"left\">death</td></tr><tr><td/><td/><td align=\"left\">GO:0008219</td><td align=\"left\">4*</td><td align=\"left\">cell death</td></tr><tr><td/><td/><td align=\"left\">GO:0012501</td><td align=\"left\">5</td><td align=\"left\">programmed cell death</td></tr><tr><td/><td/><td align=\"left\">GO:0006915</td><td align=\"left\">6</td><td align=\"left\">apoptosis</td></tr><tr><td/><td/><td align=\"left\">GO:0043067</td><td align=\"left\">5</td><td align=\"left\">regulation of programmed cell death</td></tr><tr><td/><td/><td align=\"left\">GO:0042981</td><td align=\"left\">6</td><td align=\"left\">regulation of apoptosis</td></tr><tr><td align=\"center\">D</td><td align=\"center\">response to stress [##REF##12748210##4##] GO:0006950</td><td align=\"left\">GO:0006950</td><td align=\"left\">4</td><td align=\"left\">response to stress</td></tr><tr><td align=\"center\">E</td><td align=\"center\">macromolecule metabolic process [##REF##12748210##4##] GO:0043170</td><td align=\"left\">GO:0006493</td><td align=\"left\">9</td><td align=\"left\">O-linked glycosylation</td></tr><tr><td/><td/><td align=\"left\">GO:0043170</td><td align=\"left\">4</td><td align=\"left\">macromolecule metabolism</td></tr><tr><td/><td/><td align=\"left\">GO:0044260</td><td align=\"left\">5</td><td align=\"left\">cellular macromolecule metabolism</td></tr><tr><td/><td/><td align=\"left\">GO:0019222</td><td align=\"left\">4*</td><td align=\"left\">regulation of metabolism</td></tr><tr><td align=\"center\">F</td><td align=\"center\">transcription [##REF##16503242##6##] GO:0006350</td><td align=\"left\">GO:0006350</td><td align=\"left\">6</td><td align=\"left\">transcription</td></tr><tr><td/><td/><td align=\"left\">GO:0045449</td><td align=\"left\">6</td><td align=\"left\">regulation of transcription</td></tr><tr><td/><td/><td align=\"left\">GO:0019219</td><td align=\"left\">5*</td><td align=\"left\">regulation of nucleo-base, -side, -tide and nucleic acid metabolism</td></tr><tr><td/><td/><td align=\"left\">GO:0006355</td><td align=\"left\">7</td><td align=\"left\">regulation of transcription, DNA-dependent</td></tr><tr><td/><td/><td align=\"left\">GO:0006351</td><td align=\"left\">7</td><td align=\"left\">transcription, DNA-dependent</td></tr><tr><td align=\"center\">G</td><td align=\"center\">cell cycle process [##REF##16503242##6##] GO:0022402</td><td align=\"left\">GO:0000082</td><td align=\"left\">7</td><td align=\"left\">G1/S transition of mitotic cell cycle</td></tr><tr><td/><td/><td align=\"left\">GO:0000132</td><td align=\"left\">11</td><td align=\"left\">mitotic spindle orientation</td></tr><tr><td align=\"center\">H</td><td align=\"center\">mevalonate transport [##REF##12527563##8##] GO:0015728</td><td align=\"left\">GO:0015728</td><td align=\"left\">8</td><td align=\"left\">mevalonate transport</td></tr></tbody></table></table-wrap>" ]

[]

[]

[]

[]

[]

[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional file 1</title><p>Principle component analysis (PCA) of subject co-morbidities. PCA was performed using the combined significant gene list and visualized in the context of COPD, Diabetes, CAD class, and smoking status. As expected, the top component of variation is associated with smoking status. Additionally, it does not appear associated with the remaining variables. To formally test this hypothesis, the PC1 loadings were tested for association with each of the 4 clinical variables. Smoking status was found to be significantly associated with PC1 (p &lt; 0.001). However, none of the remaining clinical variables were associated with the top component of variation (COPD p = 0.91; CAD p = 0.15; Diabetes p = 0.55) indicating that this gene list is not strongly associated with these disease states.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional File 2</title><p><bold>Complete gene list of 109 genes identified by SAM and GSA</bold>. Differentially expressed genes identified by SAM and GSA demonstrate the up-regulation of 34 genes and the down-regulation of 75 genes in subjects with high versus low plasma cotinine. The table includes the gene symbol, gene name, Genbank Accession ID, Agilent Probe ID and the mean fold change in gene expression in high <italic>versus </italic>low plasma cotinine subjects.</p></caption></supplementary-material>" ]

[ "<table-wrap-foot><p>CAD = Coronary Artery Disease, SD = Standard Deviation, L = liter, fL = femtoliter, dL = deciliter, G = gram, pG = picogram, COPD = Chronic Obstructive Pulmonary Disease. For Student's T-test, automated cell counting values were recalculated as values per gram or liter, and log2 normalized prior to determination of p-Value.</p><p>* Fisher's Exact Test shows significant differences between low and high cotinine at p = 0.0315 (2-tail)</p></table-wrap-foot>", "<table-wrap-foot><p>Top 3 gene sets (71 total genes) identified by GSA comparing gene expression profiles of subjects with high plasma cotinine versus low plasma cotinine, showing the names of differentially expressed gene sets as defined by Gene Set Enrichment Analysis [##UREF##7##70##] with accompanying p-value and FDR.</p></table-wrap-foot>", "<table-wrap-foot><p>* Node is not a child of the parent node for this group</p></table-wrap-foot>" ]

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[ "<title>Background</title>", "<p>Maps of disease distribution are an essential tool for optimizing the allocation of resources for malaria interventions [##UREF##0##1##,##REF##17147467##2##]. There have been a number of attempts to develop malaria transmission maps at different geographic scales based on expert opinion [##UREF##1##3##,##UREF##2##4##]; deterministic biological models driven by the conceptual relationship between transmission and environmental covariates [##REF##10322323##5##]; and empirical transmission models based on entomological inoculation rates [##REF##15155980##6##,##REF##11832960##7##] or human infection prevalence data [##REF##17176583##8##, ####REF##17892584##9##, ##REF##14693661##10##, ##REF##16827704##11##, ##REF##16357113##12##, ##REF##18686238##13##, ##REF##16987415##14##, ##REF##11679126##15##, ##REF##15941419##16##, ##REF##10326100##17####10326100##17##]. These methods suffer several limitations: expert opinion maps are subjective; deterministic models ignore the secular effects of expanded coverage of interventions that supersede the influence of climate on the epidemiology of malaria and do not quantify uncertainty around model results. Where studies have used observational data to predict malaria distributions, most have used historical data collected opportunistically from secondary sources [##REF##14693661##10##,##REF##11679126##15##,##REF##15941419##16##] that did not involve random sampling and/or a sampling framework optimized for spatial analysis.</p>", "<p>Arguably the greatest need for malaria maps is at the periphery of stable, endemic areas where decisions about the delivery of standard suites of interventions, such as those promoted by the Roll Back Malaria (RBM) initiative to support malaria control in high transmission areas, may become less appropriate or cost-efficient. In areas of perceived low malaria risk there is little empirical information on the risks and intensity of transmission. As such the semi-arid regions of the horn of Africa remain less well described epidemiologically compared to the rest of malaria endemic sub-Saharan Africa (SSA) and there are no contemporary national maps of the extents of malaria risk. The Malaria Atlas Project (MAP) while maintaining a global remit in its efforts to improve the cartography of malaria [##REF##17147467##2##] is equally committed to developing national mapping initiatives with country partners, where the data available can support rigorous cartography. Somalia represents the first such example.</p>", "<p>A <italic>Plasmodium falciparum </italic>malaria prevalence map for Somalia is presented here using Bayesian geostatistical analysis of community-based parasite prevalence survey data. The data used in this analysis have several unique features that minimize some of the problems of using retrospectively assembled data: first the community data were derived from random sample surveys undertaken as part of national malaria or nutritional surveys; second all the data were collected using similar methodologies; and finally all the data represent contemporary infection prevalence between 2005 and 2007.</p>" ]

[ "<title>Methods</title>", "<title>Country context</title>", "<p>Somalia is located in the horn of Africa with a predominantly semi-arid climate and is transected by two major seasonal rivers, the Shabelle and the Juba (Figure ##FIG##0##1##). Somalia is divided into three zones: South-Central; North-West (Somaliland); and North-East (Puntland) (Figure ##FIG##0##1##). The country has been without a functioning national government for almost two decades. Several international relief agencies and non-governmental organizations, however, support the provision of public services including health services [##UREF##3##18##]. While not internationally recognized, the three zones are all self-declared states, each with independent \"ministries of health\". In collaboration with these ministries of health, the United Nations Children Fund (UNICEF), as the principal recipient of money from the Global Fund to Fight Aids TB and Malaria (GFATM), together with World Health Organization (WHO), are responsible for supporting malaria control activities. These activities include: training health workers and equipping health facilities; supply of anti-malarials; distribution of insecticide-treated nets; and funding of entomological and parasitological surveillance [##UREF##4##19##].</p>", "<p>The earliest malariometric surveys undertaken in Somalia were in the North-West in 1946 which reported a highly varying prevalence distribution of <italic>P. falciparum </italic>ranging from 0 to 17% across three clusters of villages [##UREF##5##20##]. Between the 1940s and 2005 there were only three malaria infection surveys across five villages in the Lower Shabelle area of the south-central zone [##UREF##6##21##, ####REF##3055452##22##, ##REF##2696079##23####2696079##23##]. Based on limited entomological data, malaria transmission is thought to be supported almost entirely by <italic>Anopheles arabiensis </italic>[##REF##13693304##24##, ####UREF##7##25##, ##UREF##8##26####8##26##].</p>", "<title>Assembling the survey data on parasite prevalence</title>", "<p>Following the dearth of <italic>P. falciparum </italic>parasite rate (<italic>Pf</italic>PR) surveys over the last 50 years, community-based surveys of <italic>Pf</italic>PR have become a routine undertaking across the country since 2005. These surveys have been embedded in two major activities. First, a national malaria indicator survey was conducted by the WHO between January and February 2005 in the south-central and north-west zones [##UREF##9##27##] and in July 2005 in the north-east [##UREF##10##28##]. A stratified multi-stage random sampling strategy was adopted. Within each zone all regions were sampled and out of 120 districts in these regions, 88 were selected at random. Randomly selected villages within each district were surveyed successively until the required number of respondents of all ages (at least 845 per region) was achieved. Second, the United Nations Food Agricultural Organization-Food Security Analysis Unit (FAO/FSAU) completed 18 independent cluster sample surveys between March and November 2007. Malaria parasitology in all age-groups was included in these routine nutritional surveys at the request of UNICEF. In each survey a stratified multi-stage cluster sampling design was adopted where the sampling frame of a selected district was based on three livelihood definitions (pastoral, agro-pastoral, and riverine) [##UREF##11##29##,##REF##18461178##30##] within which 30 rural communities and 30 households within each community were selected at random.</p>", "<p>In all surveys, evidence of parasitaemia was determined using <italic>P. falciparum </italic>specific Rapid Diagnostic Tests (RDTs). WHO used ParaHIT – f™ Device (Span Diagnostics Limited, Surat, India) while FSAU used Paracheck Pf™ (Orchid Biomedical Systems, Goa, India). The purpose of the survey was explained to each household head or adult representative from whom informed consent was then sought prior to undertaking parasitological tests. All individuals who tested positive for infection were treated with nationally recommended first-line therapy [##UREF##12##31##]. An inclusion criterion of a minimum of 40 individuals per community was used to select villages to include in the analysis to minimize random variation inherent in small samples [##UREF##13##32##,##REF##16531119##33##].</p>", "<p>Survey data from all three sources were combined into a single database. Where communities had been surveyed more than once, the survey with the largest sample size was selected. A detailed search was undertaken to establish a set of spatial coordinates for each community. For some of the later surveys undertaken by FSAU, global positioning systems (GPS) were used to provide a longitude and latitude. For the remaining settlements a combination of electronic gazetteers [##UREF##14##34##,##UREF##15##35##] and other nationally derived UN sources of longitude and latitude [##UREF##16##36##] were used to locate the community. Finally, the location of each settlement was verified by using Google Earth (Google, Seattle, USA) to visually inspect whether the coordinates matched evidence of human settlement. Those settlements for which no reliable source of the coordinates could be obtained were excluded from the analysis.</p>", "<p>The assembled <italic>Pf</italic>PR data locations were not distributed evenly across Somalia with a higher concentration in the South-Central zone. Exploratory analysis showed different spatial autocorrelation structures for the two zones (Figure ##FIG##1##2##) and a single geostatistical model for the whole country was deemed inappropriate. To allow representative models to be developed in each region, the data were split and analysis was carried out separately for the north (north-west and north-east states combined) and the south (south-central state) (Figure ##FIG##1##2##).</p>", "<title>Outlier detection</title>", "<p>Geostatistical methods are particularly sensitive to outlying values that exert a significant effect on predictions. Extreme outliers were therefore identified and excluded using a spatial filter. The method assumes that the probability of an unusually large <italic>Pf</italic>PR value being a genuine 'outlier' is larger if (a) it is in a neighbourhood of generally much smaller values and/or (b) the neighbourhood is generally uniform. A spatial filtering algorithm was implemented that incorporated these heuristic considerations (Additional File ##SUPPL##0##1##).</p>", "<title>Selection of covariates</title>", "<p>Climatic and survey covariates were considered for inclusion in the spatial prediction model. The following four climatic variables were considered, each of which was resampled to 5 × 5 km resolution to be consistent with the prediction grid. 1) The <italic>enhanced vegetation index </italic>(EVI) derived from the global Moderate Resolution Imaging Spectroradiometer (MODIS) satellite imagery for the period 2001–2005 [##UREF##17##37##]. Temporal Fourier analysis was undertaken to derive a global EVI index [##REF##18183289##38##]. These data were available for each month at 1 × 1 km spatial resolution and obtained from a global archive developed recently by the Spatial Ecology and Epidemiology Group of the Department of Zoology, University of Oxford [##REF##18183289##38##]. Scaled EVI values ranged from 0–1, representing no, to complete vegetation cover. 2) <italic>Precipitation and temperature </italic>data described as the average monthly precipitation and temperature (minimum and maximum) at 1 × 1 km spatial resolution were downloaded from the WorldClim website [##UREF##18##39##]. These climate surfaces were developed through interpolation of global meteorological data collected from 1950–2000 [##UREF##19##40##]. 3) <italic>Distance to permanent water bodies </italic>was derived for each survey location using information provided by Africover [##UREF##20##41##] and those of marshes, flood plains and intermittent wetland from the Global Lakes and Wetlands databases [##UREF##21##42##]. 4) The effect of month of survey was assessed because of the observed temporal heterogeneity of <italic>Pf</italic>PR data. February was selected as the \"reference month\" for both zones as this was the earliest calendar month in which surveys were undertaken.</p>", "<p>The annual mean of each environmental covariate (derived from the monthly data) was extracted at each survey location using ArcGIS 9.1 (ESRI Inc., USA). To assess the effects of the covariates on observed PfPR, non-spatial binomial logistic regression models were implemented in Stata/SE Version 10 (Stata Corporation, College Station, TX, USA). With PfPR as the dependent variable, bivariate binomial logistic regression models were fitted and covariates with Wald's P &gt; 0.2 were excluded from subsequent analyses. Collinearity among all remaining covariates was assessed and if a pair had a correlation coefficient &gt; 0.9, the variable with the highest value of Akaike Information Criterion (AIC) was discarded. To select which of the two temperature variables (maximum and minimum) to include into the multivariate model, the one with lowest value of AIC was chosen [##REF##17892584##9##]. A non-spatial binomial multivariate logistic regression was then fitted, starting with a saturated model, and then seeking a parsimonious model using backwards variable elimination with an exit criterion of Wald's <italic>P </italic>&gt; 0.2. Variables that exhibited non-linear relationships with PfPR were dichotomized at the median.</p>", "<title>Bayesian geostatistical models</title>", "<p>Bayesian geostatistical (kriging) techniques provide a framework for predicting (interpolating) values of a variable of interest at unobserved locations given a set of spatially distributed data, incorporating spatial autocorrelation and computing uncertainty measures around model predictions [##UREF##22##43##,##REF##15690999##44##]. Spatial autocorrelation in the Somalia <italic>Pf</italic>PR data was therefore first evaluated by computing empirical variograms, a graphical summary of spatial autocorrelation structure, separately for the south and the north. Different variogram structures were observed for the two zones indicating that a single stationary model for the whole country was inappropriate. Comparison of the variograms suggested greater heterogeneity of observed parasite prevalence data in the south than in the north consistent with expert opinion of the transmission dynamics across the country [##UREF##8##26##]. Consequently models were constructed separately for each zone. Bayesian binomial generalized linear geostatistical models [##UREF##22##43##] were implemented in each zone with the spatial component modelled as a stationary Gaussian process with mean of zero and covariance structure defined by a powered exponential function [##UREF##23##45##]. Because survey data were modelled as a conditionally binomial variable, given the underlying Gaussian process, the variance due to sample size was accounted for implicitly. The models were implemented in WinBUGS Version 1.4 (MRC Biostatistics Unit, Cambridge, UK). The models were constructed with and without the covariates in order to compare differences in model fit. Model fit was based on the deviance information criterion (DIC). Models with a smaller DIC value (with a difference &gt;5) were considered to represent a better compromise between parsimony and fit [##UREF##24##46##]. The rate of decay of correlation between points (ϕ) with distance and the variance of the spatial process (σ²) were also recorded. The form of these models is shown in Additional File ##SUPPL##1##2##.</p>", "<p>Predictions at non-sampled locations (defined over a regular 5 × 5 km grid overlaying the entire country) were made using the <italic>spatial.unipred </italic>function in WinBUGS which solves the model equation at each prediction location given the values of each covariate at the prediction location and the distance between prediction locations and observed data locations. Coefficients and model diagnostics were estimated using Markov Chain Monte Carlo (MCMC) simulations. The posterior probability distributions were used to classify prediction points to an endemicity class. Probability of class membership was computed as an additional measure to identify areas of high model uncertainties. For presentation purposes prediction maps from the best-fit model were combined for both zones. Continuous and categorical representations of predicted prevalence and probability maps were produced. The categorical classes of <italic>Pf</italic>PR selected were 0–&lt;5% (low risk); 5–39% (medium risk); and ≥40% (high risk) and are based on a review of endemicity classification that would be most suitable as a guides for the likely impact of existing interventions [##REF##18387849##47##].</p>", "<title>Model validation</title>", "<p>A spatially de-clustered random sampling strategy was implemented to generate validation sets that could be considered spatially representative of the prediction space. Thiessen polygons, which enclose the area closest to a given point, were defined around each survey location. A 10% sample or a minimum of 30 survey locations (whichever was larger) were then drawn randomly for the north and the south with each data point having a probability of selection proportional to the area of its Thiessen Polygon. This meant data located in densely surveyed regions had a lower probability of selection than those in sparsely surveyed regions [##UREF##25##48##]. The Bayesian geostatistical models were then repeated without the validation dataset. Predicted <italic>Pf</italic>PR values from the Bayesian geostatistical models were compared to actual <italic>Pf</italic>PR values observed at the validation locations using the mean error (ME), mean absolute error (MAE) and the area under the curve (AUC) of the receiver operating characteristic (ROC). ME is a measure of the bias of predictions (the overall tendency to over or under predict) whilst MAE is a measure of overall precision (the average magnitude of error in individual predictions) and AUC is a measure of discriminatory ability of predictions with respect to a true prevalence threshold (observed endemicity classes). AUC values greater than 0.9 indicate excellent discrimination; &gt;0.7 moderate discrimination; &gt;0.5 poor discrimination; and &lt;0.5, the model does not discriminate any more successfully than random allocation of test status [##REF##16553932##49##,##UREF##26##50##].</p>", "<title>Ethical approval</title>", "<p>Ethical approval was provided through permission by the Ministry of Health Somalia, Transitional Federal Government of Somalia Republic, Ref: MOH/WC/XA/146./07, dated 02/02/07. Informed verbal consent was sought from all participating households and individuals.</p>" ]

[ "<title>Results</title>", "<title>Sample description</title>", "<p>A total of 500 independent community data locations with sample size ≥ 40 were assembled from the two survey sources (Table ##TAB##0##1##). Of these, 48 (9.6%) communities with sample size ≥ 40 could not be spatially positioned and were excluded from the analysis. Of the remaining 452 survey locations, 115 (25%) were located in the north and 337 (75%) in the south (Table ##TAB##0##1## and Figure ##FIG##0##1##). Two locations in the north and one in the south were detected as outliers (Table ##TAB##0##1## and Figure ##FIG##1##2##). The mean <italic>Pf</italic>PR of the data, excluding the outliers, was 2.8% (95% CI: 2.1–4.6, n = 10,468) for the north and 11.4% (95% CI: 10.4–14.2, n = 20,011) for the south.</p>", "<title>Non-spatial bivariate and multivariate analysis of covariates</title>", "<p>Both minimum and maximum temperatures exhibited non-linear relationships with <italic>Pf</italic>PR and were dichotomized at the median. No pairs of covariates demonstrated collinearity. During the non-spatial bivariate logistic regression analysis EVI, precipitation, maximum and minimum temperature, distance to water and survey month all displayed a highly significant association with <italic>Pf</italic>PR and met the initial entry criteria for the non-spatial multivariate logistic models for both the north and south (Table ##TAB##1##2##). Minimum rather than maximum temperature was selected for inclusion. When the selected variables were examined together in the multivariate model, EVI was not significantly associated with <italic>Pf</italic>PR in the north (p = 0.223) or the south (p = 0.395) (Table ##TAB##1##2##). Excluding EVI and maximum temperature, all remaining variables were entered into the multivariate Bayesian geostatistical model (Table ##TAB##2##3##).</p>", "<title>Bayesian geostatistical models</title>", "<title>North</title>", "<p>The Bayesian geostatistical model without covariates in the north had <italic>σ</italic>²(variance of spatial process) with a posterior median of 4.35 (95% credible interval (CI): 2.71, 7.14); <italic>ϕ </italic>(rate of decay of spatial correlation) of 8.90 (95% CI: 3.11, 12.75); a DIC value (measure of model fit) of 326; a ME (measure of model bias) of 3.83% <italic>Pf</italic>PR; and a MAE (measure of model precision) of 4.12% <italic>Pf</italic>PR (Table ##TAB##2##3##). The results of the multivariate Bayesian geostatistical model showed that none of the selected covariates remained significant (odds 95% CI included 1) after accounting for spatial correlation (Table ##TAB##2##3##). The DIC of the multivariate model was 323 and only marginally lower than that of the model without covariates, implying that the inclusion of the covariates did not improve overall model fit in the north. However, the covariates did account for some of the spatial variation in the data with spatial variance (<italic>σ</italic>²) decreasing from 4.35 to 3.70 (Table ##TAB##2##3##). Although the model with covariates exhibited lower bias (ME = 2.56% <italic>Pf</italic>PR) it also had marginally lower precision (MAE = 4.75% <italic>Pf</italic>PR) compared to the model without covariates. AUC values were similar for both models and indicated acceptable overall fit for endemicity class &lt;5% <italic>Pf</italic>PR (AUC values &gt; 0.70) but less so for endemicty class 5–39% <italic>Pf</italic>PR (AUC values of &lt; 0.70). There were insufficient data points in the validation set to compute AUC values for the endemicity class ≥ 40% <italic>Pf</italic>PR (Table ##TAB##2##3##).</p>", "<title>South</title>", "<p>In the south the posterior median variance of the spatial process for the model without covariates was 7.14 (95% Bayes CI: 5.00, 8.76) and that of the rate of spatial decay parameter was 4.79 (95% Bayes CI: 2.11, 6.79). For the model with covariates the month of survey, annual average maximum temperature and precipitation all remained significant explanatory factors for <italic>Pf</italic>PR (Table ##TAB##2##3##). Odds of <italic>P. falciparum </italic>infection were higher in March (OR: 4.06, 95% CI 2.20–7.63) relative to February; and with increasing precipitation (OR: 1.41, 1.07–1.94). Higher minimum temperatures (OR: 0.83, 0.67–0.96) and a survey month of November relative to February (OR 0.48, 0.23–0.96) both reduced the odds of <italic>P. falciparum </italic>infection (Table ##TAB##2##3##). The inclusion of the covariates improved model fit with DIC of 1,429 compared to 1,454 for the model without covariates. There were no clear differences, however, between the models with and without covariates in the other parameters of model assessment with values of ME (3.65% vs 4.14% <italic>Pf</italic>PR); MAE (5.00% vs 5.06% <italic>Pf</italic>PR) and AUC values (&lt;5% <italic>Pf</italic>PR: 0.91 vs 0.87; 5–39% <italic>Pf</italic>PR: 0.81 vs 0.78; ≥ 40% <italic>Pf</italic>PR: 0.51 vs 0.56). Similar to the multivariate model in the south, most of the spatial residual variation remained unexplained by the covariates.</p>", "<p>Overall, the models for the south (with or without covariates) had higher spatial variances and spatial autocorrelation occurred over larger distances compared to the north (Table ##TAB##2##3##). In addition, models in the south exhibited better model fit with AUC values higher across all endemicity classes probably due to greater availability and better distribution of data in this zone (Figure ##FIG##1##2##).</p>", "<title>Predicted (posterior median) <italic>Pf</italic>PR maps</title>", "<p>To maintain consistency for comparison, and because the inclusion of the covariates did account for some degree of spatial variation in the <italic>Pf</italic>PR data, the predictions based on the multivariate geostatistical models were retained for both zones (Figures ##FIG##2##3a## and ##FIG##2##3b##). Point estimates of <italic>Pf</italic>PR (based on the posterior median) for the prediction locations ranged from 0–9% (median = 0%) in the north. The majority of the area in this zone had predicted a <italic>Pf</italic>PR of &lt;5% with a small number of locations in Puntland and on the south-western border between Somaliland and Ethiopia having <italic>Pf</italic>PR of &gt;5–9% (Figure ##FIG##2##3b##). In the south, point estimates of <italic>Pf</italic>PR for prediction locations ranged from 0–52% (median 5%), with high <italic>Pf</italic>PR locations occurring in the densely populated farmlands between the Juba and Shabelle rivers (Figure ##FIG##2##3b##). The lower and upper 95% credible intervals posterior median <italic>Pf</italic>PR are shown in Additional File ##SUPPL##2##3##. Interestingly, predicted <italic>Pf</italic>PR was lower along the two rivers, particularly the Shabelle River, compared to the area in between. Overall, predictions of endemicity class membership in the north were associated with lower probabilities compared to those in the south indicating a higher degree of model uncertainty (Figure ##FIG##2##3c##).</p>" ]

[ "<title>Discussion</title>", "<p>There has been little historical description of the basic epidemiology of malaria transmission in Somalia. In 2002, an application was made to and successfully approved by the GFATM to support the funding of a suite of interventions and strategies managed by a consortium of non-government and governmental agencies across the three main zones of Somalia [##UREF##27##51##]. This application, similar to other successful applications and RBM policies in neighbouring, higher-intensity transmission countries of Kenya [##UREF##28##52##], Tanzania [##UREF##29##53##] and Uganda [##UREF##30##54##] involved a monitoring &amp; evaluation component to investigate intervention coverage and <italic>P. falciparum </italic>infection rates. In Somalia rapid, sample malaria intervention and parasitological surveys of communities have now become part of a routine component of rolling nutritional surveillance surveys across the country [##UREF##31##55##]. Consequently, despite being a country without a functioning research capacity and a fragile health system, Somalia is now one of the 87 <italic>P. falciparum </italic>endemic countries worldwide with the largest series of infection prevalence data [##REF##18303939##56##,##UREF##32##57##].</p>", "<p>Simple summaries of the data suggest that large parts of the country, particularly in the north, have very low human infection prevalence (Table ##TAB##0##1##). These summaries, however, mask spatial heterogeneities in risk that are important for better targeting of interventions and maintaining aggressive surveillance. A Bayesian geostatistical approach to predict <italic>Pf</italic>PR throughout Somalia is used here. In the north, the inclusion of the survey and environmental covariates appeared not to make a significant difference to model fit, while in the south they improved the model fit. Predictions of endemicity class membership made in the north were associated with lower prediction probabilities and generated generally lower AU-ROC values (Table ##TAB##2##3## &amp; Figure ##FIG##2##3c##). This greater prediction uncertainty in the north is due largely to the comparatively fewer empirical data points compared to the south (Figure ##FIG##1##2##). This disparity was essentially driven by the population distribution: approximately 65% of Somalia's population live in the South and communities in the North are more scattered in isolated settlements [##UREF##33##58##].</p>", "<p>Although the environmental covariates selected for inclusion in the Bayesian geostatistical model were significantly associated with <italic>Pf</italic>PR when examined in the non-spatial multivariate model (Tables ##TAB##1##2## &amp;##TAB##2##3##), none remained significant when spatial correlation was accounted for in the north and only precipitation and temperature remained significant in south. Overall the inclusion of these covariates accounted for a relatively small proportion of spatial variation suggesting that other unmeasured factors might be influencing the spatial distribution of malaria prevalence. These factors might include proximity to artificial breeding sites such as wells, dams, boreholes and seasonal streams and/or the use of interventions to prevent malaria at the household level. It has recently been demonstrated that in southern Somalia, the use of insecticide treated nets (ITN) reduced the prevalence of infection by as much as 54% [##REF##18461178##30##]. Mapping the household or community levels of ITN use at high spatial resolutions is not currently feasible at a national scale. Similarly, the mapping of fluctuating, localized vector breeding sites would require very detailed spatial reconnaissance and infection mapping at finer scales than is currently possible using public domain covariate data at national scales. Furthermore, communities where sample sizes were less than 40, most of which could not be geo-located, were excluded from the analysis and these might have resulted in information loss for some areas of Somalia. Although the difference, in terms of mean parasite prevalence, was minimal between the excluded and included surveys, future analysis should include all data regardless of sample sizes given the Bayesian analytical approach implicitly adjusts for sample size.</p>", "<p>Despite the constraints described above, the use of Bayesian geostatistics to model <italic>Pf</italic>PR does provide a valuable method to define sub-national spatial variation in prevalence, and a baseline against which future changes in prevalence can be quantified intervention coverage expands. Under such a scenario the value of the environmental covariates might be expected to wane further, particularly in areas of very low transmission intensity where the environment currently supports homogenously low transmission conditions. The similar levels of performance observed between the univariate and multivariate models for the north of Somalia may be evidence of this view. In addition, the relatively higher coverage of ITN among the communities closest to the two rivers in the south might explain the lower predicted prevalence in their immediate vicinity consistent with the observational data and reported effectiveness of ITN [##REF##18461178##30##].</p>", "<p>Population density or a derived categorisation of urbanisation, with known influences on malaria transmission [##UREF##34##59##,##REF##15589793##60##], would have been a worthy candidate covariate for testing in this study and in determining accurately the population at risk against varying malaria endemicity. However, the reliability of settlement and population data in Somalia is highly questionable. The last national census was undertaken in 1971 and the displacement and migration over the last 20 years of civil unrest has been substantial. Development agencies and non-governmental agencies working in Somalia continue to update a semi-quantitative database of settlement locations and population counts but its fidelity is unknown. The absence of an accurate national census also hampers the linkage of spatial malaria risk to populations-exposed to risk. Notwithstanding the precision and scale of calculating populations at risk, aggregated district-level estimates of population in 2004 across the 120 districts of Somalia have been used and assigned each district the dominant <italic>Pf</italic>PR risk class. From these numbers it can be estimated that approximately 75% of Somalia's estimated 7.4 million people live in areas that support unstable or very low <italic>Pf</italic>PR (0–5%) transmission and less than 0.1% live in areas classified as high, intense transmission (<italic>Pf</italic>PR &gt; 40%). Areas of low <italic>Pf</italic>PR include many communities where infection prevalence was observed as zero (Table ##TAB##0##1##). In these locations it is assumed that these observations represent a statistical zero (i.e. resulting from a limited sample in areas of very low transmission) rather than implying a true absence of infection risk [##REF##18303939##56##]. This is important to highlight because routine sample surveys in such areas demand considerably larger samples [##UREF##23##45##,##REF##5074687##61##] or the use of serological markers of parasite exposure [##REF##15792998##62##] to truly exclude the possibility of transmission.</p>", "<p>In communities exposed to low <italic>Pf</italic>PR, such as the majority of the population in Somalia, the risk of disease is low and spread across all age-groups. These are fundamentally different epidemiological conditions to areas of high transmission where functional immunity is developed early in life and a higher disease burden is experienced in young children and pregnant women [##REF##15759000##63##, ####REF##12521262##64##, ##REF##9186382##65##, ##REF##15275204##66####15275204##66##]. Tailoring the existing intervention recommendations in the Somalia National Malaria Strategy [##UREF##35##67##] to the spatial transmission patterns shown in Figure ##FIG##2##3## will be a challenge to the agencies providing malaria control services nationwide.</p>" ]

[ "<title>Conclusion</title>", "<p>The use of routine, nationwide surveillance of infection prevalence is key to monitoring the changing epidemiology of malaria in all countries scaling up coverage of malaria preventative strategies. Including RDTs in on-going community-based health surveillance is a cost-effective means of assembling this information. The use of geostatistical methods can help focus surveillance efforts and define those areas where uncertainty exists, guiding future sampling [##REF##16553932##49##,##REF##15635963##68##]. Coupled with better estimates of where people live, these should provide the basis for informed estimates of disease burden [##REF##15759000##63##] and how these might change with changing infection-risk exposure. Somalia has a range of political and economic barriers that might limit the success of a strategic, epidemiologically driven malaria control programme. It has been possible to demonstrate, however, that the foci of greatest disease risk are predominantly concentrated in one area in the South and that infection risks are very low in the northern reaches of the country. Moreover, although the density of survey sites and hence the uncertainty of the modelled output varies spatially, also it has been demonstrated that, despite constant civil disturbance, routine survey data can be assembled to inform strategic decision making. Finally, areas where model uncertainties are greatest, predominantly in the north of the country, should be the focus of any future parasitological surveys to improve further the precision of the prevalence maps.</p>" ]

[ "<p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/2.0\"/>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>", "<title>Background</title>", "<p>Maps of malaria distribution are vital for optimal allocation of resources for anti-malarial activities. There is a lack of reliable contemporary malaria maps in endemic countries in sub-Saharan Africa. This problem is particularly acute in low malaria transmission countries such as those located in the horn of Africa.</p>", "<title>Methods</title>", "<p>Data from a national malaria cluster sample survey in 2005 and routine cluster surveys in 2007 were assembled for Somalia. Rapid diagnostic tests were used to examine the presence of <italic>Plasmodium falciparum </italic>parasites in finger-prick blood samples obtained from individuals across all age-groups. Bayesian geostatistical models, with environmental and survey covariates, were used to predict continuous maps of malaria prevalence across Somalia and to define the uncertainty associated with the predictions.</p>", "<title>Results</title>", "<p>For analyses the country was divided into north and south. In the north, the month of survey, distance to water, precipitation and temperature had no significant association with <italic>P. falciparum </italic>prevalence when spatial correlation was taken into account. In contrast, all the covariates, except distance to water, were significantly associated with parasite prevalence in the south. The inclusion of covariates improved model fit for the south but not for the north. Model precision was highest in the south. The majority of the country had a predicted prevalence of &lt; 5%; areas with ≥ 5% prevalence were predominantly in the south.</p>", "<title>Conclusion</title>", "<p>The maps showed that malaria transmission in Somalia varied from hypo- to meso-endemic. However, even after including the selected covariates in the model, there still remained a considerable amount of unexplained spatial variation in parasite prevalence, indicating effects of other factors not captured in the study. Nonetheless the maps presented here provide the best contemporary information on malaria prevalence in Somalia.</p>" ]

[ "<title>Abbreviations</title>", "<p>AIC: Akaike Information Criterion; AUC-ROC: Area under the curve of the receiver operating characteristic; DIC: Deviance Information Criterion; EVI: Enhanced Vegetation Index; FAO/FSAU: Food Agricultural Organization-Food Security Analysis Unit (FSAU); GFATM: Global Fund to Fight Aids TB and Malaria; ITN: Insecticide treated nets; MAE: Mean absolute error; MAP: The Malaria Atlas Project; MCMC: Markov Chain Monte Carlo; ME: Mean Error; MODIS: Moderate Resolution Imaging Spectroradiometer; <italic>Pf</italic>PR: <italic>Plasmodium falciparum </italic>parasite rate; RDT: Rapid Diagnostics Test; SSA: sub-Saharan Africa; UNICEF: United Nations Children Fund; WHO: World Health Organization.</p>", "<title>Authors' contributions</title>", "<p>AMN was responsible for data cleaning, analysis, interpretation and production of the final manuscript and revisions, ACC contributed to the data analysis, interpretation and production of final manuscript, PWG contributed to the data analysis, interpretation and production of final manuscript, GM was responsible for the study design, supervision of data collection, cleaning and contributed to the preparation of the final manuscript, MB provided the necessary interface with community leaders, and the Ministry of Health for approval and was responsible for the data collection, cleaning and contributed to the preparation of the final manuscript, TS assisted in the survey design, supported the field investigations, provided the interface with local ministry of health and helped in the preparation of the manuscript, SIH contributed to overall scientific direction interpretation and preparation of the final manuscript and revisions, RWS was responsible for overall scientific management, analysis, interpretation and preparation of the final manuscript and revisions.</p>", "<title>Supplementary Material</title>" ]

[ "<title>Acknowledgements</title>", "<p>The authors are grateful to the WHO-MERLIN and FAO/FSAU survey team for their invaluable supervision and support during the field surveys and subsequent data entry and cleaning and Bruno Moonen of MERLIN specifically for helping with training for the parasite survey. We thank Priscilla Gikandi and Victor Alegana for additional data cleaning and geo-referencing. We are also grateful to Anand Patil and Andy Tatem for statistical and GIS advise and to Simon Brooker, Carlos Guerra and Emelda Okiro for their comments on the manuscript.</p>", "<p>Funding for the WHO-MERLIN 2005 surveys were provided by the UN Trust Fund for Human Security and the GFATM. FAO/FSAU funded training of assessment teams, data collection, paid enumerators and data entry clerks for the 2007 surveys. The FAO/FSAU nutrition surveillance project is funded primarily by OFDA-USAID and receives support from UNICEF, SIDA and EC for conducting nutrition assessments in Somalia. RDTs and anti-malarial treatment were provided by UNICEF through GFATM funding (SOM-202-G01-M-00). AMN is supported by the Wellcome Trust as a Research Training Fellow (#081829). SIH is supported by the Wellcome Trust as Senior Research Fellow (#079091). RWS is supported by the Wellcome Trust as Principal Research Fellow (#079081). AMN, SIH and RWS acknowledge the support of the Kenyan Medical Research Institute. The funders did not have a role in study design, data collection and analysis, decision to publish, or preparation of manuscript. This work forms part of the output of the Malaria Atlas Project (MAP: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.map.ox.ac.uk\"/>), principally funded by the Wellcome Trust, U.K.</p>" ]

[ "<fig position=\"float\" id=\"F1\"><label>Figure 1</label><caption><p>Map of Somalia showing the self-declared states of Puntland, Somaliland and South Central; their capital cities; and the two main rivers of Juba and Shabelle.</p></caption></fig>", "<fig position=\"float\" id=\"F2\"><label>Figure 2</label><caption><p><bold>Map and variograms of north and south of Somalia showing the distribution PR data locations (the X-axis shows distance as degrees latitude and longitude).</bold> The data were distributed as model (n = 113 north; n = 336 south) and outlier (n = 2 north; n = 1 south) locations. Variograms are of the data after outliers were removed.</p></caption></fig>", "<fig position=\"float\" id=\"F3\"><label>Figure 3</label><caption><p><bold>Maps of north and south of Somalia showing: a) posterior median <italic>Pf</italic>PR; b) endemicity classes based on the posterior probability of <italic>Pf</italic>PR class membership <italic>Pf</italic>PR; c) probability that a prediction location is correctly classified to an endemicity class: &lt;5%; 5–39%; and ≥40% <italic>Pf</italic>PR.</bold> Probabilities &lt; 0.33 are considered no better than chance class assignment. In Somalia probabilities were all ≥ 0.45.</p></caption></fig>" ]

[ "<table-wrap position=\"float\" id=\"T1\"><label>Table 1</label><caption><p>Summary of Somalia survey data by source for the North and South zones.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"3\"><bold>Survey source</bold></td></tr><tr><td align=\"left\"><bold>Zone</bold></td><td align=\"center\"><bold>FAO/FSAU</bold><break/><bold> n (%)</bold></td><td align=\"center\"><bold>WHO</bold><break/><bold> n (%)</bold></td><td align=\"center\"><bold>Total</bold><break/><bold> n (%)</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>North</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Number survey locations sampling with 40+ people</td><td align=\"center\">64</td><td align=\"center\">61</td><td align=\"center\">125</td></tr><tr><td align=\"left\">Number geo-referenced</td><td align=\"center\">55 (85.9%)</td><td align=\"center\">60 (98.4%)</td><td align=\"center\">115 (92.0%)</td></tr><tr><td align=\"left\">Number identified as outliers</td><td align=\"center\">0 (0.0%)</td><td align=\"center\">2 (3.3%)</td><td align=\"center\">2 (1.6%)</td></tr><tr><td align=\"left\">Number selected for model*</td><td align=\"center\">55 (85.9%)</td><td align=\"center\">58 (95.1%)</td><td align=\"center\">113 (90.4%)</td></tr><tr><td align=\"left\"> Number of surveys with zero <italic>Pf</italic>PR**</td><td align=\"center\">32 (50.0%)</td><td align=\"center\">26 (42.6%)</td><td align=\"center\">58 (46.4%)</td></tr><tr><td align=\"left\"> Population sample size</td><td align=\"center\">5,213</td><td align=\"center\">5,255</td><td align=\"center\">10,468</td></tr><tr><td align=\"left\"> Number <italic>Pf</italic>PR positive</td><td align=\"center\">97</td><td align=\"center\">196</td><td align=\"center\">293</td></tr><tr><td align=\"left\"> Mean (Median) <italic>Pf</italic>PR (%)</td><td align=\"center\">1.8 (0.0)</td><td align=\"center\">3.7 (1.1)</td><td align=\"center\">2.8 (0.0)</td></tr><tr><td align=\"left\"> IQR <italic>Pf</italic>PR (%)</td><td align=\"center\">(0.0, 3.1)</td><td align=\"center\">(0.0, 4.0)</td><td align=\"center\">(0.0, 4.0)</td></tr><tr><td align=\"left\"><bold>South</bold></td><td/><td/><td/></tr><tr><td align=\"left\">Number survey locations sampling 40+ people</td><td align=\"center\">311</td><td align=\"center\">64</td><td align=\"center\">375</td></tr><tr><td align=\"left\">Number geo-referenced</td><td align=\"center\">279 (89.7%)</td><td align=\"center\">58 (90.6%)</td><td align=\"center\">337 (89.9%)</td></tr><tr><td align=\"left\">Number identified as outliers</td><td align=\"center\">1 (0.3%)</td><td align=\"center\">0 (0.0%)</td><td align=\"center\">1 (0.3%)</td></tr><tr><td align=\"left\">Number selected for model*</td><td align=\"center\">278 (89.4%)</td><td align=\"center\">58 (90.6%)</td><td align=\"center\">336 (89.6%)</td></tr><tr><td align=\"left\"> Number of surveys with zero <italic>Pf</italic>PR**</td><td align=\"center\">73 (23.5%)</td><td align=\"center\">23 (35.9%)</td><td align=\"center\">96 (25.6%)</td></tr><tr><td align=\"left\"> Population sample size</td><td align=\"center\">16,048</td><td align=\"center\">3,963</td><td align=\"center\">20,011</td></tr><tr><td align=\"left\"> Number <italic>Pf</italic>PR positive</td><td align=\"center\">2,081</td><td align=\"center\">208</td><td align=\"center\">2,289</td></tr><tr><td align=\"left\"> Mean (Median) <italic>Pf</italic>PR (%)</td><td align=\"center\">13.0 (4.0)</td><td align=\"center\">5.2 (2.0)</td><td align=\"center\">11.4 (3.0)</td></tr><tr><td align=\"left\"> IQR <italic>Pf</italic>PR (%)</td><td align=\"center\">(0.0,10.0)</td><td align=\"center\">(0.0, 6.0)</td><td align=\"center\">(0.0, 9.0)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T2\"><label>Table 2</label><caption><p>Non-spatial bivariate and multivariate analysis of the association of survey and environmental covariates with <italic>Pf</italic>PR in north and south of Somalia.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td/><td align=\"center\" colspan=\"2\"><bold>Bivariate model</bold></td><td align=\"center\" colspan=\"2\"><bold>Multivariate model</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Zone</bold></td><td align=\"center\"><bold>Odds ratio (95% </bold><break/><bold>Confidence</bold><break/><bold> Interval)</bold></td><td align=\"center\"><bold>P-value</bold></td><td align=\"center\"><bold>Odds ratio (95%</bold><break/><bold>Confidence</bold><break/><bold> Interval)</bold></td><td align=\"center\"><bold>P-value</bold></td></tr><tr><td colspan=\"5\"><hr/></td></tr><tr><td align=\"left\"><bold>North</bold></td><td/><td/><td/><td/></tr><tr><td align=\"left\">Average annual Enhanced Vegetation Index</td><td align=\"center\">1.03 (1.06–1.11)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">1.97 (0.24–1.63)</td><td align=\"center\">0.223</td></tr><tr><td align=\"left\">Average annual precipitation</td><td align=\"center\">0.98 (0.97–0.99)</td><td align=\"center\">0.002</td><td align=\"center\">1.04 (1.02–1.08)</td><td align=\"center\">0.002</td></tr><tr><td align=\"left\">Average annual minimum temperature</td><td/><td/><td/><td/></tr><tr><td align=\"left\">&lt;median of 20.4°C</td><td align=\"center\">Ref</td><td align=\"center\">-</td><td align=\"center\">Ref</td><td align=\"center\">-</td></tr><tr><td align=\"left\">&gt;median of 20.4°C</td><td align=\"center\">2.02 (1.59–2.58)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">1.35 (1.01–1.80)</td><td align=\"center\">0.045</td></tr><tr><td align=\"left\">Average annual maximum temperature</td><td/><td/><td/><td/></tr><tr><td align=\"left\">&lt;median of 32.4°C</td><td align=\"center\">Ref</td><td align=\"center\">-</td><td align=\"center\">Ref</td><td align=\"center\">-</td></tr><tr><td align=\"left\">&gt;median of 32.4°C</td><td align=\"center\">2.63 (2.04–3.39)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">-</td><td align=\"center\">-</td></tr><tr><td align=\"left\">Distance to water features (km)</td><td align=\"center\">0.61 (0.49–.76)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">0.68 (0.46–0.99)</td><td align=\"center\">0.049</td></tr><tr><td align=\"left\">Survey month</td><td/><td/><td/><td/></tr><tr><td align=\"left\"> February</td><td align=\"center\">Ref</td><td align=\"center\">-</td><td align=\"center\">Ref</td><td align=\"center\">-</td></tr><tr><td align=\"left\"> July</td><td align=\"center\">2.83 (2.24–3.59)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">3.24 (2.37–4.44)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\"> September</td><td align=\"center\">0.08 (0.04–0.16)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">0.10 (0.04–0.19)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\"> November</td><td align=\"center\">1.66 (1.29–2.14)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">1.58 (1.13–2.22)</td><td align=\"center\">0.008</td></tr><tr><td align=\"left\"><bold>South</bold></td><td/><td/><td/><td/></tr><tr><td align=\"left\">Average annual Enhanced Vegetation Index</td><td align=\"center\">1.09 (1.05–1.19)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">1.60 (0.54–4.70)</td><td align=\"center\">0.215</td></tr><tr><td align=\"left\">Average annual precipitation</td><td align=\"center\">1.03 (1.02–1.04)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">1.02 (1.03–1.04)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\">Average annual minimum temperature</td><td/><td/><td/><td/></tr><tr><td align=\"left\">&lt;median of 22.1°C</td><td align=\"center\">Ref</td><td align=\"center\">-</td><td align=\"center\">Ref</td><td/></tr><tr><td align=\"left\">&gt;median of 22.1°C</td><td align=\"center\">0.43 (0.39–0.48)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">0.61 (0.55–0.68)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\">Average annual maximum temperature</td><td/><td/><td/><td/></tr><tr><td align=\"left\">&lt;median of 33.6°C</td><td align=\"center\">Ref</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">-</td></tr><tr><td align=\"left\">&gt;median of 33.6°C</td><td align=\"center\">2.15 (1.96–2.36)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">-</td><td align=\"center\">-</td></tr><tr><td align=\"left\">Distance to water features (km)</td><td align=\"center\">1.04 (1.01–1.07)</td><td align=\"center\">0.005</td><td align=\"center\">0.84 (0.81–0.87)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\">Survey month</td><td/><td/><td/><td/></tr><tr><td align=\"left\"> February</td><td align=\"center\">Ref</td><td align=\"center\">-</td><td align=\"center\">-</td><td align=\"center\">-</td></tr><tr><td align=\"left\"> March</td><td align=\"center\">3.57 (3.25–3.92)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">7.62 (6.30–9.22)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\"> May</td><td align=\"center\">1.11 (0.95–1.30)</td><td align=\"center\">0.194</td><td align=\"center\">6.03 (4.68,7.79)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\"> June</td><td align=\"center\">0.73 (0.65–0.82)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">1.71 (1.43–2.04)</td><td align=\"center\">0.001</td></tr><tr><td align=\"left\"> November</td><td align=\"center\">0.18 (0.14–0.24)</td><td align=\"center\">&lt;0.001</td><td align=\"center\">0.33 (0.25–0.45)</td><td align=\"center\">&lt;0.001</td></tr><tr><td align=\"left\"> December</td><td align=\"center\">1.10 (0.99–1.22)</td><td align=\"center\">0.056</td><td align=\"center\">1.62 (1.41–1.87)</td><td align=\"center\">&lt;0.001</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"T3\"><label>Table 3</label><caption><p>Summary output of Bayesian geostatistical models for the north and south of Somalia.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><td align=\"left\"><bold>Model/Variables</bold></td><td align=\"center\"><bold>North</bold></td><td align=\"center\"><bold>South</bold></td></tr></thead><tbody><tr><td align=\"left\"><bold>Bayesian geostatistical model (no covariates)</bold></td><td/><td/></tr><tr><td align=\"left\"><italic>α </italic>(Intercept)</td><td align=\"center\">-4.62 (-5.44, -4.33)</td><td align=\"center\">-2.9 (-3.37, -2.27)</td></tr><tr><td align=\"left\"><italic>ϕ </italic>(Decay of spatial correlation (degrees latitude and longitude))</td><td align=\"center\">8.90 (3.11, 12.75)</td><td align=\"center\">4.79 (2.11, 6.97)</td></tr><tr><td align=\"left\"><italic>σ</italic>²(Variance of spatial process)</td><td align=\"center\">4.35 (271, 7.14)</td><td align=\"center\">7.14 (5.00, 8.76)</td></tr><tr><td align=\"left\">DIC</td><td align=\"center\">326</td><td align=\"center\">1,454</td></tr><tr><td align=\"left\">ME (% <italic>Pf</italic>PR)</td><td align=\"center\">3.83</td><td align=\"center\">4.14</td></tr><tr><td align=\"left\">MAE (% <italic>Pf</italic>PR)</td><td align=\"center\">4.12</td><td align=\"center\">5.06</td></tr><tr><td align=\"left\">AU-ROC*</td><td/><td/></tr><tr><td align=\"left\"> &lt;5% <italic>Pf</italic>PR</td><td align=\"center\">0.72 (0.64, 0.86)</td><td align=\"center\">0.87 (0.72, 0.91)</td></tr><tr><td align=\"left\"> 5–39% <italic>Pf</italic>PR</td><td align=\"center\">0.66 (0.51, 0.80)</td><td align=\"center\">0.78 (0.66, 0.85)</td></tr><tr><td align=\"left\"> ≥ 40% <italic>Pf</italic>PR</td><td align=\"center\">NA</td><td align=\"center\">0.56 (0.37, 0.73)</td></tr><tr><td align=\"left\"><bold>Bayesian geostatistical model (with covariates)</bold></td><td/><td/></tr><tr><td align=\"left\"><italic>α </italic>(Intercept)</td><td align=\"center\">-4.62 (-5.23, -4.10)</td><td align=\"center\">-2.86 (-3.79, 2.27)</td></tr><tr><td align=\"left\"><italic>ϕ </italic>(Decay of spatial correlation)</td><td align=\"center\">10.35 (4.70, 12.88)</td><td align=\"center\">5.78 (2.95, 6.99)</td></tr><tr><td align=\"left\"><italic>σ</italic>²(Variance of spatial process)</td><td align=\"center\">3.70 (2.17, 7.14)</td><td align=\"center\">5.00 (3.70, 6.70)</td></tr><tr><td align=\"left\">DIC</td><td align=\"center\">323</td><td align=\"center\">1,429</td></tr><tr><td align=\"left\">ME (% <italic>Pf</italic>PR)</td><td align=\"center\">2.56</td><td align=\"center\">3.65</td></tr><tr><td align=\"left\">MAE (% <italic>Pf</italic>PR)</td><td align=\"center\">4.75</td><td align=\"center\">5.00</td></tr><tr><td align=\"left\">AU-ROC*</td><td/><td/></tr><tr><td align=\"left\"> &lt;5% <italic>Pf</italic>PR</td><td align=\"center\">0.75 (0.64, 0.91)</td><td align=\"center\">0.91 (0.87, 0.99)</td></tr><tr><td align=\"left\"> 5–39% <italic>Pf</italic>PR</td><td align=\"center\">0.64 (0.43, 0.84)</td><td align=\"center\">0.81 (0.70, 0.94)</td></tr><tr><td align=\"left\"> ≥ 40% <italic>Pf</italic>PR</td><td align=\"center\">NA</td><td align=\"center\">0.51 (0.32, 0.83)</td></tr><tr><td/><td align=\"center\" colspan=\"2\"><bold>Odds ratio, (95% Bayes credible interval)</bold></td></tr><tr><td align=\"left\">Month of survey</td><td/><td/></tr><tr><td align=\"left\"> Feb</td><td align=\"center\"><bold>Ref</bold></td><td align=\"center\"><bold>Ref</bold></td></tr><tr><td align=\"left\"> Mar</td><td align=\"center\">-</td><td align=\"center\">4.06 (2.20, 7.63)</td></tr><tr><td align=\"left\"> Jun</td><td align=\"center\">-</td><td/></tr><tr><td align=\"left\"> Jul</td><td align=\"center\">3.25 (0.91, 11.36)</td><td align=\"center\">0.87 (0.48,1.46)</td></tr><tr><td align=\"left\"> Sep</td><td align=\"center\">0.2 (0.02, 1.74)</td><td/></tr><tr><td align=\"left\"> Nov</td><td align=\"center\">1.31 (0.33, 4.36)</td><td align=\"center\">0.48 (0.23, 0.96)</td></tr><tr><td align=\"left\"> Dec</td><td align=\"center\">-</td><td align=\"center\">1.95 (0.91, 3.90)</td></tr><tr><td align=\"left\">Annual average minimum temperature</td><td/><td/></tr><tr><td align=\"left\"> &lt;median of 20.4/22.1 (North/South)°C</td><td align=\"center\"><bold>Ref</bold></td><td align=\"center\"><bold>-</bold></td></tr><tr><td align=\"left\"> &gt;median of 20.4/22.1(North/South)°C</td><td align=\"center\">1.12 (0.84, 1.33)</td><td align=\"center\">0.83 (0.67,0.96)</td></tr><tr><td align=\"left\">Annual average precipitation</td><td align=\"center\">1.70(0.53, 5.44)</td><td align=\"center\">1.41 (1.07, 1.94)</td></tr><tr><td align=\"left\">Distance to water features (km)</td><td align=\"center\">1.22 (0.53, 2.81)</td><td align=\"center\">0.79 (0.74, 1.29)</td></tr></tbody></table></table-wrap>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"S1\"><caption><title>Additional File 1</title><p>A method for identifying outliers. Statistical outliers were identified using a spatial filtering algorithm that implemented the following procedure in turn for each datum <italic>p</italic>(<italic>x</italic>_{<italic>i</italic>}) at location <italic>x</italic>_{<italic>i</italic>}.</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S2\"><caption><title>Additional File 2</title><p>The Bayesian model form developed in WinBUGS without covariates. The univariate Bayesian geostatistical models</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"S3\"><caption><title>Additional File 3</title><p>Maps of north and south of Somalia.</p></caption></supplementary-material>" ]

[ "<table-wrap-foot><p>*Sample size, number positive and the mean, median and IQR of <italic>Pf</italic>PR include those surveys reporting zero number positive for <italic>Pf</italic>PR.</p><p>**At the margins of stable malaria transmission, zero <italic>P. falciparum </italic>parasites in a sample is not necessarily an indication of true absence of parasites but could also be due to low sample sizes or test equipment with low sensitivity [##REF##16531119##33##]. Given this difficulty in distinguishing true zero from sample zero at low <italic>Pf</italic>PR, surveys reporting zero prevalence were included in the analysis.</p><p>IQR = interquartile range; <italic>Pf</italic>PR = <italic>Plasmodium falciparum </italic>parasite rate; FAO/FSAU = Food and Agricultural Organization – Food Security Analysis Unit; WHO = World Health Organization.</p></table-wrap-foot>", "<table-wrap-foot><p>Ref = reference or base outcome</p></table-wrap-foot>", "<table-wrap-foot><p>DIC = deviance information criterion (measure of model fit); ME = mean error (measure of overall bias); MAE = mean absolute error (measure of overall precision); AU-ROC = areas under receiver operating characteristic. Values in parentheses are 95% Bayes credible intervals.</p><p>*There were only two survey locations in the validation set that had <italic>Pf</italic>PR ≥ 40% in the north region meaning reliable AU-ROC values could not be computed for this model.</p></table-wrap-foot>" ]

[ "<graphic xlink:href=\"1475-2875-7-159-1\"/>", "<graphic xlink:href=\"1475-2875-7-159-2\"/>", "<graphic xlink:href=\"1475-2875-7-159-3\"/>" ]

[ "<media xlink:href=\"1475-2875-7-159-S1.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>", "<media xlink:href=\"1475-2875-7-159-S2.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>", "<media xlink:href=\"1475-2875-7-159-S3.doc\" mimetype=\"application\" mime-subtype=\"msword\"><caption><p>Click here for file</p></caption></media>" ]

{ "acronym": [], "definition": [] }

[ "<title>1. INTRODUCTION</title>", "<p>Herpes simplex virus type 2 (HSV-2) is one of the most\nprevalent sexually transmitted infections worldwide [##REF##12353183##1##]. HSV-2 is extremely\ncommon in countries where HIV is endemic, and coinfects over 75% of\nHIV-infected individuals living in or originating from these countries, but the\nHSV-2 seroprevalence is also over 50% in HIV-infected individuals from the\ndeveloped world [##UREF##0##2##, ##UREF##1##3##]. Atypical or\nunusual manifestations of genital herpes are not uncommon in HIV-coinfected\nindividuals, and persistent anogenital lesions due to HSV-2 were among the\nfirst opportunistic infections described in those with the acquired\nimmunodeficiency syndrome (AIDS) [##REF##6272110##4##]. Opportunistic infections may transiently\nworsen during the period of immune reconstitution following the initiation of\nhighly active antiretroviral therapy (HAART), a phenomenon known as immune\nreconstitution inflammatory syndrome (IRIS), with HSV-2 reactivation accounting\nfor up to half of IRIS cases [##REF##16392092##5##]. However, the median time from initiating\nHAART to developing IRIS is three months, and IRIS is rare once stable immune\nreconstitution has been achieved [##REF##16392092##5##]. Antiviral-resistant\nHSV is uncommon, although more frequently encountered among immunocompromised\nindividuals compared to immunocompetent persons [##REF##15494896##6##].</p>", "<p>Hypertrophic anogenital lesions are a rare complication of\nHSV-2 in HIV-coinfected men and women [##REF##12890233##7##–##REF##12852374##10##], generally occurring in the\ncontext of significant immune deficiency or during IRIS [##REF##11737324##11##], and these lesions\nmay be very difficult to diagnose and treat. We present the case of a 35\nyear-old, HIV-infected woman with a recalcitrant hypertrophic vulvar lesion due\nto HSV-2. The development and prolonged persistence of this lesion after\nnear-complete immune reconstitution on HAART implies that there are significant\nresidual defects in host HSV-2 immune control, and that these may have\nimportant clinical implications for patients and their care providers.</p>" ]

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[ "<title>3. DISCUSSION</title>", "<p>We have presented a case of an African\nfemale patient with recalcitrant hypertrophic genital HSV-2, which was\nclinically resistant to standard antiviral therapy and to intravenous\nfoscarnet, but which responded promptly to topical 5% imiquimod. An initial\n“typical” genital herpes outbreak occurred in the context of rapid immune\nrecovery, and was quite compatible with IRIS, as has been described in Ugandan\nmen starting therapy [##REF##11737324##11##]. However,\nwhile this outbreak responded to standard herpes therapy, the hypertrophic\ngenital HSV-2 lesion developed and persisted despite near-complete immune\nrecovery for up to three years. The rapid clinical response that was seen to\ntopical imiquimod, an agonist of Toll-like receptor 7 (TLR7) that boosts both\nhost innate and adaptive antiviral immunity [##REF##12734440##12##], strongly implies that there\nwere clinically significant defects in host antiherpes immunity despite\nHAART-induced immune recovery.</p>", "<p>Hypertrophic or squamoproliferative\nanogenital lesions in HIV-positive individuals or those with AIDS are uncommon\nand can pose a diagnostic dilemma. The\nmost likely cause of such lesions is either neoplasia or infection, although\nthe differential diagnosis can be wide [##REF##10701208##13##]. \nIt may be difficult to determine the cause of a lesion based on\nappearance alone, and the sensitivity of various diagnostic tests can be\naffected by the reactive changes present in these lesions [##REF##12852374##10##]. Further, small biopsies may be inconclusive\nor provide misleading information [##REF##11827427##8##]. \nIt may be necessary to employ repeat biopsies and viral cultures in\ncases suspicious for HSV. In our\npatient, the first biopsy of the lesion in 2003 was positive for HSV-2, but a\nsecond biopsy performed after the lesion had recurred in 2006 was negative for\nHSV-1, HSV-2, cytomegalovirus (CMV), and spirochetes using immunohistochemical\nspecial stains, showing only chronic granulation tissue present. This reinforces that it may be difficult to\ndemonstrate virus even in a generous biopsy specimen, especially in the\npresence of chronic, extensive granulation tissue. Because this second biopsy was negative for\nHSV, it is possible that\nHSV reactivation occurred within this\nproliferative mass, rather than causing it.\nHowever, it is more likely that HSV was in fact the etiologic agent\nresponsible for the progression.</p>", "<p>The pathogenesis of proliferative\nand hypertrophic HSV lesions in the immunocompromised patient population is\npoorly understood. It is thought to be a\nreflection of the increased duration of the disease course rather than any\ninherent change in the pathogenicity of the HSV-2 itself [##REF##9366853##14##]. Early reports hypothesized that immune\ndysfunction secondary to HIV, perhaps mediated by T-helper type 2 cytokines,\nmight result in the epidermal hyperplasia [##REF##8733396##15##]. \nWhatever the etiology of these lesions, their presence has been linked\nto immune function. In HIV-infected\nindividuals, HSV-2 infection is associated with an increased number and size of\ngenital lesions relative to immunocompetent individuals [##REF##6328055##16##]. Vesicles and ulcers are typically more\nnecrotic, painful, and heal more slowly [##REF##3006227##17##]. \nFinally, as CD4 cell count drops and immune status worsens, recurrent\noutbreaks increase in frequency and severity [##REF##1361745##18##].</p>", "<p>Underlying the increased severity of\nHSV-2 disease in HIV-coinfected patients are defects in herpes-specific\nimmunity. In a recent report of herpes simplex vegetans in an HIV-infected\nindividual with severe CD4+ T cell depletion [##REF##17205432##19##], a specific defect was\ndemonstrated in the production of type I interferons by plasmacytoid dendritic\ncells in response to HSV. Dendritic cells within the epithelium of the genital\ntract itself are important in mediating protective immunity against HSV [##REF##12538655##20##],\nbut these cells are dramatically depleted in the genital mucosa of HIV-infected\nindividuals [##REF##17314521##21##]. Finally, HIV infection\nmay be associated with impaired HSV-2-specific CD8+ T cell responses, although\nthese defects improve progressively after starting HAART [##REF##17205480##22##]. In contrast, our patient had a normal CD4\ncount, an undetectable viral load, and had been on HAART for six months prior\nto the development of her lesions. The pathogenesis of progressive hypertrophic\nHSV-2 in this context, where such immune defects would be expected to\nhave improved substantially, is not clear, although a similar case has been\nreported of a rapidly growing and recurrent genital mass in an HIV-infected\nwoman on HAART with a CD4 T cell count &gt;500/mm³ [##REF##12890233##7##].</p>", "<p>Immunocompromised individuals are more\nlikely to be infected with HSV-2 that is resistant to antiviral medications \n[##REF##15494896##6##]. Resistance to acyclovir is usually associated with resistance to the other\nnucleoside analog drugs, including famciclovir and valacyclovir [##REF##12428850##9##]. In such\ncases, foscarnet or topical cidofovir may be useful, since they have different\nmechanisms of action [##REF##16898926##23##]. Unfortunately, antiviral resistance testing was not\navailable for our patient, although the prompt response of her “classical”\nulcerative genital herpes outbreak to both acyclovir and valacyclovir, with\nrapid recurrence when suppressive treatment was stopped, suggested drug\nsensitivity. However, the hypertrophic lesion was clinically resistant to\nmultiple courses of famciclovir and valacyclovir, as well as to later courses\nof intravenous foscarnet. Ultimately, there was a rapid and complete response\nto topical 5% imiquimod cream. Others have reported varied success with the use\nof imiquimod for HSV-2 therapy [##REF##17205432##19##, ##REF##12410731##24##], and this drug should be considered in\nthe armamentarium for resistant and recurrent genital HSV-2 in HIV-infected\nindividuals. Thalidomide has also been described as useful in HIV-positive\npatients with hypertrophic lesions, although this drug was not used in our case \n[##REF##17479932##25##].</p>", "<p>While uncommon, hypertrophic or\nsquamoproliferative HSV disease in the HIV-positive population can be a very\nchallenging clinical condition. It is painful, disfiguring, and difficult to\ndiagnose and treat. As demonstrated by our patient, it can be seen in\nindividuals anywhere along the spectrum of immune dysfunction. If clinically\nsuspected, repeated attempts at diagnosis with viral cultures and biopsies are\nwarranted. These lesions are often resistant to first-line antiviral treatment,\nand may require less commonly used therapies such as foscarnet, cidofovir,\nimiquimod, or thalidomide. In our case, imiquimod eventually resulted in a good\nclinical outcome.</p>" ]

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[ "<p>Recommended by Susan Cu-Uvin</p>", "<p>Most HIV-infected individuals are coinfected by <italic>Herpes simplex virus</italic> type 2 (HSV-2). HSV-2 reactivates more frequently in HIV-coinfected individuals with advanced immunosuppression, and may have very unusual clinical presentations, including hypertrophic genital lesions. We report the case of a progressive, hypertrophic HSV-2 lesion in an HIV-coinfected woman, despite near-complete immune restoration on antiretroviral therapy for up to three years. In this case, there was prompt response to topical imiquimod. The immunopathogenesis and clinical presentation of HSV-2 disease in HIV-coinfected individuals are reviewed, with a focus on potential mechanisms for persistent disease despite apparent immune reconstitution. HIV-infected individuals and their care providers should be aware that HSV-2 may cause atypical disease even in the context of near-comlpete immune reconstitution on HAART.</p>" ]

[ "<title>2. CASE REPORT</title>", "<p>The patient was a 34-year old,\nasymptomatic Zimbabwe-born woman with a positive HIV IgG ELISA on immigration\nscreening in 2002. There was no prior history of sexually transmitted\ninfections, genital or perianal ulceration, and syphilis serology was negative.\nHer absolute CD4+ T cell count at diagnosis was 156/mm³, with an\nHIV-1 RNA plasma viral load of more than 100 000 copies/mL. Antiretroviral\ntherapy was initiated one month later, in October 2002, with combivir one\ntablet twice daily and nevirapine 200 mg twice daily, together with\ntrimethoprim/sulfamethoxazole as primary prophylaxis against <italic>P. jiroveci</italic> pneumonia. By January 2003,\nher CD4+ T cell count had increased to almost 500/mm³, and her HIV\nviral load remained persistently undetectable after March 2003, at which point\ntrimethoprim/sulfamethoxazole was discontinued. In April 2003, she noted\nmultiple painful papules on the left labia, with subsequent superficial\nulceration. She had no reported sexual contacts for over a year. Her family\nphysician prescribed empiric therapy with acyclovir 400 mg three times daily\nand keflex 500 mg three times daily, followed by oral valacyclovir 500 mg twice\ndaily.</p>", "<p>In July 2003, nine months after starting\nHAART, the shallow ulcerations had resolved but a pruritic 1 × 3 cm granulomatous\nlesion developed on the left labia. This was associated with surrounding tissue\nedema, and shotty left inguinal lymphadenopathy, and progressed over the\nsubsequent two months (##FIG##0##Figure 1(a)##). There was no response to azithromycin\n1 gram weekly for 4 weeks, as empiric therapy for granuloma inguinale. Both a\nsuperficial swab and a punch biopsy of the hypertrophic lesion demonstrated\nHSV-2, with glassy nuclear chromatin, multinucleation and surrounding severe\nacute and chronic inflammation (##FIG##0##Figure 1(b)##). Topical therapy was initiated with\ntrifluridine, together with oral valacyclovir 1 gram twice daily. There was no\nclinical response, although valacyclovir discontinuation was followed by an\noutbreak of painful, scattered shallow ulcerations that were culture positive\nfor HSV-2. From November 2003 and December 2005 she was treated with 4 one-month\ncourses of intravenous foscarnet; despite a near total clinical response to the\nfirst course, the response waned progressively and the fourth course was\ncomplicated by <italic>Staphylococcus aureus</italic> cellulitis\nat the line site. Lesion cultures were repeatedly positive for HSV-2, and a\nrepeat labial biopsy in February 2006 showed only chronic granulation tissue.\nThere was no response to a trial of topical protopic (tacrolimus) in August\n2006.</p>", "<p>In September 2006 there was a prompt\nclinical response to topical imiquimod 5%, applied 3 times weekly, with\nresolution of the lesion within eight weeks. She remains asymptomatic on\noral valacyclovir 1 gram twice daily combined with topical imiquimod 5% as\nneeded (approximately one topical application every two weeks) with minor\nresidual labial scarring.</p>" ]

[ "<title>ACKNOWLEDGMENTS</title>", "<p>This paper was supported by the Canadian Institutes of Health Research (RK, Grant\nHOP-75350) and the Canadian Research Chair Programme (RK, salary support). The authors \nreport no conflicts of interest.</p>" ]

[ "<fig id=\"fig1\" position=\"float\"><label>Figure 1</label><caption><p>Clinical and microscopic appearance of hypertrophic HSV-2\nlesion.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"IDOG2008-592532.001\"/>" ]

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[ "<title>1. INTRODUCTION</title>", "<p>Erectile dysfunction following prostatectomy remains a\nsignificant quality of life issue for men undergoing prostatectomy. It is\nestimated to affect 26–100% of patients\nafter surgery [##REF##17570435##1##]. Even with advancements in understanding the anatomy of\nthe prostate and the neurovascular bundle [##UREF##0##2##], a considerable number of men\nundergoing prostatectomy will have resulting erectile dysfunction.</p>", "<p>Progress has been made in identifying the events that contribute to erectile\ndysfunction after prostatectomy. The changes of neuropraxia, ischemic and\nhypoxic insults, fibrotic remodeling, and apoptosis are all believed to\ncontribute to erectile dysfunction [##REF##18087645##3##, ##REF##17466058##4##]. These events can occur even in\nattempts at meticulous dissection to preserve the neurovascular bundle. The etiologies of cavernous nerve neuropraxia\ninclude mechanical stretch injury during retraction, ischemia from accessory\nvessel disruption in dissection, thermal injury from electrocautery use, and\ninflammation from surgical trauma. This\nneuropraxia can prevent erections, and the perpetual lack of erection can\nitself set up a cascade of deleterious processes. Chronic impotence reduces\nblood flow to the corporeal bodies, which leads to fibrosis and transformation\nof the trabecular smooth muscle through collagen [##REF##9647948##5##]. Further hypoxic insults\nalso may trigger apoptosis [##UREF##1##6##]. Therefore,\nthe goal of penile rehabilitation is to set up an environment that moderates\nthese processes in attempt of preserving penile function and earlier return of\npotency. Regimented usage of erectile\naids aims to improve the circulation of oxygen and maintain the structure of\nthe corporeal bodies.</p>", "<p>However, the research has yet to be translated into a\ncoherent clinical strategy for penile rehabilitation. As such, there are\nno currently accepted guidelines for penile rehabilitation regiments.\nCertainly, there exist several popular options that are currently in use.\nIn practice, three principle modalities of treating post-prostatectomy erectile\ndysfunction are employed. This paper will review the efficacy of these\noptions in this patient population for the purposes of penile rehabilitation.</p>" ]

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[ "<title>7. CONCLUSION</title>", "<p>Sexual potency after prostatectomy remains a significant quality of life issue\nafter prostatectomy. There exist many studies on the efficacy of the\nvarious treatment options on this patient population. There are much less data looking on the\neffects of regimented usage of PDEi, VED, and/or ICI in improving erectile\nfunction in this group, and no guidelines exist to help steer the\nclinician. We are still in need of large, randomized, controlled, clinical\ntrial with adequate, long-term followup to evaluate this question. Moreover, even after each treatment can be\nestablished to be efficacious in penile rehabilitation, the exact regiment amount and\nduration will still be open to further investigations for optimization. This is\na especially important question in dealing with phosphodiesterase inhibitors,\nwhere the cost of treatment is substantial. \nHowever, even though currently sparse, the consistent, growing body of\nevidence does support penile rehabilitation in improving return of sexual\nfunctioning. As awareness of penile\nrehabilitation increases and becomes more accepted, it is becoming more\ndifficult to conduct placebo or nonintervention controlled trial. There are\nstill several ongoing trials evaluating penile rehabilitation, including a\nlarge multicenter study examining penile rehabilitation with medicated urethral system for erection (MUSE), and several of the studies presented here will further analyze with additional data from which the urology community may further define when and how to\nimplement penile rehabilitation in post-prostatectomy men.</p>", "<p>Currently, the body of evidence does\nseem to suggest a beneficial role for penile rehabilitation after prostatectomy\nin improving return of potency. Such a program should begin with a detailed\nevaluation on the preoperative sexual performance characteristic of the patient\nand then a thorough\ndiscussion of the available rehabilitation regiments. The practitioner should consider factors that\nare important to the patient including ease of use and compliance, patient\nmotivation, conditioning, cost and patient expectations about sexual function,\nand penile length. Penile rehabilitation\nmay continue to remain investigative until more standardized clinical data\nbecomes available.</p>" ]

[ "<p>Recommended by Edward Kim</p>", "<p>\n<italic>Introduction</italic>. Post-prostatectomy erectile dysfunction affects a considerable number of men and is a significant quality of life issue. There has been a substantial amount of research on the treatment of post-prostatectomy ED, and now there is a rising interest in the concept of penile rehabilitation. The goal of penile rehabilitation is to moderate the destructive processes that occur after prostatectomy in order to preserve erectile function, either through spontaneous or assisted means. <italic>Methods</italic>. We reviewed published data and experiences of post-prostatectomy penile rehabilitation using regimented interventions of phosphodiesterase inhibitors, vacuum erectile device, and intracavernosal agents, and we present and analyze the research conducted. <italic>Results</italic>. These studies show improved objective and subjective clinical outcomes in regards to physical parameters, sexual satisfaction, and rates of spontaneous erections. <italic>Conclusion</italic>. These studies are often limited by small size, study period, and study design. There continues to be a need for large, randomized, placebo controlled trials with adequate followup to fully evaluate the efficacy and cost-effectiveness of the various proposed penile rehabilitation regiments before a clear standard can be established.</p>" ]

[ "<title>2. PHOSPHODIESTERASE 5 INHIBITORS</title>", "<p>The introduction of phosphodiesterase inhibitors (PDEi) revolutionized\nthe treatment of erectile dysfunction. Since entering the market in 1998,\nthese medications have become nearly synonymous with erectile\ndysfunction. Their ease of use and relatively safe profile have made them\npervasive in the treatment of erectile dysfunction. They have also been\nextensively investigated. A Cochrane meta-analysis looking at many large,\nrandomized clinical trials concluded that PDEi are efficacious in the treatment\nof erectile dysfunction and are generally safe [##REF##12076233##7##]. However, their role\nand their administration in penile rehabilitation after prostatectomy remain\nundefined. A number of clinical studies\nhave investigated PDEi use in this population for this intention.</p>", "<p>One of the first studies on PDEi in rehabilitation looked\nat objective data to support this use. \nSchwartz et al. conducted a study on 40 men who had undergone nerve\nsparing prostatectomy [##REF##14713808##8##]. Prior to prostatectomy, all men had\npercutaneous biopsy of cavernous tissue to serve as baseline reference. They were divided into receiving either 50 or\n100 mg of Sildenafil every other night. Participant then underwent percutaneous\nbiopsy of cavernous tissue at 6 months to compare with baseline tissue. Investigators found that the 50 mg group did\nnot experience any loss of smooth muscle compared with baseline, and the 100 mg\ngroup actually showed an increase of smooth muscle content when compared to the\nbaseline. There was no control group, and no clinical correlation between\nsmooth muscle preservation and erectile function is made in this study. A prior animal study had shown that cavernosal\nsmooth muscle does atrophy after prostatatectomy [##REF##12576876##9##], though this effect is\nsomewhat mediated with unilateral nerve sparing and it is unclear to what\nextent this atrophy would occur with nerve sparing prostatectomy.</p>", "<p>Bannowski et al. conducted a randomized trial following 43\nmen who underwent nerve-sparing radical prostatectomy [##REF##18284406##10##]. All men provided baseline International Index of Erectile Function (IIEF) scores prior\nto surgery. After catheter removal following\nsurgery, the men underwent testing for nocturnal tumescence the following\nevening measured by the rigiscan. 41 of\n43 were found to have spontaneous erections on rigiscan, and these men were\nthen randomized to receive sildenafil daily or no treatment. They were then followed and evaluated with\nIIEF at 6, 12, 24, 36, and 52 weeks. The\nresults show that the daily treatment group had significantly higher IIEF score\nby 36 and 52 weeks. Additionally, 47% of\nthe daily treatment groups were able to achieve spontaneous, unassisted\nerection sufficient for penetration. \nThis compares to 28% of the control group who were able to have such\nerections. Both groups were also allowed\nSildenafil on demand, and accounting assisted erections, 86% of the daily group\nhad erections sufficient for penetration, compared to 66% of the control\ngroup. The study made no mention of any\nparticipant drop out, and there was no placebo control. However, it does appear that daily Sildenafil\ndoes improve return of spontaneous erections and can augment response to on-demand use of Sildenafil.</p>", "<p>A stringent, randomized,\ndouble-blinded, placebo controlled study was performed by McCullough et al.\nevaluating the efficacy of daily Sildenafil in men after bilateral nerve-sparing\nradical prostatectomy [##REF##18086170##11##]. This study\nincluded 54 men with baseline normal EF and NPTR (nocturnal penile tumescence\nand rigidity with duration of rigidity &gt;55% of maximal rigidity using\npenile plethysmography). After a\npretreatment period of 4 weeks, they were then randomized to either receive\nnightly 100 mg Sildenafil (<italic>N</italic> = 18), 50 mg Sildenafil (<italic>N</italic> = 17), or placebo (<italic>N</italic> = 19). The groups were then analyzed at\n16, 28, and 40 weeks. Then after 40\nweeks, all medications were discontinued and the groups were again analyzed at\n48 weeks. At each point, the\nparticipants were evaluated by NPTR and IIEF . \nThe study found that the groups receiving daily Sildenafil were able to\nhave return of rigidity (<italic>R</italic> &gt; 55%) at seven times the nadir value compared to minimal\nimprovement in the control group. This\nimprovement of erection was also seen by the investigators for RAU (rigidity-activated unit—a time-intensity measurement that\nrepresents the area under the rigidity curve during a qualified\nevent), with the additional finding that the 100 mg group experienced continued\nimprovement after the discontinuation phase, while the 50 mg group began to\nexperience decline in RAU. Importantly,\nthe men on daily Sildenafil were five times more likely to have return of spontaneous,\nunassisted erection sufficient for intercourse compared to placebo. The authors were able to link objective\nmeasurements of erections with subjective, clinical response. They noted that tip rigidity &gt;55% clearly\nseparated responders versus nonresponders (responders defined as recovery of\nspontaneous, unassisted sufficient erections). \nThis study may mark objective validation as an important component of\nfuture clinical trials. These initial\nresults are from a subset analysis performed on men who showed normal EF and\nNPTR on baseline, and we await further data and analysis on the entire study\npatients.</p>", "<p>Taken together,\nthese studies seem to indicate that phosphodiesterase inhibitors have a role in\npenile rehabilitation for men after prostatectomy. There may also be a dose-dependent relationship between the medication and outcomes. The studies also confirm the tolerability and\nsafety of such a regiment, as discontinuation rates were very minimal and no\nadverse events were reported.</p>", "<title>3. VACUUM ERECTILE DEVICE</title>", "<p>The Vacuum erectile device assists erections by drawing blood flow into the\ncavernous sinuses through negative pressure, physically causing an\nerection. A constrictive band can also be placed at the base of the\npenis, preventing backflow and maintaining corporal pressures. This\ndirect mechanism of action can circumvent the limitation of oral agents, which\nrequires an intact and functioning neuronal connection to produce\nerections. This can be a significant factor even in men undergoing nerve\nsparing prostatatectomy, as neuropraxia still occurs and can diminish the\neffectiveness of PDEi.</p>", "<p>This\ntreatment modality can also be extended to men who have undergone nonnerve\nsparing prostatectomy, though not in the context, in penile rehabilitation with\nthe expectation of return of potency.</p>", "<p>If not for potency itself, VED usage has also been advocated due to its\npossible efficacy in preventing penile shrinkage and maintaining length.\nStudies have shown significant shrinkage of penile length, with one\nstudy finding that nearly 20% of men experience a loss of length greater than\n15% [##REF##12629384##12##]. In another study examining penile shortening after\nprostatectomy, Gontero et al. followed 126 men who had undergone prostatectomies\nand measured penile length prior to surgery, at the time of catheter removal,\nand then at 3, 6, and 12 months [##REF##17570431##13##]. They found that the greatest amount of\nshrinkage occurs in the immediate postoperative period, though shortening\ncontinues at a lesser rate throughout the entire study period. These\nauthors hypothesize that early hypoxia leads to increased expression of TGF-B\nand Collagen I and III fibers. This study also finds that the return of erectile\nfunction, defined as an IIEF of 15, was associated with mitigation of the\nshrinkage, as well as having a nerve sparing surgery. Several studies looking at the efficacy of\nvacuum erectile device in preserving erectile function have also examined\npreserved penile length as a secondary endpoint.</p>", "<p>Raina et al. randomized 109 post-prostatectomy men to\neither early VED use daily (<italic>N</italic> = 74) versus no erectogenic aid (<italic>N</italic> = 35) [##REF##16107868##14##]. The men were to use the constriction band\nonly during intercourse to maintain rigidity. Participants were followed\nwith SHIM and IIEF scores for comparison. For the group using VED, 80%\nwere able to achieve penetration with use of VED, and this group, not\nsurprisingly, had a significantly higher SHIM and IIEF group compared to no\ntreatment. The discontinuation rate was 18%, and the majority of the drop\nout was for discomfort. In the context\nof penile rehabilitation, at 9 months this study found that 17% of those\nadhering to daily VED were able to have spontaneous erections sufficient for\nerections at 9 months, compared to 11% (<italic>n</italic> = 4) of the control group that had such\nerections. In regard of the effect of\nVED on penile length, the men who adhered to VED regiment experienced less\nsubjective penile shrinkage, with 23% reporting less length compared to 85% of\nthe men who quit treatment and 65% of the control. No objective data was collected concerning\nlength. The authors conclude that early\nuse of VED with the purpose of penile rehabilitation improves sexual and\npartner satisfaction and allow for earlier return of spontaneous erections. Although,\nthe rate of return of spontaneous erection is low for both groups, those\nnumbers include men who had undergone nonnerve sparing prostatectomies. This distinction is necessary, as penile\nrehabilitation is more directed for NS men and the inclusion of nonnerve sparing\nprostatectomy patients diluted the response to rehabilitation.</p>", "<p>The timing of when to initiate VED has been questioned,\nwith some advocating an earlier intervention. Köhler et al. randomized 28\nmen to either receive early VED regiment (1 month after RP) or delayed VED\nregiment (6 months) [##REF##17822466##15##]. The regiment consisted of 10 minutes of VED\nusage without the constriction band. IIEF was measured at baseline, 1, 3,\n6, 9, and 12 months. The mean followup was 9.5 months and the results\nanalyzed at 3 and 6 months showed that the early intervention group had a statistically higher\nIIEF score. At this point, the comparison shows that VED does improve\nIIEF scores among those who use VED versus those controls that do not. At beyond 6 months, the delayed group began using of VED, and at\nshort followed up the\ntwo groups converged with no statistical difference in IIEF \ncategorization. No patients in this\nstudy had return of spontaneous erections sufficient for penetration at that\nfollowup. This paper was presented as a\npilot study, and more outcomes are expected to follow, especially data\nconcerning return of spontaneous erections. There are still some important\npoints that can be gleamed from this study. For one, the authors reported\ncomplete compliance with this regiment, suggesting this short regiment (two five-minute cycles) could\nbe feasibly implemented. Importantly,\nthese researchers also looked at penile length and found that the group\nperforming VED regiment did not experience penile shrinkage, while the group on\ndelayed VED showed significant penile shrinkage at 3 (mean loss 1.87 cm) and 6\nmonths (mean 1.82 cm). However, after\nbeginning VED in the delayed group, the loss decreased to a mean of 1 cm and no longer remained\nstatistically significant. Again, this is short-term followup data in the delayed group, and\nfurther improvement in shrinkage may still yet be seen. We still await data concerning return of\nspontaneous erection from this study.</p>", "<p>The value of VED in penile\nrehabilitation remains uncertain. Daily\nregimented use of VED requires a motivated patient and does improve sexual\nsatisfaction in those who responds. If\nthe stated goal of penile rehabilitation is the return of preexisting potency,\nthen further studies are needed to show that VED improves the rate of return of\nerection. However, VED use may also be advocated for its effects on preventing\npenile shrinkage after prostatectomy.</p>", "<title>4. INTRACORPOREAL INJECTION</title>", "<p>Intracorporeal injection of vasoactive agents increase blood flow into the\ncavernous sinuses locally, either through increasing cAMP, by antagonizing alpha-adrenergic\nreceptors, or by direct smooth muscle relaxation. Like VED, they also do\nnot require an intact, functional nervous system to produce erections.\nThus, they can also be offered in men who have undergone nonnerve sparing surgery\nand men who do not respond to oral agents.</p>", "<p>Montorsi et al. conducted a randomized trial\ninvestigating whether a regiment of intracavernosal injections improves\nerectile function in post-prostatectomy men [##UREF##2##16##]. 30 men with established\npreoperative potency were randomized to either receive a regiment of 3 times\nper week injections of alprostadil for 12 weeks versus a control group that did\nnot receive erectogenic treatment. The groups were then assessed after 3 months\nfor sexual history, for Doppler response after alprostadil administration, and\nfor nocturnal tumescence. Of the ICI\nregiment group, 80% completed the 12 weeks of treatment with a 20% drop-out\nrate and a 17% complication rate. Of\nthese men, 67% at 3 months were able to have spontaneous erections sufficient\nfor penetration. This favorably compares to 20% of the control group men that\nwere able to have such erections. The authors do note that in this group some\nstill continued to use ICI to achieve erections. However, the authors\nconsidered it a complete response since the majority of sexual encounters\noccurred without ICI use (average of one in 4.2 attempts). The study also found\nthat having normal penile hemodynamics was strongly associated with ICI\ncomplete response. The study suffers from small size and short followup.\nHowever, it was still able to show an improvement in spontaneous erections for\nregimented ICI and that regimented ICI is generally well tolerated.</p>", "<p>Mulhall et al. conducted a trial\nthat may be more clinically applicable, even though it was not randomized [##REF##16422848##17##]. In\nthis trial, post-prostatectomy men were committed to penile rehabilitation with\nSildenafil or ICI if there was no response to Sildenafil, versus no penile rehabilitation\nprogram, but they were\nnot restricted from using erectile aids. \nThe rehabilitation group used either Sildenafil or ICI three times per week. Analysis at 18\nmonths revealed that 52% of rehabilitation men were able to have functional\nerections, compared to 19% of control group men. Additionally, the rehabilitation group had\nsignificantly higher IIEF scores. These\nresults are impressive, especially considering that the control group was also\nable to use erectile aids, including both Sildenafil and ICI, though not in a\nregimented manner. The study included men with nonnerve sparing\nprostatectomies, which generally are not considered candidates for erectile rehabilitation,\nthough the authors do note that there was not a differential distribution\nbetween the groups. Additionally, the\naverage length of time before sexual consultation and therefore the start of\nrehabilitation were 4.2 months. Some would suggest that this\nlength of time is too long removed from the surgery, and the insults of\nhypoxia, fibrosis, and apoptosis may have already occurred and be irreversible\nat this point. It is also important to note that selection bias may be very\nconsiderable in this study, in that only men who prospectively committed\nthemselves were included in the rehabilitation group and the study relied on\nmainly self-reported, subjective data.</p>", "<title>5. COMBINATION THERAPIES</title>", "<p>Studies have examined the feasibility and efficacy of\nemploying two treatment modalities during penile\nrehabilitation. Nandipati et al. incorporated both intracavernosal\ntherapy and PDEi in a group of 22 men who underwent nerve sparing prostatectomy\n[##REF##16482200##18##]. All participants received Sildenafil 50 mg daily (25 mg if subjects\ncomplained of headaches). For ICI, 18\npatients received PGE 1–4 micrograms and\n4 received trimix injection, with ICI being done two to three times per\nweek. These patients were then analyzed at 3, 6, 9, and 12 months with IIEF . Doppler studies were performed for dose\noptimizations of ICI and at intervals to increase dosage for response. The study reported a mean followup of six\nmonths. At this point, investigators found that 21 of 22 patients were sexually\nactive, while 12 of the 21 were using ICI alone and 9 of 21 were using combination\ntherapy. Of the 18 patients using PGE, 12 were able to lower their dosage;\nwhile 1 of the 4 patients on trimix was able to do so. 11 of the 22 men\nhad return of spontaneous erection, though none graded the erections sufficient\nfor penetration. The authors concluded\nthat the addition of Sildenafil could reduce the amount of ICI necessary to\nachieve erections. This study had a lower rate of return of functional\nerections compared to other studies looking at nightly PDEi 1 [##REF##18086170##11##] or regimented\nICI alone [##UREF##2##16##], and this difference may be explained by the\nshorter followup and the small number of participants. Without proper study design, in such\ncombination therapy studies, it is difficult to assign particular findings to\nspecific intervention.</p>", "<title>6. NOVEL THERAPIES</title>", "<p>Other therapies outside of these three mainstream\nmodalities have been investigated for penile rehabilitation after\nprostatectomy. Recently, investigators in Korea looked at statins for treating\nerectile dysfunction after prostatectomy [##REF##17570410##19##]. The basis for this\nhypothesis stems from the known protective effect on vascular endothelium and\nincreased NO activity. The researchers randomized 50 men post-prostatectomy\nto receive 10 mg of atorvastatin for 90 days. All men were then to use\nSildenafil 50 mg per day on demand. The men all had superior function prior to\nthe surgery with IIEF 25. At 6 months of followup, the study found that\nthe statin group had more patients categorized at potent ( IIEF greater than 16)\nwith 11 in the statin group and 6 in the control group. Additionally, more men in the statin group were able\nto achieve vaginal penetration without PDEi than in the control group (8 versus\n4), though this significance did not reach statistical significance. The\nstudy was neither blinded nor placebo-controlled. It is important to note that the\ninclusion criteria were extremely stringent, and patients with significant cormidities were excluded. This may affect the\napplicability or generalizability of the results.</p>" ]

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[ "<table-wrap id=\"tab1\" position=\"float\"><label>Table 1</label><caption><p>Summary table of penile rehabilitation trials.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Authors</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Year published</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Treatment regiment</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Study design</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N</italic>\n</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Significant findings</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Schwartz\net al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2004</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">QOD\nPDEi</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No\nloss of smooth muscle in 50 mg group, gain of smooth muscle in 100 mg group</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Bannowski\net al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2008</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Daily\nPDEi</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective, randomized\ncontrol</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Treatment\ngroup had significantly higher IIEF and higher spontaneous erection rates</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">McCullough et al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2008</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Daily\nPDEi</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective,\nrandomized, placebo control</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Treatment\ngroups had higher return of rigidity, higher rate of spontaneous erections</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Raina\net al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2006</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Daily\nVED</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective,\nrandomized control</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">109</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Improved\nsexual satisfaction, higher rate of spontaneous erections</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Köhler\net al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2007</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Daily\nVED (10 mins), immediate versus delayed</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective,\nrandomized</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Delayed\nuse of VED did not affect sexual satisfaction once use began. There is no\nstatistical significance in penile shrinkage once VED started</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Montorsi et al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1999</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ICI 3\ntimes weekly</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective,\nrandomized control</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Higher\npercentage of treatment group having spontaneous erections</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mulhall et al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2005</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ICI\nor PDEi to achieve erections 3 times weekly</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective, control</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">132</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Treatment\ngroups had 2.7 times the rate of spontaneous erections, statistically higher\nIIEF scores</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nandipati et al.</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2006</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Daily\nPDEi and ICI 2-3 times week</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Assisted\nearly sexual activity and satisfaction.\nAddition of PDEi allows lower dose of ICI.</td></tr></tbody></table></table-wrap>" ]

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[ "<title>1. INTRODUCTION</title>", "<p>Radical nephrectomy has considered the optimal surgical\napproach to the management of renal cancer [##REF##18280512##1##]. Additional options include\nobservation, cryosurgery, and radiofrequency ablation [##REF##18242377##2##, ##REF##17853039##3##]. Management of the small renal mass requires multiple perspective\ndecision making on the part of the physician [##REF##17949330##4##]. Recently, partial nephrectomy has been\nshown to have efficacy in the management of select patients with small renal\nmasses (4 cm or less) [##REF##18221968##5##, ##REF##18047964##6##]. Several investigators have reported\nan expanded use of this approach with success in patients with larger renal\nneoplasms (7 cm or less). Hospital stay\nafter partial nephrectomy is usually 5–8 days [##REF##18265990##7##]. Factors limiting early discharge are\nusually pain, stress-induced major organ dysfunction (i.e., ileus, atelectasis),\ntradition, fatigue, pain, nausea, and morbidity [##REF##18163944##8##, ##REF##16640972##9##]. In other abdominal procedures, including\ncolon resection, lung transplant, and laparoscopic nephrectomy, the\nintroduction of a program comprised of optimized pain relief using nonsteroidal\nanti-inflammatories for analgesia enforced oral nutrition and mobilization, and\nrevision of traditional care principles has reduced hospital stay from 5–8 days to 220 133\ndays in some studies [##REF##17186427##10##, ##REF##12780633##11##]. The concept of fast-track (FT) surgery\nhas recently attracted more interest, but has not yet been applied in patients\nundergoing partial nephrectomy. The purpose of this study was to investigate\nthe postoperative course before and after the introduction of a fast track program\nin patients undergoing open partial nephrectomies. We sought to determine\nwhether the use of this fast track program might decrease length of hospital\nstay without sacrificing outcomes.</p>" ]

[ "<title>2. MATERIALS AND METHODS</title>", "<p>A fast\ntrack clinical pathway for open partial nephrectomy was introduced at our\ninstitution in July 2006. The pathway\nhas an established management protocol (##TAB##0##Table 1##). All patients undergoing open\npartial nephrectomy from July 2006 thus far at our institution were managed by\nthe fast track protocol, and comprise the study cohort. Patients undergoing laparoscopic partial\nnephrectomy and robotic partial nephrectomy were not included. In the event that the decision was made\nintraoperatively to perform complete nephrectomy, patients were included on an\nintention-to-treat basis. Demographic\ndata, tumor size, blood loss, transfusion, final pathology, margin status, length\nof hospital stay, and complications were captured in a prospective database,\nand compared to a control group managed consecutively immediately preceding the\ninstitution of the fast track clinical pathway.<italic/>\nAll operations were performed for a renal tumor less than 7 cm in\ngreatest diameter by the same surgical team.</p>", "<title>2.1. Preoperative preparation</title>", "<p>Patients\nwere admitted to the hospital on the day of surgery and performed all\npreparation on an outpatient basis. All\npatients were counseled preoperatively regarding the target goals outlined in\n##TAB##0##Table 1##. Aspirin and nonsteroidal\nanti-inflammatory drugs (NSAIDs) were stopped 10–14 days prior to\nsurgery. Coumadin was stopped 5 days\npreoperatively and stat protime/partial thromboplastin time obtained on the morning of surgery\nto confirm acceptable value. All patients received 3 bisacodyl\ntablets and 1 bottle of magnesium citrate at noon on the day prior to surgery. All patients\nreceived erythromycin and neomycin antibiotic per oral (PO),\n500 mg (milligram) tab, at 3 pm, 6 pm, and 9 pm on the day prior to surgery. All patients were instructed to eat lightly\nand orally hydrate on the day prior to surgery.</p>", "<title>2.2. Intraoperative</title>", "<p>This\nreport is restricted to patients undergoing open partial nephrectomy on an\nintention to treat basis. General anesthesia, oral gastric tube (removed at\ncase conclusion), and Foley catheter were used in all cases. Compression pneumatic stockings were placed\nas soon as the patient moved from the stretcher onto the operating table. The retroperitoneal flank approach was\nused. All patients were positioned with\nthe bean bag, with legs straight, hyper-extended with the kidney rest raised\nmaximally. Before draping, the surgeon\nmarked the posterior axillary line (PAL),\nthe anterior axillary line (AAL), the lateral border of the rectus muscle, and\nthe course of the 10th, 11th, and 12th ribs. Incision was made from the PAL to the AAL in the course of the 11th rib using the cautery on pure cut (setting of 30). The distal tip of the 11th rib was removed in the standard manner.<italic/>\nAn extra-pleural/extra-peritoneal approach to the kidney was used. The\nkidney was explored, and vessel loop passed to tag the ureter, renal artery,\nand renal vein. A double loop was passed around the vein for subsequent\nocclusion. Patients received mannitol\n12.5 gm (gram) IV (intravenous) bolus prior to manipulation of the renal vessels\nfollowed by 5 gm/hour continuous infusion for the remainder of the operation.\nRenal artery was clamped in all cases and the kidney cooled. The renal vein was\nselectively occluded as required to provide a bloodless operative field. The\ncollecting system was closed with 3–0 monocryl on SH\nneedle in all cases. Renal arteries and venules were oversewn in\nfigure of eight fashions\nwith 3–0 monocryl on SH\nneedle. Prior to the removal of the renal artery clamp, the kidney was reconstructed essentially\nobliterating the resection defect utilizing 0-Chromic suture on CT needle in\nhorizontal mattress\nfashion. This resulted in a reniform shape approximation in nearly all cases. The rib bed\nand skin were infiltrated with 0.25% marcaine (30 ml (milliliters)). The skin\nwound was closed in a subcuticular (3–0 monocryl). A <italic>#</italic>7 Jackson-Pratt drain was placed in all\ncases. In no case was a ureteral stent\nplaced.</p>", "<title>2.3. Postoperative</title>", "<p>On POD 0 (postoperative day), on the evening of surgery,\npatients received celecoxib 200 mg per oral\nin the postanesthesia care unit (PACU) with sip when awake, and daily thereafter (##TAB##0##Table 1##). Morphine sulfate was administered IV at 2 hour\nintervals as needed. Metoclopramide was\nadministered IV 10 mg every 6 hours. Famotidine was administered 20 mg IV every 12 hours. On the day of surgery, patients ambulated and\nwere encouraged to take liquids by mouth.<italic/>\nOn POD 1, diet was advanced to tray of clears, and oral pain medication\nadministered (hydrocodone/acetominophen 5–10 mg/500 mg every 4 hours as\nneeded). On day 2, the Foley catheter\nwas removed at 7 am and regular diet initiated.<italic/>\nPatients received milk of magnesia 30 cc PO at 8 am and a repeat dose in 4 hours.<italic/>\nJackson-Pratt drain fluid was sent to the laboratory for creatinine\nmeasurement and if equal to serum creatinine level, the Jackson-Pratt drain was\nremoved. The patient was assessed and\ndischarged to home on POD 3 if appropriate.</p>" ]

[ "<title>3. RESULTS</title>", "<p>A total of 33 patients were managed\nby fast track and compared to 25 control patients (##TAB##1##Table 2##). The estimated\nblood loss, transfusion rate, tumor size, pathology, and complication rate were\nsimilar between groups. There was, however, a significant difference in the\nlength of hospital stay observed between groups. Of 25 control patients, 4 (16%) achieved\ndischarge to home in &lt;3 days compared to 22 (67%) of the 33 patients\nmanaged in the fast track program.<italic/>\nOverall, fast track patients had a shorter hospital stay compared to\ncontrols (median, 3 days versus 4 days; <italic>P</italic> = .012). Of the 11 patients in\nthe fast track cohort who were discharged after the third postoperative day,\nthis was due to poor ambulation/inadequate pain control (<italic>n</italic> = 5), abdominal\nbloating (<italic>n</italic> = 3), multiple co-morbidities (<italic>n</italic> = 2), and respiratory distress\npostoperatively requiring ICU care (<italic>n</italic> = 1).</p>", "<p>Complications are worth noting to\ndetermine whether the fast track approach was harmful in any way to the study\ncohort. In the control cohort, there was\n1 patient with respiratory distress requiring ICU admission, 3 patients\nreceived blood transfusion, there were 2 conversions to complete nephrectomy,\nand 1 positive surgical margin. In the\nfast track cohort there was 1 patient with respiratory distress requiring ICU\nadmission, 2 patients required blood transfusion, there was one conversion to\ntotal nephrectomy, 1 postoperative bleed (gross hematuria) requiring selective\narterial embolization, and 1 urine leak requiring percutaneous drainage and\nureteral stent placement.</p>", "<p>The percentage of patients with\nmalignancy increased in the fast track cohort compared to control (85% versus\n76%). This may represent improved\npreoperative assessment.</p>" ]

[ "<title>4. DISCUSSION</title>", "<p>Since 1950 in the United States, there has been a\n126% increase in the incidence of renal cancer [##REF##18265990##7##, ##REF##18163944##8##]. Although there has been an increase\nin all stages of renal cancer including advanced cases (i.e., regional\nextension, distant metastases), there has been the greatest increase in those\ndiscovered incidentally [##REF##18163944##8##, ##REF##18222599##12##]. In the early 1970s, approximately\n10% of tumors were detected incidentally compared with 61% in 1988 [##REF##18163944##8##].</p>", "<p>Previous studies of other types of major surgery have shown\nthat a combined effort comprising intensive preoperative information, effective\npostoperative pain relief and enforced mobilization, and early enteral\nnutrition can accelerate postoperative recovery and decrease hospital stay [##REF##12780633##11##, ##UREF##0##13##].</p>", "<p>Investigators have recently illustrated that in elderly\nhigh-risk patients undergoing colonic resection, mean hospital stay could be\nreduced to 2-3 days [##REF##12780633##11##]. In another group of high risk\npatients undergoing open aortic surgery, mean hospital length of stay was\nreduced from a mean of 9 days to 5 days [##UREF##1##14##–##UREF##2##16##]. In a study by Harinath et al., a\ndecrease in length of stay was observed from 5 to 4 days for ileal pouch-anal\nanastomosis [##REF##12780633##11##]. The concept of fast track has also\nbeen applied to infants and children [##REF##17208572##17##]. In the urologic literature, after\nradical prostatectomy, median hospital stay in 252 consecutive patients was\nreduced to 1 day [##UREF##0##13##]. Specifically with kidney surgery,\npostoperative hospital stay after open nephrectomy was reduced to 4 days [##REF##12944188##18##]. With laparoscopy, hospital stay has\nbeen reduced to 2 days with an FT rehabilitation program [##REF##16359206##19##].</p>", "<p>In this study, with the introduction of a fast-track program,\nopen partial nephrectomy hospital stay was decreased to 3 days, compared to 4\ndays before implementation of the program. Sixty six per cent of patients\nachieved a target discharge on day 3 or less.<italic/>\nNotably, both groups did have similar characteristics as demonstrated in\n##TAB##1##Table 2##. The estimated blood loss, transfusion rate, tumor size, pathology, and\ncomplication rate were similar between groups. We suspect that based upon our\ndata the main contributing factors responsible for the decrease in hospital\nstay was a clear protocol of expectations at each stage of the recovery\nperiod. This was accomplished in the\npresent series without an apparent increase in complication rate. Most notably, fast track did not lead to an\nincrease in readmissions.</p>", "<p>It is\nimpossible to discern exactly which components of our protocol are “more\nessential” than others, and this would require selective application in future\ninvestigations. In addition, we have no proof from the present\ninvestigation that the fast track protocol is advantageous to the\npatient. The purpose of the present study was to assess the feasibility\nof such an approach and we conclude that such an approach is feasible.\nUltimately, the patients in this study decided their discharge date.\nMost patients were eager to receive discharge to home as soon as they are\nmedically safe. One patient in the fast track protocol was discharged to\nhome at her request on POD 2. She expressed regret at doing so at\nher first postoperative visit.</p>", "<p>Our readmissions\nto the hospital were as follows. One (3.0%) patient had returned to hospital for postoperative\nhemorrhage resulting in gross hematuria. This patient was managed successfully\nwith embolization. One (3.0%) patient had returned to hospital\nforurinoma. This was managed successfully with indwelling ureteral\nstent for 6 weeks and transient percutaneous drainage of urinoma. Both patients had\ncomplex resections, and it is unlikely that fast track management resulted\nin return to hospital.</p>" ]

[ "<title>5. CONCLUSIONS</title>", "<p>In the\npresent investigation, a fast track clinical pathway after open partial\nnephrectomy reduced the postoperative length of hospital stay and did not\nappear to increase the postoperative complication rate.</p>" ]

[ "<p>Recommended by J. Rubio</p>", "<p>\n<italic>Introduction</italic>. The aim of this study is to examine the feasibility of reducing postoperative hospital stay following open partial nephrectomy through the implementation of a goal directed clinical management pathway. <italic>Materials and Methods</italic>. A fast track clinical pathway for open partial nephrectomy was introduced in July 2006 at our institution. The pathway has daily goals and targets discharge for all patients on the 3rd postoperative day (POD). Defined goals are (1) ambulation and liquid diet on the evening of the operative day; (2) out of bed (OOB) at least 4 times on POD 1; (3) removal of Foley catheter on the morning of POD 2; (4) removal of Jackson Pratt drain on the afternoon of POD 2; (4) discharge to home on POD 3. Patients and family are instructed in the fast track protocol preoperatively. Demographic data, tumor size, length of stay, and complications were captured in a prospective database, and compared to a control group managed consecutively immediately preceding the institution of the fast track clinical pathway. <italic>Results</italic>. Data on 33 consecutive patients managed on the fast track clinical pathway was compared to that of 25 control patients. Twenty two (61%) out of 36 fast track patients and 4 (16%) out of 25 control patients achieved discharge on POD 3. Overall, fast track patients had a shorter hospital stay than controls (median, 3 versus 4 days; <italic>P</italic> = .012). Age (median, 55 versus 57 years), tumor size (median, 2.5 versus 2.5 cm), readmission within 30 days (5.5% versus 5.1%), and complications (10.2% versus 13.8%) were similar in the fast track patients and control, respectively. <italic>Conclusions</italic>. In the present series, a fast track clinical pathway after open partial nephrectomy reduced the postoperative length of hospital stay and did not appear to increase the postoperative complication rate. </p>" ]

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[ "<table-wrap id=\"tab1\" position=\"float\"><label>Table 1</label><caption><p>Fast track pathway.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Day</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Goal</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\">Preoperative</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Patient and family counseling</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Medication review</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Preparation instructions</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Postoperative day 0 (evening of surgery)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OOB at least once</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clear liquids</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\">Postoperative day 1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OOB four times</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Liquid diet</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Oral pain medications</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"5\" colspan=\"1\">Postoperative day 2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OOB ad lib</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Regular diet</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Remove Foley catheter at 7 am</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Flank drain fluid for Cr at 2 pm</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Remove drain if fluid Cr = serum Cr</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Postoperative day 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Discharge to home</td></tr></tbody></table></table-wrap>", "<table-wrap id=\"tab2\" position=\"float\"><label>Table 2</label><caption><p>Outcomes of fast\ntrack open partial nephrectomy.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" rowspan=\"1\" colspan=\"1\">Conservative group</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Fast track group</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(N)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Discharge in &lt;3 days</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age range</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">32–74</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39–73</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male/female</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18/8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22/11</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Length of stay</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Range</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3–10 days</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2–6 days</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 days</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 days</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.4 days</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.3 days</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estimated blood loss</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Range</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">50–500 cc</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">50–600 cc</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">200 cc</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">200 cc</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">228 cc</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">263 cc</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Transfusions</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Complications</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Respiratory distress</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Conversion to nephrectomy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Post operative bleed</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Urine leak</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Tumor size</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Range</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.1–6.8 cm</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.2–6.2 cm</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.5 cm</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.5 cm</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.8 cm</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.9 cm</td></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pathology</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clear Cell RCC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (68%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25 (76%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Papillary\nRCC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (9%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chromophobe\nRCC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (9%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Oncocytoma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (12%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">AML</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (6%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">—</td></tr></tbody></table></table-wrap>" ]

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[ "<title>1. INTRODUCTION</title>", "<p>In MRI, spatial information is obtained from the\nobject using magnetic field gradients, which link the Larmor frequency of the\nexcited spins to their spatial location. Thus, the received signal is the\ncontinuous Fourier transform of the object's proton densitywhere the <italic>k</italic>-space position can be calculated from the time course of the\napplied gradients. In practice, the proton density is further modulated by spin\nrelaxation, off-resonance effects, and other mechanisms all neglected here.</p>", "<p>It is well known that objects with compact support\nhave a Fourier transform with nonlimited support. For example, the Fourier\ntransform of a rectangle is composed of sinc functions in each dimension.\nBecause only a single location of the Fourier space can be measured at a time,\nit is impossible to fully sample such Fourier transform by travelling the MRI\n<italic>k</italic>-space with magnetic field gradients. Hence, there are two experimental\nrestrictions for MRI. First, the continuous Fourier transform is sampled\ndiscretely, which can be seen as a multiplication with a comb-function in frequency\nspace. In image space, this corresponds to a convolution with a reciprocally\nspaced comb-function and leads to periodic object copies with a spacing inverse\nto the sample distance in <italic>k</italic>-space. Second, the Fourier transform is sampled\nonly within a finite region around the <italic>k</italic>-space center with all other\ninformation missing.</p>", "<p>In the conventional case, a discrete Fourier\ntransformation of the finitely measured data is performed to reconstruct an\nimage. This strategy implicitly assumes that the Fourier transform is zero\neverywhere outside the sampled region. It is clear that the assumption is not\nvery appropriate for finite objects, although the corresponding reconstruction\ntotally complies with all data measured. In fact, any solution that coincides\nat the sampling positions is a valid reconstruction, because the finite\nsampling pattern opens degrees of freedom from the null space of the projection\nevoked by finite sampling. Setting this null space to zero is a simple and\nconvenient solution. Unfortunately, however, the procedure corresponds to a\nmultiplication of the true object's Fourier transform with a rect-function (in\ncase of Cartesian sampling) which, in image space, results in a convolution of\nthe true object with a sinc-function. This effect is well known as truncation\nartifact or Gibbs ringing and mainly appears\nas an oscillating overshoot of the image intensity near\ndiscontinuities [##UREF##0##1##, ##UREF##1##2##]. Although the problem may be\nreduced by increasing the measured <italic>k</italic>-space, many practical applications still\nrely on acquisitions with a relatively low-matrix resolution in at least one\nimage dimension, and therefore suffer from respective artifacts.</p>", "<p>So far, various methods have been developed to\nameliorate image disturbances due to finite sampling [##UREF##1##2##–##REF##16254953##5##]. However, in the majority of\nMRI applications and, in particular, for most commercially available MRI\nsystems, only a simple data filtering is routinely employed. In this case,\nvisual reduction of the ringing artifacts is achieved by a smearing of the\nintensity oscillations, which leads to an undesired loss of image resolution.\nAlternative methods attempt to extrapolate the measured data and thereby avoid\na sharp cut-off in <italic>k</italic>-space [##REF##2067388##6##–##REF##11700743##9##]. A key difference to the filtering approach is that\nthe actually measured data is not changed but supplemented with synthetic data—a reasonable strategy as the measured data is not incorrect but only\nincomplete. This can be achieved by exploiting a priori knowledge about the\ntrue object and, consequently, all extrapolation techniques rely on certain\nassumptions, where the existing methods follow different strategies. In this\nregard, the present work demonstrates that also the very unspecific assumption\nof a piecewise-constant object can be utilized to successfully extrapolate data\nin <italic>k</italic>-space and concomitantly reduce the ringing artifacts without compromising\nimage resolution.</p>" ]

[ "<title>3. METHODS</title>", "<p>Simulations were performed with the Shepp-Logan\nphantom, which is composed of several ellipses. Because the Fourier transform\nof a single ellipse is given by a Bessel function, an analytical Fourier\ntransform of the phantom is obtained by a superposition of respective Bessel\nfunctions. Truncation artifacts can be studied by evaluating the noncompact\nanalytical transform at the sampling positions along the trajectory, here\nyielding a matrix of 96 × 96 Fourier samples. All simulations and\nprocessing of experimental data were done offline using an in-house software\npackage written in C/C++.</p>", "<p>MRI experiments were conducted at 2.9 T (Siemens\nMagnetom TIM Trio, Erlangen, Germany) with use of a receive only 12-channel\nhead coil equipped with hardware signal combiners, yielding four receiver\nchannels with different combinations of the coil elements. Measurements were\nperformed for a water phantom and human brain in vivo, where written informed\nconsent was obtained from all subjects prior to each examination. For\ndemonstration purposes, the image acquisitions were done with a simple\nslice-selective spin-echo sequence at a 200 × 200 mm² field of view, covered by a 96 × 96 acquisition matrix. Different sequence\nsettings were used to obtain data sets with low and high level of noise, where\nthe latter was achieved by reducing the flip angle and slice thickness while\nincreasing the receiver bandwidth. Further, one data set was acquired with a\nslice-selective gradient-echo sequence, which allowed for the rapid measurement\nof a full 288 × 288 acquisition matrix.</p>", "<p>All images were reconstructed on a 288 × 288 matrix corresponding to an extrapolation\nfactor of 3. The proposed algorithm was run for a fixed number of 3000\niterations, which takes about 2-3 minutes on a standard microprocessor. In\ncases where an additional data fitting term was used, the weighting factor <italic>λ</italic> was adjusted manually to yield a reasonable\nsolution as judged by visual inspection. Zero-padded solutions with and without\nfiltering were calculated for comparison. Here, a simple Lanczos sigma filter,\nthat is, multiplication with a sinc-function, was applied, where the window\nwidth was selected such that the sinc-function's first null coincides with the\nborder of the measured <italic>k</italic>-space. Although other filters might perform better, it\nserves to demonstrate the general problem related to data filtering. In\naddition, a two-dimensional version of the extrapolation method described by\nConstable and Henkelman [##REF##2067388##6##] was implemented with a window width of <italic>P</italic> = 2 for the edge-preserving sigma filter. In our\nimplementation, the filter parameter Δ was selected according to Δ = <italic>c</italic> · <italic>I</italic>,\nwhere <italic>I</italic> denotes the intensity of the pixel to be\nfiltered and <italic>c</italic> is a global coefficient that was set to <italic>c</italic> = 0.1 based on visual inspection. Finally, all\nimages were magnified and cropped to improve the visibility of the artifacts.</p>" ]

[ "<title>4. RESULTS</title>", "<p>\n##FIG##1##Figure 2## shows different reconstructions of the\nShepp-Logan phantom (left column) together with the respective Fourier\ntransforms (right column). It is clearly visible that the zero-padded solution\n(zero) suffers from severe ringing artifacts around all edges of the phantom.\nThe extent of the measured <italic>k</italic>-space can be seen in its Fourier transform. Most\nringing artifacts disappear after filtering (filter), however, at the expense\nof a significant loss of image resolution. In contrast, the image reconstructed\nwith the proposed method (TV) is neither affected by ringing artifacts nor by\nblurring, and it presents with considerably\nsharper edges relative to the zero-padded solution. Its Fourier transform\nreveals that the measured data has been properly extrapolated into the\nuncovered areas of <italic>k</italic>-space. For comparison, a full reconstruction from 288 × 288 samples is shown in the bottom row (full).</p>", "<p>\n##FIG##2##Figure 3## demonstrates the application of the method to\nexperimental data obtained for a phantom (left column) and a human brain in\nvivo (right column) in comparison to zero-padded (zero) and filtered\nzero-padded solutions (filter). Again, the ringing artifacts obtained for zero\npadding (indicated by arrows) are significantly reduced when using\nTV-constrained data extrapolation with only first-order (TV) or additionally\nsecond-order derivatives (TV2). The blocky appearance of the TV reconstruction\nbecomes much more smoother for the TV2 approach, although both solutions (TV\nand TV2) look somewhat more blocky than the zero-padding solution.</p>", "<p>In ##FIG##3##Figure 4##, the proposed approach (TV2) is compared\nto a 2D version of the extrapolation method by Constable and Henkelman (comp)\nfor the Shepp-Logan phantom (left column) and an experimental study of the\nhuman brain in vivo (right column). It can be seen that the performance of the\nproposed extrapolation method is slightly better for the simulated data, while\nboth approaches yield an effective suppression of ringing artifacts for the\nexperimental data (note that the alternative method leads to a slight denoising\nof the image). However, in our implementation, the algorithm by Constable and\nHenkelman tends to be sensitive to the parameter selection for the initial\nfilter that is used to detect true edges of the object, whereas a selection of\nrespective parameters is not needed in the TV-based approach.</p>", "<p>\n##FIG##4##Figure 5## shows reconstructions of the Shepp-Logan\nphantom from noisy data using zero-padding (zero), the proposed extrapolation\napproach (TV), and its combination with denoising (TVdns). While the basic\nextrapolation approach leads to a reduction of truncation artifacts also for\nnoisy data, it does not reduce the noise patterns. However, when extending the\nTV penalty to the measured data, the method effectively flattens noise patterns\nin addition to the suppression of ringing artifacts.</p>", "<p>Finally, corresponding reconstructions from\nexperimental data with a high degree of noise are shown in ##FIG##5##Figure 6##. Here, a\ncombination of first- and second-order derivatives was used for the TV\ncalculation. As in the simulations, the proposed method leads to a reduction of\ntruncation artifacts (TV2), while the extension to data fitting yields an\nadditional edge-preserving denoising (TV2dns).</p>" ]

[ "<title>5. DISCUSSION</title>", "<title>5.1. Accuracy and limitations</title>", "<p>Both simulations and experiments demonstrate that\nTV-constrained data extrapolation effectively reduces truncation artifacts due\nto finitely sampled MRI acquisitions. Usually, the images exhibit a more blocky\nappearance compared to zero-padding. However, it should be noted that the\nsmoothness observed for zero-padding originates to a significant degree from\nthe convolution with the sinc-function. As a consequence, a sharp edge of the\nobject is mapped as a rather smooth pattern, which might appear more familiar\nto the viewer than a blocky image, but strictly represents an image artifact.\nHence, the extrapolation technique may even lead to a slight gain of resolution\ndue to a sharpening of the point-spread function, following from the\nreciprocity property of the Fourier transformation. This effect can be best\nseen in ##FIG##5##Figure 6## when comparing the borders of the dark brain vessels obtained\nfor zero padding (zero) with the proposed method (TV2).</p>", "<p>Residual image artifacts are explained by multiple\nreasons. First, the method is based on the assumption that the true object is\npiecewise constant, which is only approximately valid for real-world objects.\nIn the presence of additional experimental effects like flow artifacts, the\nassumption might be even less appropriate. Hence, the extrapolation performance\ndepends on the object's conformance with the assumption that it is\npiecewise-constant. Moreover, if the true object contains strongly varying\npatterns, the algorithm may erroneously soften such patterns by supplementing\nrespective high frequencies. On the other hand, in the majority of cases, the\nassumption of a piecewise-constant object seems to be more appropriate than\nthat of all conventional reconstructions, namely, a Fourier transform of the\nobject that is zero outside the sampled <italic>k</italic>-space area.</p>", "<p>Second, the proposed method synthesizes only a finite\nnumber of additional frequencies, whereas an infinite number of <italic>k</italic>-space samples\nwould be required to completely eliminate all truncation effects. In practice,\nhowever, it turned out that there is no perceivable benefit of extrapolating by\na factor of higher than three. The reason is that the method yields an implicit\nfiltering of the extrapolated data: assuming that the extrapolation procedure\nwould recover the unmeasured <italic>k</italic>-space samples exactly, then a new truncation\neffect would arise at the extended border and again lead to ringing artifacts\nin image space (though with a higher oscillation frequency). Because this would\nincrement the TV value, the method automatically lowers outer frequencies during\nthe extrapolation procedure to prevent the upcoming of new ringing artifacts.\nHence, the extrapolated values diverge categorically from the true frequencies\nwhich, in this case, is a rather desirable feature as the prime target is to\nreduce visually annoying ringing artifacts rather than to gain\nsuper-resolution.</p>", "<p>Third, if a completely artifact-free reconstruction of\nthe object would be available, then respective frequency samples could be\ncalculated with a discrete Fourier transformation of the given image.\nInterestingly, these samples would diverge from the experimentally measured\nfrequencies, because image pixels are discrete and, thus, the Fourier transform\nof the image is periodic such that outer frequencies from neighboring copies\n(of the true object's noncompact Fourier transform) overlap. This is different\nfrom the experimental situation where the object is continuous and the outer\nfrequencies are missing instead of overlapping. Consequently, an artifact-free\ndiscrete reconstruction can only be obtained if the samples used for the\nreconstruction specifically diverge from the measured frequencies. A complete\nartifact removal, therefore, requires to alter the measured frequencies instead\nof keeping them unchanged. Unfortunately, the\ninformation how the samples have to\nbe adjusted is not available, so that in practice a data fitting term might be\nthe best solution when a complete removal of ringing artifacts is needed.\nHowever, this might cause a loss of object detail as described in the theory\nsection.</p>", "<title>5.2. Implementation issues</title>", "<p>The modulus function in the TV formula (##FORMU##2##2##) has a\nfundamental role for the success of TV-based image processing. Because\nsubtraction of neighboring pixels—performed before taking the modulus—can\nbe seen as applying a difference operator to the estimate, TV minimization\nyields a solution with minimum L1-norm in the difference basis. Due to the\nspecific character of the modulus function, this solution tends to be sparse in\nthe difference basis: it has few large jumps and most differences between\nneighboring pixels are near zero, which directly translates into a\npiecewise-constant image (and explains the edge preserving character of\nTV-based denoising). If the modulus would be replaced by a square function,\nthen the optimizer would try to find a minimum L2-norm solution with minimal\njumps between all neighboring pixels. This corresponds to a globally smooth\nimage, which is usually undesired due to a loss of sharp edges. While it is\nrather simple to obtain a minimum L2-norm solution as its cost function is\nstrictly convex, finding a minimum L1-norm solution is much more challenging;\nand many optimization algorithms fail if directly applied to the TV problem.\nOne major reason is that the derivative of the modulus function is just ± 1,\nwhich does not help to guess a reasonable step size toward the function's\nminimum. However, it turned out that the CG-Descent algorithm is capable to\nhandle the problem as it comprises a powerful line-search procedure, but it is\nprobably not the optimal method for finding the solution. In particular, the\nconvergence tends to be somewhat sensitive to the scaling of the data. In order\nto ensure convergence, it was, therefore, necessary to introduce a scaling\nfactor that limits the modification strength for each iteration and to run the\nalgorithm in turn for a high number of iterations (e.g., 3000 iterations as\narbitrarily chosen here). Nevertheless, this issue should not be seen as a\ndrawback of the proposed extrapolation approach itself, but rather as a\ntechnical aspect of the optimization method utilized in this proof-of-principle\nstudy. Employing a dedicated algorithm for TV minimization should render a\nscaling factor unnecessary and significantly improve the convergence rate.\nAlthough such enterprise promises reconstructions in a fraction of the current\nprocessing time, it is outside the scope of the present study.</p>" ]

[ "<title>6. CONCLUSION</title>", "<p>The present work demonstrates that the simple\nassumption of a piecewise-constant object can be exploited to extrapolate\nmeasured data in <italic>k</italic>-space. This allows for a significant reduction of ringing\nartifacts that arise from data truncation in <italic>k</italic>-space. In contrast to commonly\nused filtering approaches, the method does not degrade the spatial resolution\nof the reconstructed image and rather leads to a mild resolution enhancement\ndue to sharpening of the point-spread function. If the measured data is\nseriously contaminated by noise, an extended approach offers edge-preserving\ndenoising by slightly altering the measured data in addition to supplementing\nsynthetic data. Both variants can be implemented as a pure postprocessing\nprocedure and are also applicable for partial Fourier acquisitions. Therefore,\nno modification of the MRI sequence is required. While the current\nimplementation suffers from a relatively high computational load, the use of a\ndedicated TV optimization algorithm promises a processing speed suitable for\nroutine applications.</p>" ]

[ "<p>Recommended by David Wilson</p>", "<p>The finite sampling of <italic>k</italic>-space in MRI causes spurious image artifacts, known as Gibbs ringing, which result from signal truncation at the border of <italic>k</italic>-space. The effect is especially visible for acquisitions at low resolution and commonly reduced by filtering at the expense of image blurring. The present work demonstrates that the simple assumption of a piecewise-constant object can be exploited to extrapolate the data in <italic>k</italic>-space beyond the measured part. The method allows for a significant reduction of truncation artifacts without compromising resolution. The assumption translates into a total variation minimization problem, which can be solved with a nonlinear optimization algorithm. In the presence of substantial noise, a modified approach offers edge-preserving denoising by allowing for slight deviations from the measured data in addition to supplementing data. The effectiveness of these methods is demonstrated with simulations as well as experimental data for a phantom and human brain in vivo.</p>" ]

[ "<title>2. THEORY</title>", "<p>\n##FIG##0##Figure 1## compares the one-dimensional profile of a\nrectangle reconstructed by Fourier transformation from only 96 Fourier samples\nto that of the original function. It clearly illustrates severe ringing\nartifacts, although the true function is piecewise constant and free of any\noscillations. Such oscillations can be quantified using the total variation\n(TV), which sums the modulus of jumps between all neighboring pixels of a\nreconstructed image <italic>I</italic>(<italic>x</italic>,<italic>y</italic>)The TV concept was initially\nintroduced to image processing by Rudin et al. [##UREF##2##10##] for denoising applications\nbecause noise patterns create a high TV value relative to that of a noise-free\nimage, and they become particularly reduced when modifying the image in such a\nway that the TV value is minimized. As a specific property, edges are preserved\nduring this procedure, and thus TV minimization emerged as one the most popular\ndenoising techniques. In recent years, the TV concept is attracting strong\ninterest in the field of compressed sensing [##UREF##3##11##] because for specific sampling techniques, the TV\nvalue can be utilized to identify and to remove artifacts from undersampling,\noffering a remarkable reduction of the measurement time [##REF##17534903##12##]. In a similar manner,\ntruncation artifacts lead to an increased TV value relative to that of the true\nobject, so that the TV may also be taken as a measure of the artifact strength\nfor finite <italic>k</italic>-space sampling, which has been recognized by Landi et al. as\nwell [##UREF##4##13##]. Therefore,\nthe proposed idea is to add a set of synthetic frequencies to the measured data ,\nwhich is specifically chosen such that the TV value of the image reconstructed\nfrom the combination of the measured and synthetic data is minimizedwhere <italic>ℱ</italic> denotes the discrete Fourier transformation.\nInterestingly, by searching for the set of synthetic frequencies ,\nthe unmeasured <italic>k</italic>-space data is recovered to a certain degree if the assumption\nof a piecewise-constant object is appropriate.</p>", "<p>Estimation of the synthetic data can be achieved by\nminimizing (##FORMU##5##3##) with a nonlinear numerical optimization technique. The present\nproof-of-principle implementation used the CG-Descent algorithm [##UREF##5##14##], which is a recent variant\nof the nonlinear conjugate gradient method that allows to rather efficiently\nsolve large-scale problems. The algorithm can be used in a black-box manner,\nrequiring only the evaluation of a cost function and its gradient for given\nestimate vectors .\nThe cost function is needed to quantify the goodness of a given estimate (i.e.,\nit is small for a good estimate and large otherwise), and for the problem\ndefined in (##FORMU##5##3##) it simply has the formThe gradient of the cost\nfunction corresponds to the derivative of this function with respect to all\ncomponents of the estimate vector .\nBecause the discrete Fourier transformation is a unitary operation, it can be\nevaluated conveniently by calculating the gradient of the TV term in the image\ndomain (i.e., estimating a vector that describes how the TV value changes for\nmodifications of the individual pixels), followed by an inverse Fourier\ntransformation to the frequency domain.</p>", "<title>2.1. Extended TV formulation</title>", "<p>Calculation of the TV value according to (##FORMU##2##2##) uses only\nthe first-order derivative of the image with respect to its <italic>x</italic>- and\n<italic>y</italic>-directions. This value is minimized if an image consists of areas with\nconstant signal intensity, so that the extrapolation procedure yields a\nsolution primarily with constant areas. While desirable for truly flat objects\nlike numerical phantoms, it tends to create images with a slightly blocky or\npatchy appearance for real-world objects. Therefore, it is advisable to\nadditionally include second-order derivatives into the TV term, which then\nallows for intensity gradients in the images and yields more naturally looking\nsolutionsHere, is a weighting factor which can be used to\ntune the images between a slightly more blocky looking and a slightly smoother\nappearance. For the reconstructions presented, it was set to <italic>σ</italic> = 0.77 based on the considerations by Geman and Yang [##REF##18290044##15##].</p>", "<title>2.2. Edge-preserving denoising</title>", "<p>In practice, experimental MRI data can be\nsignificantly contaminated by Gaussian noise. While the aforementioned approach\nis still able to reduce visible truncation artifacts under these circumstances,\nit does not reduce image noise because the measured <italic>k</italic>-space data remains\nunchanged. On the other hand, an additional denoising may be achieved by\nloosing the fixed bound on the measured data, that is by introducing a data\nfitting term. In this case, the algorithm not only adds synthetic frequencies\nto obtain a TV minimization, but is also allowed to find a solution that\nslightly diverges from the measured data, which yields an effective\nedge-preserving denoising. Therefore, the estimate vector has to be extended such that it contains both\nsynthesized frequencies as well as frequencies from the measured part of\n<italic>k</italic>-space, which is indicated by writing instead.</p>", "<p>In the denoising case, the cost function takes the\nformwhere ⊖ denotes an operation that calculates the\nresidual between the measured values and the corresponding entries of the\nestimate, which are now contained in the vector .\nFurther, <italic>λ</italic> is a weighting factor that allows to select\nthe desired denoising strength. While a low weight permits considerable\ndivergences from the measured values and, thus, leads to an effective removal\nof noise, it can also cause a loss of object detail if selected too low.\nTherefore, the weight has to be adjusted with respect to the signal-to-noise\nratio of the measurement sequence, where a reasonable strategy is to estimate a\nfixed value once for each protocol by computing a set of test images with\ndifferent <italic>λ</italic> values and selecting the value yielding the\ndesired degree of denoising.</p>", "<title>2.3. Phase variations</title>", "<p>Although the basic physical quantity measured by MRI,\nthat is, the spin-density modulated by relaxation or saturation effects, should\nbe real-valued and nonnegative in theory, inherent experimental phase\nvariations usually cause the observed object to be complex-valued. Moreover,\nmodern MRI systems often use multiple receive coils with complex-valued\nsensitivity profiles, yielding differently modulated views of the object. As a\nconsequence, spatially varying transitions between the real and imaginary\ncomponent occur as well as local intensity changes, which conflict with the\nassumption of a piecewise-constant quantity and prevent a direct application of\nthe TV constraint. Therefore, some mechanism is required to cope with the phase\nvariations and the multicoil scenario.</p>", "<p>In this proof-of-principle study, phase variations\nwere removed in a preprocessing step by performing a Fourier transformation of\nthe data from each coil and calculating the sum-of-squares of all channels in\nthe image domain. Subsequently, an inverse Fourier transformation of the sum-of-squares\ndata was performed to obtain a combined data set with real-valued and\nnonnegative values in the image domain, which enables a calculation of the TV\nvalue using only the real part of the image. While this simple technique turned\nout to be sufficient for demonstrating a removal of truncation artifacts by\nTV-constrained data extrapolation, routine applications will probably require a\nmore sophisticated procedure, in particular when combined with advanced\ntechniques such as parallel imaging and when using complex coil configurations\nwith more localized sensitivities of the individual receiver elements.</p>" ]

[]

[ "<fig id=\"fig1\" position=\"float\"><label>Figure 1</label><caption><p>(Top) One-dimensional profile of a rectangle\nreconstructed by Fourier transformation from 96 Fourier samples (solid line) in\ncomparison to the true function (dotted). While the true function is piecewise\nconstant, the Fourier reconstruction exhibits severe ringing artifacts due to\ntruncation of the Fourier coefficients, which causes an increased total\nvariation (TV) value. (Bottom) Magnified view.</p></caption></fig>", "<fig id=\"fig2\" position=\"float\"><label>Figure 2</label><caption><p>(Left) Images of the numerical Shepp-Logan\nphantom (96 × 96 samples, 288 × 288 reconstruction matrix) and (right)\ncorresponding <italic>k</italic>-space representations reconstructed using zero-padding (zero),\nfiltered zero-padding (filter), and the proposed extrapolation method (TV). For\ncomparison, a data set with a fully sampled 288 × 288 matrix is shown (full). Arrow = truncation\nartifact.</p></caption></fig>", "<fig id=\"fig3\" position=\"float\"><label>Figure 3</label><caption><p>Spin-echo images (96 × 96 samples, 288 × 288 reconstruction matrix) of (left) a phantom\n(TR/TE = 4000/8 ms, BW 243 Hz/pixel, FA 70°,\n3 mm slice) and (right) a human brain in vivo (TR/TE = 4000/25 ms, BW 180 Hz/pixel, FA 90°,\n2 mm slice) using zero-padding (zero), filtered zero-padding (filter), the\nproposed extrapolation method with first-order (TV), and additionally\nsecond-order derivatives (TV2). Arrows = truncation artifacts.</p></caption></fig>", "<fig id=\"fig4\" position=\"float\"><label>Figure 4</label><caption><p>(Left) Images of the Shepp-Logan\nphantom (96 × 96 samples, 288 × 288 reconstruction matrix) and (right)\ngradient-echo images of the human brain in vivo (TR/TE = 500/8 ms, BW 80 Hz/pixel, FA 30°,\n2 mm slice) reconstructed using zero-padding (zero), the extrapolation method\nby Constable and Henkelman (comp), and the proposed method with first- and\nsecond-order derivatives (TV2). For comparison, reconstructions from fully\nsampled 288 × 288 matrices are shown (full). Arrows = truncation\nartifacts.</p></caption></fig>", "<fig id=\"fig5\" position=\"float\"><label>Figure 5</label><caption><p>Images of the numerical Shepp-Logan phantom\nreconstructed from noisy data (96 × 96 samples, 288 × 288 reconstruction matrix) using zero-padding\n(zero), the proposed extrapolation method (TV), and the proposed method\ncombined with denoising (TVdns). Arrow = truncation artifact.</p></caption></fig>", "<fig id=\"fig6\" position=\"float\"><label>Figure 6</label><caption><p>Spin-echo images of (top) a phantom (TR/TE = 4000/100 ms, BW 789 Hz/pixel, FA 50°,\n1 mm slice) and (bottom) a human brain in vivo (TR/TE = 4000/15 ms, BW 401 Hz/pixel, FA 70°,\n1 mm slice) reconstructed from noisy data (96 × 96 samples, 288 × 288 reconstruction matrix) using zero-padding\n(zero), the proposed extrapolation method (TV2), and the proposed method\ncombined with denoising (TV2dns). Arrows = truncation artifacts.</p></caption></fig>" ]

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stretchy=\"false\">)</mml:mo><mml:mo> </mml:mo><mml:mo>=</mml:mo><mml:mstyle><mml:mrow><mml:munderover><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>y</mml:mi><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:munderover></mml:mrow></mml:mstyle><mml:mtext> </mml:mtext><mml:mstyle><mml:mrow><mml:munderover><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>x</mml:mi><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:munderover></mml:mrow></mml:mstyle><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mo>−</mml:mo><mml:mn>1,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mo> </mml:mo><mml:mo>+</mml:mo><mml:mo> </mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mn>.</mml:mn></mml:mrow></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M4\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>v</mml:mi><mml:mo>→</mml:mo></mml:mover></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M5\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>y</mml:mi><mml:mo>→</mml:mo></mml:mover></mml:mrow></mml:math></inline-formula>", "<disp-formula id=\"eq3\"><label>(3)</label><mml:math id=\"M6\"><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover><mml:mo> </mml:mo><mml:mo>=</mml:mo><mml:mo> </mml:mo><mml:munder><mml:mrow><mml:mtext>arg</mml:mtext><mml:mtext>min</mml:mtext></mml:mrow><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover></mml:mrow></mml:munder><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mtext>TV</mml:mtext><mml:mo minsize=\"1em\" maxsize=\"1em\">(</mml:mo><mml:mi>ℱ</mml:mi><mml:mo stretchy=\"false\">{</mml:mo><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover><mml:mo>⊕</mml:mo><mml:mover accent=\"true\"><mml:mrow><mml:mi>y</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover><mml:mo stretchy=\"false\">}</mml:mo><mml:mo minsize=\"1em\" maxsize=\"1em\">)</mml:mo><mml:mtext> </mml:mtext><mml:mn>,</mml:mn></mml:mrow></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M7\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>v</mml:mi><mml:mo>→</mml:mo></mml:mover></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M8\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>v</mml:mi><mml:mo>→</mml:mo></mml:mover></mml:mrow></mml:math></inline-formula>", "<disp-formula id=\"eq4\"><label>(4)</label><mml:math id=\"M9\"><mml:mrow><mml:mi>Φ</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover><mml:mo stretchy=\"false\">)</mml:mo><mml:mo> </mml:mo><mml:mo>=</mml:mo><mml:mo> </mml:mo><mml:mtext>TV</mml:mtext><mml:mo minsize=\"1em\" maxsize=\"1em\">(</mml:mo><mml:mi>ℱ</mml:mi><mml:mo minsize=\"0.75em\" maxsize=\"0.75em\">{</mml:mo><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover><mml:mo>⊕</mml:mo><mml:mover accent=\"true\"><mml:mrow><mml:mi>y</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover><mml:mo minsize=\"0.75em\" maxsize=\"0.75em\">}</mml:mo><mml:mo minsize=\"1em\" maxsize=\"1em\">)</mml:mo><mml:mtext> </mml:mtext><mml:mn>.</mml:mn></mml:mrow></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M10\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>v</mml:mi><mml:mo>→</mml:mo></mml:mover></mml:mrow></mml:math></inline-formula>", "<disp-formula id=\"eq5\"><label>(5)</label><mml:math id=\"M11\"><mml:mrow><mml:mtable columnalign=\"left\" width=\"auto\"><mml:mtr><mml:mtd/><mml:mtd><mml:mtext>T</mml:mtext><mml:msub><mml:mrow><mml:mtext>V</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo> </mml:mo><mml:mo>=</mml:mo></mml:mtd></mml:mtr><mml:mtr><mml:mtd/><mml:mtd><mml:mrow><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mstyle><mml:mrow><mml:munderover><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>y</mml:mi><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:munderover></mml:mrow></mml:mstyle><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mstyle><mml:mrow><mml:munderover><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>x</mml:mi><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:munderover><mml:mtext> </mml:mtext><mml:mi>σ</mml:mi><mml:mo>⋅</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mo>−</mml:mo><mml:mn>1,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">|</mml:mo></mml:mrow></mml:mstyle><mml:mo> </mml:mo><mml:mo>+</mml:mo><mml:mo> </mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd/><mml:mtd><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mo>⁢</mml:mo><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mi>σ</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>⋅</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mo>−</mml:mo><mml:mn>1,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:mo>⋅</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo> </mml:mo><mml:mo>+</mml:mo><mml:mo> </mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mo> </mml:mo><mml:mo>+</mml:mo><mml:mo> </mml:mo><mml:mn>1,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">|</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd/><mml:mtd><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:mo>⋅</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo> </mml:mo><mml:mo>+</mml:mo><mml:mo> </mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo> </mml:mo><mml:mo>+</mml:mo><mml:mo> </mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">|</mml:mo></mml:mtd></mml:mtr><mml:mtr><mml:mtd/><mml:mtd><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mrow><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mo>−</mml:mo><mml:mn>1,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>−</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mn>,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd/><mml:mtd><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mo>+</mml:mo><mml:mi>I</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>x</mml:mi><mml:mo>−</mml:mo><mml:mn>1,</mml:mn><mml:mo> </mml:mo><mml:mi>y</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mo stretchy=\"false\">)</mml:mo><mml:mn>.</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M12\"><mml:mrow><mml:mi>σ</mml:mi><mml:mo>∈</mml:mo><mml:mo>[</mml:mo><mml:mtable><mml:mtr><mml:mtd><mml:mn>0</mml:mn></mml:mtd><mml:mtd><mml:mn>1</mml:mn></mml:mtd></mml:mtr></mml:mtable><mml:mo>]</mml:mo></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M13\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>v</mml:mi><mml:mo>→</mml:mo></mml:mover></mml:mrow></mml:math></inline-formula>", "<inline-formula><mml:math id=\"M14\"><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>v</mml:mi><mml:mo>→</mml:mo></mml:mover><mml:mi>d</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>", "<disp-formula id=\"eq6\"><label>(6)</label><mml:math id=\"M15\"><mml:mrow><mml:mi>Φ</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>d</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">)</mml:mo><mml:mo> </mml:mo><mml:mo>=</mml:mo><mml:mo> </mml:mo><mml:mi>λ</mml:mi><mml:mo>⋅</mml:mo><mml:mo minsize=\"0.75em\" maxsize=\"0.75em\">∥</mml:mo><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>d</mml:mi></mml:mrow></mml:msub><mml:mo>⊖</mml:mo><mml:mover accent=\"true\"><mml:mrow><mml:mi>y</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover><mml:mrow><mml:msubsup><mml:mrow><mml:mo minsize=\"0.75em\" maxsize=\"0.75em\">∥</mml:mo></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup></mml:mrow><mml:mo> </mml:mo><mml:mo>+</mml:mo><mml:mo> </mml:mo><mml:mtext>TV</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>ℱ</mml:mi><mml:mo stretchy=\"false\">{</mml:mo><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mo>→</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>d</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">}</mml:mo><mml:mo stretchy=\"false\">)</mml:mo><mml:mtext> </mml:mtext><mml:mn>,</mml:mn></mml:mrow></mml:math></disp-formula>", "<inline-formula><mml:math id=\"M16\"><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>v</mml:mi><mml:mo>→</mml:mo></mml:mover><mml:mi>d</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>" ]

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[ "<graphic xlink:href=\"IJBI2008-184123.001\"/>", "<graphic xlink:href=\"IJBI2008-184123.002\"/>", "<graphic xlink:href=\"IJBI2008-184123.003\"/>", "<graphic xlink:href=\"IJBI2008-184123.004\"/>", "<graphic xlink:href=\"IJBI2008-184123.005\"/>", "<graphic xlink:href=\"IJBI2008-184123.006\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>1. INTRODUCTION</title>", "<p>Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone,\nare highly effective for the treatment of type 2 diabetes and are widely\nprescribed. Unfortunately, fluid retention has emerged as the most common and\nserious side effect of TZDs and has become the most frequent cause of\ndiscontinuation of therapy. The incidence of TZD-induced fluid retention ranges\nfrom 7% in monotherapy and to as high as 15% when combined with insulin [##REF##10926309##1##–##REF##11197573##3##]. The fluid\nretention is often presented as peripheral edema, which can progress into pulmonary\nedema and congestive heart failure. TZD use leads to a 6-7% increase in\nblood volume in healthy volunteers [##UREF##1##4##, ##UREF##2##5##]. This blood\nvolume expansion can dilute the red blood cell concentration, producing a\nreduced hematocrit. In fact, changes in hematocrit have been used as a\nsurrogate marker for TZD-induced plasma volume expansion. The fluid retention\nis often resistant to loop diuretics but is reversed by withdrawing the drug. Many\naspects of TZD-induced fluid retention have been covered by excellent review\narticles [##REF##18276926##6##–##UREF##3##12##]. This review will emphasize renal sodium retention and vascular\nhyperpermeability as prominent mechanisms of TZD-induced fluid retention. We\nwill also introduce several possible treatment strategies.</p>" ]

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[ "<title>5. CONCLUSIONS</title>", "<p>The fluid retention and rapid body weight\ngain induced by TZD treatment are caused by increased fluid reabsorption in the\ndistal nephron as well as increased vascular permeability in adipose tissues (see ##FIG##2##Figure 3##).\nThe molecular mechanisms of the effects of TZDs in renal collecting duct and in\nblood vessels remain unknown. Despite documentation of ENaC as a molecular target\nof TZDs in the collecting duct, increasing evidence indicates ENaC-independent\nmechanisms that may involve changes in paracellular transport. PKCß is shown to\nmediate TZD-induced vascular permeability in adipose tissues. More studies are\nrequired for determination of the signaling pathway responsible for PPAR<italic>γ</italic>-dependent\ntissue-specific activation of PKCß. Currently,\nthere are no effective therapies for the side effects of TZDs except drug\nwithdrawal. A number of potential treatment strategies that target collecting\nduct sodium transport (amiloride) and vascular permeability (PKC inhibitors)\nhave been developed from animal studies and should be evaluated by future clinical\ntrials.</p>" ]

[ "<p>Recommended by Jane Pinaire</p>", "<p>Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor subtype <italic>γ</italic> (PPAR<italic>γ</italic>) activators that are clinically used as an insulin sensitizer for glycemic control in patients with type 2 diabetes. Additionally, TZDs exhibit novel anti-inflammatory, antioxidant, and antiproliferative properties, indicating therapeutic potential for a wide variety of diseases associated with diabetes and other conditions. The clinical applications of TZDs are limited by the common major side effect of fluid retention. A better understanding of the molecular mechanism of TZD-induced fluid retention is essential for the development of novel therapies with improved safety profiles. An important breakthrough in the field is the finding that the renal collecting duct is a major site for increased fluid reabsorption in response to rosiglitazone or pioglitazone. New evidence also indicates that increased vascular permeability in adipose tissues may contribute to edema formation and body weight gain. Future research should therefore be directed at achieving a better understanding of the detailed mechanisms of TZD-induced increases in renal sodium transport and in vascular permeability.</p>" ]

[ "<title>2. RENAL MECHANISM</title>", "<p>The kidney is the key regulator of electrolyte balance and water conservation. Fluid\nretention at the renal level is suggested by evidence that TZD-induced edema is\nassociated with reduced urinary sodium and water excretion. Song et al.\nreported that chronic three-day administration of rosiglitazone to Sprague Dawley\nrats significantly reduced urine volume (by 22%) and sodium excretion (by 44%) [##REF##14593089##13##]. These findings lead us to speculate that\nrenal mechanisms play a major role in TZD-induced fluid retention. TZDs may\ncause renal fluid reabsorption directly by affecting tubular transport, renal\nsodium retention, and vascular hyperpermeability or indirectly by affecting\nrenal hemodynamics or processes. Yang et al. examined the effect of a PPAR<italic>γ</italic>\nagonist, GI262570 (farglitazar), on the glomerular filtration rate, effective\nrenal plasma flow, and renal filtration fraction in chronically\ncatheter-implanted conscious rats [##REF##12960690##14##]. In this\nstudy, glomerular filtration rate was determined by using fluorescein\nisothiocyanate (FITC)-inulin and renal blood\nflow by using para-aminohippurate (PAH). A 10-day infusion of GI262570\ndecreased hematocrit, hemoglobin, and serum albumin (all <italic>P</italic> &lt; .05), indicating volume expansion, but did not alter\nglomerular filtration rate, effective renal plasma flow, or renal filtration\nfraction. This indicates that PPAR<italic>γ</italic> agonist-induced volume expansion is not\nrelated to changes in renal hemodynamics [##REF##12960690##14##]. This\nobservation is reinforced by a human study in which the six-week administration\nof pioglitazone to healthy volunteers led to sodium retention without a significant\neffect on glomerular filtration rate or renal blood flow [##REF##15001599##15##]. This lack\nof change in renal hemodynamics is, however, not universally reported. The three-day\nadministration of rosiglitazone in Sprague Dawley rates induced a 35% reduction\nin creatinine clearance, an indirect measure of the glomerular filtration rate [##REF##14593089##13##]. It is\nunclear whether or not this discrepancy is related to differences in glomerular\nfiltration rate measurement techniques or other experimental protocols.</p>", "<p>The lack of solid evidence to support\nthe alteration of renal hemodynamic parameters following treatment with PPAR<italic>γ</italic>\nligands suggests the possibility of a direct influence on tubular transport processes.\nThe regulation of NaCl reabsorption in the kidney can occur at the level of sodium\ntransport proteins lining the renal epithelia. These sodium transporters\ninclude basolateral Na-K-ATPase, and the following apical transporters that\nvary with individual nephron segments: the sodium hydrogenexchanger\nsubtype III (NHE3) and the sodium phosphate cotransporter subtype II (NaPi-2) in the proximal\nconvoluted tubule, the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2\nor BSC1) in the thick ascending limb, the thiazide-sensitive Na-Cl cotransporter (NCC or TSC)\nin the distal convoluted tubule, and the amiloride-sensitive sodium channel\n(ENaC) in the collecting duct. The major water channel proteins (aquaporins,\nAQPs) in the kidney include AQP1-4, of which AQP1 and AQP2 function\non the apical membrane, and AQP3 and AQP4 on the basolateral\nmembrane [##REF##8743483##16##]. The study of Song et al. is the first to\nprovide a comprehensive examination of the effects of PPAR<italic>γ</italic> agonists on various\nrenal sodium and water transport proteins [##REF##14593089##13##]. In that study, a three-day rosiglitazone\ntreatment increasedthe whole kidney protein level of the <italic>α</italic>-1 subunit of Na-K-ATPase, NKCC2,\nNHE3, AQP2, and AQP3 [##REF##14593089##13##]. These findings suggest that increases in\nsodium transport may occur in the proximal convoluted tubule and the thick\nascending limb.</p>", "<p>The collecting duct reabsorbs approximately 2-3% of the filtered\nsodium load primarily through ENaC, which is comprised of three subunits, <italic>α</italic>, <italic>β</italic>,\nand <italic>γ</italic>. These proteins are vital to day-to-day adjustment of sodium reabsorption\nand are regulated by the hormones aldosterone and insulin [##REF##10751213##17##–##REF##10510339##19##]. A key\nmediator of aldosterone activation of ENaC is serum and glucocorticoid\nregulated kinase 1 (SGK1) [##REF##12923071##20##, ##REF##12110505##21##]. Activated SGK1 prevents ENaC degradation by inactivating the ubiquitin\nligase Nedd4-2 [##REF##16192418##22##]. Nedd4-2\ninteracts with the PY motif of ENaC leading to endocytosis and degradation of\nthe channel [##REF##16192418##22##]. Prior to\nthe conditional knockout (KO) studies, three major lines of evidence indicated\nthat the activation of sodium transport processes in the distal nephron may\nunderlie TZD-induced fluid retention. First, within the kidney, PPAR<italic>γ</italic> is highly expressed in the renal medullary\ncollecting duct, with lower expression levels in glomeruli, proximal tubules,\nand microvasculature. This was demonstrated by both RT-PCR and microdissection as well as by in situ hybridization techniques [##REF##9435691##23##–##REF##10600944##25##]. Second, in\na cultured human cortical collecting duct (CCD) cell line, PPAR<italic>γ</italic> agonists\nincreased levels of cell surface <italic>α</italic>-ENaC. This is paralleled by an increase in SGK1 mRNA, which is abolished by pretreatment with\na specific PPAR<italic>γ</italic> antagonist, leading to increased levels of cell surface <italic>α</italic>-ENaC.\nElectrophoretic mobility shift assays further suggest that these effects are\ncaused by the binding of PPAR<italic>γ</italic> to a specific response element in the SGK1 promoter [##REF##12923071##20##]. Third, in vivo evidence shows that GI262570 stimulates\nsodium and water reabsorption from the distal nephron in Sprague Dawley rats [##REF##15475592##26##]. This\nevidence comes from increases in plasma sodium and chloride concentrations with\nconcomitant decreases in plasma potassium concentration. Reciprocal changes in\nplasma NaCl and potassium levels are typically seen as a consequence of renal\nmineralocorticoid activation promoting NaCl reabsorption and potassium\nsecretion in the distal nephron [##REF##15475592##26##]. Additionally,\nmRNA levels for a group of genes involved in distal nephron sodium and water\nabsorption in the kidney medulla are changed with GI262570 treatment [##REF##15475592##26##].</p>", "<p>The involvement of the distal nephron in TZD-induced fluid retention has been assessed in two independent\nstudies using mice with a collecting duct-specific deletion of PPAR<italic>γ</italic> (CD PPAR<italic>γ</italic> KO) \n[##UREF##4##27##, ##REF##15956187##28##]. In both studies, the expression of Cre recombinase was driven by an\nAQP2 promoter highly specific to the collecting duct. In these two studies, the\nexperimental approaches for assessment of fluid retention were quite different:\na combination of hematocrit, plasma aldosterone levels, and Evans blue (EB) dye-based\nmeasurement of plasma volume in one study (see ##FIG##0##Figure 1##) [##REF##15956187##28##] and\ndetermination of total water content in the other [##UREF##4##27##]. Remarkably,\nboth studies reported a similar phenotype in that the conditional PPAR<italic>γ</italic>\nknockout mice proved to be resistant to the rosiglitazone- or\npioglitazone-induced body weight gain and plasma volume expansion found in mice\nexpressing PPAR<italic>γ</italic> in the collecting duct. As shown in ##FIG##0##Figure 1##, a nine-day\nrosiglitazone treatment induced a gradual and significant increase in body\nweight in floxed mice when compared to untreated floxed controls (2.74 ± 0.25 versus 1.05 ± 0.16 gram, on day 9, <italic>P</italic> &lt; .05). In contrast,\nbody weight gains between rosiglitazone-treated and untreated CD PPAR<italic>γ</italic> KO mice\nwere not significantly different (0.90 ± 0.25 versus 0.81 ± 0.19\ngram, on day 9, <italic>P</italic> &gt; .05). Rosiglitazone treatment in the control mice\ninduced plasma volume expansion, which was reflected by a significantly\ndecreased hematocrit and plasma aldosterone levels as well as by a 32.2%\nincrease in plasma volume as assessed by the EB dye technique. In contrast,\nrosiglitazone-treated CD PPAR<italic>γ</italic> KO mice exhibited nonsignificant trends toward\nchange in these parameters (see ##FIG##1##Figure 2##). These two studies also provided\nevidence that exposure of primary collecting duct cells to PPAR<italic>γ</italic> ligands leads\nto increased sodium transport as assessed by measurements of ²²Na⁺ flux and transepithelial resistance.</p>", "<p>Guan et al. examined the effects of pioglitazone on the expression of <italic>α</italic>-, ß-, and <italic>γ</italic>-ENaC\nsubunits in cultured inner medullary collecting duct (IMCD) cells [##UREF##4##27##]. Notably, within one hour following treatment\nof IMCDs with pioglitazone (1 <italic>μ</italic>M), <italic>γ</italic>-ENaC mRNA expression\nincreased roughly 10 folds\nbefore gradually diminishing. This stimulatory effect appeared to be specific\nfor <italic>γ</italic>-ENaC mRNA, because <italic>α</italic>-ENaC and ß-ENaC mRNA levels did not show any change\nin response to treatment with pioglitazone. Interestingly, PPAR response elements (PPREs) are identified in intron 1 but not in the\n5′ flanking region of the <italic>γ</italic>-ENaC gene. Chromatin immunoprecipitation (ChIP) of\ngenomic DNAisolated from cultured\nmouse IMCDs revealed a physical interaction between PPAR<italic>γ</italic> and <italic>γ</italic>-ENaC genomic DNA. Somewhat unexpectedly, the PPAR<italic>γ</italic> binding site\nwas shown to be located outside intron 1 of the <italic>γ</italic>-ENaC gene. Overall, these\ndata support <italic>γ</italic>-ENaC as a direct target gene of PPAR<italic>γ</italic> in the collecting duct\ncells, although the exact mechanism remains to be elucidated.</p>", "<p>However, the role of ENaC as a direct target of PPAR<italic>γ</italic> has not always been demonstrable. Nofziger et al.\nreported that, in collecting duct cell lines, PPAR<italic>γ</italic> agonists failed to enhance\nbasal or insulin-stimulated sodium transport as assessed by measurement of\nshort-circuit current (Isc) [##REF##16170524##29##]. This study\nalso did not find that PPAR<italic>γ</italic>-induced changes in the amount of SGK1 transcript or protein expression. Additionally,\nthere is no solid evidence for major changes in renal expression of any of the\nENaC subunits in response to PPAR<italic>γ</italic> ligands in vivo [##REF##14593089##13##, ##REF##15475592##26##, ##REF##17211457##30##]. More recently, Vallon et al. reported that collecting duct-specific gene\ninactivation of <italic>α</italic>-ENaC in the mouse does not attenuate the rosiglitazone-induced\nbody weight gain [##UREF##5##31##]. In this\nstudy, the Hoxb-7 promoter was used to inactivate <italic>α</italic>-ENaC in the collecting duct,\nwhile leaving ENaC expression in the cortical connecting tubule (CNT) intact [##REF##12925696##32##]. As\nexpected, in the floxed control mice, rosiglitazone treatment (320 mg/kg diet) rapidly\nincreased body weight (ΔBW day 11: 4.5 ± 0.8% versus 1.1 ± 0.6%, <italic>P</italic> &lt; .05) and\nlowered hematocrit (44 ± 1.0% versus 47 ± 1%, <italic>P</italic> &lt; .0005), while rosiglitazone treatment increased body weight (ΔBW: 7.3 ± 0.9% versus 0.9 ± 0.7%, <italic>P</italic> &lt; .0005) and lowered hematocrit (42 ± 2% versus 47 ±\n1%, <italic>P</italic> &lt; .05) in <italic>α</italic>-ENaC collecting\nduct knockout mice. These data may argue against collecting duct ENaC playing a\nsignificant role in mediating the adverse effect of rosiglitazone. However,\ninvolvement of ENaC activity in the CNT cannot be ruled out. To resolve this issue, AQP2-Cre mice could be used to\ninactivate ENaC in the entire collecting duct system.</p>", "<p>The negative results discussed above\nprompt consideration of alternative mechanisms for explaining PPAR<italic>γ</italic>-mediated\nincreases in distal tubular fluid reabsorption. There is a significant amiloride-insensitive\ncomponent in the rosiglitazone-induced increases in sodium transport [##REF##15956187##28##]. The\npossibility exists that increased reabsorption may occur by way of a\nparacellular route. For example, PPAR<italic>γ</italic> may regulate the tight junction leading\nto altered permeability to sodium or other electrolytes. In an in vitromodel of differentiating normal human urothelial (NHU) cells, PPAR<italic>γ</italic> activation in conjunction with epidermal growth factor receptor (EGFR) blockade led to the de novo expression\nof claudin 3 mRNA and protein and downregulation of claudin 2 transcription [##REF##16688762##33##]. These\nresults suggest a role for PPAR<italic>γ</italic> and EGFR signaling\npathways in regulating the tight junction formation in NHU cells. There is an\nintriguing possibility that a similar mechanism may operate in renal epithelial\ncells. Another possible mechanism is that PPAR<italic>γ</italic> may regulate transport of ions other than sodium. Further studies\nare clearly needed to explore not only ENaC-dependent, but also\nENaC-independent mechanisms, for TZD-activated fluid reabsorption in the distal\nnephron.</p>", "<title>3. VASCULAR MECHANISM</title>", "<p>PPAR<italic>γ</italic> is expressed in the vascular system [##REF##10696077##34##], including endothelial\ncells [##REF##9610365##35##, ##REF##9920814##36##], vascular\nsmooth muscle cells (VSMC) [##REF##9655393##37##] as well as monocyte/macrophages\n[##REF##9748221##38##, ##REF##9422508##39##]. Several\nlines of evidence suggest that PPAR<italic>γ</italic> regulates various aspects of vascular\nfunction, including capillary permeability. Increased capillary permeability\nleads to extravasation of fluid and is thought to contribute to edema in\npatients treated with TZDs. Donnelly et al. were the first to examine the\ndirect effect of rosiglitazone on endothelial barrier function using an in vitro system of pulmonary artery\nendothelial cell monolayers. Transendothelial albumin flux was measured using EB\ndye-labeled albumin. They found that exposure to high concentrations of rosiglitazone\nfor four hours increased transendothelial albumin flux dose-dependently, with a\nnoticeable effect at 10 <italic>μ</italic>M and a maximal effect at 100 <italic>μ</italic>M. This\nhyperpermeability response to high concentrations of rosiglitazone was fully\nreversible by washing rosiglitazone off the monolayer. After incubation for 24\nto 48 hours, the effect of rosiglitazone began to subside. High concentrations\nof rosiglitazone (0.1–1 mM) are also\nneeded to induce a vasodilator effect in isolated arteries [##REF##14744930##40##]. Future\nstudies, ideally employing gene knockout mice, may determine the extent of\nPPAR<italic>γ</italic> mediation of the vascular response to high concentrations of TZDs. The\nmechanism of TZD-induced capillary permeability is not well characterized but\nmay involve a number of factors, notably vascular endothelial growth factor (VEGF),\nnitric oxide, and protein kinase C, each of which is discussed below.</p>", "<p>VEGF is a potent cytokine that augments vascular permeability in tumors, healing wounds, retinopathies, many\nimportant inflammatory conditions, and certain physiological processes, such as\novulation and corpus luteum formation [##REF##9893348##41##]. VEGF is estimated to be 50 times more potent than histamine in\nenhancing vascular permeability [##REF##9893348##41##]. The gene transfer of naked plasmid DNA encoding the 165-amino acid isoform of VEGF in patents with peripheral artery\ndisease causes peripheral edema [##REF##10836914##42##]. Evidence\nsuggests an involvement of VEGF in TZD-induced edema. The study of Emorto et\nal. was the first to report that plasma levels of VEGF are significantly \nincreased in troglitazone-treated subjects (120.1 ± 135.0 pg/mL)\ncompared with those treated with diet alone (29.2 ± 36.1 pg/mL), sulfonylurea\n(25.8 ± 22.2 pg/mL), or insulin (24.6 ± 19.0 pg/mL). The effect of\ntroglitazone on increased VEGF levels was further supported by plasma VEGF\nlevels in five patients before treatment (20.2 ± 7.0 pg/mL), after three months\nof troglitazone treatment (83.6 ± 65.9 pg/mL), and three months after\ndiscontinuation (28.0 ± 11.6 pg/mL). These authors further demonstrated that\ntroglitazone, as well as rosiglitazone, at the plasma concentrations observed\nin patients, increased VEGF mRNA levels in 3T3-L1 adipocytes. The finding suggests that PPAR<italic>γ</italic> activation may\ndirectly stimulate expression of VEGF that leads to tissue edema. However, it\nis puzzling that several other studies show that PPAR<italic>γ</italic> negatively regulates\nVEGF signaling. In transformed and primary endometrial cells rosiglitazone or 15-deoxy-delta\n12,14-prostaglandin J₂ (15d-PGJ₂) decreased VEGF protein\nsecretion [##UREF##6##43##]. In\ntransiently transfected Ishikawa cells, rosiglitazone repressed VEGF gene\npromoter-luciferase activation with an IC [##REF##9655393##37##] approximately 50 nM. By using\ntruncated and mutated VEGF promoter constructs, this study further revealed\nthat the PPAR<italic>γ</italic>-regulated domain is a direct repeat (DR)-1 motif −443 bp\nupstream of the transcriptional start site [##UREF##6##43##]. Similarly,\nrosiglitazone attenuated VEGF-induced proliferation and migration of human\npulmonary valve endothelial cells (HPVECs) [##REF##16840174##44##].\nRosiglitazone also antagonized VEGF-induced nuclear factor translocation in activated T cells subtype c1 (NFATc1) [##REF##16840174##44##]. Furthermore,\nrosiglitazone markedly decreased VEGF-induced tube formation and cell migration\nin human umbilical vein endothelial cells [##REF##16297938##45##]. Taking these studies together, it seems likely that PPAR<italic>γ</italic> exerts a dual\neffect on VEGF signaling, possibly depending on cell type.</p>", "<p>Nitric oxide (NO) is a ubiquitous,\nnaturally occurring molecule found in a variety of cell types and organ\nsystems. Endothelial cells are rich in NO, which has been shown to regulate\nmany aspects of vascular function, including vascular permeability. Polikandriotis\net al. report that 15d-PGJ₂ and ciglitazone increase cultured\nendothelial cell NO release without increasing the expression of endothelial\nnitric oxide synthase (eNOS) [##UREF##7##46##]. This study\nprovided further evidence that PPAR<italic>γ</italic> activation leads to eNOS ser1177\nphosphorylation [##UREF##7##46##]. It seems\nplausible that the stimulation of eNOS-derived NO may contribute to TZD-induced\nedema. St-Pierre et al. examined the effect of rosiglitazone on muscle\nvasopermeability and NO system in the fructose-fed rat model [##REF##15130775##47##]. In this\nstudy, extravasation of EB dye in vivo\nin specific muscle groups was used to assess vascular permeability. Fructose-fed\nrats treated with rosiglitazone had a 30–50% increase in extravasation\nof EB in the the Rectus femoris, soleus, gastrocnemius lateralis, vastus\nlateralis, and tibialis cranialis skeletal muscles [##REF##15130775##47##]. In\nhomogenates of skeletal muscles (vastus lateralis) from fructose-fed rats,\nrosiglitazone resulted in a significant increase in nitric oxide synthase (NOS)\nactivity and eNOS immunoreactive content compared to the control animals [##REF##15130775##47##].\nUnexpectedly, the immunoreactive level of the most abundant muscle NOS\nisoforms, neuronal NOS (nNOS), remained unchanged.</p>", "<p>Protein kinase C (PKC) plays a major role in determining vascular permeability through\nphosphorylation of the cytoskeleton proteins that form the tight intercellular\njunction [##REF##10854687##48##–##REF##1522136##51##]. In the study of Sotiropoulos et al., rosiglitazone treatment\nselectively activated PKC in fat and retinal tissues in parallel with the\nincreased vascular permeability in these tissues [##REF##16672634##52##]. The activation of PKC is evaluated by\ndetermining the enzyme activity together with tissue levels of diacylglycerol (DAG), a\nstrong PKC activator [##REF##16672634##52##]. These investigators tested the effect\nof PKC<italic>β</italic> inhibition and gene knockout but did not determine specific PKC\nisoforms. They found that posttreatment with ruboxistaurin (RBX), a PKC<italic>β</italic> inhibitor,\neffectively attenuated the increases in capillary permeability, water content,\nand weight of epididymal fat, as well as the increase in body weight associated\nwith rosiglitazone treatment; this finding was also confirmed by using PKC<italic>β</italic> KO mice\n[##REF##16672634##52##].</p>", "<title>4. POTENTIAL THERAPIES</title>", "<title>4.1. Inhibition of sodium transport in the collecting duct</title>", "<p>The use of diuretics for management of TZD-induced fluid retention has been evaluated\nby several case reports [##UREF##0##2##, ##REF##12396745##53##] and, \nrecently, by a controlled trial [##REF##17093067##54##]. Most case reports show that the edema\nis refractory to a loop diuretic (furosemide) and that the symptoms resolve\nonly after discontinuation of TZD. The recent controlled trial involved 381\npatients with type 2 diabetes. It examined the effect of three diuretics that\nact with different mechanisms on rosiglitazone-induced body weight gain and\nplasma volume [##REF##17093067##54##]. The diuretics included furosemide,\nwhich inhibits the Na-K-Cl cotransporter in the thick ascending limb of the loop\nof Henle, hydrochlorothiazide (HCTZ), which acts to inhibit the Na-Cl\ncotransporter in the distal convoluted tubule, and spironolactone (SPIRO), which\nis an ENaC inhibitor in the collecting duct. The degree of fluid retention in\nthis study was evaluated by measuring changes in the hematocrit as an index of\nchanges in plasma volume, body weight, total body water, and extracellular\nfluid changes determined by noninvasive bioelectrical impedance with an Akern\nsoft tissue analyzer. SPIRO and HCTZ both effectively reduced fluid retention\nand body weight while furosemide had only a limited effect. The effectiveness\nof SPIRO may be attributable to the ability of this diuretic to interfere with\nthe sodium retaining action of PPAR<italic>γ</italic> in the collecting duct. It is unclear\nwhether the same mechanism can explain the action of HCTZ. Thiazide diuretics act\nprimarily in the proximal part of the distal convoluted tubules where they\ninhibit Na⁺/Cl⁻ cotransport [##REF##3631283##55##, ##REF##7840252##56##], but they are also reported to inhibit\nsalt and water reabsorption in the medullary collecting duct [##REF##6876566##57##]. The reason for the lack of diuretic response\nof TZD-treated diabetics to furosemide is not entirely clear, but one possible\nexplanation might be the lack of distal effect of this loop diuretic. Another possibility\nis that TZD-induced fluid retention may be associated with impaired transport machinery\nin the thick ascending limb. Possibly secondary to the volume expansion, the\nplasma level of atrial natriuretic factor (ANF)\nis elevated in TZD-treated diabetics [##REF##17093067##54##]. ANF inhibits NaCl reabsorption in the loop of Henle as well as in other sites of\nnephron through the activation of guanylyl cyclase\nreceptors that release cyclic GMP [##REF##9551429##58##]. It also\nremains possible that PPAR<italic>γ</italic> may negatively affect NaCl transport in the loop of\nHenle.</p>", "<p>The experimental evidence favoring\nENaC as a potential target of PPAR<italic>γ</italic> in the distal nephron seems to provide a\nrationale for the use of amiloride as a specific ENaC inhibitor for treatment\nof TZD-induced fluid retention. Unfortunately, amiloride was not included in\nthis clinical trial [##REF##17093067##54##]. In the mouse, pretreatment with\namiloride effectively prevents body weight gain and fluid retention produced by\npioglitazone. However, in the rat model, posttreatment with amiloride\nunexpectedly exacerbates the fluid retention induced by farglitazar. It is\nunclear whether this discrepancy between the studies is due to species\ndifferences, PPAR<italic>γ</italic> ligand activity, or the different timing of amiloride\ntreatment.</p>", "<title>4.2. Combination of a PPAR<italic>γ</italic> and a PPAR<italic>α</italic> agonist</title>", "<p>Boden et al. examined the effect of the\ncombined use of rosiglitazone and fenofibrate in patients with type 2 diabetes [##REF##17192489##59##]. Compared with rosiglitazone alone,\nrosiglitazone/fenofibrate proved significantly more effective in lowering\nfasting free fatty acid levels and tended to be more effective in achieving plasma\nglucose control. Interestingly, rosiglitazone/fenofibrate completely prevented\nthe increase in body weight and body water content associated with\nrosiglitazone. This study is the first to show that the combined use of a PPAR<italic>γ</italic>\nand a PPAR<italic>α</italic> agonist can prevent rosiglitazone-induced fluid retention. The\ninvestigators did not propose a mechanism to explain this phenomenon. The two\nPPAR isoforms occur in different locations along the nephron. PPAR<italic>α</italic> mRNA is found predominately in the cortex and is\nspecifically localized in the proximal convoluted tubule (PCT). PPAR<italic>γ</italic> is\nabundant in the renal inner medulla, specifically localized to the inner\nmedullary collecting duct [##REF##9435691##23##, ##REF##10600944##25##]. The\ndifference in nephron localization does not seem to favor the direct interaction\nbetween the two PPAR isoforms. However, it remains possible that low PPAR<italic>α</italic>\nactivity in the collecting duct may antagonize the sodium-retaining action of\nPPAR<italic>γ</italic>. Future studies are needed to investigate whether an interaction occurs\nin the collecting duct or another location.</p>", "<p>Dual PPAR<italic>α</italic>/<italic>γ</italic> agonists\nhave been developed by several pharmaceutical companies, and some have\nundergone or are currently undergoing clinical trials [##REF##16503761##60##–##REF##16239637##62##]. Unfortunately,\nmuraglitazar, the first dual PPAR<italic>α</italic>/<italic>γ</italic> agonist, has been associated with an\nexcessive incidence of major adverse cardiovascular events, including myocardial\ninfarction, stroke and transient ischemic attack, chronic heart failure and\ndeath [##REF##16239637##62##]. This\nfinding raises significant safety concerns about the dual agonists as well as the combination of a PPAR<italic>γ</italic> and a PPAR<italic>α</italic> agonist. In the study of\nBoden et al., rosiglitazone/fenofibrate appeared to be well tolerated [##REF##17192489##59##]. The safety issues may be related to\nthe ratio of PPAR<italic>γ</italic> to PPAR<italic>α</italic>. The ratios are fixed for the dual agonists, but can\nbe varied by changing the proportion of PPAR<italic>γ</italic> and PPAR<italic>α</italic> agonists. It should be\npointed out that Boden's study was limited to a small number of patients and a\nshort period of treatment [##REF##17192489##59##]. The safety issue regarding the\ncombined use of a PPAR<italic>γ</italic> and PPAR<italic>α</italic> agonist needs to be carefully evaluated in\nlarger-scale and longer-term clinical trials as well as animal studies.</p>", "<title>4.3. Inhibition of protein kinase C</title>", "<p>There is functional evidence suggesting\nthe involvement of vascular permeability in TZD-induced body weight gain and\nfluid retention [##REF##16672634##52##]. Therefore, targeting vascular\npermeability may provide a potential therapeutic strategy for this side effect\nof the TZDs. In an animal study, the use of a PKC<italic>β</italic> inhibitor, RBX, to target\nvascular permeability effectively attenuated the increases in TZD-induced body\nweight gain [##REF##16672634##52##]. Is there any safety issue related to\nRBX? In the animal models tested, including Zucker and lean fatty rats, and mice,\nRBX reduced rosiglitazone-induced capillary permeability, but had no\nsignificant effect on the baseline capillary permeability without rosiglitazone\ntreatment. In this short-term animal study, the compound appears to be well\ntolerated. Another positive note is that RBX is being used in clinical trials\nfor diabetic microvascular complications. In these trials, as well as in animal\nstudies, RBX shows promise for treatment of diabetic retinopathy and nephropathy\nwithout noticeable side effects [##REF##17693752##63##, ##UREF##8##64##].</p>" ]

[ "<title>ACKNOWLEDGMENTS</title>", "<p>Portions of this work were funded by NIH grants RO-1 HL079453,\nRO-1 DK066592, R21 DK069490, and Veterans Affairs Merit Review (to T. Yang).</p>" ]

[ "<fig id=\"fig1\" position=\"float\"><label>Figure 1</label><caption><p>Body weight gains in untreated and rosiglitazone (RGZ)-treated PPAR<italic>γ</italic>\n^f/f mice (a) and CD PPAR<italic>γ</italic> knockout mice\n(b) (adapted from [##REF##15956187##28##]).*, <italic>P</italic> &lt; .05\nversus vehicle at the corresponding time point.</p></caption></fig>", "<fig id=\"fig2\" position=\"float\"><label>Figure 2</label><caption><p>Changes in plasma volume in PPAR<italic>γ</italic>\n^f/f and CD PPAR<italic>γ</italic> knockout mice following rosiglitazone\n(RGZ) treatment (adapted from [##REF##15956187##28##]). (a) Hematocrit (Hct) in PPAR<italic>γ</italic>\n^f/f and\nCD PPAR<italic>γ</italic> knockout mice before and after RGZ treatment. (b) Plasma aldosterone\nlevels in PPAR<italic>γ</italic>\n^f/f and CD PPAR<italic>γ</italic> knockout mice following RGZ\ntreatment. (c) Determination of plasma volume in PPAR<italic>γ</italic>\n^f/f and CD\nPPAR<italic>γ</italic> KO mice by the Evans blue (EB) dye technique.</p></caption></fig>", "<fig id=\"fig3\" position=\"float\"><label>Figure 3</label><caption><p>The mechanism for thiazolidinedione- (TZD-) induced\nedema. In the renal collecting duct, PPAR<italic>γ</italic> activation\nincreases sodium reabsorption through ENaC-dependent and independent\nmechanisms. In the blood vessels of adipose tissues, PPAR<italic>γ</italic> ligands activate PKCß, VEGF, and NO, which together lead to\nincreased endothelial permeability. The increased renal sodium retention at the\nlevel of the collecting duct in conjunction with increased vascular permeability\nmay determine edema development.</p></caption></fig>" ]

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[ "<graphic xlink:href=\"PPAR2008-943614.001\"/>", "<graphic xlink:href=\"PPAR2008-943614.002\"/>", "<graphic xlink:href=\"PPAR2008-943614.003\"/>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Pneumococcal disease is a major cause of mortality and morbidity worldwide. In the United States alone, <italic>S. pneumoniae</italic> was responsible for 3 000 cases of meningitis, 500 000 cases of pneumonia, and 7 000 000 cases of otitis media each year before the introduction of conjugate vaccination in 2000 ##REF##10081505##[1]##.</p>", "<p>A heptavalent pneumococcal conjugate vaccine has been available for a few years. This vaccine covers the 7 most frequently carried pneumococcal serotypes in the United States, which are also among the most invasive serotypes. Since its introduction, a marked decrease in the incidence of invasive pneumococcal disease (IPD) has been reported ##REF##12724479##[2]##. Because of herd immunity, this decrease is not restricted to vaccinated children and has been observed in the entire population ##REF##12724479##[2]##, ##REF##12706665##[3]##.</p>", "<p>Nevertheless, several factors may undermine the efficacy of the vaccine. Several studies have reported evidence of replacement of vaccine serotypes by non-vaccine serotypes in vaccinated populations ##REF##16140686##[4]##, ##REF##17456820##[5]##. Mathematical models have suggested that this may in the long term cancel the impact of vaccination on pneumococcal colonization rates ##REF##15155223##[6]##, ##REF##10341170##[7]## and lessen its impact on IPD incidence ##REF##15962556##[8]##.</p>", "<p>Another worrying factor is the suspicion that some highly invasive or antibiotic resistant pneumococci may have a propensity to switch their capsular type, thereby evading vaccine-conferred immunity ##REF##16517889##[9]##–##REF##18171247##[11]##. Several studies have indeed reported the emergence in vaccinated populations of new pneumococcal clones expressing non-vaccine serotypes, but genetically closely related to vaccine serotypes ##REF##16517889##[9]##, ##REF##18171247##[11]##–##REF##15551214##[14]##. In particular, since the introduction of conjugate vaccination, serotype 19A has emerged as the predominant invasive pneumococcal serotype in the United States ##REF##18419539##[15]##. Molecular epidemiology analyses suggest that this increase is largely attributable to the expansion of capsular switch variants ##REF##17529860##[16]##. However, evidence of capsular switch has also been reported in <italic>S. pneumoniae</italic> before the vaccination era, and the influence of the conjugate vaccine remains unclear ##REF##16517889##[9]##, ##REF##9466257##[17]##, ##REF##15583299##[18]##. Furthermore, little is known on the actual frequency at which pneumococci may switch their capsular type ##REF##15583299##[18]##, ##REF##12517877##[19]##.</p>", "<p>In this study, we first review 4 studies presenting longitudinal data on pneumococcal carriage in the population to estimate the frequency of “natural” vaccine-independent capsular switch. Since it is suspected that conjugate vaccination may favour selection of capsular switch in vaccine-covered or related serotypes ##REF##16517889##[9]##, ##REF##15551214##[14]##, we specifically investigate this possibility from those of the studies which also provide data on the vaccination status of individuals.</p>", "<p>Secondly, we use the gathered information in a mathematical model of pneumococcal transmission to quantify the possible changes induced by capsular switch on the incidence of invasive pneumococcal disease in vaccinated populations.</p>" ]

[ "<title>Methods</title>", "<title>Review on pneumococcal capsular switch</title>", "<p>In this article, we distinguish between “natural” capsular switch, which is the propensity for a pneumococcal strain to switch its capsular type independently of its environment, and vaccine-selected capsular switch, which results from the selective pressure of conjugate vaccination on pneumococcal strains. The vaccine-selected capsular switch phenomenon is added to the natural capsular switch phenomenon in vaccinated individuals.</p>", "<title>Search strategy and selection criteria for the review</title>", "<p>Data for the review were identified by a systematic search of Medline and references from relevant articles. Search terms were “longitudinal” (or “birth”, “scheduled”, “month*”, “week*”, “age*”), “surveillance” (or “cohort”, “trial”, “monitor*”, follow*”), “colonization” (or “carriage”), “isolate*” (or “sample*”), “serotype*” (or “genetic*”, “multilocus”, “capsul*”, “molecular”) and “pneumoniae” (or “pneumococc*”).</p>", "<p>English and French language papers were reviewed. We included only those studies which provided detailed longitudinal data on pneumococcal carriage in a population with information on serotypes and MLST genotypes of carried isolates at all sampling times, as well as reports on observed capsular switches. Conversely, we excluded studies which did not provide detailed enough data.</p>", "<title>Frequency of vaccine-independent “natural” capsular switch</title>", "<p>Capsular switch events were defined in all studies as the consecutive isolation in persistently colonized individuals of two isolates that were identified by MLST as closely related, but that expressed different serotypes. The frequency of capsular switch in a study was estimated as the ratio of the number of observed switches by the total time during which switches may have occurred.</p>", "<p>In order to obtain the denominator, which represented the time at risk for capsular switch, we computed the duration of observed persistent pneumococcal colonization for all children included in the study. For any given child, this was defined as the sum of all durations between 2 consecutive positive samplings in the course of the study. The time at risk was then calculated as the total duration of persistent pneumococcal colonization, cumulated over all children.</p>", "<title>Investigation of vaccine-selected capsular switch</title>", "<p>Vaccine-selected capsular switch events were defined as the consecutive isolation in persistently colonized vaccinated individuals of a vaccine and a non-vaccine isolate (in that order) that were identified by MLST as belonging to the same subtype. The frequency of vaccine-selected capsular switch was defined as the weekly probability for a vaccine-type strain colonizing a vaccinated individual to be selected after it has switched its capsular type in order to evade vaccine-conferred immunity.</p>", "<title>Mathematical model of pneumococcal transmission in a vaccinated population</title>", "<p>We developed a mathematical model of pneumococcal colonization in a partially vaccinated community (##FIG##0##Figure 1##). At any given time, individuals reside in a compartment according to their carrier status and to the characteristics of the pneumococcal strain they may carry. The model depends on several parameters, which are listed with their values in ##TAB##0##Table 1##.</p>", "<title>Capsular switch modeling</title>", "<p>The model did not account for all switching, but only for “excess” vaccine-selected capsular switch, which may not be compensated. That is, we hypothesized that conjugate vaccination aided the selection of switched variants of the seven strains included in its formulation. The “natural” switch phenomenon, which affects all pneumococcal strains regardless of vaccination, needs not be explicitly modeled as switches to and from vaccine strains should compensate.</p>", "<p>We investigated a frequency of vaccine-selected capsular switch in vaccinated individuals ranging from 0 (no vaccine-selected capsular switch) to 10⁻²/week.</p>", "<title>Model structure</title>", "<p>The model structure is depicted in ##FIG##0##Figure 1##. The seven serotypes covered by the heptavalent conjugate vaccine (vaccine serotypes, namely serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, representing 58% of all strains in Europe) are distinguished from other serotypes (non-vaccine serotypes, representing 42% of all strains). Non-vaccine serotypes deriving from the capsular transformation of vaccine serotypes are further separated from “wild-type” non-vaccine serotypes. The vaccine is supposed to be 100% effective against colonization with vaccine serotypes.</p>", "<p>Individuals in the model reside in one of 7 carriage states at any given time: non carriers; carriers of vaccine serotypes (V); carriers of non-vaccine serotypes deriving from the capsular switch of vaccine serotypes (S); carriers of “wild-type” non-vaccine serotypes (N); and multiple carriers who are colonized with two different isolates (VS, VN and SN). Because the model also takes into account the vaccination status, meaning that individuals can be either vaccinated or not vaccinated, as well as two age classes (&lt;2 years old and &gt;2 years old), there are 28 compartments in all.</p>", "<title>Colonization modeling</title>", "<p>Both the first acquisition of a pneumococcal strain and the acquisition of a second strain occurred following an infectious contact with a colonized individual. The frequency of these infectious contacts depended on the age and colonization status of the host, as well as on the strain involved.</p>", "<p>Observed colonization rates with vaccine and non-vaccine serotypes in the pre-vaccine era suggest a reduced fitness in the latter, which might be expressed by a lower transmissibility ##REF##17426010##[20]##. Here, we supposed a 0.98 ratio between the frequencies of infectious contacts involving non-vaccine and vaccine serotypes. Switched serotypes were supposed to have the same fitness as vaccine serotypes.</p>", "<p>In order to model competition, the probability of acquisition of a particular serotype was reduced by 50% in hosts already carrying another serotype ##REF##10341170##[7]##.</p>", "<p>The frequencies of infectious contacts with vaccine strains in uncolonized children and adults were calibrated to reflect observed colonization rates in the pre-vaccine era (40% in children under 2 years old ##REF##14986245##[21]##, 20% for “adults” over 2 years old ##REF##9521139##[22]##; 58% of vaccine serotypes).</p>", "<p>The mean duration of colonization was supposed to be the same for all types of strains in the model. The duration of pneumococcal colonization was estimated from the literature at 13.8 weeks in children &lt;2 years old, and at 4.4 weeks in adults ##REF##16897668##[23]##.</p>", "<title>Invasiveness modeling</title>", "<p>We simplified our description by assuming that all vaccine serotypes were invasive, whereas only a portion of wild-type non-vaccine serotypes were. We also hypothesized that capsular switch did not affect invasiveness, implying that non-vaccine serotypes deriving from the capsular transformation of vaccine serotypes remained invasive.</p>", "<title>From colonization to invasive disease</title>", "<p>Invasive pneumococcal disease (IPD) incidence was presumed to occur in a portion of incident colonization cases with invasive strains.</p>", "<p>A recently published study investigated serotype-specific invasiveness from longitudinal data on pneumococcal acquisition and infection in children &lt;2 years old ##REF##16897668##[23]##. In this study, serotype-specific attack rates were defined as the ratio of the incidence of invasive pneumococcal disease to the incidence of acquisition and were calculated to determine the expected number of IPD cases for 100,000 acquisitions of each pneumococcal serotype. We used these serotype-specific attack rates to compute the attack rate of vaccine serotypes (13 IPD cases/100,000 acquisitions), as well as the attack rate of invasive non-vaccine serotypes (25 IPD cases/100,000 acquisitions) and the attack rate of non-invasive non-vaccine serotypes (3.25 IPD cases/100,000 acquisitions) in children. For adults, it is thought that immunological history induces a reduction in attack rates ##REF##17426010##[20]##, ##REF##14976594##[24]##. We calculated adult attack rates by dividing children attack rates by 4.5, in order to reproduce the difference between observed incidences in children and adults in age-specific data on IPD ##REF##11120930##[25]##.</p>", "<p>We then determined the number of IPD cases/100,000 per annum from model predictions by multiplying the number of acquisitions of each strain type during a given year by the corresponding mean attack rate in each age class.</p>", "<title>Sensitivity analysis</title>", "<p>In order to evaluate the robustness of our results, we performed a sensitivity analysis of the model using the Latin hypercube sampling-partial rank correlation coefficients technique ##UREF##0##[26]##.</p>" ]

[ "<title>Results</title>", "<title>Review on pneumococcal capsular switch</title>", "<title>Selected studies (##TAB##1##Table 2##)</title>", "<p>Our systematic search of Medline retrieved 147 original articles. Four epidemiological studies providing data on capsular switch were found consistent with our requirements ##REF##12517877##[19]##, ##REF##7706816##[27]##–##REF##15634953##[29]##.</p>", "<p>The first study ##REF##12517877##[19]## aimed specifically at estimating the frequency of serotype change. A birth cohort of a 100 infants was followed for 2 years. There was no evidence for any change of serotype due to capsular switch.</p>", "<p>The second study ##REF##7706816##[27]## reported results from routine surveillance in a day care centre over 7 years; one capsular switch event was reported during an outbreak of colonization involving 14 children between May 1990 and December 1991.</p>", "<p>In the third study ##REF##9666000##[28]##, 19 children were monitored during the first 2 years of their life; one episode of capsular switch was reported.</p>", "<p>The fourth and most recent study ##REF##15634953##[29]## was a randomized double-blind trial with the 7-valent conjugate pneumococcal vaccine involving 383 children who were followed for 6 months; 3 episodes of capsular switch were reported.</p>", "<title>Estimation of the “natural” vaccine-independent switch frequency</title>", "<p>Switch frequencies estimated from the 4 selected studies are summarized in ##TAB##0##Table 1## and vary between 0 and 8.10⁻³/week. We also computed a random effects pooled estimate of the switch frequency from all available data in the 4 studies, which included a total of 5 reported switches for an overall cumulated duration of persistent colonization of 5044 weeks (DerSimonian and Laird meta-analysis ##REF##3802833##[30]##). The estimated frequency was 1.5×10⁻³/week (95% confidence interval, [4.6×10⁻⁵/week–4.8×10⁻³/week]).</p>", "<title>Investigation of vaccine-selected “excess” capsular switch</title>", "<p>Only one of the 4 selected studies provided data on the vaccination status of colonized individuals ##REF##15634953##[29]##. Two more studies of the impact of conjugate vaccination on pneumococcal colonization were identified ##REF##15750461##[31]##, ##REF##17955441##[32]##; these 2 studies looked at capsular switch but did not provide full individual data.</p>", "<p>Neither of these studies provided any major evidence for an increased frequency of strains with suspected capsular changes among pneumococci isolated from the nasopharyngeal samples of vaccinees. In the last identified study ##REF##17955441##[32]##, possible capsular switching from a non-vaccine serotype toward a vaccine serotype was detected once among 197 isolates collected over 1 year in vaccinated children.</p>", "<p>As a whole, the available data was too scarce to allow any conclusions to be drawn regarding the possibility of an increased switch frequency due to the selective immunological pressure of vaccination.</p>", "<title>Mathematical model: validation and predictions</title>", "<p>We simulated the introduction of the heptavalent conjugate vaccine with 90% coverage, under various scenarios regarding the actual frequency of vaccine-selected capsular switch in vaccinated individuals. ##FIG##1##Figure 2## depicts time changes in the colonizing pneumococcal population in the post-vaccine era, with a frequency of vaccine-selected capsular switch fixed at 0 (##FIG##1##Figure 2a##), 10⁻⁴/week (##FIG##1##Figure 2b##) and 10⁻³/week (##FIG##1##Figure 2c##). ##FIG##2##Figure 3## provides the yearly incidence of IPD in children 5 years after the introduction of the vaccine for vaccine-selected capsular switch frequencies between 0 and 10⁻²/week; the pre-vaccination incidence is also presented as a reference.</p>", "<title>“Long-term” pneumococcal population</title>", "<p>Following the introduction of the vaccine, colonization with vaccine-type isolates decreased while colonization with non-vaccine-type isolates increased (##FIG##1##Figure 2##). Both the speed and the extent of this replacement increased with the frequency of vaccine-selected capsular switch. For a 10⁻³/sem frequency of vaccine-selected capsular switch, the overall prevalence of carriage in the population had regained its pre-vaccine level after 10 years of simulated vaccination (##FIG##1##Figure 2c##).</p>", "<p>Switched strains constituted more than 30% of all invasive strains for vaccine-selected capsular switch frequencies over 10⁻⁴/week. However, non-invasive strains still represented at least half of all pneumococcal strains, even for high selected switch frequencies in vaccinated individuals (data not shown).</p>", "<p>Simulations with a less extensive vaccination coverage led to similar results provided the coverage remained over 40%.</p>", "<title>Comparison of model predictions with observed data</title>", "<p>In most European countries, the reported incidence of IPD in the pre-vaccination era was between 10 and 24 cases/100,000 per annum among children ##REF##11120930##[25]##, ##REF##10619740##[33]##. This is consistent with the 18.7 IPD cases/100,000 children predicted by the model in the absence of vaccination (##FIG##2##Figure 3##).</p>", "<p>After 5 years of vaccination, the predicted annual incidence was reduced by more than 40% in children (##FIG##2##Figure 3##), and more than 30% in the general population (data not shown). This is consistent with observed reductions in European vaccinated populations ##REF##17457623##[34]##, ##UREF##1##[35]##.</p>", "<title>Predictions on the impact of capsular switch</title>", "<p>Without any vaccination-selected switch, the expected IPD incidence was reduced by 48% after 5 years of vaccination. For switch frequencies up to 10⁻²/week, this reduction remained significant at over 40% (##FIG##2##Figure 3##).</p>", "<p>However, vaccine-selected switch in vaccinated individuals led to excess IPD cases. ##TAB##2##Table 3## provides the expected number of supplemental IPD cases in children, cumulated over the 10 years following the introduction of conjugate vaccination. A high vaccine-selected switch frequency (over 7,5.10⁻³/week) induced 3 excess IPD cases per 100,000 children over these 10 years.</p>", "<title>Sensitivity analysis</title>", "<p>The results of the sensitivity analysis are given in ##TAB##3##Table 4##. The most critical parameters for predicting the incidence of IPD in the post-vaccine era were the duration of colonization in adults and the rate of infectious contacts between adults. Other important parameters were the rate of infectious contacts between children and adults, as well as the attack rate of non-vaccine serotypes.</p>" ]

[ "<title>Discussion</title>", "<p>Combining literature review and mathematical modelling, we investigated the potential impact of capsule switching on the efficacy of conjugate pneumococcal vaccination. Although there was not enough statistical power to quantify precisely the vaccine-selected capsular switch frequency among vaccinated individuals, we found that this phenomenon should not lessen significantly the reduction in IPD incidence induced by the vaccine. This is in part due to competition between switched isolates and other non-vaccine isolates in vaccinated populations.</p>", "<title>Discussion of the switch frequency estimation</title>", "<p>Estimating the switch frequency from published studies only should be challenged, as available data are scarce and exhibit several limitations, such as being restricted to antibiotic-resistant populations, specific age groups or invasive infections ##REF##15583299##[18]##.</p>", "<p>Because the data was so scarce, we could only estimate the switch frequency with a wide confidence interval. Moreover, it is possible that this frequency depends on the pneumococcal serotype or on characteristics of the human host (such as age), but we were unable to investigate this hypothesis.</p>", "<p>Finally, it could be argued that there is no direct proof that the serotype changes reported in the studies we selected are indeed due to capsular switch, as many events can go unobserved between two consecutive samplings. This is all the more true when samplings occur many months apart. For instance, in the study by Bogaert and colleagues ##REF##15634953##[29]##, it is possible that the observed consecutive colonization at a 6-month interval with different serotype variants of closely related strains merely suggests the recruitment of a second isolate with identical genotype. Conversely, it is also possible that some capsular switch events may have gone undetected in this study. It would be interesting to use a data augmentation technique such as Markov Chain Monte Carlo on the data from these studies in order to obtain more robust estimates of the switch frequency ##REF##16445857##[36]##.</p>", "<title>Discussion of model hypotheses</title>", "<title>Vaccine-independent capsular switch vs. vaccine-selected capsular switch</title>", "<p>We hypothesized that in the absence of vaccine pressure, switches to and from vaccine strains should be balanced. This allowed us not to take into account vaccine-independent capsular switch in our model. Although it seems possible that the underlying genetic nature of different switches may have specific directionality, our balance hypothesis is supported by the fact that, in a given location, the frequency distribution of particular pneumococcal serotypes or serogroups in IPD has remained nearly constant over several decades before conjugate vaccination ##REF##17426010##[20]##.</p>", "<p>In vaccinated individuals however, non-vaccine isolates which result from the natural switch of a vaccine isolate may persist under selective pressure due to vaccination, whereas vaccine isolates will not. Therefore, we included the possibility for vaccine-selected capsular switch in vaccinated individuals in our model.</p>", "<title>Range of values for the vaccine-selected switch frequency</title>", "<p>The emergence of new clones expressing non-vaccine serotypes has been observed in countries where the vaccine was widely used ##REF##15551214##[14]##. However, although this raises suspicions that conjugate vaccination might increase the frequency of capsule switching from vaccine-covered types, quantification is not yet possible from epidemiological data. Here, we supposed that vaccination could not do much more than double the overall “natural” propensity of pneumococci to switch their capsule. Based on our estimation of the frequency of natural capsular switch between 5.10⁻⁵/week and 5.10⁻³/week, this led us to investigate vaccine-selected switch frequencies in vaccinated individuals between 0 and 10⁻²/week.</p>", "<p>In order to be as conservative as possible, we also investigated vaccine-selected switch frequencies up to 0.1/week, although we chose not to present this data in this article. For such high frequencies, we expect switched isolated to be responsible for nearly half of IPD cases 5 years after the introduction of the vaccine. However, because this increase in switched isolates should for the most part occur as replacement of non-vaccine isolates, rather than add to the overall colonization, the impact on IPD incidence may remain limited. For instance, vaccine-selected capsular switch should not lead to more than 5 excess IPD cases per 100,000 children over the 10 years following the introduction of conjugate vaccination.</p>", "<title>Capsular type and pathogenicity</title>", "<p>A major model hypothesis is that capsular switch does not affect the pathogenicity of a given pneumococcal strain, implying that a highly virulent vaccine strain would still be as virulent if it switched its capsular type to express a non-vaccine serotype. However, this is a widely debated issue, as earlier studies have shown complicated interactions between capsular type and other genes in determining pneumococcal virulence.</p>", "<p>The capsule has long been identified as a virulence factor by virtue of its antiphagocytic activity, and acapsular mutants are known to be avirulent ##UREF##2##[37]##. Several molecular epidemiology studies have shown a clear association between the pneumococcal capsule and the ability of pneumococci to cause invasive disease ##REF##12717624##[38]##, ##REF##9498451##[39]##. However, in two recent studies, isolates belonging to the same clone but with different capsules because of serotype switch were found to have the same disease potential ##REF##14976594##[24]##, ##REF##15043881##[40]##.</p>", "<p>According to several experimental studies based on the construction of chimeric pneumococcal mutants with a switched capsular type, the capsule type is not the only determinant of pathogenicity ##REF##12010976##[41]##, ##REF##8168944##[42]##. Hence the virulence of a switched strain may not be predictable from that of the virulence of the original strain. Moreover, other genetic factors might be involved in colonization and in non-invasive pneumococcal disease ##REF##14688083##[43]##.</p>", "<p>In this study, we aimed at evaluating the potential impact of capsular switch on the long-term effectiveness of current pneumococcal conjugate vaccines, based on the suspicion that the constant usage of these vaccines may aid the expansion of serotype switch variants. In that context, our hypothesis that switched vaccine-type pneumococcal strains retained their higher virulence despite expressing non-vaccine serotypes constituted a worst-case scenario.</p>", "<title>Multiple colonization and competition</title>", "<p>Colonization studies show that many people carry more than one pneumococcal type at the same time, although multiple colonization is less frequent than would be expected if each serotype circulated independently of the others ##REF##2639508##[44]##. Because the most probable mechanism for vaccine-selected capsular switching involves multiple colonization of a vaccinated host, we allowed for dual colonization in our model, but we also hypothesized that there was some level of competition. We modelled competition by a reduction in the probability of acquisition of a second strain in an already colonized host ##REF##10341170##[7]##. Although interference between pneumococcal strains is documented ##REF##10812233##[45]##, there was little data to estimate this competition parameter. Based on the sensitivity analysis where we investigated reductions in acquisition probability ranging from 25% to 75%, this parameter does not appear to be the most critical for predicting IPD incidence in the post-vaccine era.</p>", "<title>Fitness</title>", "<p>In the pre-vaccine era, the portion of pneumococcal colonization attributable to vaccine serotypes seems to have remained stable over time at a level &gt;50% in various parts of the world, although it varied geographically. In a context of multiple colonization, this strongly suggests that vaccine serotypes have an increased fitness for colonization. This fitness may be expressed either as increased carriage duration or as higher transmissibility. However, in an earlier study based on longitudinal data, we found little to no differences in the mean duration of carriage and transmission rates of vaccine serotypes and nonvaccine serotypes groups ##REF##16445857##[36]##. Here, we included in the model a slightly reduced transmissibility for non-vaccine serotypes; even with identical carriage durations, this allowed us to reproduce the observed differences in prevalence before the introduction of the vaccine.</p>", "<title>Discussion of model predictions</title>", "<p>Because we evaluated the invasive attack rates of pneumococci from European data ##REF##16897668##[23]##, we used European data on IPD incidence to validate model predictions. This incidence is significantly inferior to that reported in the United States, as a 4 to 7-fold difference has been reported between the United States and Europe in the pre-vaccination era ##REF##11120930##[25]##, ##REF##10619740##[33]##. This may arise from different medical practices between countries, such as admission thresholds and blood culture rates ##REF##11120930##[25]##. Model parameters could easily be adjusted to reflect US IPD incidence, provided data on serotype-specific attack rates in the US became available.</p>", "<p>According to our predictions, the reduction in IPD incidence after 5 years of vaccination should only be around 40–50% in children, whereas observations in the United States showed a 69% reduction in children under 2 years old after 3 years of vaccination ##REF##12724479##[2]##. Reasons for this apparent discrepancy are twofold. First, again, we focused on the European rather than US context; this means that the initial serotype coverage of the heptavalent vaccine was noticeably less favourable (58% of carried serotypes instead of approximately 80%). Second, as evidenced in previous work ##REF##15155223##[6]##, our model predicts the replacement of vaccine serotypes by non-vaccine serotypes. Despite differing attack rates, this should be expected to lessen the impact of vaccination on IPD incidence within a five-year timeframe ##REF##15962556##[8]##. Recent data showing a re-increase in non-vaccine IPD incidence in the US support these predictions ##REF##17456820##[5]##, ##REF##14993532##[46]##, ##UREF##3##[47]##.</p>", "<title>Conclusions</title>", "<p>Despite the significant short-term impact of conjugate pneumococcal vaccination reported in the US, it has been predicted from mathematical models that non-vaccine serotypes may come to replace vaccine serotypes, with implications for the long-term effectiveness of the vaccines. This is supported by recent data from various countries ##UREF##3##[47]##. However, suspicions that pneumococci expressing vaccine serotypes may be incited to switch their capsule, thereby evading vaccine immunity while retaining their invasiveness and their high levels of antibiotic resistance, may be even more worrying ##REF##18020702##[12]##. Indeed, this could lead to a re-increase in highly resistant IPD in vaccinated populations.</p>", "<p>The new conjugate vaccines with increased valence which should be available in years to come might–at least in the short term-help lessen the extent and speed of serotype replacement ##REF##15155223##[6]##. On the other hand, the potential impact of vaccine-selected capsular switch could only increase with the number of serotypes covered by the vaccine.</p>", "<p>Here, we show that, based on the limited data available, the extent of this phenomenon should remain quite limited. This is in agreement with recent data from the US ##REF##17955441##[32]##. Nevertheless, this study stresses the major importance of collecting surveillance data in vaccinated populations which will allow for the detection of any switch event from vaccine to non-vaccine serotypes, as well as for the quantification of the frequency of these events. This should also be kept in mind when designing and analysing clinical trials for new pneumococcal vaccines with higher valence.</p>" ]

[ "<title>Conclusions</title>", "<p>Despite the significant short-term impact of conjugate pneumococcal vaccination reported in the US, it has been predicted from mathematical models that non-vaccine serotypes may come to replace vaccine serotypes, with implications for the long-term effectiveness of the vaccines. This is supported by recent data from various countries ##UREF##3##[47]##. However, suspicions that pneumococci expressing vaccine serotypes may be incited to switch their capsule, thereby evading vaccine immunity while retaining their invasiveness and their high levels of antibiotic resistance, may be even more worrying ##REF##18020702##[12]##. Indeed, this could lead to a re-increase in highly resistant IPD in vaccinated populations.</p>", "<p>The new conjugate vaccines with increased valence which should be available in years to come might–at least in the short term-help lessen the extent and speed of serotype replacement ##REF##15155223##[6]##. On the other hand, the potential impact of vaccine-selected capsular switch could only increase with the number of serotypes covered by the vaccine.</p>", "<p>Here, we show that, based on the limited data available, the extent of this phenomenon should remain quite limited. This is in agreement with recent data from the US ##REF##17955441##[32]##. Nevertheless, this study stresses the major importance of collecting surveillance data in vaccinated populations which will allow for the detection of any switch event from vaccine to non-vaccine serotypes, as well as for the quantification of the frequency of these events. This should also be kept in mind when designing and analysing clinical trials for new pneumococcal vaccines with higher valence.</p>" ]

[ "<p>Analyzed the data: LT PYB. Wrote the paper: LT. Initiated the research on this theme: DG. Revised the results critically: DG. Participated in the development of the model: LO PYB. Performed the literature search: LT. Developed the mathematical model and analyzed its predictions: LT.</p>", "<title>Background</title>", "<p>Despite the dramatic decline in the incidence of invasive pneumococcal disease (IPD) observed since the introduction of conjugate vaccination, it is feared that several factors may undermine the future effectiveness of the vaccines. In particular, pathogenic pneumococci may switch their capsular types and evade vaccine-conferred immunity.</p>", "<title>Methodology/Principal Findings</title>", "<p>Here, we first review the literature and summarize the available epidemiological data on capsular switch for <italic>S. pneumoniae</italic>. We estimate the weekly probability that a persistently carried strain may switch its capsule from four studies, totalling 516 children and 6 years of follow-up, at 1.5×10⁻³/week [4.6×10⁻⁵–4.8×10⁻³/week]. There is not enough power to assess an increase in this frequency in vaccinated individuals. Then, we use a mathematical model of pneumococcal transmission to quantify the impact of capsular switch on the incidence of IPD in a vaccinated population. In this model, we investigate a wide range of values for the frequency of vaccine-selected capsular switch. Predictions show that, with vaccine-independent switching only, IPD incidence in children should be down by 48% 5 years after the introduction of the vaccine with high coverage. Introducing vaccine-selected capsular switch at a frequency up to 0.01/week shows little effect on this decrease; yearly, at most 3 excess cases of IPD per 10⁶ children might occur due to switched pneumococcal strains.</p>", "<title>Conclusions</title>", "<p>Based on all available data and model predictions, the existence of capsular switch by itself should not impact significantly the efficacy of pneumococcal conjugate vaccination on IPD incidence. This optimistic result should be tempered by the fact that the selective pressure induced by the vaccine is currently increasing along with vaccine coverage worldwide; continued surveillance of pneumococcal populations remains of the utmost importance, in particular during clinical trials of the new conjugate vaccines.</p>" ]

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[ "<fig id=\"pone-0003244-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003244.g001</object-id><label>Figure 1</label><caption><title>Model Structure within one age class.</title><p>Individuals can be either uncolonized (U), colonized with vaccine serotypes (V), non-vaccine serotypes resulting from the capsular switch of a vaccine serotype (S) or wild-type non-vaccine serotypes (N). Dual colonization is allowed (VS, VN or SN). The model is structured into two age classes (children and adults). A portion of children are vaccinated, and vaccination immunity may last into adulthood. Vaccinated individuals can only acquire colonization with non-vaccine-type pneumococci. Switch events may occur in vaccinated individuals colonized with both vaccine and non-vaccine serotypes (VN) and are depicted by a bold arrow.</p></caption></fig>", "<fig id=\"pone-0003244-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003244.g002</object-id><label>Figure 2</label><caption><title>Time changes in the pneumococcal population in the post-vaccine era: global colonization rate (bold line), colonization with vaccine serotypes (dashed line), non-vaccine serotypes (dotted lines), and dual colonization with vaccine and non-vaccine serotypes (full line), a) with a frequency of vaccine-selected capsular switch _← = 0, b) with a frequency of vaccine-selected capsular switch _← = 10⁻⁴/week, and c) with a frequency of vaccine-selected capsular switch _← = 10⁻³/week.</title></caption></fig>", "<fig id=\"pone-0003244-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003244.g003</object-id><label>Figure 3</label><caption><title>Invasive Pneumococcal Disease (IPD) incidence in children &lt;2 years old (per 100,000 children, per year) 5 years after the introduction of a conjugate vaccine, as a function of the frequency of capsular switch selected in vaccinated individuals, for a 90% vaccination coverage.</title><p>The origin of the cases (vaccine strains, non-vaccine strains, switched strains) is specified, and the expected incidence without vaccination is also depicted as a reference.</p></caption></fig>" ]

[ "<table-wrap id=\"pone-0003244-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003244.t001</object-id><label>Table 1</label><caption><title>Estimation of the switch frequency (weekly probability for a persistently carried pneumococcal strain to switch its capsule).</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Study</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of reported switches</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cumulated persistent carriage duration (weeks, estimated)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estimated switch frequency (weeks⁻¹)</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(Barnes et al) ##REF##7706816##[27]##\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">121</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.2×10⁻³\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(Sluijter et al)<xref ref-type=\"table-fn\" rid=\"nt101\">*</xref>\n##REF##9666000##[28]##\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">169</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.9×10⁻³\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(Meats et al) ##REF##12517877##[19]##\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3852</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(Bogaert et al) ##REF##15634953##[29]##\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3068</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.7×10⁻⁴\n</td></tr><tr><td colspan=\"3\" align=\"left\" rowspan=\"1\">\n<italic>Random effects pooled estimate from the 4 studies </italic>\n##REF##3802833##[30]##\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.5×10⁻³ [4.6×10⁻⁵–4.8×10⁻³]</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003244-t002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003244.t002</object-id><label>Table 2</label><caption><title>Model parameters.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Parameter</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estimated value</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Reference</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean duration of pneumococcal colonization in children</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.8 weeks</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n##REF##16897668##[23]##\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean duration of pneumococcal colonization in adults</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.4 weeks</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"table-fn\" rid=\"nt102\">**</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rate of infectious contacts among children</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.4 weeks⁻¹\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"table-fn\" rid=\"nt102\">**</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rate of infectious contacts between children and adults</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.25 weeks⁻¹\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"table-fn\" rid=\"nt102\">**</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rate of infectious contacts among adults</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.26 weeks⁻¹\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"table-fn\" rid=\"nt102\">**</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Reduction of the acquisition probability in colonized individuals</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"table-fn\" rid=\"nt102\">**</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Non-vaccine isolates transmissibility-vaccine isolates transmissibility ratio (fitness parameter)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.98</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n##REF##16445857##[36]##\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Attack rates in children (IPD cases per 100,000 acquisitions)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n##REF##16897668##[23]##\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">vaccine serotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 IPD/100,000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">nonvaccine serotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.5 IPD/100,000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Attack rates in adults (IPD cases per 100,000 acquisitions)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n##REF##16897668##[23]##, ##REF##11120930##[25]##\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">vaccine serotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 IPD/100,000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">nonvaccine serotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.5 IPD/100,000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean duration of vaccine immunity after 2 years old</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10 years</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n##REF##12450706##[48]##\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccine coverage</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0–100%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccine-selected capsular switch frequency</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0–10⁻² weeks⁻¹\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003244-t003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003244.t003</object-id><label>Table 3</label><caption><title>IPD cases due to capsular switch. Number of IPD cases due to switched pneumococcal strains, per 100,000 children under 2 years old, cumulated over the 10 years following the introduction of conjugate vaccination, as a function of the frequency of capsular switch selected in vaccinated individuals.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccine-selected switch frequency (weeks⁻¹)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of IPD cases (for 100,000 children) due to switched strains, cumulated over 10 years</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0–2×10⁻⁴\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3×10⁻⁴–1.6×10⁻³\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.7×10⁻³–5.8×10⁻³\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.9×10⁻³–10⁻²\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td></tr></tbody></table></alternatives></table-wrap>", "<table-wrap id=\"pone-0003244-t004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003244.t004</object-id><label>Table 4</label><caption><title>Sensitivity analysis of the incidence of IPD 5 years after the introduction of conjugate vaccination. Vaccination coverage is fixed at 90% and the frequency of vaccine-selected capsular switch is fixed at 10⁻³/week. Critical parameters are in bold.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Parameter</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">PRCC<xref ref-type=\"table-fn\" rid=\"nt104\">a</xref>\n</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean duration of pneumococcal colonization in adults</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.925</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean duration of pneumococcal colonization in children</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.223</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rate of infectious contacts among children</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.048</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rate of infectious contacts between children and adults</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.469</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rate of infectious contacts among adults</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.896</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Reduction of the acquisition probability in colonized individuals</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.265</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Non-vaccine isolates transmissibility-vaccine isolates transmissibility ratio (fitness parameter)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.071</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Attack rate of vaccine serotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.134</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Attack rate of non-vaccine serotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.369</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean duration of vaccine immunity after 2 years old</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.060</td></tr></tbody></table></alternatives></table-wrap>" ]

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[ "<table-wrap-foot><fn id=\"nt101\"><label>*</label><p>Regarding the second study ##REF##9666000##[28]##, it is to be noted that although the article provided the results of all positive samplings in chronological order for all children, it did not specify the corresponding sampling times. We therefore had to approximate the overall persistent colonization duration by supposing that all successive positive samplings occurred consecutively at a 1 month interval.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt102\"><label>**</label><p>Value resulting from a calibration of the model in order to reproduce observed age-specific data on pneumococcal colonisation.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt103\"><label/><p>Vaccination coverage is fixed at 90%.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"nt104\"><label>a</label><p>Partial rank correlation coefficients (PRCC) indicate the degree of monotonicity between a specific input variable and a particular outcome variable. The sign of the PRCC indicates the qualitative relationship between input and output variables. The magnitude indicates the importance of uncertainty in estimating the value of the input variable in contributing to the imprecision in predicting the value of the outcome variable.</p></fn></table-wrap-foot>", "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>The authors have no support or funding to report.</p></fn></fn-group>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Models of genetic regulatory networks hold the promise of a deeper understanding of two fundamental processes in biology. First, the relationship between genotype and phenotype in each individual depends on the dynamic behavior of genes interacting with each other and their environment. Second, natural selection acts on the resulting phenotypes produced by this interaction, thus the response to selection and the long-term course of evolution depend on how variation in network properties can be altered by mutation and recombination. Of particular interest is understanding the connection between these two processes, as our assumptions about how these networks are formed affect how they operate <italic>at a time</italic>, and simultaneously how they can change <italic>over time</italic>. As with all modeling efforts, constructing these models requires a balance between simple, general, and easily interpreted models on the one hand, and more complex, specific, and predictive models on the other. Here we present what we call a <italic>publish-subscribe</italic> model of gene regulation. This model adds a layer of complexity to an existing simple model, Kauffman's <italic>NK</italic> networks ##REF##5803332##[1]##, ##UREF##0##[2]##. Our model produces networks that operate similarly to those in the <italic>NK</italic> model–a number of regulatory genes affect each other, producing a series of activation states that stabilizes to a point or cyclic attractor. What differs is the fashion in which the regulatory connections are made, and hence how they can evolve. The changes we introduce allow for independently mutable regulatory and transcribed regions of a gene, and for regulatory connections to be made via intermediary products. This enables significantly different evolutionary dynamics (for example, significant neutral change can take place) and allows the network dynamics to change in different environments, as the intermediary products can be exogenously introduced. The “environment” of the network may be the external environment or neighboring cells in a multicellular organism. The focal network may also be a module within the total genetic network of an organism ##REF##10578108##[3]##, in which case its environment includes other components of that larger network. We explore some consequences of these changes for the properties of single networks and the evolution of populations of networks.</p>", "<p>The <italic>NK</italic> model has been used to explore the properties and dynamic behavior of genetic networks (e.g. ##UREF##0##[2]##, ##UREF##1##[4]##, ##REF##17188715##[5]##). This model represents a set of <italic>N</italic> genes, where the activation of these genes is represented by a binary state that is expressed (1) or not expressed (0). Each gene is influenced by <italic>K</italic> other genes. Whether or not a gene is expressed at time <italic>t</italic> is decided by a Boolean operation on the previous expression state (at time <italic>t</italic>−1) of the <italic>K</italic> other genes that influence it. In the absence of stochasticity or perturbation, the activation of these <italic>N</italic> genes moves through a series of expression states depending on the initial conditions, ending up in either a stable state or periodic attractor. The entire state space can be described, and each possible attractor enumerated, by starting the network in each of its 2<italic>^N</italic> possible states and constructing a directed graph in which the nodes are possible states of the network and the edges are transitions among them. These transitions depend only on the connections between the genes and the specific Boolean rules associated with each gene.</p>", "<p>The use of discrete, Boolean rules for gene regulation appears justified as a first approximation to data from living organisms ##REF##10910359##[6]##–##REF##15041638##[8]##. In a real network, the interactions among genes are mediated by gene products, transcription factors, signaling pathways, cellular machinery, and diffusion processes ##UREF##3##[9]##. In the <italic>NK</italic> network model, all of these processes are collapsed into the edges linking one gene to another. This may be a good assumption in part because biological networks must be somewhat environmentally robust, i.e. buffered against perturbations and stochasticity ##REF##12183631##[10]##, ##REF##17543998##[11]##. This may preclude, for example, dependence on sensitively fine-tuned levels of gene expression. Thus simple <italic>NK</italic> networks seem to capture many of the fundamental dynamics of genetic networks.</p>", "<p>However, the assumption of these simple gene-to-gene connections may affect our understanding of the two basic questions raised above. Consider the first issue, the relationship between genotype and phenotype. We wish to know, for example, combinations of parameters for which networks exhibit a certain behavior (e.g. ##UREF##3##[9]##). Randomly generated <italic>NK</italic> networks can provide an estimate, but models including other parts of the genetic regulatory process may widen the volume of parameter space in which solutions are found ##REF##12362429##[12]##, or change our understanding of the effect on network properties of processes such as gene duplication ##UREF##4##[13]##.</p>", "<p>Consider also the second issue, the evolution of populations of networks. Evolution is often envisaged by constructing a fitness landscape, a multidimensional surface defined by fitness as a function of genotype (or phenotype), where a single “step” on the surface is equivalent to a one locus mutation of the genotype ##UREF##5##[14]##. Our assessment of the ruggedness of the landscape, and therefore the ability of populations to evolve toward global optima rather than remain on isolated local peaks, depends on the details of the model. In particular, what constitutes a single mutational step determines the structure of variation available to evolution. So we must consider not just how these networks operate, but also how changes in the genotype affect fitness, for this will be crucial to constructing the statistical properties of the fitness landscape. The simplest type of mutation in <italic>NK</italic> networks is the addition or removal of a single connection (or “edge”) between genes in the network (e.g. ##UREF##0##[2]##). Changes in such regulatory influence are often represented as changes to values in a connection matrix (e.g. ##UREF##3##[9]##). Of course, simulated evolution of a population of <italic>NK</italic> networks can proceed by multiple such changes in a single generation or by other types of mutation, such as gene duplication or loss (e.g. ##UREF##1##[4]##), and this has been a productive avenue for research on network evolution. Nonetheless, our view of the landscape of possible network configurations–whether it has a single or multiple adaptive peaks ##UREF##0##[2]## or how connected is the “metagraph” of networks possessing some quality like robustness ##REF##15041638##[8]##–depends on which networks are connected to each other by a single mutational step.</p>", "<p>In our model, we explicitly consider the process of gene regulation by introducing gene products that mediate the regulatory connections among genes. These gene products may represent proteins, or they may be any of a variety of non-protein regulatory molecules whose role is just beginning to be understood ##REF##17846637##[15]##. Each gene is separated into a coding region, which produces a gene product, and a regulatory region, to which gene products may bind. The coding region of each gene acts as a binary switch, either expressed or not in each time step. Whether a gene is expressed–whether the coding region produces its product–depends on the products that are bound to its regulatory region and a set of Boolean rules that translates the binding state of the regulatory region into the expression state of the coding region. The regulatory connections are therefore not specified directly, but rather are an upshot of the correspondence between coding regions and regulatory regions.</p>", "<p>For instance, the coding region of a particular gene might produce some product φ. Any gene that has the binding site for φ in its regulatory region will then be regulated by that gene, and also any other gene that produces product φ. This has an effect on the range of variation in network behavior and on what constitutes a single mutational step. We can think of coding regions that contain a conserved DNA motif ##REF##17510665##[16]## as transmitting or <italic>publishing</italic> a signal on a certain channel, and regulatory binding sites which bind this motif as <italic>subscribing</italic> on that same channel. If a publisher (coding region) stops transmitting on a channel, then all subscribers (regulatory binding sites) tuned to that channel will be affected. Likewise, if a subscriber is tuned to a channel over which multiple publishers are sending signals, it will be affected by each of these multiple signals. In this way, the equivalent of several connections among genes in the network can be created or destroyed by a single genetic mutation. What constitutes a single step on the adaptive landscape is now significantly different than a model that directly connects or disconnects the regulatory interactions by adding or removing an edge or changing the weight in a connection matrix.</p>", "<p>It is worth noting that what we have described here as publish-subscribe has a relevant parallel in the area of modern software construction (indeed, that is where we derived the name) ##UREF##6##[17]##, ##UREF##7##[18]##. The move from directly connecting two interacting parts of a software application to connecting them via this more indirect manner has an important result. The two processes are now decoupled, as new upstream processes may influence any processes subscribed to the right message, and likewise new downstream processes can react to a message by simply subscribing to it. This particular kind of “design pattern” ##UREF##7##[18]## ensures that, although the system remains operationally equivalent to one with direct connections, it is far easier to implement changes that re-use the available structure. We might say that implementing the system in this way makes it more “evolvable”, in the sense that modifications are easier to make, and have less chance of having a catastrophic effect. In a similar manner, moving from a model where connections are made directly, to one where the interactions occur indirectly through such a publish-subscribe paradigm, will have important implications in how the system may evolve.</p>", "<p>Below we describe the model formally. We derive some basic properties of the structure and dynamic behavior of the networks, both by sampling randomly constructed networks and by analytic means. We then consider how this publish-subscribe view of gene regulatory interactions drives the potential of populations of networks to evolve in response to different regimes of selection.</p>" ]

[ "<title>Methods</title>", "<p>We consider a conceptually simple model of a genetic regulatory network consisting of <italic>N</italic> genes, each of which includes a regulatory region and a coding region (##FIG##0##Fig. 1##). The regulatory region consists of a number of binding sites, to which specific gene products may bind. Let us denote the regulatory region of the <italic>i</italic>th gene by ρ<italic>_i</italic> and its coding region by π<italic>_i</italic>. We define ρ<italic>_i</italic> and π<italic>_i</italic> as sequences of length <italic>l</italic> and 1, respectively:\n</p>", "<p>Each element <italic>x_ik</italic> (where <italic>k</italic> = 1,…,(<italic>l</italic>+1)) for the sequence of a gene is chosen from an alphabet Π containing <italic>r</italic> letters with uniform probability 1/<italic>r</italic>. So if our alphabet Π = {0,1,2,3}, and length <italic>l</italic> = 3, then one possible gene would be (1,1,3,2). Here ρ = (1,1,3) and π = 2. A network consists of <italic>N</italic> such genes.</p>", "<p>Interaction between two genes is mediated by gene products. If the <italic>i</italic>th gene produces a product <italic>x</italic> that matches a binding site in the <italic>j</italic>th gene, then the <italic>i</italic>th gene may regulate the expression of the <italic>j</italic>th gene. We denote the possible interaction (adjacency) matrix by <italic>w</italic>, with elements\n</p>", "<p>The in- and out-degree of a gene are calculated by summing the elements of the adjacency matrix, respectively:Below we show both numerical and analytic estimations of in- and out-degree distributions ##UREF##8##[19]##. Note that in estimating in- and out-degree distributions, we do not consider the particular set of Boolean rules governing the activity state of each gene. For instance, a given letter <italic>x</italic> may occur in the regulatory sequence of gene <italic>i</italic>. All genes containing <italic>x</italic> in their coding region (i.e., genes that may produce the corresponding product) are considered to be connected to gene <italic>i</italic> in the calculations of degree below. As with <italic>NK</italic> networks, this is the case even if the particular Boolean rules for gene <italic>i</italic> imply that the presence of that product has no effect on the activity state of gene <italic>i</italic>.</p>", "<p>In each time step a set of products <italic>R</italic>(<italic>t</italic>) is present, where <italic>R</italic>(<italic>t</italic>)⊂Π. Each binding site in the regulatory region ρ<italic>_i</italic> is bound if the matching gene product is present (i.e. if <italic>x</italic>\n_ik⊂<italic>R</italic>(<italic>t</italic>)). Products are not consumed when they bind; thus the product from a single gene is sufficient for binding the regulatory regions of several genes (effectively, we ignore quantities of gene products). We denote the entire binding state for a gene at time <italic>t</italic> as the vectorwhere <italic>b_ik</italic> denotes the binding state (either bound or not) of the <italic>k</italic>th site in the binding region,\n</p>", "<p>Note that this state <italic>B_i</italic>(<italic>t</italic>) may be equivalent to some other binding state <italic>B_j</italic>(<italic>t</italic>) if the <italic>j</italic>th gene has the same values in its binding region. It may also be equivalent to <italic>B_i</italic>(<italic>t</italic>−<italic>s</italic>) if the same products were present at time <italic>t</italic>−<italic>s</italic>. This binding state is used to determine whether or not the gene is active, and whether the corresponding value in the coding region will produce a product at time <italic>t</italic>+1.</p>", "<p>The value <italic>B_i</italic>(<italic>t</italic>) locates a unique entry in a Boolean table that returns a value representing whether the corresponding gene is active or not. This table is common to all genes in the network (and all networks if there is a population of networks evolving). We will denote the table as Ψ. This table contains all possible combinations of values in a binding region and their possible bound state. Providing a global table of each particular Boolean response to a combination of bound products provides a realistic degree of stability to the system: two genes with identical regulatory regions presented with the same set of intermediary products will always do the same thing. The activity state of gene <italic>i</italic> at the following time step is read from this table as\n</p>", "<p>The activity state σ<italic>_i</italic> is binary, taking values of either 1 or 0. If σ<italic>_i</italic>(<italic>t</italic>+1) = 1, the product <italic>x_i</italic> will be produced by gene <italic>i</italic>, so that <italic>x_i</italic>⊂<italic>R</italic>(<italic>t</italic>+1). If σ<italic>_i</italic>(<italic>t</italic>+1) = 0, then <italic>x_i</italic> will not be produced by gene <italic>i</italic>, but the identical product <italic>x</italic> may be produced by another gene. The activation state of the network at time <italic>t</italic> is given by Σ(<italic>t</italic>) = (σ<italic>₁</italic>(<italic>t</italic>),…, σ<italic>_N</italic>(<italic>t</italic>)). In constructing the table Ψ, the value 1 is assigned to each σ<italic>_i</italic> with probability <italic>p</italic>, so that <italic>p</italic> gives a measure of the overall probability of gene activity.</p>", "<p>This model results in several possible regulatory patterns: for instance, multiple genes with the same product have identical regulatory effects, genes may regulate themselves (e.g. genes A and F in ##FIG##1##Fig. 2##), and products can have either inhibitory or activating effects (e.g. the effect of product 6 on gene A versus gene B in ##FIG##1##Fig. 2##). Because there is a finite number of genes and gene activity is binary, there is a finite number of states of the network. Therefore, given a set of starting conditions and no stochasticity, the network reaches a stable attractor. The attractor can be a single state (i.e. the same set of gene products in each time step) or a cycle (the same sets of products produced at regular intervals). ##FIG##1##Fig. 2## illustrates a period-3 attractor over the entire network, with some genes (D and F) in a stable state.</p>", "<p>Because each gene's activation σ<italic>_i</italic>(<italic>t</italic>) is binary, the dynamics of any particular model network is much like the <italic>NK</italic> model, in that many binary states determine a single downstream gene's state by a set of Boolean operations. What differs is how the regulatory connections are constructed, and thus how they might evolve. How we initiate the network also differs. Rather than setting it into a particular state, its initial conditions are defined by the introduction of an initial set of products <italic>R</italic>(0). Note that this means that, although there are 2<italic>^N</italic> possible states of the network, not all of these states may be strictly reachable. There may be no combination of products that can produce a particular activation state Σ. In the course of simulations, we may activate any state and see what products it produces. But driving the dynamics of the network purely by introducing gene products already places a constraint on possible states that the network can enter. Finally, mediating connections among genes by using gene products means that a network can operate in an “environment” of exogenous gene products that influence its dynamic behavior. This environment may be stable or temporally variable, as we illustrate below.</p>" ]

[ "<title>Results</title>", "<title>Basic Properties of the Network</title>", "<p>The interactions between a set of genes in the model described above can be represented as a directed graph, where the nodes represent genes and the edges represent connections among genes in the publish-subscribe model. The edges are directed because of the way we define the regulatory and coding regions of our genes. For instance, the product <italic>x_i</italic> of gene <italic>i</italic> may affect the activity state of gene <italic>j</italic> at the next time step, but not vice versa. Thus each gene may affect “downstream” genes and simultaneously be affected by “upstream” genes. The number of upstream and downstream genes connected to a particular gene is the in-degree and out-degree (respectively) of that gene. Each network can be characterized by its in- and out-degree distribution–the frequency distribution of in- and out-degree across all genes, or nodes in the network.</p>", "<p>Degree distributions are important indicators of the organizational principles underlying networks and have been the focus of network theory approaches to gene regulation. The in- and out-degree distributions of real transcriptional regulatory networks exhibit different functional forms. In-degree typically displays an exponential decay and is restricted to a narrow interval, while the out-degree distribution typically has a broad tail ##REF##11967534##[20]##–##REF##17551581##[25]##. It has been shown ##REF##17551581##[25]## that in- and out-degree distributions together are sufficient to reproduce most of the global topological properties of genetic regulatory networks such as degree-degree correlation ##REF##11736611##[26]## and clustering coefficient ##UREF##11##[27]##. Degree distributions are also considered to be important in determining the resistance of networks to perturbations (robustness) and the ability of populations of networks to evolve (evolvability) ##REF##17188715##[5]##. With these motivations, here we derive the directed degree distributions to provide better insight on the properties of our model networks. We have calculated these distributions both numerically and analytically. Numerical results were calculated from frequency distributions of a large number of networks, each generated by randomly and independently assigning letters from the alphabet Π to each regulatory and coding site, while keeping the alphabet size, regulatory region length, and total network size constant. Below we present results for relatively small values of alphabet size (<italic>r</italic> = 10), regulatory region size (<italic>l</italic> = 3), and total network size (<italic>N</italic>). These parameter values, particularly alphabet size and total network size, are likely to be much smaller than those measured in actual genetic networks ##REF##17510665##[16]##, ##REF##12399584##[21]##, ##REF##14657375##[28]##, but they provide a starting point for exploring the behavior of the publish-subscribe model. Our goal is to compare our results to the more basic <italic>NK</italic> model, so that our conclusions can be tied to the addition of explicit gene products. Because of the modularity found in empirical gene networks, we can envision smaller networks as modules operating in the context of a larger organismal network; in this context, the “environment” of exogenous gene products that we consider below represents other interacting modules of the overall network.</p>", "<p>For relatively small values of <italic>N</italic>, both in-degree and out-degree distributions shift to the right as <italic>N</italic> increases (##FIG##2##Fig. 3##). In other words, as <italic>N</italic> increases, the number of genes with products corresponding to a binding site of gene <italic>i</italic> increases (in-degree), and the number of genes with binding sites corresponding to the product of gene <italic>i</italic> increases (out-degree). To explore the large-<italic>N</italic> limit, ##FIG##3##Fig. 4## shows in-degree and out-degree distributions for large networks (<italic>N</italic> = 1000). In the large-<italic>N</italic> limit, such that all sequences of length <italic>l</italic> are likely to be realized, the out-degree distribution approaches a single binomial distribution (##FIG##3##Fig. 4A##). In contrast, the in-degree distribution approaches a superposition of binomial distributions, with separate peaks corresponding to the number of different letters contained in a sequence of length <italic>l</italic> = 3 randomly sampled with replacement from the finite alphabet Π (##FIG##3##Fig. 4B##). For example, the smallest peak in ##FIG##3##Fig. 4B## is the result of genes whose three binding sites contain the same letter <italic>x</italic>, and the largest peak is the result of genes with a different letter at each of the three binding sites.</p>", "<p>To calculate the out-degree distribution analytically, first we determine the probability of finding a given letter <italic>x</italic> in a randomly chosen sequence of length <italic>l</italic>, which is given byThis equals the probability of the product of gene <italic>i</italic> occuring in the regulatory sequence of gene <italic>j</italic>. Thus in the large-<italic>N</italic> limit, out-degree <italic>k_out</italic> is binomially distributed:The mean and variance of this distribution are given byThis analytic solution for out-degree distribution closely matches the numerical estimate (##FIG##3##Fig. 4A##).</p>", "<p>An analytic solution for the in-degree distribution is more complex, being in fact a superposition of binomial distributions. This is because a regulatory sequence of length <italic>l</italic>, chosen from a finite alphabet of size <italic>r</italic>, may contain duplicate letters. Let <italic>I</italic> be the number of different letters <italic>x</italic> occurring in a regulatory sequence, so that 1≤<italic>I</italic>≤min(<italic>l</italic>,<italic>r</italic>), and let ω(<italic>I</italic>) be the number of possible sequences containing exactly <italic>I</italic> different letters <italic>x</italic>. The total number of possible regulatory sequences is . The value ω(<italic>I</italic>) can be directly calculated in terms of the parameters <italic>r</italic> and <italic>l</italic>. Denote the multiplicity of letter <italic>x_i</italic> in a sequence of length <italic>l</italic> by <italic>n</italic>(<italic>x_i</italic>). Given <italic>I</italic> and <italic>l</italic> there are two constraints on <italic>n</italic>(<italic>x_i</italic>):For a set of <italic>I</italic> different letters with multiplicities {<italic>n</italic>(<italic>x_i</italic>)}, the number of possible sequences is a multinomial coefficientCombining equations (9) and (10) we get the number of regulatory sequences containing exactly <italic>I</italic> different letters:If we sum over the multiplicities in equation (11), we getNote that ω(<italic>I</italic>) also gives us the number of possible tuples <italic>B_i</italic>(<italic>t</italic>) in the table Ψ:For regulatory sequences with <italic>I</italic> different letters, the in-degree distribution iswhere <italic>I</italic>/<italic>r</italic> is the probability that a randomly selected gene product <italic>x</italic> matches one of the <italic>I</italic> different letters in the regulatory sequence. The mean and variance of this distribution areThe total in-degree distribution is thus:where ω(<italic>I</italic>)/ω is the probability that a randomly selected regulatory sequence contains <italic>I</italic> different letters. This analytical solution closely matches the numerical estimate (##FIG##3##Fig. 4B##).</p>", "<title>State Space</title>", "<p>Although a graph representing the regulatory interactions between genes tells us something about the structure of possible interactions in the network, the full dynamics of a particular network–what that network does–can be represented by exploring its state space. A network activation state space contains all possible activation states that the network can take, and the transitions between each of them.</p>", "<p>For a given number of genes <italic>N</italic>, there is a total of Ω = 2<italic>^N</italic> possible activation states of the network. For a finite network size <italic>N</italic>, the state space is also finite. Starting from an initial state, the system will eventually return to a previously visited state. Thereafter it will follow stable or cyclic behavior, if no stochasticity or exogenous gene products are introduced. The set of states that constitutes a cycle is called an <italic>attractor</italic>, and the number of states it contains is the <italic>attractor length</italic>. All the states converging to an attractor constitute its <italic>basin of attraction</italic>, and the number of states in a basin of attraction is the <italic>basin size</italic>. The state space of a network can be represented as a graph (##FIG##4##Fig. 5##), just as the possible regulatory links among genes can be. But these two graphs are very different things. For example, the in-degree of a gene is the number of other genes that may regulate it; the in-degree of a particular state of the network is the number of states at time <italic>t</italic> that will end up at that state at time <italic>t</italic>+1. We call the in-degree of a network state the <italic>precursor number</italic> of that state. Below we consider these characteristics of the state space of networks of size <italic>N</italic> = 10.</p>", "<p>In a randomly constructed network, the vast majority of network states have no precursor (##FIG##4##Fig. 5##). Such states are unreachable by the network, unless they are used to initiate the network in a simulation. An immediate consequence of this fact is that the average <italic>transient time</italic> that it takes to reach an attractor starting from an arbitrary state is very short compared to the state space size Ω. To make these statements clear we have calculated the probabilities <italic>P_p</italic>(<italic>n_p</italic>) and <italic>P_τ</italic>(τ) that an arbitrary state has <italic>n_p</italic> precursors and transient time τ, respectively. These quantities are displayed in ##FIG##5##Fig. 6## for a network of size <italic>N</italic> = 10. It should be noted that <italic>P_p</italic>(0) increases as <italic>N</italic> increases (not shown) and <italic>P_p</italic>(<italic>n_p</italic>) may have any value between 0 and Ω. Note also that the mode of the transient time distribution shifts to the right as <italic>N</italic> increases (not shown).</p>", "<p>We consider also the basin size distribution, <italic>P_s</italic>(<italic>n_s</italic>), which is the probability of having a basin of attraction of size <italic>n_s</italic> (##FIG##5##Fig. 6C##). <italic>P_s</italic>(<italic>n_s</italic>) is concentrated on values <italic>n_s</italic> = Ω/2^m, <italic>m</italic> = 0,1,…,∞, and decreases dramatically as <italic>m</italic>→∞. This means that in an arbitrary realization we may observe only the peaks at <italic>n_s</italic> = Ω or <italic>n_s</italic> = Ω/2. The case of <italic>n_s</italic> = Ω corresponds to a network with a single attractor whose basin of attraction encompasses the entire network. This pattern is similar to that found in <italic>NK</italic> networks when <italic>K</italic> is relatively small (e.g., <italic>K</italic> = 1) ##UREF##12##[29]##.</p>", "<p>\n##FIG##6##Fig. 7## shows the distribution of number of attractors, <italic>P_a</italic>(<italic>n_a</italic>), and the probability that a given attractor has length <italic>l_a</italic>, <italic>P_l</italic>(<italic>l_a</italic>), in randomly constructed networks of size <italic>N</italic> = 10. Note that <italic>P_a</italic>(1) and <italic>P_l</italic>(1) decrease as <italic>N</italic> increases (not shown). Below we evolve populations of networks using selection on attractor number and attractor length. The distributions shown in ##FIG##6##Fig. 7## for randomly constructed networks illustrate the range of variation in these properties available to evolution in a randomly generated population, and they provide a benchmark against which to measure the efficacy of evolution to find relatively small regions of network space where fitness is maximized. ##FIG##7##Fig. 8## shows the mean values for attractor number (<italic>n_a</italic>), attractor length (<italic>l_a</italic>), transient time (<italic>τ</italic>), and attractor basin size (<italic>n_s</italic>), over a range of small values of <italic>N</italic>. Note that the first three of these measures increase roughly linearly with <italic>N</italic>, while basin size increases exponentially. Thus basin size increases roughly proportional to state space size Ω, which itself is an exponential function of <italic>N</italic>. It had been believed that the average number of attractors of <italic>NK</italic> networks increased as the square root of system size ##UREF##12##[29]##, but recent numerical studies ##UREF##13##[30]## have shown that this quantity increases linearly with <italic>N</italic>, as it does in our model.</p>", "<title>Evolution of the Networks</title>", "<p>In this section we use simulations to explore what sort of networks can be produced by selecting for a particular property in a population of networks. In the following simulations we restricted the changes to point mutations (changes in single letters in either the regulatory or coding regions of genes), and modeled the evolution of an asexual population. The model could also be extended to include recombination among genomes, and other types of mutations such as gene duplications and deletions (e.g. ##UREF##4##[13]##), but we leave this for a later time. To begin, we selected on two network properties: attractor length and number of attractors. Given that attractors form the basis of any subsequent control of gene expression, it is important to show the lability these properties have under a simple selective regime. Such network traits may also relate to fitness in biological systems by corresponding to the identity and behavior of different cell types in multicellular organisms ##UREF##0##[2]##, or alternative states of a genetic network module ##UREF##14##[31]##. Here they provide a simple first test of how the networks might evolve, and the resulting evolved networks provide an interesting comparison with the randomly sampled networks studied above.</p>", "<p>In both cases we generated an initial population of 100 networks, analyzed the state space of each network and assigned it a fitness equal to either the number of states in the largest attractor or the total number of attractors in the state space. We then generated a new, non-overlapping generation of 100 networks. Each network in the new generation was produced, without recombination, from a single parent drawn randomly from the previous generation. The probability that a network was selected as a parent was directly proportional to its fitness. Each reproductive event included a single random point mutation in the network's genome, with each site in either regulatory or coding regions having an equal probability of mutation. We repeated this procedure for 100 generations. The state space of the fittest networks resulting from selection for attractor size and attractor number are shown in ##FIG##8##Fig. 9##.</p>", "<p>Selecting on these particular properties resulted in some very atypical networks. The results of these simulations were strikingly different from randomly generated networks, such as those depicted in ##UREF##0##[2]## or in ##FIG##4##Fig. 5##. For instance, the maximum attractor length in a sample of 40,000 randomly generated networks was 31 (##FIG##6##Fig. 7A##). In contrast, simulated evolution was able to produce an attractor length of 254 in less than 100 generations. Similarly, selection for attractor number produced a network with 112 attractors, far greater than the maximum of 17 in the sample of 40,000 networks shown in ##FIG##6##Fig. 7B##. The large number of possible graphs in this network model means that random sampling to estimate distributions of network properties may fail to capture evolutionarily important parts of the space of all possible networks. Furthermore, it appears that such atypical networks can reliably be reached in relatively few generations, even when the range of variation available to selection is constrained to single point mutations as it was in these simulations.</p>", "<p>Surprisingly, despite their rapid evolution in the character subject to selection (attractor length and number, respectively), these evolved networks did not seem atypical in other respects. Their in-degree and out-degree distributions, shown in ##FIG##9##Fig. 10##, were very close to the expectation for randomly generated networks of their size (<italic>N</italic> = 10; ##FIG##2##Fig. 3##). The dramatic changes in attractor length and number were not the result of concomitant changes in degree distribution. This independence of network properties is further illustrated in ##FIG##10##Fig. 11##. Fitness did not increase smoothly, but rather made occasional large jumps. In contrast, genotypic change occurred more steadily over the course of the simulation. Many genetic changes were neutral with respect to attractor length. In addition, length or number of the other attractors in the network's state space changed without affecting length of the longest attractor; these are phenotypic changes that were also neutral with respect to fitness. Neither genotypic change nor change in other phenotypic traits was a reliable predictor of change in fitness in these simulations, despite the relative simplicity of the trait being selected.</p>", "<title>Evolution in an Environment</title>", "<p>We have been treating networks as though they operated in isolation, subject only to the gene products produced by the network itself. Because intermediary products control the activation of genes in our model, the introduction of any exogenous products can influence the downstream activation and resultant attractor of the network. This gives our model an important additional property over the <italic>NK</italic> model: the state space, including the number and type of attractors, is a property of a particular network <italic>combined with</italic> a particular environment.</p>", "<p>A network with no exogenous input has a single state space. However, if we assume that our environment provides a constant set of products, not produced by the focal network itself, but still able to bind and regulate the functioning of the network, the state space for any single network now depends on the particular environment of exogenous products in which the network operates (##FIG##11##Fig. 12##). Under constant environmental conditions the network will settle into one attractor, depending on the starting point. When environmental conditions change, the state that was previously in an attractor may shift to the edge of a basin, and the network may move to a new state. The introduction or removal of different products can have many effects on the state space, such as changing the number of attractors, the size of their basins, or the set of expression states contained in their basins. The maximum number of possible environments is 2<italic>^r</italic>, where <italic>r</italic> is the number of possible letters in the alphabet <italic>Π</italic>. Thus the number of state space graphs corresponding to a single network may be as large as 2<italic>^r</italic>.</p>", "<p>One property of a network is the degree to which these state spaces are similar, or fall into broad groups. This similarity may be considered a measure of the environmental robustness of the network. If the network continues to act relatively unchanged (the attractors remain constant) in various environments (differing exogenous inputs), then the network operation is robust to these changes. Although robustness in Boolean networks can be thought of in this way, our model permits us to explore a much more dynamic sense of robustness (in contrast with ##UREF##3##[9]##, for example). The environments in which genetic networks operate are both sources of noise and sources of important signals, either from the external environment, from other parts of a multicellular organism, or from other modules in the organism's overall genetic network ##UREF##14##[31]##. Fitness depends on responding appropriately to the signals and ignoring the noise. Viewed in this way, what must be robust is the <italic>reaction norm</italic> of the network–its ability to react in a plastic and appropriate manner under various environments by distinguishing signal from noise.</p>", "<p>We simulated evolution in a series of simple environments, in which fitness was determined by their ability to respond “appropriately.” If some <italic>indicator</italic> product was present in the environment, a network had high fitness if it produced some other <italic>functional</italic> product. If another indicator product was present, the network was fit if it produced a second, different, functional product. A network had high fitness by doing the right thing at the right time: in environment <italic>A</italic>, produce product <italic>a</italic>, and in environment <italic>B</italic>, produce product <italic>b</italic>. Doing the right thing implies not doing the wrong thing also–producing product <italic>b</italic> in environment <italic>A</italic> reduced fitness, and a network that simply produced <italic>a</italic> and <italic>b</italic> constitutively did not have high fitness. We selected on networks' ability to respond correctly to two different environments that alternated over time.</p>", "<p>We evolved a population of 100 networks of size <italic>N</italic> = 10. Each network was exposed to the first environment for 10 time steps, and then switched to the second environment for another 10 time steps. The networks were then returned to the original environment. This environment switching continued until the network had been exposed to each environment 5 times. Fitness was calculated as the number of correct functional products produced, minus the number of incorrect functional products, summed across all time steps. Gene products that were not the functional product in either environment did not affect fitness.</p>", "<p>In addition to this alternation of environmental signals, we tested the ability of networks to evolve robustness to environmental noise. In the stable environment, the only exogenous products were the indicator products. In this simulation the evolved networks quickly behaved exactly as required, changing their required output in the presence of different indicator products. In the noisy environment, the indicator product was present with 2 other products, randomly chosen at each time step. Achieving a high fitness under noisy conditions was more difficult to evolve, and the networks remained at lower fitnesses throughout the simulation under noisy conditions. However, we found that a network that had evolved in a noisy environment would often perform perfectly in a stable environment.</p>", "<p>What sort of difference is there between a network evolved in a stable environment and one evolved in a noisy environment? We tested this by subjecting the fittest network from each simulation to a number of trials (10,000) in a noisy environment. The sample distributions generated are shown in ##FIG##12##Fig. 13##. We assessed both the original network (steps = 0) and a sample of 1-, 2-, and 3-step mutants from this network. This gives us some idea of the fitness of the networks in the local mutational neighborhood, and thus an indication of the ruggedness of the fitness landscape close to the peak on which the evolved network sits. Evolving the networks in a noisy environment did indeed produce a more consistently environmentally robust network, shown by both the relative positions and the widths of the peaks in the frequency distributions in ##FIG##12##Fig. 13##. The decline in fitness with increasing numbers of mutations away from the original network is similar for the networks evolved in both stable and noisy environments. Thus these networks have roughly equivalent mutational robustness. In terms of the fitness landscape, the fitness peaks to which the networks have evolved in both stable and noisy environments are somewhat intermediate between broad plateaus and precipitous spires, which would allow for some near-neutral variation to persist in mutation/selection balance.</p>" ]

[ "<title>Discussion</title>", "<p>Simple models of genetic networks have led to general conclusions about the properties of network architecture and how they affect network evolution ##REF##5803332##[1]##, ##UREF##0##[2]##, ##UREF##1##[4]##, ##REF##17188715##[5]##, ##REF##17316697##[32]##. At the same time, a growing number of technological and analytical tools allow the direct measurement of regulatory networks in natural systems ##UREF##15##[33]##–##UREF##17##[39]##, so that a number of empirical networks have been described in detail ##REF##12399584##[21]##, ##REF##14657375##[28]##, ##UREF##18##[40]##, ##REF##17237218##[41]##. In seeking to connect these growing fields, modeling efforts can proceed by adding layers of complexity and assessing the degree to which features of the model better approximate empirical results. Here we have added a degree of complexity to simple <italic>NK</italic> networks, using a publish-subscribe view of gene regulation. Although our model shares some basic similarities with the <italic>NK</italic> model, we have found some tantalizing differences in both the properties of single networks and in the evolution of populations of networks.</p>", "<p>First, the pattern of degree distributions from randomly constructed networks in our model is substantially different from that of previous models. In Kauffman's ##REF##5803332##[1]## original <italic>NK</italic> model, each gene has exactly <italic>K</italic> inputs and in-degree distribution is therefore a Dirac delta function. In randomly constructed networks under the “standard” <italic>NK</italic> model ##UREF##1##[4]##, regulatory inputs to each gene are assigned independently with a given probability, resulting in unimodal binomial (or equivalently for large <italic>N</italic>, Poisson) distributions for in- and out-degree. In scale-free networks, in-degree distribution follows a power law <italic>P</italic>(<italic>k</italic>)∼<italic>k</italic>\n^−γ while out-degree follows a Poisson distribution, or vice versa ##UREF##1##[4]##. In contrast, our publish-subscribe model produces an in-degree distribution that is multimodal due to the superposition of binomial distributions with different mean values. The fact that in-degree and out-degree distributions differ in form from each other in our model also contrasts with the standard <italic>NK</italic> model. This qualitatively different pattern is a consequence of the matching rule between the different nodes, i.e. between the coding and regulatory sequences. Thus, although the networks in our model exhibit similar dynamics to those of Boolean <italic>NK</italic> networks, the distributions of basic network properties differ as a result of the publish-subscribe regulatory framework. A network model based on a similar matching rule was able to reproduce global topological properties of the yeast gene regulatory network ##REF##17551581##[25]##. These properties include not only degree distributions, but also other network descriptors such as clustering coefficient, rich-club coefficient, degree-degree correlation, and <italic>k</italic>-core decomposition.</p>", "<p>This divergence from previous models is echoed as well by the networks evolved in our simulations. Generally, degree distribution is believed to be a central feature of a network and a key predictor of its dynamic behavior in other respects ##REF##17188715##[5]##. For example, the importance of scale-free degree distributions for other properties like robustness and evolvability has been established in several studies of <italic>NK</italic> networks ##UREF##1##[4]##, ##REF##12853565##[42]##. However, in our publish-subscribe model, it appears that dynamic behavior may be to some extent uncoupled from degree distribution. In the simulations above, attractor length and number evolved far outside the distribution expected from randomly generated networks, but degree distribution remained remarkably similar to random. The degree distributions of the evolved networks give us no clue to the general principles by which length and number of attractors may evolve. Conversely, degree distribution may be a poor predictor of other network properties in this model. Other topological properties (e.g. ##REF##17551581##[25]##) may be more relevant to the evolutionary dynamics of our publish-subscribe model, and this issue should be explored further. However, additional metrics that are directed toward specific tasks, such as robustness to various types of change, may be necessary to fully compare across networks and predict evolutionary dynamics.</p>", "<p>In the broader context of dynamic behavior and evolution of genetic regulatory network models, two issues have received particular attention: evolvability and robustness. A critical component of evolvability is the presence of neutral variation in a population ##UREF##19##[43]##–##UREF##20##[46]##. Evolution in our network model produces neutral variation in genotype, as seen in ##FIG##10##Fig. 11##, that has no immediate effect on either phenotype or fitness. From an adaptive landscape perspective, this neutral change can be seen as meanderings along neutral ridges in the landscape ##REF##21238086##[47]##, ##UREF##21##[48]##. The importance of this neutral variation is its effect on the fitness of subsequent mutations. In our model, as in natural systems ##REF##21238076##[49]##, genes often interact epistatically, so that the fitness effect of a single mutation depends on the allelic states of other loci. Thus the genetic background against which a mutation arises may determine whether it is favored by selection, and therefore whether it sweeps to fixation and increases the average fitness of the population as a whole. Neutral mutations change the genetic background that determines both the sign and the magnitude of the fitness effects of subsequent mutations.</p>", "<p>Our network model illustrates the mechanism of neutral variation in the publish-subscribe view of gene regulation. For example, regulatory binding sites may mutate to a state for which there is not currently a matching gene product being produced. At the time, this mutation may be neutral, with no effect on the phenotype of the network. However, this mutation has created a new subscriber, ready to receive a signal from a publisher, or coding region. Such a mutation in a publisher may occur in the future, and thus a new connection is made between two genes. In addition, the number of transcriptional regulators (gene products) is limited in our model ##REF##17846637##[15]##, ##REF##16185862##[50]##. As a result, multiple neutral mutations in the form of publishers (or subscribers) tuned to the same signal can accumulate as neutral changes with no effect on fitness. When a single mutation in a subscriber (or publisher) shifts to the matching signal, multiple new connections are formed. The effect on phenotype, and perhaps fitness, as a result of this single mutation is magnified by the presence of existing variation. In fact the ability of mutations to have broader effects on phenotype in this way may be an important component of evolvability ##UREF##22##[51]##.</p>", "<p>In our simulations we explored the evolution of environmental robustness, which is the ability of a network to perform (i.e., maintain high fitness) in the face of a noisy environment. Incorporating the ability for networks to react to the local environment enables us to explore a number of possibilities. Here, we have emphasized that robustness can be a dynamic, rather than a static, property of networks. The publish-subscribe model allows us to evolve networks whose reaction norm is robust under noisy environments. The shift from a static to a dynamic conception of robustness may have important implications. Consider an idea introduced by Kauffman ##UREF##0##[2]##, in which the attractors in genetic networks are viewed as analogous to cell types in a multicellular organism ##REF##15903968##[52]##. For the <italic>NK</italic> model, the attractor into which a network falls is fixed for a particular genetic network and the starting conditions. In multicellular development, however, the environment is, in part, other cells, and the process of differentiation may be driven by dynamic interactions between cells rather than the isolated properties of a single cell ##UREF##23##[53]##. The evolution of this plastic response to the local cellular environment, and the evolution of its subsequent robustness, may be a key element in understanding the emergence of multicellularity ##REF##12492416##[54]##. Alternatively, the focal network may be a module of a larger genetic network, and organismal fitness may depend on the network's ability to respond appropriately to signals from other modules.</p>", "<p>A large number of issues could be explored further with the publish-subscribe model. First, in our estimates of degree distribution, we considered two genes to be connected if the coding region of one gene matched a site in the regulatory region of the other. However, this ignores the particular Boolean rules of expression for the second gene, whose expression state may not actually depend on the first gene's product; in fact, whether this dependence is present may itself depend epistatically on the expression states of yet other genes ##UREF##0##[2]##. Calculation of degree distribution in this expanded sense soon gets quite complicated, although it may be necessary for more direct comparisons to empirical data, such as gene co-expression networks or expression time series ##UREF##16##[34]##.</p>", "<p>Second, we assumed here that a single coding region produces a sufficient concentration of gene product to bind any number of matching regulatory sites. The consequences of this assumption, or alternatively of competition among binding sites for limited gene product copies, could be explored further. Relaxing this assumption would not change the observed patterns of degree distribution of networks, according to the rules by which we calculated it. However, it would introduce an element of stochasticity into the activation of genes at each time step if single gene products were to bind to either one or another regulatory site with some probability between 0 and 1. As a result, our conception of the state space of a network would also change. Under the current assumption, the out-degree of any node in the state space network is one, but relaxing this assumption would produce some states with probabilistic edges connecting to multiple other states. This would result in an additional concept of robustness that could be explored: the robustness of attractors to stochastic shifts outside of their attractor basin as a result of the stochastic binding of gene products.</p>", "<p>Third, one could explore the consequences of variation in several of the parameters. Our goal here was to explore the properties of the simplest publish-subscribe model, so in our evolutionary simulations we held alphabet size, regulatory region length, and total network size constant. Varying these parameters across networks may have implications for measures of network topology and for the evolutionary dynamics of populations of networks. Regulatory region length could also vary across nodes within a network; in a network model similar to ours such variation produced similar qualitative behavior but improved the fit to empirical data on topological descriptors from yeast networks ##REF##17551581##[25]##. Change in this parameter has also been implicated in the evolution of organismal complexity ##UREF##24##[55]##. Among other effects, longer regulatory regions would provide a larger mutational target for regulatory versus coding regions. It remains an outstanding question to what extent changes in regulatory versus coding regions play different roles in phenotypic evolution ##REF##17492956##[56]##, ##REF##16530225##[57]##, and the publish-subscribe model explicitly separates the two. We plan to address this issue in future work. In our simulations, we used networks of relatively small size (<italic>N</italic> = 10), which can be thought of as modules within a larger network. However, simulations of larger networks, particularly in the noisy or fluctuating environments that we described, could be used to address the evolution of modularity itself; that is, do networks evolve some degree of internal separation of components that partition the response to environmental signals? Alternatively, can the behavior of larger networks be adequately represented by studies of smaller networks?</p>", "<p>Finally, we addressed network evolution solely in the context of single-step mutations. The publish-subscribe model could easily be extended to address other types of mutations, such as gain or loss of binding sites in regulatory regions, gene duplication and divergence ##UREF##4##[13]##, or whole genome duplication. Nonetheless, our results suggest that the publish-subscribe model holds remarkable evolutionary potential even when mutation is restricted to single steps.</p>", "<p>Our publish-subscribe model of genetic regulatory networks adds a layer of complexity to the common <italic>NK</italic> networks by making the gene regulation process more explicit, and by using a rule system for matching gene products to regulatory sites that affect the expression state of other genes. In this way it is similar to yet more complex models. Examples include the Artificial Genome class of models ##UREF##25##[58]##–##REF##16095624##[61]##, which create an information sequence analogous to DNA, and content-based networks ##UREF##8##[19]##, ##REF##17551581##[25]##, ##UREF##27##[62]##, where the focus is on the topological properties of the networks rather than their dynamics. The production of new, more complex, variants on well-studied models in biology can often aid in two ways. First, the introduction of new parameters might suggest that there is behavior outside the scope of the simpler model. Second, the introduction might allow us to ask different questions. The publish-subscribe model appears to do both.</p>" ]

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[ "<p>Conceived and designed the experiments: BC DB PAH. Performed the experiments: BC DB PAH. Analyzed the data: BC DB PAH. Contributed reagents/materials/analysis tools: BC DB PAH. Wrote the paper: BC DB PAH.</p>", "<p>We present a simple model of genetic regulatory networks in which regulatory connections among genes are mediated by a limited number of signaling molecules. Each gene in our model produces (publishes) a single gene product, which regulates the expression of other genes by binding to regulatory regions that correspond (subscribe) to that product. We explore the consequences of this publish-subscribe model of regulation for the properties of single networks and for the evolution of populations of networks. Degree distributions of randomly constructed networks, particularly multimodal in-degree distributions, which depend on the length of the regulatory sequences and the number of possible gene products, differed from simpler Boolean <italic>NK</italic> models. In simulated evolution of populations of networks, single mutations in regulatory or coding regions resulted in multiple changes in regulatory connections among genes, or alternatively in neutral change that had no effect on phenotype. This resulted in remarkable evolvability in both number and length of attractors, leading to evolved networks far beyond the expectation of these measures based on random distributions. Surprisingly, this rapid evolution was not accompanied by changes in degree distribution; degree distribution in the evolved networks was not substantially different from that of randomly generated networks. The publish-subscribe model also allows exogenous gene products to create an environment, which may be noisy or stable, in which dynamic behavior occurs. In simulations, networks were able to evolve moderate levels of both mutational and environmental robustness.</p>" ]

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[ "<p>We thank G. Broderick, A. Erzan, faculty at the Santa Fe Institute and Istanbul Technical University, and one anonymous reviewer for useful discussion and comments.</p>" ]

[ "<fig id=\"pone-0003245-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g001</object-id><label>Figure 1</label><caption><title>Schematic diagram of the network model.</title><p>Shown are six genes, each with a regulatory region of length 2 and a coding region (underlined). Arrows represent possible interactions, i.e. directed edges in the network. Below one gene is the Boolean rule set specific to that gene. A “−“ indicates that the binding site is not bound by the corresponding product, and a “+” indicates that it is bound. The gene is then either expressed (“on”) or not (“off”). In this case, if product 7 but not product 0 is present at time <italic>t</italic>, the binding state of the regulatory region of this gene corresponds to the second row of the Boolean table. As a result, the gene is expressed and product 0 is present at time <italic>t</italic>+1. Because 0 occurs in both the regulatory region and the coding region of this gene, it is self-regulating and will not be expressed at time <italic>t</italic>+2.</p></caption></fig>", "<fig id=\"pone-0003245-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g002</object-id><label>Figure 2</label><caption><title>Diagram of four time steps in a 6-gene network.</title><p>In the initial conditions, products 9 and 0 are present. Filled boxes represent expressed genes, dotted arrows represent binding of products to regulatory regions, and solid arrows represent production of gene products. From these initial conditions, this network enters a stable period-3 cyclic attractor. Boolean tables are not shown.</p></caption></fig>", "<fig id=\"pone-0003245-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g003</object-id><label>Figure 3</label><caption><title>Degree distributions for small networks.</title><p>(A) Out-degree and (B) in-degree distributions are shown for networks of size <italic>N</italic> = 5 to <italic>N</italic> = 10. Each distribution is constructed from 10⁶ independent, randomly generated networks with parameter values <italic>r</italic> = 10 and <italic>l</italic> = 3.</p></caption></fig>", "<fig id=\"pone-0003245-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g004</object-id><label>Figure 4</label><caption><title>Numerical and analytic degree distributions for large networks.</title><p>(A) Out-degree and (B) in-degree distributions for networks of size <italic>N</italic> = 1000. Numerical distributions are constructed from 10⁶ independent, randomly generated networks with parameter values <italic>r</italic> = 10 and <italic>l</italic> = 3.</p></caption></fig>", "<fig id=\"pone-0003245-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g005</object-id><label>Figure 5</label><caption><title>State space of a randomly generated network.</title><p>The state space of a network can be represented as a directed graph. Each point (node) represents an expression state of the network, and lines (edges) connecting them represent transitions from one time step to the next. This network has <italic>N</italic> = 10 genes, and therefore 1024 states. The network has three attractors (open circles), of which one is a single steady state where an identical set of gene products is present at each time step, and the other two are cyclic attractors of period 2 and 4, respectively.</p></caption></fig>", "<fig id=\"pone-0003245-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g006</object-id><label>Figure 6</label><caption><title>Precursor number, transient time, and basin size for networks of size <italic>N</italic> = 10.</title><p>(A) Frequency distribution of precursor number across network states, estimated from 40,000 randomly generated networks, on a log scale. Note that <italic>P</italic>(0)≈0.96 has been suppressed, meaning that the large majority of states have no precursor. (B) Frequency distribution of transient time, estimated from 40,000 randomly generated networks. The maximum value of τ is 31 in this number of realizations. (C) Frequency distribution of attractor basin size, defined as the number of states that lead to a given attractor, estimated from 40,000 randomly generated networks of size <italic>N</italic> = 10. Note the peaks at Ω/2<italic>ⁿ</italic>, <italic>n</italic> = 0,1,2,…, where Ω = 1024 is the total number of states in each network.</p></caption></fig>", "<fig id=\"pone-0003245-g007\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g007</object-id><label>Figure 7</label><caption><title>Length and number of attractors in networks of size <italic>N</italic> = 10.</title><p>(A) Frequency distribution of length of attractors, estimated from 40,000 randomly generated networks. The maximum attractor length in this sample was 31. (B) Frequency distribution of the number of attractors in each network, estimated from 80,000 randomly generated networks. The maximum number of attractors was 17 in this sample.</p></caption></fig>", "<fig id=\"pone-0003245-g008\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g008</object-id><label>Figure 8</label><caption><title>Attractor properties as a function of network size in small networks.</title><p>The average attractor number (A), attractor length (B), and transient time (C) increase linearly as a function of network size <italic>N</italic>, while average basin size (D) increases exponentially.</p></caption></fig>", "<fig id=\"pone-0003245-g009\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g009</object-id><label>Figure 9</label><caption><title>State spaces of evolved networks.</title><p>(A) State space of a network evolved in a population of 100 networks after 100 generations of selection for large attractor size. The attractor shown has length 254. Here <italic>N</italic> = 10, <italic>l</italic> = 3, and <italic>p</italic> = 0.5. (B) State space of a network evolved under selection for many attractors. This network has 112 attractors. All other parameter values as in (A).</p></caption></fig>", "<fig id=\"pone-0003245-g010\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g010</object-id><label>Figure 10</label><caption><title>In- and out-degree distributions of evolved networks.</title><p>Shown are (A) in-degree and (B) out-degree distributions for the evolved network in ##FIG##8##Fig. 9A##, the result of selection on attractor size, and (C) in-degree and (D) out-degree distributions for the network in ##FIG##8##Fig. 9B##, the result of selection on attractor number. These distributions may be compared to the random expectations for <italic>N</italic> = 10 in ##FIG##2##Fig. 3##.</p></caption></fig>", "<fig id=\"pone-0003245-g011\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g011</object-id><label>Figure 11</label><caption><title>Genotype, phenotype, and fitness in a single evolving lineage.</title><p>Shown are three network properties over 100 generations in the lineage leading to the network shown in ##FIG##8##Fig. 9A##. Genotypic divergence (dotted line) is the number of letters <italic>x</italic> in the network sequence, either in regulatory or coding regions, different from the ancestor. Changes in phenotype (open circles) are points at which the attractors of the network change, whether or not this results in a change in length of the longest attractor. Fitness (solid line) is the length of the longest attractor in the network state space. Note that changes in either genotype or phenotype may be effectively neutral, without corresponding changes in fitness, and that large changes in fitness can occur with relatively small changes in genotype.</p></caption></fig>", "<fig id=\"pone-0003245-g012\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g012</object-id><label>Figure 12</label><caption><title>State space of a single network subject to different environmental conditions.</title><p>Shown are the state spaces of a single network of size <italic>N</italic> = 10 under four different environments. Each environment represents a different set of gene products that are constantly present (e.g. exogenously produced) and available to bind to regulatory regions in the network. Note that a single network can vary in both the number and size of attractors depending on the environment.</p></caption></fig>", "<fig id=\"pone-0003245-g013\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003245.g013</object-id><label>Figure 13</label><caption><title>Fitness of evolved networks in noisy environments.</title><p>Frequency distributions of fitness for single evolved networks subjected to 10,000 trials in a noisy environment. Solid lines indicate the fitness of the evolved network, while dashed lines indicate the fitness of networks that are 1, 2, or 3 mutational steps away from the evolved network. (A) Fitness of the network produced by evolution in a stable environment. (B) Fitness of the network produced by evolution in a noisy environment.</p></caption></fig>" ]

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[ "<disp-formula><label>(1)</label></disp-formula>", "<disp-formula><label>(2)</label></disp-formula>", "<disp-formula><label>(3)</label></disp-formula>", "<disp-formula><label>(4)</label></disp-formula>", "<disp-formula><label>(5)</label></disp-formula>", "<disp-formula><label>(6)</label></disp-formula>", "<disp-formula><label>(7)</label></disp-formula>", "<disp-formula><label>(8a)</label></disp-formula>", "<disp-formula><label>(8b)</label></disp-formula>", "<inline-formula></inline-formula>", "<disp-formula><label>(9)</label></disp-formula>", "<disp-formula><label>(10)</label></disp-formula>", "<disp-formula><label>(11)</label></disp-formula>", "<disp-formula><label>(12)</label></disp-formula>", "<disp-formula><label>(13)</label></disp-formula>", "<disp-formula><label>(14a)</label></disp-formula>", "<disp-formula><label>(14b)</label></disp-formula>", "<disp-formula><label>(15)</label></disp-formula>" ]

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[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>All 3 authors: Santa Fe Institute Complex Systems Summer School Fellowship. BC: Cambridge-Templeton Consortium Research Grant. PH: National Institutes of Health National Research Service Award Ruth L. Kirschstein Postdoctoral Fellowship. Funders had no direct role in any part of the study or manuscript preparation, review, or approval.</p></fn></fn-group>" ]

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[ "<title>Introduction</title>", "<p>Sex pheromones play important roles in the reproductive behaviors of many organisms. These compounds are important for finding a mate, appealing to the mate for successful copulation and also for avoiding inappropriate mates for reviews see ##REF##11092827##[1]##, ##REF##8209251##[2]##. In <italic>Drosophila</italic>, hydrocarbon pheromone profiles also provide more subtle information about a potential mate, e.g. the sexual status of females: their maturation level and/or whether they are previously mated. While both mature virgin and mated females contain aphrodisiac pheromones, mated females have aversive compounds which have been acquired from the male during copulation. Males detect these components and adjust their behavior, showing a reduced level of courtship to copulated females ##REF##218208##[3]##. The hydrocarbon profile of a very young female (∼8 h-old) contains a mixture of saturated and unsaturated hydrocarbons and is very different from that of a mature female (4–5 day-old) ##REF##15694302##[4]##, ##REF##24276194##[5]##. We previously found that a male recognizes the differences between mature and immature females and can produce trainer-type specific courtship suppression upon training with virgin females under conditions in which copulation is prevented ##REF##15694302##[4]##. This type of courtship suppression relies on males' formation of an association between volatile maturation-specific compounds and the aversiveness of the failure to copulate, causing a reduction in courtship only toward the type of female used as a trainer.</p>", "<p>One of the courtship parameters that is modulated in this learning paradigm is courtship initiation ##REF##15694302##[4]##. Courtship was first described by Sturtevant back in 1915 ##UREF##0##[6]##, and now is considered to be initiated in response to appropriate olfactory and visual cues emitted by the potential mate, and consists of male orientation, chasing and tapping ##UREF##1##[7]##. Lack of both visual and olfactory information reduces initiation to very low levels ##REF##15694302##[4]##, ##REF##3092798##[8]##. Once courtship is started, gustatory information from the target female contributes, accelerating the courtship ritual and stimulating wing vibrating, licking, curling abdomen and mounting. To date, only one chemosensory receptor, <italic>Gr68a</italic>, has been reported as a putative female pheromone receptor in <italic>Drosophila</italic>. <italic>Gr68a</italic> encodes a gustatory receptor expressed in approximately 10 male-specific bristles of the male's foreleg. Intriguingly, blocking neurotransmitter release by expressing tetanus toxin or RNA interference of the receptor gene under control of a <italic>Gr68a</italic> promoter upstream of a sequence encoding yeast-derived GAL4, lowered both copulation success and wing vibration ##REF##12971900##[9]##. These findings suggested that the neurons in which <italic>Gr68a</italic> regulatory sequence is active are involved in information processing of pheromonal cues during late stages of courtship after the male contacts the female. In the current study, we examined whether this group of neurons (which we find includes a wide variety of mechanosensory cells in addition to the previously identified chemosensory cells) also has a role in courtship initiation. The existence of problems at both late and early stages of courtship in <italic>TNT/Gr68a</italic> flies has allowed us to uncover a previously unappreciated role for mechanosensation in initiation of courtship.</p>" ]

[ "<title>Materials and Methods</title>", "<title>Animal maintenance</title>", "<p>Flies were raised on autoclaved cornmeal-yeast-sucrose-agar food, containing the mold inhibitor Tesgosept, in a 12 h light/dark cycle at 25°C. Males and females were anesthetized with CO₂ on the day of eclosion then used immediately as immature flies or separated by sex and aged for 4 or 5 days. Experimental males were housed in individual tubes. Mated females were prepared by putting 3-day-old females with males. Only females, which copulated for ≥14 min were used the next day. Decapitated flies were prepared by cutting their heads off with fine scissors just before use. For surgical manipulation of the auditory system, aristae of wild-type males were either fixed to the third antennal segment with a small amount of wax or partially amputated with fine scissors (less than one quarter of the arista was left).</p>", "<title>Fly strains</title>", "<p>\n<italic>Canton-S</italic> was used as the wild-type strain. Transgenic lines <italic>Gr68a-GAL4</italic>, <italic>UAS-TNT</italic> and <italic>UAS-TNT^IN</italic> were provided by H. Amrein at Duke University. <italic>UAS-mCD8GFP</italic> was obtained from Bloomington Stock Center and jumped onto the second chromosome for convenience. An auditory mutant, <italic>5D10</italic> and its genetic control strain, 40A-G13 were kindly provided by D. Eberl at University of Iowa.</p>", "<title>Behavioral assays</title>", "<p>All behavior experiments were performed under dim red lights (&gt;700 nm) unless otherwise noted in a Harris environmental room (25°C, 70% humidity). Wet filter paper (Whatman, 42 ashless) was put in each chamber to maintain humidity.</p>", "<p>A 4 or 5-day-old male was placed with a decapitated female “courtship object” in a single-pair-mating chamber (8 mm in diameter, 3 mm in depth) and its courtship performance was videotaped with a digital camcorder (Sony, DSR-PD150) for 10 min. A courtship index (CI) was calculated as the proportion of time (×100%) a male displayed courtship action during the 10 min observation period. Courtship latency was the time lag to the first courtship display (courtship orientation) after pairing. A latency value of 600 sec was recorded when no courtship was performed during the 10 min observation. Bout duration is the mean duration of each consecutive courtship sequence between breaks. ≥20 males were tested for each genotype.</p>", "<p>For courtship conditioning, a male was put together with a trainer female, either an immature female or a decapitated mature virgin for 60 min. Immediately after training, males were transferred into a clean chamber and paired with a decapitated mature female “tester” for 10 min. Sham trained males were kept alone in the mating chamber for the first hour then paired with a tester. Memory index is calculated by dividing CI at test (CI_test) by the mean of sham CIs (CI_sham); CI_test/mCI_sham. If memory index = 1, it indicates that there has been no learning since the courtship level of trained males is equivalent to that of sham trained males ##REF##10327242##[13]##. ≥20 males were tested for each condition.</p>", "<p>For experiments using intact females, a male was paired with a female in a slit-cell chamber (7 mm×10 mm×70 mm) under white lights or dim-red lights and latencies of courtship and copulation, time lags until the first courtship performance or the successful copulation, were recorded. A latency value 1800 sec was given when no courtship or copulation was performed during 30 min observation. 24 males were tested for each genotype and experimental condition.</p>", "<title>Pheromone preparation</title>", "<p>When introducing pheromone extracts, a two-part chamber (8 mm in diameter, 6 mm in depth) with a fine nylon mesh (Tetko, 3–180/43) was used to block direct contact with the pheromone-containing filter paper. In order to collect female pheromones, a mature female was put on a wet filter paper in a chamber for 1 h to transfer odors to the filter. For the experiment using female pheromone deposits inside a large chamber (##FIG##4##Fig. 5##), three intact females were introduced and kept for 10 min before the test then discarded before introducing a test male and a decapitated silent female.</p>", "<title>Sound preparation</title>", "<p>Fly sounds (walking and grooming) was recorded from two males and a female in a chamber with courtship song amplifier (Aktogen). Courtship sound (sine song and pulse song) was manually deleted from the recording by observing image references using QuickTime version 7.5 and Amadeus Pro version 1.2.1. The moving-fly sounds and white noise were played back through an audio speaker (Sound Force 660. Intensity was 88∼96 dB at the outside of the 3 mm thick Plexiglas chamber. Background room noise was 78 dB).</p>", "<title>Statistical analysis</title>", "<p>Each CI was subjected to arcsine square root transformation to effect an approximation of normal distribution, using JMP software version 5.0.1.2 for the Macintosh. ANOVA with each indicated condition as the main effect was performed on the transformed data. Posthoc analysis was done using Fisher's PLSD test. Bars in figures represent means±SEM with levels of significance indicated by *<italic>P</italic> significant = 〈&lt;0.05.</p>", "<title>Imaging</title>", "<p>Animals expressing <italic>UAS-mCD8-GFP</italic> under control of the <italic>Gr68a-GAL4</italic> driver were observed with a Olympus BX-51W fluorescence microscope. Brains were dissected and imaged on a Leica TCS SP2 mounted on a Leica DMIRE2 inverted microscope without fixation.</p>" ]

[ "<title>Results</title>", "<title>Courtship behavior of <italic>TNT/Gr68a</italic> mutant males with immobile females is normal</title>", "<p>Before examining the role of <italic>Gr68a</italic> neurons in courtship conditioning, we reexamined their role in basic courtship under our standard experimental conditions. In a previous study by Bray and Amrein ##REF##12971900##[9]##, blocking the output of <italic>Gr68a</italic> neurons caused reduced courtship levels, bout initiation rate and copulation success when intact <italic>w¹¹¹⁸</italic> females were used as the courtship object in a 300 mm² chamber with room light. For courtship conditioning, in addition to female pheromones, an active courtship performance by the male is thought to be essential during the training session since without a courtship target, exposure to the female extracts alone does not cause modification of subsequent behavior ##REF##15694302##[4]##, ##REF##6422921##[10]##. In some cases, the mobility of the target female can contribute to apparent courtship defects; e.g. courtship level could appear low if the male has visual or locomotion defects that affects his ability to track the moving female, or copulation success could be reduced if the male's courtship is defective and does not stimulate the female to become receptive. To examine the courtship enthusiasm of <italic>Gr68a</italic> males separate from their tracking ability and performance quality, we employed a decapitated immobile wild-type female as a courtship target in a 50 mm² chamber. We also observed behavior under dim red lights, which limits the visual cues available to the male, since visual cues are mostly positive and sometimes dominate over subtle changes and/or defects in other sensory inputs ##REF##3092798##[8]##, ##REF##10706600##[11]##. Males with defective <italic>Gr68a</italic> neuron function were prepared by crossing <italic>Gr68a-GAL4</italic> and <italic>UAS-TNT</italic> to express tetanus toxin (TNT), a protease that blocks vesicle fusion ##REF##8100740##[12]##. A <italic>TNT/Gr68a</italic> male was put together with a decapitated mature female and its courtship level (courtship index, CI), courtship latency, and duration of each courtship bout were measured for a 10 min observation period (##FIG##0##Fig. 1##). As controls, we also examined males expressing an inactive toxin (TNT^IN), and <italic>+/TNT</italic> and <italic>Gr68a/+</italic> heterozygotes.</p>", "<p>As shown in ##FIG##0##Figure 1A##, <italic>TNT/Gr68a</italic> males performed a significantly lower level of courtship (54±5%, black bar) than wild-type males (83±3%, open bar). Courtship latency, the time lag to the first courtship display after pairing with the female, was significantly extended (##FIG##0##Fig. 1B##, 192.0±29.0 sec for <italic>TNT/Gr68a</italic>, compared with 28.8±7.2 sec for wild-type). In contrast, bout duration, the average length of each courtship bout, was not affected (<italic>P</italic>&gt;0.05), implying that the mutant males have a defect in finding the immobile female in the dark, not a defect in maintaining courtship once it begins. The specificity of this finding for <italic>Gr68a</italic> cell function, however, was not confirmed since all other control strains except the <italic>+/Gr68a</italic> heterozygous control also yielded similarly low CIs (40%∼55%). No significant difference was found between any combination of <italic>TNT/Gr68a</italic>, <italic>TNT^IN/Gr68a</italic>, <italic>TNT/+</italic> and <italic>TNT^IN/+</italic>, and between wild-type and <italic>+/Gr68a</italic> males, indicating that the courtship defects of <italic>TNT/Gr68a</italic> males under our experimental conditions resulted primarily from the genetic background of the <italic>UAS-TNT</italic> lines. We conclude that <italic>Gr68a</italic> neurons do not play a critical role in initiating and performing courtship with an immobile female in the absence of visual input.</p>", "<title>Discrimination of maturation stage of trainer females in courtship conditioning is intact in <italic>TNT/Gr68a</italic> males</title>", "<p>Males show a high level of initial courtship to a virgin female, but when copulation is prevented over the course of an hour, they lose interest in virgins of that age, and courtship remains reduced up to four hours- this learning has been terms “trainer type-specific courtship suppression” ##REF##15694302##[4]##. When a wild-type male is trained with a very young immature female, he will show suppression of courtship toward an immature female ##REF##15694302##[4]##, but not to a decapitated mature virgin (mVd) tester (##FIG##1##Fig. 2 left, *control##). In courtship conditioning, virgin female pheromones can be employed as associative cues, so that addition of pheromone extract over a mesh barrier during training changes the specificity, producing courtship suppression with mature female testers too. The extract alone, in the absence of a courtship object produces no suppression (##FIG##1##Fig. 2 left##). In order to examine whether <italic>Gr68a</italic> neurons are involved in the discrimination of female age, <italic>TNT/Gr68a</italic> males were examined for behavior modification in this paradigm. As shown in ##FIG##1##Figure 2 right panel##, the mutant male produced normal age-specific courtship suppression. Training with a mature virgin produced suppression of mature virgin courtship, but training with an immature female did not change courtship of mature females (+control). Addition of mature female extract over a mesh barrier produced courtship suppression to the mature female. These results indicate that the <italic>TNT/Gr68a</italic> mutant males were able to sense and discriminate the age-specific pheromones of mature and immature females to produce trainer-specific courtship suppression. These cues have been shown to be volatile in nature ##REF##15694302##[4]##, so these data imply that <italic>TNT/Gr68a</italic> males have normal olfaction, at least for pheromonal cues.</p>", "<title>Suppression of mated female courtship by <italic>TNT/Gr68a</italic> males is normal</title>", "<p>It is well established that a mated female elicits less courtship than a virgin female of the same age ##REF##218208##[3]##, ##REF##10327242##[13]##. To examine whether <italic>Gr68a</italic> neurons are required for the perception of these aversive components of the mated female pheromone, <italic>TNT/Gr68a</italic> mutant males were assayed for their ability to discriminate between virgin and mated females. A mated female that had copulated 24 h before the experiment was decapitated to eliminate active rejection behavior ##REF##5956584##[14]##, put together with a <italic>TNT/Gr68a</italic> male, and courtship behavior recorded under dim red lights. Compared to a decapitated mature virgin female (mV), the mated female (M) elicited significantly less courtship from the <italic>TNT/Gr68a</italic> mutant (M: 27±6%, mV: 55±9%, <italic>P</italic>&lt;0.05). This suggests that <italic>Gr68a</italic> neurons are not essential to perceive the aversiveness of mated females, and again imply that <italic>TNT/Gr68a</italic> males have normal olfactory processing of pheromones.</p>", "<title>Courtship behavior of <italic>TNT/Gr68a</italic> males toward intact females</title>", "<p>Our finding that <italic>TNT/Gr68a</italic> males showed the same level of courtship as genetic controls (<italic>TNT^IN/Gr68a</italic>, <italic>TNT/+</italic> and <italic>TNT^IN/+</italic>, ##FIG##0##Fig. 1##) was unexpected, given the results of Bray and Amrein ##REF##12971900##[9]##, who showed a role for these cells in both initiation of wing vibration and maintenance of courtship. This suggested that our experimental conditions might mask subnormal male behavior. Our courtship chamber is much smaller (8 mm in diameter, 3 mm in depth) than that of Bray and Amrein (4 mm×10 mm×30 mm). In this limited space, the male could be continuously exposed to positive stimuli from the target female, perhaps overwhelming a subtle change in another modality, e.g. gustation, given the nature of <italic>Gr68a</italic>'s encoded protein. To determine if assay conditions were the basis of differences between our results, we measured latencies of courtship and copulation of individual males in a large environment (7 mm×10 mm×70 mm) with an intact, mobile female. ##FIG##2##Figure 3A## shows a cumulative plot of the percentage of males that initiated courtship at each time point during a 30 min observation. Mean values of courtship latency were calculated for each genotype (##FIG##2##Fig. 3B##). In room (white) light, only 29% of <italic>TNT/Gr68a</italic> started courtship within 10 min, while over 75% of each of the controls had initiated. No courtship was observed for about a half of the <italic>TNT/Gr68a</italic> males within the 30 min observation period, causing the mean courtship latency to be significantly longer than control (##FIG##2##Fig. 3B open bars##). Percentage copulation success and the copulation latency for each genotype were also measured; only one <italic>TNT/Gr68a</italic> male (4%) copulated, while at least 75% of each control line was successful, indicating that the <italic>TNT/Gr68a</italic> initiation defect contributed to their inability to copulate. This initiation defect was surprising since it is only after initiation that males obtain gustatory information, and it implies that the <italic>TNT/Gr68a</italic> males have more than a problem with taste.</p>", "<p>One possible explanation for the initiation defect of <italic>TNT/Gr68a</italic> males could be that they have a problem in the visual system which prevents them from locating the female. If so, repeating the experiment and eliminating visual input should diminish the advantage of control males and cause them to show the same levels of courtship and copulation as <italic>TNT/Gr68a</italic>. However, the courtship latency of <italic>TNT/Gr68a</italic> males was still significantly longer than any of the controls even in dim red light (##FIG##2##Fig. 3##). Comparison of white and dim red light latencies implies that males do use visual information for initiation since turning off the lights resulted in significant increases in latency for <italic>TNT/Gr68a</italic>, <italic>TNT/+</italic> and <italic>TNT^IN/+</italic> (<italic>P</italic>&lt;0.05). None of the <italic>TNT/Gr68a</italic> males copulated during the observation period. This suggests that, while vision is important, the defect of the <italic>TNT/Gr68a</italic> flies did not result from a visual defect.</p>", "<title>\n<italic>Gr68a-GAL4</italic> is pleiotropically expressed in gustatory and mechanosensory neurons</title>", "<p>\n<italic>TNT/Gr68a</italic> males have a significantly extended courtship latency, implying that they have a defect in a modality that is required for finding a female at a distance. Other than vision, the major sensory modality that has been implicated in initiation is olfaction ##REF##3092798##[8]##. Although the ability of these flies to form trainer type-specific courtship memory (##FIG##1##Fig. 2##) under conditions that require olfactory processing suggested that they were normal for this sensory modality, we sought to determine whether <italic>Gr68a-GAL4</italic> is expressed in the olfactory organs, investigating the expression pattern of the driver by using a membrane-bound form of green fluorescent protein mCD8-GFP, ##REF##10197526##[15]##. We found no detectable expression on the surfaces of either of the olfactory organs: third antennal segment (##FIG##3##Fig. 4A, arrows##) or maxillary palp (arrowheads). Interestingly, the second segment of the antenna, which houses Johnston's organ, the fly auditory apparatus ##REF##10934246##[16]##, showed an intense signal from auditory neurons ##REF##16998934##[17]## beneath the cuticular sheath (##FIG##3##Fig. 4A′##).</p>", "<p>\n<italic>Gr68a-GAL4</italic> was originally reported to have expression in chemosensory neurons of male-specific gustatory bristles on forelegs ##REF##12971900##[9]##. This expression was confirmed (##FIG##3##Fig. 4B##). Female didn't show the male-like foreleg gustatory bristle expression, but they did have multiple GFP-positive cells in the forelegs (##FIG##3##Fig. 4C##), including three with intensive expression at the base of the leg near where chordotonal organs are located (arrow). This expression pattern was characteristic of all legs of the female and was also seen in the midleg and hindleg of males. A closer look of the male midleg showed cell bodies at the root of mechanosensory bristles (##FIG##3##Fig. 4D arrow##). The male genitalia contained intense expression at the base of clasper teeth and on the anal plate (##FIG##3##Fig. 4E,F##). The labellum also exhibited non-sex-specific expression of <italic>Gr68a</italic> at the tip (##FIG##3##Fig. 4G##) in a region which houses cell bodies of many gustatory neurons ##REF##11257221##[18]##. GFP signals were also found at the wing root, the wing margin and on the female abdomen (##FIG##3##Fig. 4H,I##).</p>", "<p>In the brain, expression of <italic>Gr68a-GAL4</italic> is seen in antennal projections that go to a pair of almond-shaped structures known as the antennal mechanosensory and motor center AMMC, ##REF##16998934##[17]##, which resides next to the suboesophageal ganglion (SOG). Posterior to the SOG, <italic>Gr68a</italic> processes form a commissure connecting the bilateral AMMCs (##FIG##3##Fig. 4J##). No expression was detected in antennal lobe (AL), which receives olfactory information from the third antennal segment and maxillary palp. In aggregate, we see no evidence of expression of <italic>Gr68a-GAL4</italic> in olfactory structures, but we do find that it is highly expressed in mechanosensory and auditory neurons as well as gustatory structures.</p>", "<title>Female movement stimulates male courtship</title>", "<p>The expression of <italic>Gr68a-GAL4</italic> in the second antennal segment and its projection to AMMC in the brain suggests that <italic>TNT/Gr68a</italic> males could have an auditory defect. It is known that courting males listen to their own courtship song (wing vibration) and that this boosts courtship vigor and allows the male to “fine tune” his song ##REF##5004809##[19]##, ##UREF##2##[20]##. Deaf mutants cannot optimize the frequency of their wing vibration to make high quality courtship song and therefore exhibit low courtship success ##REF##9405700##[21]##. While this may contribute to low copulation success of <italic>TNT/Gr68a</italic> males, it cannot explain their increase in courtship latency, since this epoch of courtship occurs before song production. How could an auditory defect affect courtship latency? Does female activity generate specific sounds that attract males? Investigators ##UREF##3##[22]##, ##UREF##4##[23]## have recorded a wing-flicking “rejection signal” produced by young females, but did not detect any mature female-generated male-attracting sounds with their recording devices set at 20–2000 Hz, ##UREF##4##[23]##. It is however possible that female movement (foot-steps, grooming or wing flicking) could generate subtle sounds that alert the male to the presence of the female.</p>", "<p>In order to test this idea, we prepared two kinds of quiet females: wingless females and decapitated females. The wingless female makes walking and head-grooming sounds, but no wing-grooming, wing-flicking or flight sounds (“mute”). The decapitated female is very still and does not make any active movements except occasional grooming (“silent”). In a large chamber, a wild-type male was put together with either an intact, wingless or decapitated female and was observed for courtship initiation. ##FIG##4##Figure 5A (top panel)## shows that wild-type males had no problem finding wingless females in dim red lights and started courtship as early as with intact females. On the other hand, the lack of active movement caused by decapitation significantly prolonged the courtship latency when there was no visual information (##FIG##4##Fig. 5B##), suggesting that active movement of both intact and wingless females helped the male to notice the target female. When visual cues are available (white light, ##FIG##4##Fig. 5A lower panel##), males easily find the decapitated silent female, suggesting that the strong positive cue provided by the visual system overcame the lack of female movement.</p>", "<p>One possible way in which female activity could enhance initiation is if chemicals released by active females and distributed in the chamber allowed males to use gustatory sensation to “taste” the female's footprints. To test this, we pre-scented the chamber with three active females, but this did not decrease courtship latency toward a decapitated immobile female. It is clear that olfaction is extremely important in initiation in the dark, but these experiments rule out a chemosensory cue as being an explanation for the difference in initiation of courtship toward mobile and immobile targets.</p>", "<p>How does female movement stimulate courtship initiation? Does it work as a navigator to help the male to locate the position of the female? Or is it a trigger, which alerts the male to the potential presence of the female, stimulating some sort of searching behavior? In order to assess these possibilities, we provided “fly sounds” to the male in the presence of a decapitated female by placing the courtship chamber over a speaker and replaying a recoding of flies walking around in a chamber. If mechanical stimuli trigger searching, this noise should stimulate courtship initiation. On the other hand, if the test male employs the movement noise of a female to position and/or chase her, noise played through a speaker won't rescue the delayed courtship initiation toward a silent female and might even disrupt the test male's ability to locate the position of the real target female. We found that fly noise enhanced courtship initiation, causing the mean latency of courtship toward a silent female to be significantly shorter than with a silent female alone (##FIG##4##Fig 5##, <italic>P</italic>&lt;0.0001). The courtship latency toward a decapitated immobile female paired with fly sound was even shorter than that with a mobile female (<italic>P</italic>&lt;0.05). Courtship initiation was enhanced also by addition of a wingless noise maker male into a chamber (<italic>P</italic>&lt;0.05, data not shown), implying that both males and females can emit non-sex-specific mechanosensory signals that alert a male to the presence of another animal in proximity and stimulates him to search for a female in a large field.</p>", "<p>The ability of fly sound to stimulate the male to look for a female could reflect either a specific recognition of “fly sounds” or an alteration in the male's attentional state. To discriminate between these possibilities, we played white noise to a male in the presence of a decapitated silent female. White noise was equipotent in stimulating initiation (##FIG##4##Fig. 5##, <italic>P</italic>&gt;0.8 for comparison to fly noise), indicating that mechanosensory signals are likely to act by increasing the males state of alertness instead of being recognized as specific indicators of the presence or location of another fly.</p>", "<title>Mechanosensory defects in <italic>TNT/Gr68a</italic> mutant males</title>", "<p>Given the expression of <italic>Gr68a-GAL4</italic> in mechanosensory neurons, it is possible that the <italic>TNT/Gr68a</italic> males cannot detect the female-movement mechanosensory signal, and this therefore delays courtship initiation in the dark. In order to examine this possibility, <italic>TNT/Gr68a</italic> males were paired with a silent female. As shown in ##FIG##5##Figure 6##, there was no significant difference between <italic>TNT/Gr68a</italic> mutant and <italic>TNT^IN/Gr68a</italic> controls (<italic>P</italic>&gt;0.6), indicating that the <italic>TNT/Gr68a</italic> mutant has no disadvantage when asked to find an immobile female. This suggests that the initiation defect of <italic>TNT/Gr68a</italic> males is specific for mobile females and that TNT expression in <italic>Gr68a-GAL4</italic> auditory and/or other mechanosensory neurons is the cause of poor performance in courtship initiation. This experiment also excludes a possibility that the <italic>TNT/Gr68a</italic> male has a defect in detecting volatile pheromones via <italic>Gr68a</italic>-positive gustatory neurons as a cause of delayed initiation since his performance is equal to that of the control male under conditions in which he must use olfaction to locate the target.</p>", "<title>Function of aristae in courtship initiation</title>", "<p>\n<italic>TNT/Gr68a</italic> mutant males showed a defect in finding active females and a delay in courtship initiation in dark conditions (##FIG##2##Fig. 3##). <italic>Gr68a-GAL4</italic> is pleiotropically expressed in many parts of fly body (##FIG##3##Fig. 4##). Which <italic>Gr68a</italic>-positive cells are responsible for the female detection? One of the strongest areas of <italic>Gr68a-GAL4</italic> expression is found in Johnston's organ, the main auditory organ for detection of wing-vibrating courtship song. The song signal is first sensed by an arista attached on the third segment of antenna then transmitted to Johnston's organ inside the second segment ##REF##11418847##[24]##. In <italic>Drosophila melanogaster</italic>, <italic>fruitless</italic>, a sex-determination transcription factor, is expressed in most of the auditory neurons of Johnston's organ ##REF##15959468##[25]##, implying that audition plays an important role in sexual behavior. When a female is exposed to conspecific courtship song, she reduces her locomotion to accept copulation ##UREF##5##[26]##. On the other hand, a male, listening to the courtship song of other males nearby, increases his locomotion and performs enhanced courtship ##REF##9405700##[21]##, ##UREF##5##[26]##.</p>", "<p>In order to examine whether males use the arista-Johnston's organ auditory system to detect the moving-female signal, we measured courtship behavior of an auditory mutant, <italic>5D10</italic>\n##REF##9405700##[21]##, and found a defect in courtship initiation in the dark, with a mean latency of 553±185 sec, which was significantly lower than that of its genetic control line, 40A-G13 (control 149±17 sec, <italic>P</italic>&lt;0.005), supporting a role of the auditory system in courtship initiation. We also surgically manipulated arista function and compared courtship responses to those of intact males. Aristae of wild-type males were either fixed to the third antennal segment with a small amount of wax or partially amputated with fine scissors. The males with waxed-arista showed a significantly longer latency of courtship initiation (829±240 sec) than intact males courting intact females (435±193 sec, <italic>P</italic>&lt;0.05). The level corresponded to that of intact males courting decapitated silent females. This was consistent with a freely moving arista being essential for the female-movement detection. However, in this manipulation, the wax also covered some surface area of the third antennal segment so that it is possible that olfactory function was also disrupted, which may cause delayed courtship initiation. Therefore, we next cut most (more than three quarters) of both aristae off with fine scissors. The males with the partial aristae (1/4 arista) showed a normal level of courtship latency compared to intact males (331±100 sec, <italic>P</italic>&gt;0.5). This implies that the full length arista is not essential for motion-signal detection. It is not clear whether the remaining quarter of arista is sufficient for the function or arista itself is not required for this signal detection. It is also possible that Johnston's organ receives mechanosensory signals from other bristles on the second segment and not just the arista. Since <italic>Gr68a-GAL4</italic> is broadly expressed in mechanosensory neurons, it also remains possible that attention-getting auditory stimuli maybe sensed by cells other than those in Johnston's organ.</p>" ]

[ "<title>Discussion</title>", "<p>\n<italic>Drosophila</italic> males respond to multimodal cues to locate and choose an appropriate mating partner ##REF##3092798##[8]##, ##REF##3114743##[27]##. For dissection of the role of specific sensory modalities, one therefore needs to adjust the experimental conditions e.g. chamber size, illumination and target mobility, in order to preclude strong stimulatory input in other modalities masking subthreshold responses in interest ##REF##15694302##[4]##, ##REF##3092798##[8]##, ##REF##9364084##[28]##. In <italic>TNT/Gr68a</italic> males, chambers of different sizes led to different courtship performance. In a small chamber designed for learning and memory analysis, <italic>TNT/Gr68a</italic> males generated average levels of courtship (##FIG##0##Fig. 1##), while in a medium sized chamber, courtship was initiated normally but was maintained only for short period, implying a gustatory defect in the mutants ##REF##12971900##[9]##. On the other hand, in a large chamber, mutant males also had difficulty finding females, delaying courtship initiation (##FIG##2##Fig. 3##). The delayed initiation was a surprising outcome since the <italic>TNT/Gr68a</italic> was reported as a gustatory mutant in the earlier study. Is it possible that a gustatory defect affects courtship performance prior to contact with the female? We investigated the possibility that gustation could be used before initiation (i.e. that the male was tasting the female's pheromone “footprints” in the chamber before he touched her) by pre-scenting a chamber with mobile females before introducing a decapitated silent female, but this did not enhance courtship initiation of wild-type males (##FIG##4##Fig. 5##). Also, the <italic>TNT/Gr68a</italic> mutant males had no comparable defect in finding “silent” females (##FIG##5##Fig. 6##). These data led us to suspect that there were non-gustatory defects in <italic>TNT/Gr68a</italic> males and perform comprehensive anatomical characterization of <italic>Gr68a-GAL4</italic> driving a membrane-bound GFP. It was clearly demonstrated that <italic>Gr68a-GAL4</italic> has high expression in auditory and other mechanosensory neurons and in the primary auditory processing area of the brain (##FIG##3##Fig. 4##). The earlier study missed this pleiotropy, perhaps because a soluble GFP was used as a marker for <italic>Gr68a-GAL4</italic> expression ##REF##12971900##[9]##. This supposition, however, leads to the question of whether this GAL4 line is a faithful reporter of <italic>Gr68a</italic> expression, and whether this receptor has a role in mechanosensation. Further analysis using antibodies to the Gr68a protein or a mutant in the gene will be needed. In any case, the robust expression of this GAL4 line stimulated us to uncover a novel role of mechanosensation in courtship initiation.</p>", "<p>In this study, we, for the first time, found that acoustic (or perhaps seismic) signals provided by active females stimulate fast localization of the female for courtship initiation. This stimulation of initiation is likely to be due to a change in the attentional state of the male, since noises emitted by either male and female flies or even white noise enhanced initiation. If noise makes the male more alert, he is likely to move around the chamber and to encounter and sense the silent female. <italic>TNT/Gr68a</italic> males failed to detect movement signals and had delayed courtship initiation, suggesting that neural function of <italic>Gr68a</italic>-mechanosensory organs (this broad category includes Johnston's organ, bristles and chordotonal organs) was required for signal detection. However, it is not clear yet whether the signal is transmitted through air as an acoustic signal or as a seismic signal propagating via the chamber floor. The intense expression of <italic>Gr68a-GAL4</italic> in Johnston's organ and AMMC strongly suggests that the auditory system is blocked in the mutant males. Supporting this possibility, an auditory mutant, <italic>5D10</italic>\n##REF##9405700##[21]## showed delayed courtship initiation in dark. The failure of truncation of the aristae to decrease courtship latency, however, suggests that there may be contributions from other mechanosensory organs to the noise-dependent increase in attention which enhances courtship initiation. Further analysis will be needed to determine whether the mechanosensory system that increases arousal employs an arista-antenna-rotation mechanism ##REF##11418847##[24]## as in courtship song detection, or a footstep-sound-transmitted-as-floor-vibration mechanism as do crickets or spiders ##REF##15321061##[29]##, ##REF##15252876##[30]##.</p>" ]

[]

[ "<p>Conceived and designed the experiments: AE LG. Performed the experiments: AE. Analyzed the data: AE. Wrote the paper: AE LG.</p>", "<p>Finding a mating partner is a critical task for many organisms. It is in the interest of males to employ multiple sensory modalities to search for females. In <italic>Drosophila melanogaster</italic>, vision is thought to be the most important courtship stimulating cue at long distance, while chemosensory cues are used at relatively short distance. In this report, we show that when visual cues are not available, sounds produced by the female allow the male to detect her presence in a large arena. When the target female was artificially immobilized, the male spent a prolonged time searching before starting courtship. This delay in courtship initiation was completely rescued by playing either white noise or recorded fly movement sounds to the male, indicating that the acoustic and/or seismic stimulus produced by movement stimulates courtship initiation, most likely by increasing the general arousal state of the male. Mutant males expressing tetanus toxin (TNT) under the control of <italic>Gr68a-GAL4</italic> had a defect in finding active females and a delay in courtship initiation in a large arena, but not in a small arena. <italic>Gr68a-GAL4</italic> was found to be expressed pleiotropically not only in putative gustatory pheromone receptor neurons but also in mechanosensory neurons, suggesting that <italic>Gr68a</italic>-positive mechanosensory neurons, not gustatory neurons, provide motion detection necessary for courtship initiation. <italic>TNT/Gr68a</italic> males were capable of discriminating the copulation status and age of target females in courtship conditioning, indicating that female discrimination and formation of olfactory courtship memory are independent of the <italic>Gr68a</italic>-expressing neurons that subserve gustation and mechanosensation. This study suggests for the first time that mechanical signals generated by a female fly have a prominent effect on males' courtship in the dark and leads the way to studying how multimodal sensory information and arousal are integrated in behavioral decision making.</p>" ]

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[ "<p>We thank D. Eberl and H. Amrein for strains; J. Hall for insightful comments; A. Kamikouchi, M. Koganezawa, C. Pikielny, R. Greenspan and J.-F. Ferveur for helpful discussion and encouragement. We also appreciate P. Katz and J. Knierim for providing a quiet room to record moving-fly sounds at the MBL Neural Systems and Behavior course.</p>" ]

[ "<fig id=\"pone-0003246-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003246.g001</object-id><label>Figure 1</label><caption><title>Basal courtship behavior of <italic>TNT/Gr68a</italic> mutant males under standard memory assay conditions.</title><p>Courtship index (CI, A), courtship latency (B) and bout duration of each courtship (C) were measured for the experimental male (<italic>TNT/Gr68a</italic>, black bar), paired with an immobile, decapitated wild-type female in dim red light. As controls, males expressing an inactive form of TNT (<italic>TNT^IN/Gr68a</italic>, hatched bar), mono-transgenic heterozygous flies (<italic>TNT/+</italic>, <italic>TNT^IN/+</italic>, <italic>+/Gr68a</italic>, gray bars) and wild-type (<italic>+/+</italic>, open bar) were also examined. Different capital letters were given above each bar when values were significantly different (<italic>P</italic>&lt;0.05). Behavior was recorded in a round 8 mm diameter ×3 mm depth chamber.</p></caption></fig>", "<fig id=\"pone-0003246-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003246.g002</object-id><label>Figure 2</label><caption><title>Trainer type-specific courtship conditioning of <italic>TNT/Gr68a</italic> males.</title><p>Males received 60 min training by exposure to; an immature virgin female (iV), iV and mature virgin (mV) pheromone extract over mesh barrier, mV extract alone or a decapitated mV (mVd), then transferred to a clean chamber and tested with mVd. CI of the tester female was standardized by mean CI of sham to calculate a memory index (see <xref ref-type=\"sec\" rid=\"s4\">Materials and Methods</xref>). All experiments took place in dim red light. Each genotype is compared to own control. * (for wild type) and + (for <italic>TNT/Gr68a</italic>) indicate statistically significant differences (<italic>P</italic>&lt;0.05). Behavior was recorded in a round 8 mm diameter ×3 mm depth chamber.</p></caption></fig>", "<fig id=\"pone-0003246-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003246.g003</object-id><label>Figure 3</label><caption><title>Courtship initiation and copulation success of the <italic>TNT/Gr68a</italic> male in large arena with or without visual cues.</title><p>(A) Percentage of males that performed the first display of courtship orientation (left) and succeeded mating (right) in either white light (upper panel) or dim red light (lower panel) at each time point during 30 min observation period. (B) Mean values of courtship latency and copulation latency. Statistical significance is represented by capital letters for latencies under white light and small letters for dim-red light above each bar (<italic>P</italic>&lt;0.05). Behavior was recorded in a rectangular chamber with dimensions 70 mm×10 mm×7 mm.</p></caption></fig>", "<fig id=\"pone-0003246-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003246.g004</object-id><label>Figure 4</label><caption><title>Expression pattern of <italic>Gr68a-GAL4</italic>.</title><p>(A) External appearance of the GFP expression on a head. Arrows and arrowheads indicate antennal third segment and maxillary palps respectively. (A′) Close-up of the second segment of the antenna. (B) Male forelegs. (C) Female foreleg. (D) Close-up of a male midleg. A cell body (arrowhead) is marked by GFP at base of mechanosensory bristle (arrow). (E) Root of male clasper teeth on the genitalia (arrow) and (F) male anal plate (arrow). (G) Tip of proboscis. (H) Root (arrowhead) of chemosensory bristles (arrow) of the wing margin. (I) Ventral view of female abdomen. (J) Frontal view of a dissected brain, showing antennal lobe (AL), suboesophageal ganglion (SOG), antennal mechanosensory and motor center (AMMC) and optic lobe (OL).</p></caption></fig>", "<fig id=\"pone-0003246-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003246.g005</object-id><label>Figure 5</label><caption><title>Courtship is stimulated by the active movement of the target female.</title><p>Wild-type males were paired with the indicated type of female. (A) Courtship initiation was assayed for wild-type, paired with a wingless (“mute”) or a decapitated (“silent”) wild-type female (upper panel) or a “silent” female and additional putative stimulatory cues (lower panel). (B) Mean values of courtship latency in each courtship condition. Mechanosensory stimulus, not excess amounts of female deposits, stimulates courtship initiation. Statistical significance is represented for comparison to the data indicated by each arrow (*<italic>P</italic>&lt;0.05). Behavior was recorded in a rectangular chamber with dimensions 70 mm×10 mm×7 mm.</p></caption></fig>", "<fig id=\"pone-0003246-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003246.g006</object-id><label>Figure 6</label><caption><title>The <italic>TNT/Gr68a</italic> males have no disadvantage in finding decapitated “silent” females.</title><p>(A) Courtship initiation was examined for the <italic>TNT/Gr68a</italic> mutant males, when paired with the decapitated “silent” females in dim-red light. (B) There was no significant difference between courtship latencies of the <italic>TNT/Gr68a</italic> mutant and the <italic>TNT^IN/Gr68a</italic> control males (ND). Behavior was recorded in a rectangular chamber with dimensions 70 mm×10 mm×7 mm.</p></caption></fig>" ]

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[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>This work was supported by NIH grant R01 GM54408 to LCG. The Brandeis confocal imaging facility is supported by NIH grants P30 NS045713 and S10 RR16780. The sound recording room was provided by MBL Neural Systems and Behavior course supported by NIMH R25 MH059472.</p></fn></fn-group>" ]

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[ "<title>Introduction</title>", "<p>The germ cells of sexually reproducing organisms have a unique role in generating genetic diversity and transmitting genetic information from one generation to the next. Establishment of the germline in mammals involves the induction of germ cells from pluripotent epiblast cells through the action of extra-embryonic ectoderm-derived bone morphogenetic proteins and occurs comparatively late in development, commencing around day 6.25 days post coitum (dpc) in mice ##REF##10049358##[1]##–##REF##15937476##[4]##. At around 12.5 dpc -13.5 dpc the sexually dimorphic germ cells become committed to develop along either a male or a female pathway and start to initiate sex-specific differentiation ##REF##11874911##[5]##. Although there are numerous differences in the differentiation of the germline and in the timing and regulation of meiosis between the sexes, the fundamental events of meiosis that increase genetic diversity and reduce ploidy of the gametes are common to both.</p>", "<p>The main group of genes that have been shown to be required for mammalian meiosis are those involved in the recombination and synapsis of homologous chromosomes. Mice carrying loss-of-function mutations in these genes, such as <italic>Atm</italic>, <italic>Dmc1</italic>, <italic>γH2AX, Mlh1</italic>, <italic>Msh5</italic>, <italic>Rec8</italic>, <italic>Rad51</italic>, <italic>Smc1β</italic>, <italic>Spo11</italic>, <italic>Sycp1</italic>, <italic>Sycp2</italic>, <italic>Sycp3, Syce2</italic> and <italic>Tex14</italic>, typically exhibit defects in chromosome synapsis in both sexes, although male and female germ cells can exhibit different responses to these defects ##REF##17674147##[6]##–##REF##18158299##[8]##.</p>", "<p>A second group of genes that are required for progression through meiosis are those involved in repression of transposable genetic elements. Retrotransposons, for example long interspersed repeats (LINEs), short interspersed repeats (SINEs), and endogenous retroviruses such as intracisternal A-particles (IAPs) are the major class of transposable genetic elements in mammals and comprise around 37.5% of the mouse genome ##REF##12466850##[9]##. To allow new transposition events to propagate through subsequent generations, retrotransposons have evolved to be active in the germline. Accordingly, germ cells appear to have evolved mechanisms to reduce the mutational load of retrotransposon activity. Mutations in genes involved in mediating DNA methylation-dependent transcriptional repression of retrotransposons cause increased expression of retrotransposons and defects in chromosome synapsis during meiosis in either male or female germ cells ##REF##17115026##[10]##,##REF##15318244##[11]##. For example Dnmt3L is a catalytically inactive member of the DNA methyltransferase family that is expressed in foetal germ cells but is absent by 6 days post partum (dpp) ##REF##15318244##[11]##. Male mice null for this gene do not methylate dispersed repeat DNA during foetal germ cell development, and express LINEs and a class of endogenous retroviruses known as intracisternal A-particles (IAPs) in the germline ##REF##15318244##[11]##. <italic>Dnmt3L</italic> mutant male mice also exhibit meiotic abnormalities that result in a loss of post-meiotic germ cells in the testis ##REF##15318244##[11]##. The <italic>Dnmt3L</italic> mutant phenotype suggests that epigenetic changes that occur in foetal germ cells can cause meiotic defects later in germ cell development. A second example is provided by the murine piwi-related genes, which encode germline-specific proteins that are associated with a class of small germline-specific piwi-interacting RNAs (piRNAs) and are also required to repress retrotransposons during spermatogenesis ##REF##17446352##[12]##–##REF##18381894##[14]##. <italic>Mili</italic> and <italic>Miwi2</italic> both appear to be involved in de novo methylation of LINE and IAP elements during germ cell development in male embryos, and both mutants exhibit reduced DNA methylation and increased expression of LINE and IAP element in the testis, and defects in chromosome synapsis during meiosis in male germ cells ##REF##17446352##[12]##–##REF##18381894##[14]##. The mechanism by which increased expression of retrotransposons results in the defects in chromosome synapsis seen in these mutant mice is unknown, but the phenotype of these mutant mice suggests that repression of transposable genetic elements is required to allow germ cells to progress through meiosis.</p>", "<p>A set of testis expressed (<italic>Tex</italic>) genes has been identified in a subtractive hybridisation screen for genes expressed in spermatogonia but not somatic tissue ##REF##11279525##[15]##. One of these genes, <italic>Tex19.1</italic> (AAH53492.1), was found in a screen for potential RNA-targets of the germline-specific RNA binding protein Dazl by immunoprecipitation and microarray analysis ##REF##16278232##[16]##. Further unpublished work in this laboratory and work recently published by Kuntz et al. ##REF##18096721##[17]## confirmed an earlier report that <italic>Tex19.1</italic> is a “pluripotent cell expressed gene” ##REF##12923297##[18]##. Humans and primates possess a single <italic>Tex19</italic> gene in their genome, but in rodents a recent duplication has produced a two-gene family arranged as divergently transcribed genes separated by 29kb of DNA ##REF##18096721##[17]##. While expression of murine <italic>Tex19.1</italic> is restricted to pluripotent stem cells and developing germ cells, <italic>Tex19.2</italic> is expressed in the testis somatic tissues and does not appear to be restricted to germ cells or pluripotent stem cells in mice ##REF##18096721##[17]##.</p>", "<p>The expression pattern of <italic>Tex19.1</italic> suggests that the protein could have an important role in pluripotency or germ cell function. Since the sequence of Tex19.1 gives no clue to the biochemical function of this protein we decided to take a genetic approach to determining the function of this gene in the germline. In this paper we report that targeted deletion of <italic>Tex19.1</italic> in mice results in upregulation of endogenous retrovirus expression in testicular germ cells, perturbed chromosome synapsis during meiosis, and impaired spermatogenesis.</p>" ]

[ "<title>Materials and Methods</title>", "<title>Generation of <italic>Tex19.1</italic> Knockout Mice</title>", "<p>\n<italic>Tex19.1</italic> knockout mice were generated by replacing the <italic>Tex19.1</italic> open reading frame with a neomycin selection cassette by homologous recombination in E14 embryonic stem cells ##UREF##0##[19]##. Homologous regions were cloned by PCR from E14 embryonic stem cell genomic DNA using primers listed in Supplementary ##SUPPL##0##Table S1##. The <italic>Tex19.1</italic> targeting vector was linearised, electroporated into E14 embryonic stem cells, and neomycin-resistant clones screened for the desired integration event by PCR and Southern blot. <italic>Tex19.1^+/−</italic> ES cells were used to generate <italic>Tex19.1^−/−</italic> knockout mice by blastocyst injection and breeding as described ##UREF##0##[19]##. Mice were genotyped by multiplex PCR using primers listed in Supplementary ##SUPPL##0##Table S1##. Phenotypic analysis was performed on mice with a 129/Ola x CD1 mixed genetic background. Generation and analysis of <italic>Tex19.1^−/−</italic> knockout mice was performed under a UK Home Office project licence with approval from an institutional ethics committee.</p>", "<title>Antibody Production</title>", "<p>Anti-Tex19.1 antibodies were raised in rabbits using the synthetic peptide ⁷⁸ESEQEPGPEQDAWRG⁹² (Eurogentec). This peptide was designed to be specific to Tex19.1 and is not present in the Tex19.2 protein sequence. Antibodies were affinity purified from sera using the immunising peptide immobilised on a Sulfolink column (Pierce) according to manufacturer's instructions.</p>", "<title>Immunohistochemistry</title>", "<p>Testes were recovered from mice, fixed at 4°C overnight in 4% paraformaldehyde in phosphate buffered saline (PBS) and embedded in paraffin wax. 6 µm-thick sections were dewaxed in xylene, rehydrated, and antigen retrieval performed by boiling slides for 15 minutes in 0.01 M sodium citrate, pH 6.0. Sections were blocked, incubated with rabbit anti-Tex19.1 primary antibody at 1∶50, and bound antibody detected using the DAKOvision ABC diaminobenzidine (DAB) kit as described by the manufacturer (DakoCytomation). For peptide competition, anti-Tex19.1 antibodies were pre-incubated with 5 nM immunising peptide. For immunostaining of cultured cells, cells were fixed for 30 minutes at room temperature with 3.7% formaldehyde in PBS, then blocked with PBS containing 5% serum and 0.01% Tween-20. Cells were incubated with rabbit anti-Tex19.1 primary antibody at 1∶100, then fluorescently labelled secondary antibodies at 1 µg/mL (Invitrogen). DNA was counterstained with 2 µg/mL DAPI.</p>", "<title>Nuclear/Cytoplasmic Fractionation</title>", "<p>E14 embryonic stem cells or 13 dpp postnatal testes were lysed in cytoplasmic lysis buffer (10 mM Hepes pH 7.6, 3 mM MgCl₂, 40 mM KCl, 50 mM β-glycerophosphate, 5% glycerol, 0.5% Igepal CA-630, 2 mM NaF, 1 mM Na₃VO₄, 2 mM DTT and protease inhibitors) for 5 minutes on ice and the whole cell lysate centrifuged for 5 minutes at 1000g at 4°C. The nuclear pellet was resuspended in Laemmli buffer, boiled for 5 minutes and sonicated to disrupt genomic DNA. The cytosolic supernatant was mixed with Laemmli buffer and boiled for 5 minutes. Equivalent proportions of each fraction were separated by SDS-PAGE then Western blotted.</p>", "<title>Western Blot</title>", "<p>Testis was homogenised in Laemmli buffer, boiled for 5 min and sonicated to disrupt genomic DNA. Western blotting was performed using standard procedures ##UREF##1##[20]##. Tex19.1 was detected with rabbit anti-Tex19.1 polyclonal antibodies used at a 1∶200 dilution, mouse anti-Gapdh antibodies (Abcam) were used at 1∶1000, mouse anti-HP1α antibodies (Chemicon) at 1∶2500, and rabbit anti-histone H3 antibodies (Abcam) at 1∶20000. Peroxidase-conjugated secondary antibodies and enhanced chemiluminescence were used to detect primary antibodies.</p>", "<title>Southern Blotting</title>", "<p>Non-radioactive Southern blots were performed using a digoxigenin-labeled DNA probe generated using primers listed in Supplementary ##SUPPL##0##Table S1##, and alkaline phosphatase-conjugated anti-digoxigenin antibodies, essentially as described by the manufacturer (Roche).</p>", "<title>RT-PCR and Quantitative PCR</title>", "<p>Testis RNA was isolated with Trizol (Invitrogen) according to the manufacturer's protocol and reverse transcription performed with Superscript III (Invitrogen) on 1 µg RNA per reaction using oligo dT primer. Primers for RT-PCR are listed in Supplementary ##SUPPL##0##Table S1##.</p>", "<p>For quantitative PCR (qPCR), random-primed cDNA was generated from total RNA using Superscript III (Invitrogen). qPCR was performed using SYBR Green PCR System (Applied Biosystems) and a PTC-200 thermal cycler equipped with a Chromo4 continuous fluorescence detector and Opticon Monitor software (MJ Research). Primers for qPCR are listed in Supplementary ##SUPPL##0##Table S1##. Five technical replicates were performed for each biological sample, and the relative changes in gene expression determined using the ΔΔ^−2Ct method as described ##REF##11846609##[21]##. As <italic>Tex19.1</italic> is expressed in the germ cells in the testis the Sertoli cell marker <italic>Sdmg1</italic>\n##REF##18321981##[22]## was used to normalise cDNAs prepared from different animals to reduce the probability that the cDNAs were being normalised to a transcript whose level could be influenced by loss of <italic>Tex19.1.</italic>\n</p>", "<title>Testis Weight and Sperm Count</title>", "<p>Both testes from each adult animal (6–36 weeks old) were weighed, and the mean testis weight was used for statistical comparison. For sperm count one epididymis from each animal was homogenised in 1 mL 1% sodium citrate and incubated for 5 minutes at room temperature to allow the debris to settle. Sperm in the supernatant was then counted with a hemocytometer.</p>", "<title>Histology</title>", "<p>Testes were fixed for 4–6 hours in Bouin's solution (Sigma-Aldrich) at room temperature, then embedded in wax. For histological analysis 6 µm sections were dewaxed with xylene, rehydrated, then stained with hematoxylin and eosin.</p>", "<title>Immunostaining of Meiotic Chromosome Spreads</title>", "<p>Immunostaining of chromosome spreads from meiotic spermatocytes was performed essentially as described ##REF##15944401##[23]##. Briefly, testes were homogenized in PBS and 0.1 mL of cells were incubated in 0.5 mL 5% sucrose on a microscope slide for 1 hour. Cells were lysed with 0.1 mL 0.05% Triton-X-100 for 10 minutes, and fixed with 0.8 mL of fixing solution (2% paraformaldehyde, 0.02% SDS in PBS) for 1 hour. The slides were then washed, blocked with 5% serum, 0.1% Tween in PBS and incubated with primary antibodies for 1 hour. Mouse anti-Sycp3 antibodies (Abcam) were used at a 1∶2000 dilution, rabbit anti-Sycp1 antibodies (Abcam) at 1∶250, rabbit anti-γH2AX antibodies (Upstate Biotechnology) at 1∶200, and mouse anti-Rad51 antibodies (Upstate Biotechnology) at 1∶125. Fluorescently labelled secondary antibodies were used at 1 µg/mL (Invitrogen), and DNA was stained with 2 µg/mL DAPI.</p>", "<title>Metaphase I Analysis of Spermatocytes</title>", "<p>Chromosome spreads for metaphase I analysis were prepared as described in ##UREF##2##[24]##. Briefly, testes were incubated for 20 minutes in 1% sodium citrate, minced with scissors, and the cells harvested by centrifugation. Cells were then washed and resuspended in fixing solution (3∶1 methanol∶glacial acetic acid), dropped onto slides, and the resulting chromosome spreads were stained with Giemsa solution. 100 metaphase I spreads were scored per animal, and two animals scored for each genotype.</p>", "<title>In Situ Hybridisation and Northern Blotting</title>", "<p>A 460 bp fragment of the MMERVK10C endogenous retrovirus was amplified by RT-PCR from <italic>Tex19.1^−/−</italic> mutant testes using primers listed in Supplementary ##SUPPL##0##Table S1## and cloned into pBluescript II SK+ (Stratagene). Sense and anti-sense digoxigenin-labelled riboprobes were generated using T3 and T7 RNA polymerase according to the supplier's instructions (Roche). In situ hybridisation on 6 µm wax sections of Bouin's-fixed testis tissue was performed essentially as described ##REF##10753512##[25]## using 100 ng/mL digoxigenin-labelled probe and a hybridisation temperature of 50°C. Bound probe was detected with alkaline phosphatase-conjugated anti-digoxigenin antibodies (Roche) and BCIP/NBT precipitating stain (Vector Labs), then sections counterstained with nuclear fast red according to manufacturer's instructions. The antisense MMERVK10C digoxigenin-labelled riboprobe was also used for non-radioactive Northern blotting of 1 µg testis RNA as described ##REF##18321981##[22]##.</p>" ]

[ "<title>Results</title>", "<title>Expression of Tex19.1 Protein during Spermatogenesis</title>", "<p>Spermatogenesis in the adult testis involves the differentiation of a small pool of spermatogonial stem cells into large numbers of mature sperm. Within the testis spermatogenesis takes place in the seminiferous tubules where the mitotic spermatogonia reside at the outermost edge of the tubule, and progressive stages of differentiation are found as layers of meiotic spermatocytes then haploid spermatids located more and more centrally towards the lumen of the tubule ##UREF##3##[26]##. Published RT-PCR expression data for <italic>Tex19.1</italic> in purified spermatogenic cell populations suggests that <italic>Tex19.1</italic> expression is highest in mitotic spermatogonia, decreases as the spermatocytes progress through meiosis, and is present at low levels in round spermatids ##REF##16118233##[27]##. To establish the expression pattern of Tex19.1 protein in male testis during spermatogenesis we raised anti-peptide antibodies to Tex19.1. Immunohistochemistry on mouse testis shows strong cytoplasmic expression of Tex19.1 in spermatogonia that is downregulated as these cells differentiate and progress through meiosis (##FIG##0##Figure 1A–C##). We were able to detect cytoplasmic Tex19.1 protein in some meiotic spermatocytes (##FIG##0##Figure 1C##), but not in others (##FIG##0##Figure 1B##) suggesting that Tex19.1 protein expression is switched off as the germ cells proceed through meiosis. The expression of Tex19.1 protein in spermatogonia and spermatocytes is consistent with that observed for <italic>Tex19.1</italic> mRNA by in situ hybridisation (##SUPPL##0##Figure S1A–C##).</p>", "<p>Our finding that Tex19.1 is present in the cytoplasm of spermatogonia and early spermatocytes in the adult testis is not consistent with the published nuclear localisation of Tex19.1 protein in embryonic stem cells ##REF##18096721##[17]##. We confirmed that our anti-Tex19.1 antibody detects Tex19.1 and not a cross-reacting antigen by blocking the anti-Tex19.1 immunohistochemistry signal by competition with the immunising peptide, and by immunohistochemistry on <italic>Tex19.1^−/−</italic> knockout testes (##SUPPL##0##Figure S1D–F##). We were also able to detect a predominantly cytoplasmic subcellular localisation of Tex19.1 by immunostaining germ cells isolated from 14.5 dpc embryonic testes (##SUPPL##0##Figure S1G##). Again the cytoplasmic anti-Tex19.1 immunostaining could be competed with the immunising peptide, and was absent in germ cells from <italic>Tex19.1^−/−</italic> knockout embryos (##SUPPL##0##Figure S1H, I##). These data suggest that the anti-Tex19.1 antibody used in this present study specifically recognises endogenous Tex19.1 in germ cells, and that at least some Tex19.1 is present in the cytoplasm of spermatogonia and early spermatocytes in adult mouse testes.</p>", "<p>To investigate whether the discrepancy between the cytoplasmic localisation of Tex19.1 in germ cells presented in this study and the nuclear localisation of Tex19.1 in embryonic stem cells described previously ##REF##18096721##[17]## is caused by the difference between the cell types studied we performed immunostaining for Tex19.1 on embryonic stem cells. In contrast to the previous report ##REF##18096721##[17]##, we found that Tex19.1 is predominantly cytoplasmic in embryonic stem cells (##FIG##0##Figure 1D–F##).</p>", "<p>To exclude the possibility that our anti-Tex19.1 antibody is unable to detect a nuclear population of Tex19.1 due to loss or masking of the epitope during the immunohistochemical procedures we biochemically fractionated 13 dpp prepubertal testes and embryonic stem cells into nuclear and cytoplasmic fractions. On Western blots the 42 kDa Tex19.1 band was barely detectable in the nuclear fraction, but was easily detectable in equivalent loadings of the whole cell lysate and cytoplasmic fractions (##FIG##0##Figure 1G,H##). The observed size of the anti-Tex19.1 band in the Western blots (42 kDa) correlates well with the predicted molecular weight of Tex19.1 (40.4 kDa). This 42 kDa band appears to be endogenous Tex19.1 as it is not present in testes from <italic>Tex19.1^−/−</italic> knockout animals (##FIG##1##Figure 2E##). The biochemical fractionation of embryonic stem cells and testes therefore confirms the predominantly cytoplasmic subcellular localisation of Tex19.1 that we have observed by immunohistochemistry and immunostaining. Taken together the data presented here strongly suggests that Tex19.1 is a predominantly cytoplasmic protein in embryonic stem cells and germ cells.</p>", "<p>Germ cells in many species possess specialised cytoplasmic structures termed nuage that are implicated in RNA metabolism. Although Tex19.1 appears to be a predominantly cytoplasmic germ cell protein, the subcellular localisation and cell-type distribution of Tex19.1 appears to be distinct from the nuage component Tdrd1 ##REF##17038506##[28]## (##SUPPL##1##Figure S2##). Thus Tex19.1 does not appear to be a novel component of nuage. As the subcellular localisation of Tex19.1 does not provide any major insight into what the cellular function of this protein might be, and the Tex19.1 protein sequence does not contain any functional domains to illuminate the potential biochemical function of this protein, we decided to take a genetic approach to analyse the function of <italic>Tex19.1</italic> in the germline.</p>", "<title>Generation of <italic>Tex19.1^−/−</italic> Knockout Mice</title>", "<p>In order to investigate the function of <italic>Tex19.1</italic> in germ cell development we generated <italic>Tex19.1^−/−</italic> knockout mice. The <italic>Tex19.1</italic> open reading frame was replaced with a neomycin-resistance cassette by homologous recombination in embryonic stem cells (##FIG##1##Figure 2A##), and the targeted deletion confirmed by Southern blotting (##FIG##1##Figure 2B##). The heterozygous <italic>Tex19.1^+/−</italic> embryonic stem cells were used to generate chimaeric mice by blastocyst injection, and the <italic>Tex19.1⁻</italic> mutant allele bred to homozygosity (##FIG##1##Figure 2C##). <italic>Tex19.1^−/−</italic> homozygous pups were born from heterozygous crosses at a sub-Mendelian frequency (72 wild-type, 131 heterozygous, 40 homozygous pups born from heterozygous matings, significant deviation from expected Mendelian 1∶2∶1 ratio, χ²-test p&lt;0.01). The low rate of recovery of <italic>Tex19.1^−/−</italic> homozygous animals at birth indicates that some <italic>Tex19.1^−/−</italic> homozygous embryos are lost during embryonic development.</p>", "<p>To confirm that the <italic>Tex19.1⁻</italic> mutant allele removes <italic>Tex19.1</italic> mRNA and protein we performed RT-PCR on <italic>Tex19.1^−/−</italic> testis cDNA with <italic>Tex19.1</italic>-specific primers (##FIG##1##Figure 2D##) and Western blotting on <italic>Tex19.1^−/−</italic> testis protein extract with anti-Tex19.1 antibodies (##FIG##1##Figure 2E##). Both methods show that <italic>Tex19.1</italic> is not expressed in the testes of <italic>Tex19.1^−/−</italic> homozygous mice. We conclude that the <italic>Tex19.1⁻</italic> allele that we have produced is a null allele and that <italic>Tex19.1</italic> function is ablated in the <italic>Tex19.1^−/−</italic> homozygous mice.</p>", "<p>The surviving <italic>Tex19.1^−/−</italic> knockout mice are apparently healthy, with overtly normal morphology and behaviour. However both male and female <italic>Tex19.1^−/−</italic> knockout mice have reduced fertility. <italic>Tex19.1^−/−</italic> knockout females have a mean litter size of 5.0±2.2 SD, n = 9 compared to a litter size of 10.6±2.9, n = 23 for <italic>Tex19.1^+/−</italic> heterozygous females (Student's t-test p&lt;0.01). The reduced fertility in <italic>Tex19.1^−/−</italic> homozygous females is consistent with expression of <italic>Tex19.1</italic> in embryonic ovaries ##REF##18096721##[17]##, and a detailed analysis of the cause of the subfertility in the <italic>Tex19.1^−/−</italic> knockout female mice will be published elsewhere. Similarly, <italic>Tex19.1^−/−</italic> knockout male mice are also severely subfertile when test-mated with wild-type female mice. Although one out of the eleven <italic>Tex19.1^−/−</italic> knockout males tested for fertility was able to sire offspring, the remaining <italic>Tex19.1^−/−</italic> knockout males were infertile. The sterile <italic>Tex19.1^−/−</italic> knockout male mice were apparently able to mate with the wild-type females to produce a copulation plug, but these females did not give birth to any pups. <italic>Tex19.1^−/−</italic> knockout male mice have smaller testes (##FIG##2##Figure 3A, B##), with the median testis weights of adult animals reduced from 111 mg in <italic>Tex19.1^+/+</italic> wild type and <italic>Tex19.1^+/−</italic> heterozygous mice to 42.5 mg in <italic>Tex19.1^−/−</italic> knockout littermates (Mann Whitney U-test, p&lt;0.01). Furthermore, the median epididymal sperm count is reduced to 1.3×10⁵ in <italic>Tex19.1^−/−</italic> knockout mice from 1.3×10⁷ in wild type and heterozygous littermates (Mann Whitney U-test, p&lt;0.01, ##FIG##2##Figure 3C##) suggesting that spermatogenesis is defective in the <italic>Tex19.1^−/−</italic> knockout testes. We have not been able to detect any difference in testis weight or sperm count between <italic>Tex19.1^+/+</italic> wild-type and <italic>Tex19.1^+/−</italic> heterozygous animals.</p>", "<p>The extent of the spermatogenesis defect in the <italic>Tex19.1^−/−</italic> knockout males varied between individual animals. When sperm was counted, a strong reduction was observed for most of the animals, but for the single fertile animal the sperm count was close to normal levels (##FIG##2##Figure 3C##). A similar variation in testis weight was also evident amongst <italic>Tex19.1^−/−</italic> knockout animals (##FIG##2##Figure 3B##). This phenotypic variation was not influenced by the age of the mice at the time of analysis. Age-matched adult mice were analysed at 6 weeks, 3 months, 6 months and 9 months during the course of this study, and there appeared to be no correlation between the severity of the phenotype and the age at which the adult mice were examined. Rather phenotypic variation was observed in adult mice at all ages (R.O. and I.R.A., data not shown). The outbred component of the genetic background of these mice may contribute to this variation.</p>", "<title>\n<italic>Tex19.1^−/−</italic> Knockout Spermatocytes Exhibit Defects in Progression through Meiosis</title>", "<p>We investigated the spermatogenesis defect in <italic>Tex19.1^−/</italic>\n⁻ knockout mice further by examining the testis histology in these animals. We did not detect any overt differences in testis histology between <italic>Tex19.1^+/+</italic> wild-type and <italic>Tex19.1^+/−</italic> heterozygous animals. However, <italic>Tex19.1^−/−</italic> knockout testes have considerably narrower seminiferous tubules than their wild-type or heterozygous littermates due to a reduction in the number of post-meiotic germ cells (##FIG##3##Figure 4A, B##). This phenotype was also subject to some heterogeneity. In animals with a more severe phenotype all postmeiotic cell-types were missing and the most advanced meiotic cells were in pachytene stage (##FIG##3##Figure 4C, D##). In animals with a less severe phenotype a proportion of cells were able to complete meiosis and haploid cells could be detected, although often in comparatively low numbers (##FIG##3##Figure 4C, E##). Like the testis weight and sperm count phenotypes described in the previous section, there appeared to be no correlation between the severity of the testis histology phenotype and the age at which the adult mice were analysed (from 6 weeks to 9 months).</p>", "<p>To test whether the reduction in the number of post-meiotic germ cells in <italic>Tex19.1^−/−</italic> knockout testes arises from a decrease in the spermatogonial mitotic divisions or apoptosis of differentiating germ cells, we counted the number of B-type spermatogonia, early meiotic cells and apoptotic cells in testis sections from <italic>Tex19.1^+/−</italic> heterozygous and <italic>Tex19.1^−/−</italic> knockout animals. B-spermatogonia and early meiotic cells were identified by their location and histological appearance in the seminiferous tubules ##UREF##3##[26]##, and apoptotic cells were identified using the TUNEL assay to label fragmented chromatin. Whereas the number of B-type spermatogonia and early meiotic cells did not differ between <italic>Tex19.1^+/−</italic> heterozygous and <italic>Tex19.1^−/−</italic> knockout testes (R.O., data not shown), TUNEL staining showed an increase in the number of dying cells in adult <italic>Tex19.1^−/−</italic> knockout testis (##SUPPL##2##Figure S3A, B, N##). In more severe <italic>Tex19.1^−/−</italic> knockout seminiferous tubules, TUNEL-positive cells were found within or next to layers of meiotic germ cells (##SUPPL##2##Figure S3C##), but even at high magnification the nuclear morphology of these TUNEL-positive cells was not distinct enough to allow their developmental stage to be unambiguously identified (##SUPPL##2##Figure S3H–J##). In less severe <italic>Tex19.1^−/−</italic> knockout seminiferous tubules, TUNEL-positive cells could also be found between the layers of meiotic germ cells and post-meiotic round spermatids (##SUPPL##2##Figure S3D##). At higher magnification, some of these TUNEL-positive cells could be identified as metaphase I spermatocytes (##SUPPL##2##Figure S3E–G##).</p>", "<p>In order to further define the point during spermatogenesis when the <italic>Tex19.1^−/−</italic> knockout cells are dying we examined the synchronous first wave of spermatogenesis that occurs in prepubertal mice. The first wave of spermatogenic germ cells initiates meiosis at around 10 dpp in the prepubertal testis, and progresses through the pachytene stage of meiosis from around 14 to 20 dpp to produce the first post-meiotic round spermatids around 21 dpp, and mature sperm at around 31 dpp ##REF##874003##[29]##,##REF##14996999##[30]##. Analysis of apoptosis (##SUPPL##2##Figure S3N##) and testis histology (##SUPPL##3##Figure S4##) at various stages of prepubertal testis development revealed no overt differences in testis histology and no statistically significant increase in apoptosis at 16 dpp in <italic>Tex19.1^−/−</italic> knockout testes. However by 19–22 dpp, a reduction in the number of meiotic and post-meiotic germ cells and an increase in the frequency of cell death are both evident in <italic>Tex19.1^−/−</italic> knockout testes (##SUPPL##2##Figures S3N##, ##SUPPL##3##S4##). In 22 dpp testes, clusters of TUNEL-positive cells can be seen within the layer of pachytene germ cells that line the lumen of the seminiferous tubule suggesting that at least some apoptosis is occurring at the pachytene stage of meiosis (##SUPPL##2##Figure S3K–M##). The high level of apoptosis in the <italic>Tex19.1^−/−</italic> knockout testes increases by 29–31 dpp to the level seen in adult testes (##SUPPL##2##Figure S3N##). This data suggests that the reduction in the number of post-meiotic germ cells and increased levels of apoptosis seen in the adult <italic>Tex19.1^−/−</italic> knockout testes is at least partly due to some <italic>Tex19.1^−/−</italic> knockout germ cells initiating apoptosis during the pachytene stage of meiosis, and some <italic>Tex19.1^−/−</italic> knockout germ cells initiating apoptosis during metaphase I.</p>", "<p>Although the vast majority of the <italic>Tex19.1^−/−</italic> null testes examined contained differentiating germ cells, two of thirty analysed knockout animals had an extremely severe phenotype with one testis that completely lacked germ cells. One of these agametic testes was isolated from a 31 dpp prepubertal mouse (##FIG##3##Figure 4F##) suggesting that this extreme phenotype is indicative of defects occuring during embryonic or early post-natal germ cell development rather than a progressive loss of spermatogonial stem cells in an ageing adult testis. However, as only a small number of testes exhibited this phenotype, we were not able to study this extreme phenotype further and instead focused on the meiotic phenotype evident in the vast majority of the <italic>Tex19.1^−/−</italic> mutant testes.</p>", "<title>Chromosome Synapsis during Male Meiosis Is Impaired in the Absence of <italic>Tex19.1</italic>\n</title>", "<p>We next attempted to determine the cause of the increased apoptosis in <italic>Tex19.1^−/−</italic> null testes. Defects in homologous chromosome synapsis or homologous recombination during meiotic prophase can cause apoptosis in late pachytene spermatocytes ##REF##15051947##[31]##,##REF##17997150##[32]##. Therefore we used immunocytochemistry on meiotic chromosome spreads to analyse chromosome synapsis and homologous recombination in <italic>Tex19.1^−/−</italic> knockout testes. In order to analyse chromosome synapsis, meiotic chromosome spreads were stained using Sycp3 as a marker for lateral elements of meiotic chromosomes and Sycp1 as a marker for synapsed homologous chromosomes ##REF##17674147##[6]##. In wild-type pachytene cells the autosomal chromosome axes stain completely for both markers, whereas the X and Y sex chromosomes remain largely asynapsed with only a small area of Sycp1 staining in the pseudo-autosomal region (##FIG##4##Figure 5A##). In contrast, about half the pachytene cells in <italic>Tex19.1^−/−</italic> homozygotes have Sycp3-stained autosomal chromosomal axes that lack Sycp1 staining (##FIG##4##Figure 5B–D, I##). The asynapsed chromosomes in <italic>Tex19.1^−/−</italic> knockout cells did not appear to be arranged in homologous pairs (##FIG##4##Figure 5D##). However it is not clear whether the asynapsed chromosomes have never paired in <italic>Tex19.1^−/−</italic> knockout spermatocytes, or have paired but have subsequently fallen apart. In some of the incompletely synapsed <italic>Tex19.1^−/−</italic> knockout cells, some chromosomes appeared to form chains linked by regions of apparent non-homologous synapsis (##FIG##4##Figure 5C##, asterisk). Incompletely synapsed pachytene cells comprise less than 1% of spreads from <italic>Tex19.1^+/+</italic> wild-type or <italic>Tex19.1^+/−</italic> heterozygotous testes (##FIG##4##Figure 5I##). Thus <italic>Tex19.1^−/−</italic> knockout animals exhibit defects in homologous chromosome synapsis during male meiosis.</p>", "<p>During meiotic prophase, homologous recombination starts prior to homologous chromosome pairing and synapsis ##REF##11242108##[33]##. As progression of homologous recombination and chromosome synapsis are interdependent on each other ##REF##17674147##[6]##,##REF##15473851##[7]##, we investigated whether the chromosome synapsis defect in <italic>Tex19.1^−/−</italic> knockout spermatocytes was a consequence of an earlier defect in the initiation of homologous recombination. The appearance of DNA double strand breaks and the formation of early recombination foci during meiotic prophase can be detected by immunostaining for the phosphorylated histone γH2AX and the recombinase enzyme Rad51 respectively ##REF##11242108##[33]##,##REF##9311981##[34]##. γH2AX staining is normally present on chromatin during the leptotene and zygotene stages of early meiotic prophase. As synapsis proceeds during zygotene, the DNA double strand breaks are resolved, resulting in γH2AX staining disappearing from the autosomal chromosomes, but not the sex chromosomes. In normal <italic>Tex19.1^+/+</italic> wild-type pachytene cells, chromosome synapsis is complete and only the sex chromosomes stain for γH2AX (##FIG##4##Figure 5E##). However, the incompletely synapsed pachytene cells in <italic>Tex19.1^−/−</italic> knockout testes, exhibit strong diffuse γH2AX staining (##FIG##4##Figure 5F##). This γH2AX staining is localised to the regions of the chromosome spreads that contain the unsynapsed chromosomes (##FIG##4##Figure 5F##). Similarly, immunostaining for the early recombination foci marker Rad51, which largely disappears from autosomal chromosomes as synapsis proceeds, suggests that Rad51 foci are formed in <italic>Tex19.1^−/−</italic> knockout spermatocytes, but are not resolved or matured on the unsynapsed chromosomes (##FIG##4##Figure 5G, H##). Thus the formation of DNA double strand breaks and the assembly of early recombination foci both appear to be occurring in <italic>Tex19.1^−/−</italic> knockout spermatocytes. This suggests that the defect in meiotic chromosome synapsis that we have observed in <italic>Tex19.1^−/−</italic> spermatocytes does not appear to be a secondary consequence of impaired initiation of homologous recombination. Rather, the presence of DNA double strand breaks and early recombination foci in the unsynapsed regions of the incompletely synapsed <italic>Tex19.1^−/−</italic> pachytene spermatocytes is consistent with impaired chromosome synapsis. Furthermore, the presence of DNA double strand breaks and early recombination foci in unsynapsed regions of incompletely synapsed <italic>Tex19.1^−/−</italic> pachytene spermatocytes indicates that the unsynapsed chromosomes arise from a failure to initiate synapsis rather than premature desynapsis. The unsynapsed chromosomes in the incompletely synapsed pachytene <italic>Tex19.1^−/−</italic> knockout cells are presumably sufficient to trigger apoptosis at the pachytene checkpoint ##REF##15051947##[31]##,##REF##17997150##[32]##, and would account for the increased levels of cell death seen in pachytene stage meiotic germ cells in <italic>Tex19.1^−/−</italic> knockout testes (##SUPPL##2##Figure S3##).</p>", "<p>Although incompletely synapsed pachytene cells could explain the increased levels of cell death in the pachytene meiotic germ cells in <italic>Tex19.1^−/−</italic> knockout testes, the presence of apoptotic metaphase I spermatocytes in these animals suggests that there may be an additional defect later in spermatogenesis to account for cell death at the metaphase I stage. Around half of the <italic>Tex19.1^−/−</italic> knockout pachytene cells did not appear to have any overt defects in chromosome synapsis (##FIG##4##Figure 5I##), and would therefore presumably be able to progress to metaphase I and continue through spermatogenesis. To investigate whether there might be additional defects in chromosome behaviour at later stages of meiosis in <italic>Tex19.1^−/−</italic> knockout spermatocytes, we prepared and analysed meiotic metaphase I chromosome spreads. During metaphase I of meiosis, homologous chromosomes are held together as bivalents by chiasmata (##FIG##4##Figure 5J##). 94% of the metaphase I spreads from <italic>Tex19.1^+/−</italic> heterozygous testes contained only bivalent metaphase I chromosomes, 5% contained univalent sex chromosomes, and 1% contained univalent autosomes. However, only 34% of the metaphase I spreads from <italic>Tex19.1^−/−</italic> knockout testes contained only bivalent metaphase I chromosomes, while 56% of the spreads contained univalent sex chromosomes, and 33% contained univalent autosomes (##FIG##4##Figure 5K##). 23% of the <italic>Tex19.1^−/−</italic> knockout metaphase I spreads feature univalent autosomes and univalent sex chromosomes. Thus <italic>Tex19.1^−/−</italic> knockout testes contain increased numbers of univalent chromosomes at meiotic metaphase I that could potentially trigger apoptosis at the metaphase I checkpoint ##REF##9500548##[35]##,##REF##12217320##[36]## and account for the apoptotic metaphase I cells seen in <italic>Tex19.1^−/−</italic> knockout testes. Furthermore, the presence of univalent chromosomes in metaphase I spreads from <italic>Tex19.1^−/−</italic> knockout testes is indicative of a defect in the formation or maintenance of chiasmata in post-pachytene spermatocytes.</p>", "<title>Overexpression of Retrotransposable Elements in <italic>Tex19.1^−/−</italic> Homozygous Testes</title>", "<p>Meiotic defects similar to those present in the <italic>Tex19.1^−/−</italic> knockout testes have been observed in various different mouse mutants that carry defects in genes encoding components of meiotic chromosomes, the meiotic recombination machinery, or the synaptonemal complex ##REF##17674147##[6]##,##REF##15473851##[7]##. However, we have been unable to detect any Tex19.1 protein physically associated with meiotic chromosomes by immunostaining (R.O., data not shown), and our finding that Tex19.1 is predominantly localised to the cytoplasm rather than the nucleus suggests that Tex19.1 is unlikely to be a component of meiotic chromosomes or the synaptonemal complex. We therefore reasoned that the meiotic defects present in the <italic>Tex19.1^−/−</italic> knockout testes are unlikely to be a direct effect of Tex19.1 on meiotic chromosome structure or function but rather may be an indirect consequence of changes in meiotic gene expression.</p>", "<p>In order to detect changes in gene expression in the testis of <italic>Tex19.1^−/−</italic> knockout mice, we performed microarray analysis using an Illumina MouseWG-6 v1.1 Whole Genome Gene Expression Beadchip containing 48,318 different probes. To exclude potential differences in transcript levels due to the loss of post-meiotic germ cells in the <italic>Tex19.1^−/−</italic> testes we performed this analysis on testes from 16 dpp prepubertal mice during the first synchronous wave of spermatogenesis. At this stage of testis development, some germ cells are already in the pachytene stage of meiosis, but no obvious changes in cell composition were apparent between <italic>Tex19.1^+/+</italic> wild type and <italic>Tex19.1^−/−</italic> knockout testes (##SUPPL##3##Figure S4##). RNAs from two different 16 dpp <italic>Tex19.1^−/−</italic> knockout testes were compared with <italic>Tex19.1^+/+</italic> wild type or <italic>Tex19.1^+/−</italic> heterozygous littermates, and transcripts that had consistent and greater than three fold changes in relative gene expression between the two groups of animals were identified. The Mouse Genome Database (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.informatics.jax.org\">http://www.informatics.jax.org</ext-link>) currently lists 97 mutations that are known to give rise to meiotic arrest during spermatogenesis ##REF##18158299##[8]##. These male meiotic arrest genes include genes that encode components of meiotic chromosomes, the meiotic recombination machinery and the synaptonemal complex such as <italic>Atm</italic>, <italic>Dmc1</italic>, <italic>γH2AX, Mlh1</italic>, <italic>Msh5</italic>, <italic>Rec8</italic>, <italic>Rad51</italic>, <italic>Smc1β</italic>, <italic>Spo11</italic>, <italic>Sycp1</italic>, <italic>Sycp2</italic>, <italic>Sycp3, Syce2</italic> and <italic>Tex14</italic>. None of the male meiotic arrest genes listed in the Mouse Genome Database showed a consistent change in expression level in <italic>Tex19.1^−/−</italic> knockout testes compared to littermate controls (I.R.A., data not shown). However, analysis of the microarray data suggested that the class II LTR-retrotransposon MMERVK10C ##REF##16093699##[37]## is upregulated by around four-fold in the testis RNA from each of the 16 dpp <italic>Tex19.1^−/−</italic> knockout animals relative to their littermate controls (I.R.A., data not shown). The mouse genome contains around 16 approximately full-length copies of the MMERVK10C sequence in the genome, and a further 1200 fragments of the MMERVK10C endogenous retrovirus. Increased retrotransposon expression has been proposed to be responsible for impaired chromosome synapsis and meiotic defects during spermatogenesis in <italic>Dnmt3L</italic>, <italic>Miwi2</italic> and <italic>Mili</italic> mutant mice ##REF##15318244##[11]##–##REF##18381894##[14]##. As overexpression of the MMERVK10C retrotransposons could similarly be responsible for the meiotic defects seen in <italic>Tex19.1^−/−</italic> mutant mice we sought to determine whether MMERVK10C expression is indeed upregulated in the testis in the absence of <italic>Tex19.1</italic>.</p>", "<p>The levels of MMERVK10C expression in testis cDNA from two <italic>Tex19.1^−/−</italic> knockout animals relative to their <italic>Tex19.1^+/+</italic> wild-type littermates were each tested by quantitative PCR (##FIG##5##Figure 6A##). The Sertoli cell marker <italic>Sdmg1</italic>\n##REF##18321981##[22]## was used to normalise cDNAs from different animals. Although there was no significant change in the expression of the ubiquitously expressed β-actin gene, or the germ cell marker <italic>Dazl</italic>\n##REF##9288969##[38]##, expression of the MMERVK10C endogenous retrovirus was increased by a factor of approximately four-fold in both <italic>Tex19.1^−/−</italic> knockout animals (Student's t-test, p&lt;0.01) (##FIG##5##Figure 6A##). Expression of LINE, SINE or IAP retrotransposons showed no significant change in the absence of <italic>Tex19.1</italic> (##FIG##5##Figure 6A##).</p>", "<p>To further validate the potential upregulation of MMERVK10C transcripts in the <italic>Tex19.1^−/−</italic> knockout mice we performed Northern blots on testis RNA from the same two 16 dpp <italic>Tex19.1^−/−</italic> knockout animals and their <italic>Tex19.1^+/+</italic> wild type littermates. Using a probe derived from the <italic>env</italic> gene of the MMERVK10C endogenous retrovirus we were able to detect a predominant 3.2 kb MMERVK10C <italic>env</italic> transcript in mouse testes, and some weaker MMERVK10C <italic>env</italic> transcripts at around 4.5 kb and 7.5 kb (##FIG##5##Figure 6B##). The Northern blot profile for MMERVK10C <italic>env</italic> transcripts is comparable to that of <italic>env</italic>-containing transcripts from HERV-K endogenous retroviruses in human teratocarcinoma cell lines ##REF##8506289##[39]##. Northern blotting confirmed that the predominant 3.2 kb MMERVK10C <italic>env</italic> transcript is consistently more abundant in testes from <italic>Tex19.1^−/−</italic> knockout animals than in testes from their wild-type littermates at 16 dpp (##FIG##5##Figure 6B##).</p>", "<p>In order to determine which cell types are accumulating MMERVK10C transcripts in the <italic>Tex19.1^−/−</italic> knockout testes we performed in situ hybridisation on testis sections using a MMERVK10C <italic>env</italic> probe (##FIG##5##Figure 6C–N##). In <italic>Tex19.1^+/+</italic> wild-type and <italic>Tex19.1^+/−</italic> heterozygous testes at 16 dpp, low levels of MMERVK10C <italic>env</italic> transcripts were present in some meiotic spermatocytes (##FIG##5##Figure 6C,G##). However, MMERVK10C transcripts were generally more abundant in <italic>Tex19.1^−/−</italic> knockout testes than in testes from <italic>Tex19.1^+/+</italic> wild-type or <italic>Tex19.1^+/−</italic> heterozygous littermates at 16 dpp (##FIG##5##Figure 6D, H##). The increased levels of MMERVK10C <italic>env</italic> transcript in 16 dpp <italic>Tex19.1^−/−</italic> testes appeared to be largely due to the presence of strongly expressing cells located towards the centre of the tubules where meiotic spermatocytes are present (##FIG##5##Figure 6K, L##). Similarly in adult animals MMERVK10C <italic>env</italic> transcripts were upregulated in meiotic germ cells in the testes from adult <italic>Tex19.1^−/−</italic> knockout animals relative to their heterozygous littermates (##FIG##5##Figure 6E, F, I, J##). A total of nine different <italic>Tex19.1^−/−</italic> knockout animals at various ages were assayed for MMERVK10C expression in the testes by in situ hybridisation, and MMERVK10C expression in <italic>Tex19.1^−/−</italic> knockout testes was consistently higher that in <italic>Tex19.1^+/+</italic> or <italic>Tex19.1^+/−</italic> littermate controls. No in situ hybridisation signals were detected on testis sections using a sense MMERVK10C control probe (##FIG##5##Figure 6M, N##).</p>", "<p>Taken together, the quantitative PCR, Northern blotting and in situ hybridisation data all suggest that transcripts from the MMERVK10C endogenous retrovirus are upregulated in the meiotic spermatocytes of <italic>Tex19.1^−/−</italic> knockout testes.</p>", "<p>The upregulation of retrotransposons in <italic>Dnmt3L</italic>, <italic>Mili</italic> and <italic>Miwi2</italic> mutant mice is associated with defects in de novo DNA methylation of IAP and LINE elements in the male germline, which presumably allows increased transcription of these elements during spermatogenesis ##REF##15318244##[11]##–##REF##18381894##[14]##. In order to investigate whether the upregulation of MMERVK10C retrotransposons in <italic>Tex19.1^−/−</italic> knockout testis was caused by a similar mechanism, we investigated the DNA methylation status of CpG dinucleotides in MMERVK10C elements by bisulphite sequencing MMERVK10C elements from 16 dpp prepubertal <italic>Tex19.1^−/−</italic> knockout testes. The MMERVK10C element includes a weak CpG island overlapping the LTR and 5′untranslated region (##SUPPL##4##Figure S5##). As promoters with weak CpG islands are good candidates for regulation by DNA methylation ##REF##17334365##[40]##, we examined DNA methylation at CpG dinucleotides within this region. Sequence analysis of 30 independent clones from each of <italic>Tex19.1^+/+</italic> wild-type, <italic>Tex19.1^+/−</italic> heterozygous and <italic>Tex19.1^−/−</italic> homozygous 16 dpp testes showed that CpG dinucleotides in this region of the MMERVK10C element are predominantly methylated in the testis at this age (##SUPPL##4##Figure S5##). The MMERVK10C element was also methylated to a similarly high level in liver taken from the same animals as a somatic tissue control (##SUPPL##4##Figure S5##). The prepubertal testis is composed of approximately equal numbers of germ cells and somatic cells at 16 dpp ##REF##874003##[29]##,##REF##14996999##[30]##, therefore around half the clones analysed by bisulphite sequencing are likely to be derived from testicular germ cells and around half from testicular somatic cells. As all of the 16 dpp testis clones represented highly methylated DNA sequences (##SUPPL##4##Figure S5##), the MMERVK10C element appears to be highly methylated in both the germ cell and somatic cell compartments of <italic>Tex19.1^+/+</italic> wild-type, <italic>Tex19.1^+/−</italic> heterozygous and <italic>Tex19.1^−/−</italic> homozygous 16 dpp testes.</p>", "<p>Although we have been unable to find any evidence that the methylation status of MMERVK10C elements in the testis changes in the absence of <italic>Tex19.1</italic> (##SUPPL##4##Figure S5##), we cannot exclude the possibility that the absence of <italic>Tex19.1</italic> causes reduced DNA methylation in a subset of MMERVK10C elements in the genome, or in a subset of germ cells in 16 dpp testes. If only a subset of germ cells have altered DNA methylation at MMERVK10C elements in <italic>Tex19.1^−/−</italic> mutant testes then we estimate that this subset would need to represent less than 25% of the germ cell population to be below our detection limit in this assay (χ²-test, p&lt;0.05). Nevertheless, our observations that loss of <italic>Tex19.1</italic> causes the upregulation of MMERVK10C retrotransposon elements in the testis, but not IAP or LINE elements, combined with the absence of a detectable change in DNA methylation levels in MMERVK10C elements in <italic>Tex19.1^−/−</italic> knockout testes, suggests that <italic>Tex19.1</italic>-mediated repression of retrotransposons may involve a mechanism that is distinct from <italic>Dnmt3L</italic>/<italic>Miwi2</italic>/<italic>Mili</italic>-mediated repression of retrotransposons. Thus we conclude that <italic>Tex19.1</italic> is part of a novel genetic pathway that represses retrotransposons in the male germline.</p>" ]

[ "<title>Discussion</title>", "<title>The <italic>Tex19.1^−/−</italic> Knockout Phenotype</title>", "<p>This study describes the functional consequences of deleting the pluripotency-associated <italic>Tex19.1</italic> gene in mice. Our data shows that loss of <italic>Tex19.1</italic> causes impaired spermatogenesis and defects in chromosome synapsis during meiosis. Mutations in genes that are involved in various aspects of meiotic chromosome behaviour such as the initiation of recombination between homologous chromosomes, or the assembly of the synaptonemal complex, all typically cause defective chromosome synapsis during meiosis, and apoptosis in the male germline ##REF##17674147##[6]##,##REF##15473851##[7]##. However, although there is some similarity between these phenotypes and the <italic>Tex19.1</italic> mutant phenotype, we have been unable to detect any localisation of Tex19.1 to meiotic chromosomes by immunostaining testis sections or testis chromosome spreads (R.O., data not shown). Indeed our data suggest that Tex19.1 is a predominantly cytoplasmic protein and is therefore unlikely to play a direct role in meiotic chromosome behaviour. Thus, although <italic>Tex19.1</italic> mutant mice exhibit defects in chromosome pairing during meiosis, we do not believe that Tex19.1 is a component of meiotic chromosomes and favour the interpretation <italic>Tex19.1</italic> is influencing meiotic chromosome behaviour indirectly.</p>", "<p>Our finding that Tex19.1 is a predominantly cytoplasmic protein in germ cells and embryonic stem cells contradicts a previous study suggesting that Tex19.1 is a nuclear protein in embryonic stem cells ##REF##18096721##[17]##. The reason for the discrepancy between these studies is not yet clear. Kuntz et al. ##REF##18096721##[17]## raised monoclonal antibodies to Tex19.1 and observed nuclear staining with those antibodies in embryonic stem cells and pre-implantation embryos. The Tex19.1 peptide used by Kuntz et al. ##REF##18096721##[17]## to raise the monoclonal anti-Tex19.1 antibody is located C-terminally to the peptide that we have used to raise the anti-Tex19.1 antibodies in this study. Both peptides, and indeed the entire <italic>Tex19.1</italic> open reading frame, lie within a single exon. In our study we have shown that Tex19.1 is predominantly cytoplasmic in embryonic stem cells by immunostaining and by Western blotting of subcellular fractions. We have also shown that Tex19.1 has a predominantly cytoplasmic localisation in germ cells by immunostaining germ cells isolated from embryonic testes, by immunohistochemistry on wax sections of adult testis and by Western blotting of subcellular fractions from prepubertal testes. Furthermore we have demonstrated the specificity of our antibody in the assays that we use by immunostaining and Western blotting on material from <italic>Tex19.1^−/−</italic> knockout animals. As the cytoplasmic anti-Tex19.1 staining patterns that we present in this paper are lost in <italic>Tex19.1^−/−</italic> knockout animals, at least some of the Tex19.1 protein that is present in germ cells and embryonic stem cells is cytoplasmic. However we cannot exclude the possibility that the two different antibodies raised in these two studies recognise mutually exclusive isoforms of Tex19.1 that have different subcellular localisations. Alternatively, the discrepancy between our study and the study by Kuntz et al. ##REF##18096721##[17]## could be caused by procedural differences, or by cross-reaction of anti-Tex19.1 antibodies with an unrelated antigen.</p>", "<p>\n<italic>Tex19.1^−/−</italic> null male mice showed some phenotypic variation between individuals ranging from completely agametic testes to fertility. This phenotypic variability may be partly due to the genetic heterogeneity in the outbred component of the genetic background used for this study. However, as some germ cells are more severely affected by the loss of <italic>Tex19.1</italic> than other germ cells in the same animal, there is also some phenotypic variability in the absence of genetic variation. Furthermore, our finding that loss of <italic>Tex19.1</italic> can impair spermatogenesis even in this heterogeneous genetic background suggests that mutations in the single human homologue, <italic>TEX19</italic>, could contribute to fertility problems in human populations. The human <italic>TEX19</italic> gene contains two premature stop codons in the open reading frame that truncates the Tex19 protein from 351 residues in mouse to 164 residues in human ##REF##18096721##[17]##. The first premature stop codon in the human <italic>TEX19</italic> gene is conserved in other primates suggesting that the C-terminal region of Tex19 is dispensable for function in primates ##REF##18096721##[17]##. The significance of this major difference in structure between human and mouse is at present unclear given our current level of understanding of the mechanisms underlying the phenotype in mouse.</p>", "<p>The <italic>Tex19</italic> genomic locus has undergone a duplication event in rodents to generate two closely related divergently transcribed genes ##REF##18096721##[17]##. The mutation that we have engineered removes the entire <italic>Tex19.1</italic> open reading frame, but leaves <italic>Tex19.2</italic> intact. Therefore <italic>Tex19.2</italic> could potentially provide some functional redundancy with <italic>Tex19.1</italic>. Although <italic>Tex19.1</italic> and <italic>Tex19.2</italic> are reported to be expressed in testicular germ cells and testicular somatic cells respectively ##REF##18096721##[17]##, there appears to be a moderate upregulation of <italic>Tex19.2</italic> in <italic>Tex19.1^−/−</italic> knockout testes as judged by quantitative RT-PCR (I.R.A., data not shown). It is not clear at present whether this upregulation of <italic>Tex19.2</italic> occurs in the germ cells or somatic cells of the testis, but any upregulation of <italic>Tex19.2</italic> that is occurring does not seem to be able to fully compensate for loss of <italic>Tex19.1</italic>. Nevertheless, deletion of the entire <italic>Tex19</italic> locus may be required to rule out the possibility of some functional redundancy between these genes and may reveal additional functions for <italic>Tex19.1</italic> in the germline.</p>", "<p>This study demonstrates that <italic>Tex19.1</italic> has a function in progression through meiosis in the male germline. Characterisation of the meiotic defect in <italic>Tex19.1^−/−</italic> knockout spermatocytes indicates that homologous recombination is being initiated in the <italic>Tex19.1^−/−</italic> knockout spermatocytes but that, for some chromosomes, synapsis does not occur. As homologous recombination and chromosome synapsis progress interdependently during meiosis, it is possible that the chromosome synapsis defect that we describe in <italic>Tex19.1^−/−</italic> knockout spermatocytes is a secondary consequence of a defect in the progression of homologous recombination, or a secondary consequence of defects in the pairing between homologous chromosomes that normally precedes chromosome synapsis ##REF##15473851##[7]##. Further work is needed to dissect the molecular basis of the <italic>Tex19.1</italic> chromosome synapsis defect in more detail, and to understand if and how the upregulation of MMERVK10C retrotransposons that we detect in <italic>Tex19.1^−/−</italic> spermatocytes causes these defects in meiotic chromosome synapsis.</p>", "<title>Tex19.1 and Repression of Retrotransposons in the Germline</title>", "<p>The <italic>Tex19.1</italic> mutant phenotype bears some resemblance to the <italic>Dnmt3L</italic>, <italic>Miwi2</italic> and <italic>Mili</italic> mutant phenotypes in that they all exhibit defects in meiotic chromosome synapsis and increased expression of retrotransposons in the germline ##REF##15318244##[11]##–##REF##18381894##[14]##. However it is not yet clear whether there is a direct causal relationship between these two events. The increase in retrotransposon expression does not appear to be caused by defects in meiotic chromosome synapsis ##REF##15318244##[11]##,##REF##17395546##[13]##, but it is not clear whether or how the increase in retrotransposon expression causes the defects in meiotic chromosome synapsis in any of these mutant mice. Increased transposition of mobile genetic elements could introduce quantitative, qualitative, or temporal changes in the DNA double strand breaks normally present during early meiotic prophase that could interfere with the homologous recombination events that normally precede and initiate chromosome pairing. Support for this model comes from the observation that mutating genes involved in piRNA function in flies activates the DNA damage signalling pathway ##REF##17363252##[41]##,##REF##17199040##[42]##. Alternatively, it is possible that repression of retrotransposons is important for the fidelity of homolog pairing and synapsis during meiosis, and that increased expression of these repetitive elements either interferes with homolog recognition and synapsis, or promotes pairing between non-homologous chromosomes. A third possibility is that proteins encoded by the MMERVK10C endogenous retrovirus mediate the defects in meiotic chromosome synapsis by interfering with host cell proteins involved in meiotic chromosome behaviour or regulation of the meiotic cell cycle. In this regard it is important to note that transgenic mice expressing the rec protein derived from the HERVK human endogenous retrovirus exhibit defects in spermatogenesis ##REF##15735668##[43]##. Lastly, there may not be a direct causal relationship between retrotransposon de-repression and chromosome asynapsis. Rather the <italic>Tex19.1</italic>, <italic>Dnmt3L</italic>, <italic>Miwi2</italic> and <italic>Mili</italic> mutants may all cause defects in meiotic chromosome structure that lead to both retrotransposon de-repression and defective chromosome synapsis. Clearly further work is needed to clarify the molecular mechanism underlying the chromosome synapsis defect in the <italic>Tex19.1</italic> mutant mice presented here, and in the <italic>Dnmt3L</italic>, <italic>Miwi2</italic> and <italic>Mili</italic> mutant mice ##REF##15318244##[11]##–##REF##17395546##[13]##. However, this study provides further evidence demonstrating a correlation between de-repression of retrotransposons and impaired chromosome synapsis during mouse meiosis.</p>", "<p>Although there are gross similarities between the <italic>Tex19.1</italic> mutant phenotype and the <italic>Dnmt3L</italic>, <italic>Miwi2</italic> or <italic>Mili</italic> mutant phenotypes, there are also important differences. <italic>Dnmt3L</italic>, <italic>Miwi2</italic> and <italic>Mili</italic> are all required to repress LINE and IAP retrotransposons in the germline, and these three genes appear to converge on DNA methylation and transcriptional repression of these sequences in the genome ##REF##15318244##[11]##–##REF##18381894##[14]##. However, repression of LINE and IAP retrotransposons is not perturbed in <italic>Tex19.1^−/−</italic> knockout testes suggesting that <italic>Tex19.1</italic> is not involved in the transcriptional repression of LINE or IAP elements. Rather our data shows that transcripts from the MMERVK10C class of endogenous retroviruses accumulate in the germ cells in the absence of <italic>Tex19.1</italic>. These differences between the <italic>Tex19.1</italic> mutant phenotype and the <italic>Dnmt3L</italic>, <italic>Miwi2</italic> and <italic>Mili</italic> mutant phenotypes may reflect the existence of multiple mechanisms with different specificities to repress retrotransposons in the germline.</p>", "<p>The <italic>Tex19.1</italic> mutant phenotype is characterised by the accumulation of MMERVK10C retrotransposon transcripts, but the molecular basis for this phenotype is not yet clear. The upregulation of MMERVK10C transcripts could be caused by changes acting at any level of gene expression from the initiation of transcription to mRNA turnover. We have not been able to find any difference in the level of DNA methylation at MMERVK10C elements in <italic>Tex19.1</italic> mutant testes. This provides further evidence that <italic>Tex19.1</italic> belongs to a different genetic pathway than <italic>Miwi2</italic>, <italic>Mili</italic> and <italic>Dnmt3L</italic> for repression of retrotransposons in the germline. However, we cannot exclude the possibility that DNA methylation may be altered in a subset of MMERVK10C elements in a subset of germ cells in the <italic>Tex19.1</italic> mutant testes, and that this subset of elements is responsible for the upregulation of MMERVK10C transcripts that we describe in the <italic>Tex19.1</italic> mutant testes. An alternative model to explain the upregulation of MMERVK10C elements in <italic>Tex19.1</italic> mutant testes is that Tex19.1 could be a transcriptional repressor of MMERVK10C elements. The nuclear localisation of Tex19.1 reported by Kuntz et al. ##REF##18096721##[17]## would be consistent with this type of mechanism operating. However, although we cannot exclude the possibility that some Tex19.1 acts in the nucleus in the germ cells in the adult testes, our finding that Tex19.1 is predominantly cytoplasmic in these cells would be more consistent with Tex19.1 acting to regulate gene expression at a post-transcriptional level. We are able to detect MMERVK10C transcripts in wild-type testes (##FIG##5##Figure 6B,G##) suggesting that some MMERVK10C transcripts must escape DNA methylation or transcriptional repression, and that post-transcriptional regulation of MMERVK10C mRNA may play a role in repressing the activity of this retrotransposon. The upregulation of MMERVK10C transcripts in <italic>Tex19.1</italic> mutant testes does not appear to be the result of changes in RNA splicing as the MMERVK10C isoforms present in <italic>Tex19.1</italic> mutant testes do not appear to be qualitatively different from those present in wild-type testes. However, the accumulation of MMERVK10C transcripts in <italic>Tex19.1</italic> knockout testes would be consistent with <italic>Tex19.1</italic> promoting degradation of MMERVK10C mRNA. Investigation into the biochemical function of Tex19.1 should provide a ready test of these models and generate some insight into the molecular mechanism of <italic>Tex19.1-</italic>dependent repression of MMERVK10C endogenous retroviruses.</p>", "<p>Repression of retrotranposons in the mammalian germline requires mechanisms to distinguish retrotransposons from endogenous genes to allow repression to be targeted to the correct loci. piRNAs, a group of small RNAs that are physically associated with the piwi class of proteins, are abundant in male germ cells and some piRNAs have sequence homology to various classes of retrotransposon ##REF##18381894##[14]##, ##REF##16751777##[44]##–##REF##16766680##[46]##. The sequence homology between some piRNA molecules and retrotransposons is presumably used to target DNA methylation to retrotransposons rather than endogenous genes. Although there is good genetic evidence that the piwi class of proteins is involved in transcriptional repression of retrotransposons ##REF##17446352##[12]##–##REF##18381894##[14]##, there is also good biochemical evidence that piwi proteins and piRNAs are physically associated with the translational machinery in male germ cells ##REF##16766680##[46]##,##REF##16938833##[47]##, suggesting a role in translation or mRNA turnover. Thus piRNA-mediated repression of retrotransposons may be working at multiple levels of gene expression in male germ cells. It will be informative to investigate whether the <italic>Tex19.1</italic> pathway for repression of retrotransposons that we describe here also utilises piRNAs to target repression to MMERVK10C elements.</p>", "<p>One of the interesting aspects of the <italic>Tex19.1</italic> phenotype is that although the MMERVK10C subclass of retrotransposons is upregulated in <italic>Tex19.1</italic> mutant testes, LINE, SINE and IAP retrotransposons are not. It is not clear how <italic>Tex19.1</italic> determines specificity for the MMERVK10C element. Notably, IAP elements belong to the same subclass of endogenous retroviruses as MMERVK10C elements (class II LTR retrotransposons) but are not upregulated in <italic>Tex19.1</italic> mutant testes. Sequences within the MMERVK10C promoter or transcript could be involved in targeting <italic>Tex19.1</italic> activity to this element. Alternatively <italic>Tex19.1</italic> may have the potential to regulate a wider range of retrotransposons than we have been able to identify here, but alternative mechanisms to repress retrotransposon expression during spermatogenesis, such as DNA methylation, may limit the phenotypic effects of losing <italic>Tex19.1</italic> to a subset of its potential targets. Furthermore, as <italic>Tex19.1</italic> expression is not restricted to spermatogenesis but also occurs in primordial germ cells, oocytes and pluripotent stem cells, it will be of interest to determine if <italic>Tex19.1</italic> is involved in repressing MMERVK10C elements and other classes of retrotransposons in these cell types.</p>", "<p>In addition to its role in the germline, <italic>Tex19.1</italic> is also expressed in pluripotent cells. Like germ cells, pluripotent cells are viable targets for retrotransposon activity as any new transposition events could be propagated through successive generations. Therefore pluripotent cells presumably also need to modulate retrotransposon activity to ensure that the mutational load on the genome is not too high. Our finding that <italic>Tex19.1^−/−</italic> homozygotes are born at a sub-Mendelian frequency is consistent with a role for <italic>Tex19.1</italic> in pluripotent cells in early embryonic development. Further work is required to determine whether the loss of <italic>Tex19.1^−/−</italic> homozygotes during embryogenesis is caused by defects in pluripotent cells, and whether pluripotent cells upregulate retrotransposon expression in <italic>Tex19.1^−/−</italic> knockout embryos.</p>", "<p>The ongoing battle between retrotransposons and the host genome has important consequences for evolution, and for genetic disease. Retrotransposons that can successfully evade genome defences in germ cells and pluripotent cells will be selected for during evolution, whereas germ cells and pluripotent cells are under selective pressure to keep the mutational load on the genome at sustainable levels. The striking differences in the relative abundance of different classes of retrotransposable elements between the mouse and human genomes suggest that this conflict is ongoing during mammalian evolution ##REF##12466850##[9]##. Although low levels of mutation and retrotransposition in the germline are required to generate the genetic variation essential for evolution, high levels of mutation or retrotransposition are deleterious to the survival of a species. In humans, endogenous retroviruses with intact coding sequences comprise a very small proportion of the genome ##REF##11237011##[48]##, yet intact endogenous retroviral particles are found in human pluripotent stem cells, and in testicular germ cell tumours where the expression of endogenous retroviral proteins has been suggested to contribute to tumourigenesis ##REF##8506289##[39]##,##REF##15735668##[43]##,##REF##6726185##[49]##. Furthermore, a number of human genetic diseases are associated with de novo mutagenic retrotransposition events that disrupt the function of endogenous human genes ##REF##16440055##[50]##,##REF##15983781##[51]##. Our data suggests that <italic>Tex19.1</italic> is part of a mechanism that protects the genome from the deleterious effects of retrotransposon activity in the germline, and thereby helps to maintain genomic stability through successive generations.</p>" ]

[]

[ "<p><bold>¤a:</bold> Current address: MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen's Medical Research Institute, Edinburgh, United Kingdom</p>", "<p><bold>¤b:</bold> Current address: Wellcome Trust Centre for Stem Cell Research, Cambridge, United Kingdom</p>", "<p>Conceived and designed the experiments: RÖ NKG HJC IRA. Performed the experiments: RÖ AJC HMB RMS PRL NR IRA. Analyzed the data: RÖ RMS HJC IRA. Contributed reagents/materials/analysis tools: RÖ AJC IRA. Wrote the paper: RÖ HJC IRA.</p>", "<p>As genetic information is transmitted through successive generations, it passes between pluripotent cells in the early embryo and germ cells in the developing foetus and adult animal. <italic>Tex19.1</italic> encodes a protein of unknown function, whose expression is restricted to germ cells and pluripotent cells. During male spermatogenesis, <italic>Tex19.1</italic> expression is highest in mitotic spermatogonia and diminishes as these cells differentiate and progress through meiosis. In pluripotent stem cells, <italic>Tex19.1</italic> expression is also downregulated upon differentiation. However, it is not clear whether <italic>Tex19.1</italic> has an essential function in germ cells or pluripotent stem cells, or what that function might be. To analyse the potential role of <italic>Tex19.1</italic> in pluripotency or germ cell function we have generated <italic>Tex19.1^−/−</italic> knockout mice and analysed the <italic>Tex19.1^−/−</italic> mutant phenotype. Adult <italic>Tex19.1^−/−</italic> knockout males exhibit impaired spermatogenesis. Immunostaining and histological analysis revealed defects in meiotic chromosome synapsis, the persistence of DNA double-strand breaks during meiosis, and a loss of post-meiotic germ cells in the testis. Furthermore, expression of a class of endogenous retroviruses is upregulated during meiosis in the <italic>Tex19.1^−/−</italic> testes. Increased transposition of endogenous retroviruses in the germline of <italic>Tex19.1^−/−</italic> mutant mice, and the concomitant increase in DNA damage, may be sufficient to disrupt the normal processes of recombination and chromosome synapsis during meiosis and cause defects in spermatogenesis. Our results suggest that <italic>Tex19.1</italic> is part of a specialised mechanism that operates in the germline to repress transposable genetic elements and maintain genomic stability through successive generations.</p>", "<title>Author Summary</title>", "<p>The germ cells—eggs in females and sperm in males—are responsible for passing genetic information from one generation to the next. As any genetic changes that arise in the germ cells can be transmitted to the next generation, germ cells are a prime target for the activity of mobile genetic elements. Mobile genetic elements make up around 40% of a mammalian genome, and many of these elements are derived from retroviruses that have infected germ cells, or early embryonic precursors to germ cells, and have integrated into the genome. Here, we characterise the function of <italic>Tex19.1</italic>, a gene whose expression is restricted to germ cells and the pluripotent cells that are early embryonic precursors to germ cells. We show that when <italic>Tex19.1</italic> is deleted from mice, germ cells have problems progressing through meiosis, and sperm production is impaired. Furthermore, we show that, in the absence of <italic>Tex19.1</italic>, endogenous retroviruses are activated in male germ cells attempting to go through meiosis. Our results suggest that <italic>Tex19.1</italic> is part of a specialised mechanism that guards against mutagenic endogenous retrovirus activity in germ cells and pluripotent cells and thus helps to maintain the integrity and stability of the genome through successive generations.</p>" ]

[ "<title>Supporting Information</title>" ]

[ "<p>We acknowledge the Wellcome Trust Clinical Research Facility, Western General Hospital, Edinburgh for performing the microarray hybridisation, and thank Taiza Stumpp-Teixeira (MRC-HGU) for help with interpreting the testis histology, Shinichiro Chuma (Kyoto University, Japan) for anti-Tdrd1 antibodies, Donncha Dunican and Richard Meehan (MRC-HGU) for help and advice on analysing DNA methylation status, Ewelina Bolcun-Filas (MRC-HGU) for help and advice analysing the meiosis defect, and Richard Meehan (MRC-HGU) for comments and suggestions on the manuscript.</p>" ]

[ "<fig id=\"pgen-1000199-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pgen.1000199.g001</object-id><label>Figure 1</label><caption><title>Tex19.1 is a cytoplasmic protein expressed in spermatogonia and spermatocytes in the adult testis.</title><p>(A–C) Immunohistochemistry for Tex19.1 in adult testis. Anti-Tex19.1 staining (brown precipitate) can be seen in the cytoplasm of spermatogonia (open arrowheads in B) and early spermatocytes (filled arrowheads in C) in the seminiferous tubules but not in later stage pachytene spermatocytes (filled arrowheads in B). (D–F) Immunofluorescence for Tex19.1 in embryonic stem cells. Anti-Tex19.1 staining (green) can be seen in the cytoplasm of embryonic stem cells. DNA is counterstained in red. (G) Western blot for Tex19.1 in whole cell (WC), nuclear (N) and cytoplasmic (C) fractions from 13 dpp prepubertal testis. Subcellular fractionation was monitored using histone H3 and Gapdh as nuclear and cytoplasmic markers respectively. Tex19.1 is detected predominantly in the cytoplasm. (H) Western blot for Tex19.1 in whole cell, nuclear and cytoplasmic fractions from embryonic stem cells. Tex19.1 is predominantly cytoplasmic, and the purity of the fractions is shown by the cytoplasmic marker Gapdh and the nuclear marker HP1α.</p></caption></fig>", "<fig id=\"pgen-1000199-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pgen.1000199.g002</object-id><label>Figure 2</label><caption><title>Generation of <italic>Tex19.1^−/−</italic> knockout mice.</title><p>(A) Schematic view of the <italic>Tex19.1</italic> knockout strategy. The <italic>Tex19.1</italic> gene in the wild-type (wt) allele is replaced by a neomycin box in the knockout (ko) allele; black borders delineate the short and long arms upstream and downstream of the gene. Location of probe for Southern blot (SP), <italic>Bam</italic>HI sites and lengths of restriction fragments are indicated for both alleles. (B) Southern blot of wild type E14 embryonic stem cell genomic DNA (+/+) and a successfully targeted clone (+/−). (C) Genotyping of a wild type, a heterozygous and a homozygous animal from the <italic>Tex19.1</italic> knockout line by multiplex PCR. (D) RT-PCR from testes of wild type and knockout animals. <italic>Tex19.1</italic> transcript can be detected in wild type but not in knockout testes. <italic>Gapdh</italic> transcript can be seen in both. (E) Western blot on protein extracts from the testes of heterozygous and knockout animals: Tex19.1 is detected in heterozygous but not in knockout testes. Gapdh is shown as a loading control.</p></caption></fig>", "<fig id=\"pgen-1000199-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pgen.1000199.g003</object-id><label>Figure 3</label><caption><title>\n<italic>Tex19.1^−/−</italic> knockout mice have defects in spermatogenesis.</title><p>(A) Testes from <italic>Tex19.1^−/−</italic> knockout mice are smaller in size than testes from <italic>Tex19.1^+/+</italic> wild-type littermates (comparison of two 36-week old testes). (B) Box plot showing that median testis weights of adult animals are reduced in <italic>Tex19.1^−/−</italic> knockout mice (Mann Whitney U-test, p&lt;0.01). The genotypes and number of animals analysed are indicated. (C) Box plot showing that median epididymal sperm count is reduced in <italic>Tex19.1^−/−</italic> knockout mice (Mann Whitney U-test, p&lt;0.01).</p></caption></fig>", "<fig id=\"pgen-1000199-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pgen.1000199.g004</object-id><label>Figure 4</label><caption><title>\n<italic>Tex19.1^−/−</italic> null testes contain reduced numbers of post-meiotic germ cells.</title><p>Testis histology in <italic>Tex19.1^+/−</italic> heterozygous and <italic>Tex19.1^−/−</italic> knockout animals. (A, C) In <italic>Tex19.1^+/−</italic> heterozygotes, the testis histology is normal and the seminiferous tubules contain Sertoli cells (arrows), spermatogonia (white arrowheads), spermatocytes (broad arrowheads) and spermatids (narrow arrowheads). (B, D) <italic>Tex19.1^−/−</italic> knockout animals with a severe phenotype feature a reduced tubule diameter with a substantial reduction of cell numbers. Sertoli cells (arrows) spermatogonia (open arrowheads) and meiotic cells (broad arrowheads) are present, but few spermatids can be found. Additionally, there are cells with pyknotic appearance (asterisks). (E) In <italic>Tex19.1^−/−</italic> knockout animals with a less severe phenotype, some spermatids (narrow arrowheads) can be detected in reduced numbers. (F) In two of thirty <italic>Tex19.1^−/−</italic> knockout animals analysed one of the recovered testes was agametic and only contained Sertoli cells (arrows). A–E are from 3 month old adult animals, F is from a 31 dpp prepubertal animal.</p></caption></fig>", "<fig id=\"pgen-1000199-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pgen.1000199.g005</object-id><label>Figure 5</label><caption><title>\n<italic>Tex19.1^−/−</italic> null spermatocytes exhibit defects in chromosome synapsis during meiosis.</title><p>(A–D) Synapsis of homologous chromosomes in meiotic cells; Sycp3 (red) is present on both synapsed and unsynapsed chromosomes during early meiotic prophase whereas Sycp1 (green) is only present on synapsed chromosomes. (A) In wild type pachytene cells all autosomal chromosomes are synapsed, and only the sex chromosomes remain unsynapsed (arrowhead). (B–D) Many <italic>Tex19.1^−/−</italic> knockout cells exhibit incomplete synapsis where some chromosomes are unsynapsed (arrows) in cells that otherwise have pachytene appearance. Some unsynapsed chromosomes in <italic>Tex19.1^−/−</italic> knockout cells, appear to form chains linked by regions of apparent non-homologous synapsis (asterisk) (E, F) Distribution of the DNA double-strand break marker γH2AX (green) in meiotic cells. (E) In wild type pachytene cells DNA double strand breaks that were generated during earlier stages of meiosis have been repaired leaving γH2AX staining restricted to the sex chromosomes (arrowhead). (F) In <italic>Tex19.1^−/−</italic> knockout mice a proportion of pachytene-like cells exhibit incomplete resolution of γH2AX staining at places where incomplete synapsis is observed (arrows). (G, H) Distribution of Rad51 early recombination foci (green) in meiotic cells. (G) In normal pachytene cells, the early recombination marker Rad51 has already largely been lost from the fully synapsed autosomes but remains on the sex chromosomes (arrowhead). (H) In <italic>Tex19.1^−/−</italic> knockout cells, Rad51 foci are abundant on unsynapsed chromosomes (arrows), but have mostly been lost from synapsed chromosomes (I) Distribution of meiotic stages in Sycp3/Sycp1-stained spreads. Around 100 chromosome spreads were scored from each of five <italic>Tex19.1^−/−</italic> knockout animals and from each of five <italic>Tex19.1^+/+</italic> wild-type or <italic>Tex19.1^+/−</italic> heterozygous animals. Around half of the pachytene cells (47%) in <italic>Tex19.1^−/−</italic> knockout testes feature incomplete synapsis. (J,K) Analysis of metaphase I chromosome spreads. (J) In normal metaphase I cells from <italic>Tex19.1^+/−</italic> heterozygotes all pairs of homologous chromosomes are held together as bivalents by chiasmata. (K) Around two-thirds of the metaphase I spreads from <italic>Tex19.1^−/−</italic> knockout mice exhibited univalent autosomal (asterisk) and/or sex (X,Y) chromosomes.</p></caption></fig>", "<fig id=\"pgen-1000199-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pgen.1000199.g006</object-id><label>Figure 6</label><caption><title>Expression of the endogenous retrovirus MMERVK10C is upregulated in <italic>Tex19.1^−/−</italic> null testes.</title><p>(A) Quantitative PCR showing relative expression of retrotransposons and marker genes in testes from 16 dpp <italic>Tex19.1^−/−</italic> knockout animals relative to their <italic>Tex19.1^+/+</italic> wild-type littermates. Expression levels in cDNAs prepared from different animals were normalised to <italic>Sdmg1</italic>, animals represented in the first and third columns were littermates, and those in the second and fourth columns were littermates. Error bars indicate standard error. (B) Northern blot probed for MMERVK10C <italic>env</italic> transcripts in <italic>Tex19.1^+/+</italic> wild-type and <italic>Tex19.1^−/−</italic> knockout testes at 16 dpp. A schematic diagram showing the organisation of the 8.5 kb full-length MMERVK10C element and the predicted size and organisation of the <italic>env</italic>-containing transcripts (∼8.5 kb and ∼3.3 kb, ##REF##8506289##[39]##) is shown above the Northern blot. Littermates are indicated with a black bar. 28S rRNA is shown as a loading control. (C–L) In situ hybridisation with an antisense MMERVK10C probe (purple precipitate) in <italic>Tex19.1^+/+</italic>, <italic>Tex19.1^+/−</italic> and <italic>Tex19.1^−/−</italic> testes. Nuclei are counterstained with nuclear fast red. (C, D, G, H) Low-level expression of MMERVK10C can be seen in some seminiferous tubules in <italic>Tex19.1^+/+</italic> and <italic>Tex19.1^+/−</italic> testes. However, MMERVK10C transcripts are more abundant in testes from 16 dpp <italic>Tex19.1^−/−</italic> animals than those from heterozygous or wild-type littermates. (K, L) MMERVK10C transcripts are upregulated in meiotic spermatocytes in 16 dpp <italic>Tex19.1^−/−</italic> mutant testes. (E, F, I, J) MMERVK10C transcripts are upregulated in meiotic spermatocytes in testes from adult <italic>Tex19.1</italic>\n^−/− knockout animals relative to their <italic>Tex19.1^+/−</italic> heterozygous littermates. (M, N) Control in situ hybridisation with a sense MMERVK10C probe shows no staining in adult <italic>Tex19.1^+/−</italic> or <italic>Tex19.1^−/−</italic> testes.</p></caption></fig>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"pgen.1000199.s001\"><label>Figure S1</label><caption><p>Validation of anti-Tex19.1 antibody specificity. (A–C) In situ hybridisation for <italic>Tex19.1</italic> (purple precipitate) on adult testis sections. (A, C) In situ hybridisation with antisense <italic>Tex19.1</italic> probe gives signal in the outer region of seminiferous tubules where spermatogonia and early spermatocytes are localized. (B) Hybridisation with a sense probe does not lead to a signal. (D–F) Immunohistochemistry with anti-<italic>Tex19.1</italic> antibody (brown precipitate) on adult testis sections. (D) Anti-Tex19.1 antibodies stain spermatogonia and early spermatocytes in the outer region of the seminiferous tubules. (E) When the antibody is blocked with immunising peptide (+pep) the signal is not present. (F) Immunohistochemistry with anti-Tex19.1 antibodies on <italic>Tex19.1^−/−</italic> knockout testes gives no signal. (G–I) Immunofluorescence on single cell suspensions from 14.5 dpc male embryonic gonads. Isolated gonads were trypsinised to single cell suspensions, then attached to poly lysine-coated slides, fixed with 4% paraformaldehyde in PBS and immunofluorescence was performed as described for cell spreads. Anti-Tex19.1 primary antibody is shown in green, nuclei counterstained with DAPI are shown in red. (G) Anti-Tex19.1 staining on a suspension of 14.5 dpc embryonic male gonadal cells gives strong signal in the germ cells. This signal is predominantly localized to the cytoplasm (inset in G). (H) This signal is not present when the antibody is blocked with immunising peptide (+pep). (I) Anti-Tex19.1 antibodies give no signal on gonadal cell suspensions from a 14.5 dpc male <italic>Tex19.1^−/−</italic> knockout embryo.</p><p>(4.2 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pgen.1000199.s002\"><label>Figure S2</label><caption><p>Tex19.1 does not co-localise with the nuage marker Tdrd1 in the adult testis. Immunofluorescence staining of 6 µm thick wax sections of paraformaldehyde-fixed adult testis. (A–C) Anti-Tex19.1 antibodies (green) predominantly label the cytoplasm of spermatogonia (open arrowheads) and early spermatocytes (broad arrowheads). The anti-Tex19.1 antibodies are distributed throughout the cytoplasm of these cells. DNA is counterstained with DAPI (red). (D–F) Anti-Tdrd1 antibodies (green) label elaborate punctate cytoplasmic structures in spermatocytes (broad arrowheads) and a single cytoplasmic spot in round spermatids (narrow arrowheads). DNA is counterstained with DAPI (red).</p><p>(1.4 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pgen.1000199.s003\"><label>Figure S3</label><caption><p>\n<italic>Tex19.1^−/−</italic> knockout animals exhibit increased levels of cell death in the testis. 6 µm thick wax sections of Bouin's-fixed testes were prepared, and the TUNEL assay for cell death performed using the DeadEnd Fluorometric TUNEL System (Promega) following the manufacturer's instructions. (A–M) TUNEL positive cells (green) in testes from <italic>Tex19.1^−/−</italic> knockout animals and <italic>Tex19.1^+/−</italic> heterozygous littermates. Nuclei are counterstained with DAPI (red). Panels G, J and M are merged images of panels E and F, and H and I, and K and L respectively. TUNEL-positive metaphase I cells (arrows) can be seen in some adult seminiferous tubules (E–G). Groups of TUNEL-positive cells (asterisks) can also be seen within the pachytene spermatocyte layer (arrowheads) of seminiferous tubules in adult (H–J) and prepubertal (K–M) testes. (N) <italic>Tex19.1^−/−</italic> knockout testes have increased numbers of TUNEL-positive cells. For statistical analysis TUNEL-positive cells were counted in 25 seminiferous tubule cross-sections for each animal. At least three knockout and three wild-type or heterozygous animals were analysed at each age. Mean number of TUNEL-positive cells per 25 tubules and standard error are indicated. Mann Whitney U-test was used as a statistical test as the TUNEL positive cells are not normally distributed. <italic>Tex19.1^−/−</italic> animals exhibit a statistically significant increase in the number of TUNEL-positive cells in the seminiferous tubules of the testis in 19–22 days post partum (dpp), 29–31 dpp, and in adult animals (Mann Whitney U-test, p&lt;0.01) as indicated by asterisks.</p><p>(3.7 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pgen.1000199.s004\"><label>Figure S4</label><caption><p>Histology of <italic>Tex19.1^−/−</italic> mutant testes during prepubertal development. Testis histology of <italic>Tex19.1^−/−</italic> knockout pups during the first wave of spermatogenesis. (A, E) At 14 days post partum (dpp) some pachytene spermatocytes are present in both knockout and wild-type testes and no obvious difference can be seen between genotypes. (B, F) At 16 dpp more pachytene spermatocytes are present and there is no obvious difference between the cell types present in the testes of knockout and wild-type littermates. (C, G) By 20 dpp, the germ cells appear to be greatly reduced in number in <italic>Tex19.1^−/−</italic> knockout testes (D, H) At 29 dpp, round spermatids and some elongating spermatids are present in heterozygous testes, but these cell types are reduced in number in testes from <italic>Tex19.1^−/−</italic> knockout littermates.</p><p>(6.2 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pgen.1000199.s005\"><label>Figure S5</label><caption><p>MMERVK10C retrotransposons show no detectable change in DNA methylation status in <italic>Tex19.1^−/−</italic> knockout testes. A schematic diagram showing the genomic organisation of the 5-end of the MMERVK10C retrotransposon is shown at the top of the figure. The long terminal repeat (LTR), 5′untranslated region (5′utr) and the start of the <italic>gag</italic> open reading frame are indicated, and the region analysed by bisulphite sequencing shown below with CpG dinucleotides indicated by grey circles. The DNA methylation status of CpG dinucleotides in 30 independent clones isolated from the liver or testes from 16 dpp <italic>Tex19.1^+/+</italic> wild-type, <italic>Tex19.1^+/−</italic> heterozygous and <italic>Tex19.1^−/−</italic> homozygous animals is also shown. Black circles indicate methylated CpGs protected from bisulphite conversion, white circles indictate unmethylated CpGs. 500 ng genomic DNA from these tissues was bisulphite treated using the EZ DNA Methylation Gold kit (Zymo Research) then used as a template for nested PCR using the primers <named-content content-type=\"gene\">5′-AGGTTTATAAAAGTAGTATTAG-3′</named-content> and <named-content content-type=\"gene\">5′- ATAACAATTAAAACAATAACATA-3′</named-content>, then <named-content content-type=\"gene\">5′-TAAAAGTAGTATTAGTTTTGGG-3′</named-content> and <named-content content-type=\"gene\">5′-AAACAAACAACACAATCCCA-3′</named-content>. The resulting 480 bp PCR product was then blunt-end cloned into pBluescript II SK+ (Stratagene) and independent plasmid clones were isolated and sequenced. Around 95% of the non-CpG cytosine residues were converted to thymine in the analysed sequences indicating succesful bisulphite conversion.</p><p>(8.2 MB TIF)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pgen.1000199.s006\"><label>Table S1</label><caption><p>Primer sequences.</p><p>(0.02 MB DOC)</p></caption></supplementary-material>" ]

[ "<fn-group><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p>We are grateful to the Medical Research Council, the Deutsche Forschungsgemeinschaft (RO) and the Lister Insitute of Preventive Medicine (IRA) for funding. IRA is a Lister Research Fellow, NKG is an MRC non-clinical senior research fellow.</p></fn></fn-group>" ]

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{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>Corals of the genus <italic>Acropora</italic> are the dominant reef-builders throughout the Indo-Pacific and, although hybridization is thought to have been an important factor in their evolutionary success ##UREF##0##[1]##, there are few unambiguous examples of hybrids or hybrid species. In the Caribbean, where only three extant <italic>Acropora</italic> species are known, <italic>A. prolifera</italic> is the product of hybridization between the other two <italic>Acropora</italic> spp. ##REF##10972775##[2]##, ##REF##12065836##[3]##. The low species complexity of the Caribbean coral fauna greatly simplifies unraveling such relationships. By contrast, the extraordinary species-richness of the Indo-Pacific, where over 60 <italic>Acropora</italic> species may occur in sympatry [Wallace &amp; Muir, unpublished], greatly complicates the identification of hybrids.</p>", "<p>Allele sharing between species provides evidence for introgressive hybridization ##REF##12144658##[4]##, ##REF##11420370##[5]##, but the unknown age of many extant Indo-Pacific species ##UREF##1##[6]## makes it often difficult to distinguish between hybridization and incomplete lineage sorting (i.e. shared ancestral polymorphism) ##REF##11420370##[5]##, ##UREF##2##[7]##. For the common species on which most work to date has focused, effective (<italic>N_e</italic>) and census population sizes (N) and coalescence times are unknown but potentially large and long, respectively, therefore incomplete lineage sorting cannot be ruled out. Rare species can provide new insights into the evolution of reef corals due to their intrinsically limited population sizes and therefore very short coalescence times.</p>", "<p>\n<italic>Acropora</italic> species typically occupy reef flat, reef crest and upper reef slope habitats (i.e. 2–30 m), however, some rare species occur outside this range, and this suggests an intriguing possibility-that some rare corals may be hybrids that can occupy atypical or non-parental niches, as is the case for the Caribbean hybrid species <italic>A. prolifera</italic>\n##REF##12065836##[3]##. To address to address the question of whether rare Indo-pacific <italic>Acropora</italic> species might also be hybrids, we analyzed DNA sequence data from nuclear and mitochondrial loci in a range of rare and common <italic>Acropora</italic> species from the Indo-Pacific and Caribbean.</p>" ]

[ "<title>Materials and Methods</title>", "<title>Sample collection and census data</title>", "<p>Samples (n = 1–3 individuals per species) of 14 rare and 8 common Indo-Pacific species of <italic>Acropora</italic> (##TAB##0##Table 1##) were collected from the Great Barrier Reef (Palm Island Group), the Marshall Islands (Rongelap Atoll) and Papua New Guinea (Kimbe Bay). Skeletal and matching tissue samples were collected from all corals sampled (n = 102 corals). Each sample was initially identified by Richards and confirmed by Wallace. All samples used for molecular analyses have matching voucher specimens registered in the World Wide <italic>Acropora</italic> Collection at the Museum of Tropical Queensland (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mtq.qld.gov.au\">www.mtq.qld.gov.au</ext-link>). Voucher specimens are available for inspection on request from the museum. For the purpose of this paper, rare species are those which have been recorded at &lt;2.5% of sites for which data are available in the World Wide <italic>Acropora</italic> Database (which contains &gt;20,000 records for &gt;1,500 sites). Mean (±SE) global census sizes were estimated by &gt;multiplying the mean global reef area available to each species by its mean local abundance per unit area (Supplementary ##SUPPL##1##Table S1##). Mean global reef area was calculated as the sum of the mean regional reef habitat available for all regions in which each species is known to occur (Supplementary ##SUPPL##2##Table S2##,). The proportion of reefs and sites occupied by rare species was estimated to be 10–30% of total reef area available. For present purposes, the effective population sizes were assumed to be approximately 11% of the calculated mean global census sizes (Supplementary ##SUPPL##0##Methods S1##); this relationship is based on a comprehensive meta-analysis of data for 102 species of animals ##UREF##3##[8]##.</p>", "<title>DNA Extraction, Polymerase Chain Reaction, cloning and sequencing</title>", "<p>DNA was extracted from ∼1 cm branch fragments of individual corals as previously described ##REF##11420370##[5]##. Markers studied were the highly polymorphic single-copy nuclear <italic>Pax-C</italic> 46/47 intron and the mitochondrial DNA (mtDNA) control region, for which a reference body of data exists from various <italic>Acropora</italic> species ##REF##11420370##[5]##, ##REF##12144656##[9]##. Details of primers and procedures for PCR, cloning and sequencing are described in ##REF##11420370##[5]##, ##REF##12144656##[9]##. New sequences obtained have been lodged in GenBank under EU918202-EU918288 (mitochondrial data) and EU918771-EU918925 (nuclear intron data).</p>", "<title>Phylogenetic Analysis</title>", "<p>Sequences were manually aligned in Sequencher 4.5 against a subset of the existing <italic>Acropora Pax-C</italic> intron and mitochondrial control region sequences ##REF##10972775##[2]##, ##REF##11420370##[5]##, ##REF##12144656##[9]## before phylogenetic analysis in a Bayesian statistical framework in Mr Bayes 3.1.2 ##REF##11524383##[10]##. The dataset analysed therefore consisted of sequences from 17 rare and 15 common Indo-Pacific species <italic>Acropora</italic> species, the three Caribbean <italic>Acropora</italic> species and <italic>Isopora cuneata</italic>. Genetic distances were calculated as Kimura 2-parameter distances ##REF##7463489##[11]##. The optimal model of sequence evolution was identified using hierarchical likelihood ratio tests in MrModeltest 2.2 ##UREF##4##[12]##. The (MCMC) analyses were run for 5 million generations, with burn-in times of 20,000–50,000 (p&lt;0.05). Trees generated from the <italic>Pax-C</italic> data were rooted using sequences from <italic>Isopora cuneata,</italic> whereas the mtDNA tree was rooted with <italic>A. cervicornis</italic> as in this case the degree of divergence of the <italic>I. cuneata</italic> sequence effectively precluded unambiguous alignment. Analyses were conducted on the full alignments as the exclusion or weighting down of large indels or repeat regions was found not to significantly effect the overall topology (see also ##REF##11420370##[5]##).</p>" ]

[ "<title>Results</title>", "<p>Allele/haplotype data from nuclear and mitochondrial loci were determined for 17 rare and 15 common Indo-Pacific <italic>Acropora</italic> species as well as all 3 Caribbean species of <italic>Acropora</italic> (##TAB##0##Table 1##) and <italic>Isopora cuneata</italic>. Only samples from taxonomically unambiguous individuals were included in this study; the morphology of the corals sampled was absolutely consistent with their formal description ##UREF##1##[6]##. To avoid the possibility of sampling clonemates, corals sampled were separated by at least 10 meters. The extreme rarity of several of the species examined limited the number of samples that it was possible to examine. Plots of the number of species distribution records against rank order (##FIG##0##Figure 1a##) clearly resolve rare species, such as <italic>A. pichoni</italic> (##FIG##0##Figure 1b##), from common species, with <italic>A. valida</italic> and <italic>A. nasuta</italic> being essentially pandemic throughout the Indo-Pacific.</p>", "<title>Census Sizes</title>", "<p>Effective population sizes in reef corals are expected to be significantly smaller than census sizes for a number of reasons ##REF##21236037##[13]##. First, corals are known to undergo extreme variation in census population sizes due to perturbations such as storms and cyclones, bleaching or crown-of-thorns starfish outbreaks, which will substantially reduce effective sizes because it diminishes the proportion of the population involved in reproduction ##UREF##3##[8]##. Second, high variance in fecundity (which is again known in corals ##UREF##5##[14]##) reduces <italic>N</italic>\n_e because neither juveniles nor senescent adults take part in reproduction ##UREF##6##[15]##. Third, some <italic>Acropora</italic> species reproduce asexually by fragmentation or fission ##REF##11108587##[16]##, which again reduces <italic>N</italic>\n_e. Here we find mean (±SE) global census population sizes for rare species in this study varied from 32823 (±16412) for <italic>A. spathulata</italic> to 224 (±117) for <italic>A. rongelapensis</italic>. Based on the <italic>N_e</italic> estimate of 11% of the census population size, <italic>A. spathulata</italic> has a mean effective global population size of 3611 (±1805) and A. <italic>rongelapensis</italic>, 25 (±13) (##FIG##1##Figure 2##). Furthermore, it is likely that local population census and effective population sizes are substantially smaller than these conservative global estimates.</p>", "<title>Pax-C intron data</title>", "<p>Results of phylogenetic analyses of Pax-C intron data (##FIG##2##Figure 3##) are broadly consistent with previous results, but some details differ due to the selection of taxa. To facilitate comparison with previous analyses, clades are labeled according to published trees ##REF##11420370##[5]##, ##REF##12144656##[9]##. As in previous analyses, the basal clade contains <italic>A. longicyathus</italic>, and, in the present case, <italic>A. austera</italic>. In the present tree, a polytomy then gives rise to strongly supported clades corresponding to IIIA, IVB, IIID of previous studies; a major difference is the novel clade V which is composed exclusively of rare species with the exception of a single allele from <italic>A. valida</italic>. The nuclear tree distinguishes the Caribbean species in the highly supported clade IIID. Within the large terminal clade, two novel subclades (III F+G) were identified, containing predominantly sequences from rare species.</p>", "<title>Mitochondrial control region data</title>", "<p>Phylogenetic analyses of the mtDNA Control Region (##FIG##3##Figure 4##) were also broadly consistent with previous results and clades were labeled as in previous publications ##REF##11420370##[5]##, ##REF##12144656##[9]##. The basal clade (IA/IB) again contains <italic>A. longicyathus</italic> and <italic>A. austera</italic>, with <italic>A. tenuis</italic> added. In the present case, clade III is expanded and clade IV contracted relative to published analyses, due to differences in composition of the datasets. Clade IV includes <italic>A. aspera, A. humilis</italic> and several rare species (e.g. <italic>A. kirstyae, A. derawanensis</italic>).</p>" ]

[ "<title>Discussion</title>", "<p>In both the Pax-C and mitochondrial phylogenies many <italic>Acropora</italic> species are polyphyletic. Previous work ##REF##11420370##[5]##, ##REF##12144656##[9]## provides precedents for this pattern, which has been interpreted as evidence for interspecific hybridization. However, the Indo-Pacific species examined in these previous studies are widespread and locally common, and in these cases lineage sorting will occur slowly. As the fossil record of <italic>Acropora</italic> is extremely limited, for common and widespread species incomplete lineage sorting cannot be rigorously excluded as an alternative explanation for the observed polyphyletic patterns. However, for the rare species included in the present study, effective population sizes are so small (##FIG##1##Fig 2##) that lineage sorting will occur on very short time scales, so in contrast to the position with common species, polyphyletic patterns observed for rare species provide unequivocal evidence for hybridization.</p>", "<p>Comparison of the trees generated from nuclear and mitochondrial data (##FIG##4##Figure 5##) shows that three of the rare species studied here-<italic>A. pichoni</italic>, <italic>A. kimbeensis</italic> and <italic>A. papillare</italic>-are monophyletic for the mtDNA marker but are polyphyletic and contain highly divergent alleles at the nuclear marker, even within individual corals. The presence of species-specific mitochondrial haplotypes is unusual in <italic>Acropora</italic>\n##REF##11420370##[5]##, ##REF##12144656##[9]##. Of the 49 species studied to date, the only other <italic>Acropora</italic> species that is monophyletic in mtDNA is <italic>A. tenuis</italic> (##FIG##3##Figure 4##; however, see also below), which is known to be reproductively isolated through a difference in spawning time ##REF##11420370##[5]##.</p>", "<p>The mitochondrial phylogeny implies that the three monophyletic rare species have evolved relatively recently, because they fall within derived positions of the large terminal clade that reflects the post-Miocene Indo-Pacific speciation of <italic>Acropora</italic> (i.e. &lt;5.32 my) ##REF##11420370##[5]##, ##UREF##7##[17]##. In contrast, sequences from these three species are widely distributed throughout the nuclear tree; for example, alleles from <italic>A. papillare</italic> occur in both Clades III and V. This pattern in nuclear versus mtDNA loci can be explained by the known faster lineage sorting of mitochondrial haplotypes than alleles at single copy nuclear loci ##REF##6505980##[18]##. Unlike their more common relatives, the small effective global population sizes of these three rare species (<italic>A. pichoni</italic> = 521±125; <italic>A. kimbeensis</italic> = 1208±707; <italic>A. papillare</italic> = 284±142) effectively rules out the possibility of incomplete lineage sorting, because of their small population sizes, these rare species have very short coalescence times.</p>", "<p>There is no evidence that these rare species were historically more common. Moreover, these observed patterns–monophyly with respect to mitochondrial haplotypes accompanied by polyphyly at nuclear loci-cannot be explained as consequences of either recent population crashes or population bottlenecks. Under a population crash scenario one would expect to find divergent mitochondrial haplotypes as well as divergent nuclear alleles, whereas under a population bottleneck scenario (i.e. a crash occurring less recently) low diversity at both nuclear and mitochondrial loci is expected. These alternate possibilities can therefore be ruled out, and the most parsimonious explanation for the observed patterns of allele/haplotype distribution is that <italic>A. pichoni</italic>, <italic>A. kimbeensis</italic> and <italic>A. papillare</italic> are unidirectional hybrids.</p>", "<p>In the Caribbean, the hybrid species <italic>A. prolifera</italic> colonizes habitats that are distinct from those of the parental species ##REF##10972775##[2]##, ##REF##12065836##[3]##. Similarly, two of the three rare putative hybrid species from the Indo-Pacific, <italic>A. pichoni</italic> and <italic>A. papillare,</italic> occur in atypical habitats. Whereas the vast majority of <italic>Acropora</italic> spp. occur in relatively shallow reef flat, crest and slope habitats (2–30 m), <italic>A. pichoni</italic> occurs below 40 m and <italic>A papillare</italic>, is found in extremely shallow intertidal habitats (&lt;2 m). Specialization in extremely shallow or deep habitats is atypical for <italic>Acropora</italic> species hence our data provide support for the hypothesis that hybrid species may exploit atypical (or non-parental) niches.</p>", "<p>Other rare species occurring in small and isolated populations (e.g. <italic>A. walindii, A. loisetteae, A. derawanensis</italic> and <italic>A. jacquelineae</italic>) are polyphyletic with respect to both nuclear alleles and mitochrondrial haplotypes. Whilst these patterns are again consistent with hybridization, in these cases alternative explanations, such as recent population crashes, cannot be rigorously excluded.</p>", "<p>Two species that are geographically restricted but locally common (<italic>A. spathulata</italic> and <italic>A. tortuosa)</italic> are also monophyletic at the mitochondrial marker but polyphyletic at the nuclear marker. However, in these latter cases, incomplete lineage sorting cannot be ruled out because of the longer coalescence times for these species resulting from their larger census and predicted effective population sizes.</p>", "<p>The results presented here imply that a number of rare Indo-Pacific <italic>Acropora</italic> species are the products of recent hybridization events, and highlight the significance of hybridization in coral diversification. Whether these species have hybrid origins or have evolved and then hybridized in the absence of conspecific gametes remains to be elucidated.</p>", "<p>In summary, although it has often been assumed that small populations have a decreased potential for adaptation ##UREF##8##[19]##, our analyses imply that some rare Acroporid corals may actually have increased adaptive potential as a consequence of introgressive hybridization ##REF##16701254##[20]##, and therefore may be less vulnerable to extinction than has been assumed.</p>" ]

[]

[ "<p>Conceived and designed the experiments: ZR MJHvO DJM. Performed the experiments: ZR. Analyzed the data: ZR. Contributed reagents/materials/analysis tools: MJHvO DJM. Wrote the paper: ZR MJHvO CW BLW DJM. Specimen collection: ZR. Specimen identification: ZR CW.</p>", "<title>Background</title>", "<p>Coral reefs worldwide face a variety of threats and many coral species are increasingly endangered. It is often assumed that rare coral species face higher risks of extinction because they have very small effective population sizes, a predicted consequence of which is decreased genetic diversity and adaptive potential.</p>", "<title>Methodology/Principal Findings</title>", "<p>Here we show that some Indo-Pacific members of the coral genus <italic>Acropora</italic> have very small global population sizes and are likely to be unidirectional hybrids. Whether this reflects hybrid origins or secondary hybridization following speciation is unclear.</p>", "<title>Conclusions/Significance</title>", "<p>The interspecific gene flow demonstrated here implies increased genetic diversity and adaptive potential in these coral species. Rare <italic>Acropora</italic> species may therefore be less vulnerable to extinction than has often been assumed because of their propensity for hybridization and introgression, which may increase their adaptive potential.</p>" ]

[ "<title>Supporting Information</title>" ]

[]

[ "<fig id=\"pone-0003240-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003240.g001</object-id><label>Figure 1</label><caption><title>(a). Global abundance of the <italic>Acropora</italic> species used in this study.</title><p>These data are based on numbers of records in the World Wide <italic>Acropora</italic> Database (n = 1523 sites; ##UREF##1##[6]## and Wallace unpublished). (b). Several rare species, such as <italic>A. pichoni</italic> shown here, are likely to be unidirectional hybrids and occupy atypical habitats. Photo credit: Maria Beger.</p></caption></fig>", "<fig id=\"pone-0003240-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003240.g002</object-id><label>Figure 2</label><caption><title>Effective population size data for rare <italic>Acropora</italic> species included in this study.</title><p>Mean (±SE) global census sizes are shown as black histograms, and predicted effective population sizes as red histograms. Data for <italic>A. tortuosa</italic> are omitted, as the mean global census size for this species (Supplementary ##SUPPL##1##Table S1##) is more than two-fold higher than for <italic>A. spathulata</italic> (of those shown, the species with the largest global census size).</p></caption></fig>", "<fig id=\"pone-0003240-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003240.g003</object-id><label>Figure 3</label><caption><title>Phylogenetic analysis of PaxC data.</title><p>The figure shows the majority rule (&gt;50%) consensus tree obtained in a Bayesian analysis of nuclear sequence data for the thirty-five <italic>Acropora</italic> species included in this study, with <italic>Isopora cuneata</italic> defined as outgroup. Bayesian analyses used likelihood settings from best-fit model (HKY+G) selected by hLRT in MrModeltest 2.2 ##UREF##4##[12]##: 5 million generations; burn in  = 50,000. Numbers above branches are posterior probability values supporting the topology shown and clades are labelled according to previous ##REF##11420370##[5]##, ##REF##12144656##[9]## analyses. Numbers after species names indicate the coral colonies from which the sequences were obtained; where more than one sequence was obtained per colony, the clone identity is given after an asterisk. Note that in some cases multiple clones (sometimes from different species) had identical sequences.</p></caption></fig>", "<fig id=\"pone-0003240-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003240.g004</object-id><label>Figure 4</label><caption><title>Phylogenetic analysis of mitochondrial sequence data.</title><p>The figure shows the majority rule (&gt;50%) consensus tree obtained in a Bayesian analysis of mitochondrial sequence data for thirty-five Indo-Pacific <italic>Acropora</italic> species with the Caribbean species <italic>Acropora cervicornis</italic> defined as outgroup. Bayesian analysis used likelihood settings from best-fit model (HKY+I+G) selected by hLRT in MrModeltest 2.2 ##UREF##4##[12]##: 5 million generations; burn in  = 20,000. Numbers above branches are posterior probability values supporting the topology shown and clades are labelled according to previous ##REF##11420370##[5]##, ##REF##12144656##[9]## analyses. Numbers after species names indicate the coral colonies from which the sequences were obtained.</p></caption></fig>", "<fig id=\"pone-0003240-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003240.g005</object-id><label>Figure 5</label><caption><title>Comparison of nuclear and mitochondrial phylogenies.</title><p>Asterisks indicate posterior probability values of 100% (black) or &gt;70% (red); for clarity, asterisks are shown only at nodes affecting the positions of sequences from <italic>A. papillare</italic>, <italic>A. pichoni</italic>, <italic>A. kimbeensis</italic>, <italic>A. spathulata</italic> and <italic>A. tortuosa.</italic>\n</p></caption></fig>" ]

[ "<table-wrap id=\"pone-0003240-t001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003240.t001</object-id><label>Table 1</label><caption><title>Biological characteristics of species included in this study.</title></caption><alternatives><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Species</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Distribution</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Range</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ecological niche</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Collection location or source</td></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. walindii</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">PNG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">deep sandy reef slopes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. rongelapensis</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Micronesia/Indonesia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">deep protected sandy slopes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rongelap Atoll, RMI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. loisetteae</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Malaysia, W. Aust, Micronesia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">protected sandy lagoons</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rongelap Atoll, RMI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. pichoni</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">PNG, Micronesia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">deep submerged shelf reefs, shipwrecks</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. lokani</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SE Asia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">shallow reef flat</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. derawanensis</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SE Asia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">protected deep sandy slopes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. tenella</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SE Asia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal protected slopes, shelfs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. batunai</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indonesia, PNG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">submerged reefs, slopes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. chesterfieldensis</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chesterfield Is., Micronesia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">submerged shallow reefs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rongelap Atoll, RMI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. kimbeensis</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">PNG, Micronesia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">submerged reef flat</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. spathulata</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">GBR, PNG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">reef flat and slope</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Orpheus Island, GBR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. kirstyae</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indonesia, GBR, PNG, New Caledonia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">protected interrefal locations</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Orpheus Island, GBR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. papillare</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">W. Australia, GBR, Japan</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ultra shallow and exposed reef</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Orpheus Island, GBR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. speciosa</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SE Asia, GBR, Central Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal, protected slopes and walls</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rongelap Atoll, RMI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. jacquelineae</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indonesia, PNG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">reef slopes and submerged reefs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. caroliniana</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SE Asia-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">submerged habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Kimbe Bay, PNG</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. tortuosa</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Restricted</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Central Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal, protected sandy lagoons</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rongelap Atoll, RMI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. granulosa</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">reef slopes and walls</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rongelap Atoll, RMI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. vaughani</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">protected subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Orpheus Island, GBR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. pulchra</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">intertidal or shallow subtidal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. aspera</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">intertidal or shallow subtidal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. longicyathus</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SE Asia-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. loripes</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal shallow reef habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Rongelap Atoll, RMI</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. gemmifera</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">intertidal or shallow subtidal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. microphthalma</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Orpheus Island, GBR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. millepora</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">intertidal or shallow subtidal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. digitifera</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">intertidal or shallow subtidal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. humilis</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">intertidal or shallow subtidal</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. austera</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">shallow subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. cerealis</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">shallow subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. nasuta</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">shallow subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. valida</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Widespread</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">shallow subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic Island, GBR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. palmata</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Outgroup</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Atlantic Ocean</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2000</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. prolifera</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Outgroup</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Atlantic Ocean</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtial habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2000</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>A. cervicornis</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Outgroup</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Atlantic Ocean</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2000</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>I. cuneata</italic></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Outgroup</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Indo-Pacific</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">subtidal habitats</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">van Oppen et al. 2001</td></tr></tbody></table></alternatives></table-wrap>" ]

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[ "<supplementary-material content-type=\"local-data\" id=\"pone.0003240.s001\"><label>Methods S1</label><caption><p>Calculation of mean global census and effective population sizes</p><p>(0.07 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003240.s002\"><label>Table S1</label><caption><p>Estimates of mean global census size for rare species included in this study.</p><p>(0.12 MB DOC)</p></caption></supplementary-material>", "<supplementary-material content-type=\"local-data\" id=\"pone.0003240.s003\"><label>Table S2</label><caption><p>Regional estimates of available reef habitat post 2004.</p><p>(0.09 MB DOC)</p></caption></supplementary-material>" ]

[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>Principal funding source: Australian Research Council. Other funding: Queensland State Government (Smart State PhD funding), Winifred Violet Scott Estate, International Society for Reef Studies (fellowship to ZR)</p></fn></fn-group>" ]

[ "<graphic id=\"pone-0003240-t001-1\" xlink:href=\"pone.0003240.t001\"/>", "<graphic xlink:href=\"pone.0003240.g001\"/>", "<graphic xlink:href=\"pone.0003240.g002\"/>", "<graphic xlink:href=\"pone.0003240.g003\"/>", "<graphic xlink:href=\"pone.0003240.g004\"/>", "<graphic xlink:href=\"pone.0003240.g005\"/>" ]

[ "<media xlink:href=\"pone.0003240.s001.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003240.s002.doc\"><caption><p>Click here for additional data file.</p></caption></media>", "<media xlink:href=\"pone.0003240.s003.doc\"><caption><p>Click here for additional data file.</p></caption></media>" ]

{ "acronym": [], "definition": [] }

[ "<title>Introduction</title>", "<p>In eukaryotes, recruitment of canonical factors including the ribosome to the 7-methyl guanosine cap structure on the 5′ end of the mRNA is thought to be the primary mechanism by which translation initiation occurs##REF##215319##[1]##. Once recruited to the mRNA, the 40S ribosome scans along the 5′ leader until it encounters the initiator codon ##REF##215319##[1]##. However, initiation of a number of eukaryotic mRNAs can also occur through internal ribosomal entry sites (IRESes, for review see ##REF##15749702##[2]##), a mechanism first identified in viral RNAs ##REF##6260787##[3]##. In a manner analogous to the cap structure, a single IRES recruits the 40S ribosomal subunit to the mRNA for translation ##REF##10878768##[4]##, ##REF##12667457##[5]##.</p>", "<p>Unlike initiation from the cap structure, the subset of proteins required for initiation from a eukaryotic IRES has yet to be determined. However, the current literature suggests that most eukaryotic IRESes require non-canonical translation factors referred to as IRES transactivating factors (ITAFs) for initiation. The three isoforms of polypyrimidine tract binding protein (PTB1, 2 and 4) ##REF##11063741##[6]##\n##REF##11421360##[7]##, La protein##REF##10848591##[8]##, and upstream of n-ras protein (unr) ##REF##11313462##[9]## are examples of ITAFs required for efficient translation from eukaryotic and viral IRESes. These proteins may function as chaperones, altering or maintaining specific RNA structures that permit binding of the translational machinery (for review see ##REF##15900315##[10]##). This theory is supported by work from the Niepmann lab demonstrating that binding of PTB to the foot-and-mouth disease (FMDV) IRES enhances the recruitment of the initiation factor eIF4G and stimulates FMDV IRES activity ##REF##16314455##[11]##. Additionally, it has been shown that the eukaryotic Apaf-1 5′ leader undergoes secondary structure changes upon the binding of unr and PTB, allowing for internal initiation of translation ##REF##12667457##[5]##.</p>", "<p>Utilization of eukaryotic IRESes often occurs during cellular events that reduce cap-dependent translation. For example, during mitosis the family of eIF4E binding proteins (4E-BP) is hypophosphorylated, which enhances their ability to bind eIF4E ##REF##10882097##[12]##. The sequestering of eIF4E inhibits cap-dependent translation ##REF##6260787##[3]## and the overall level of protein synthesis is decreased ##REF##14739278##[13]##. However, during mitosis several mRNAs, including those encoding for ornithine decarboxylase (ODC) ##REF##10882097##[12]## and p58 (PITSLRE kinase) ##REF##10882096##[14]##, are translated in an IRES-dependent manner. For PITSLRE kinase, its IRES activity partially depends on the upregulation of unr expression at the G2/M junction underscoring not only the importance of the cell state, but also the importance of ITAF availability for internal initiation.</p>", "<p>Studies in model systems indicate that neural activity may also promote dependency on internal initiation of translation. In response to activity-dependent neural plasticity, Aplysia neuroendocrine cells undergo a switch from cap-dependent to cap-independent translation ##REF##12592407##[15]##. Additionally, multiple dendritically localized mRNAs exhibit IRES activity within neural cell lines ##REF##15908588##[16]## and primary neuronal cultures ##REF##11226315##[17]## suggesting they may be translated in response to neural activity.</p>", "<p>The mRNA encoding for the neurotrophin receptor TrkB is dendritically-localized ##REF##9391005##[18]## and encodes for a tyrosine kinase receptor that binds brain-derived neurotrophin factor (BDNF) (for review see ##REF##12676795##[19]##). The TrkB receptor is synthesized under diverse conditions and its activity contributes to multiple cellular processes (for review see ##REF##11520916##[20]##). In neural stem cells, the TrkB mediated pathway promotes cell proliferation ##REF##7643217##[21]##. In the adult, TrkB activity contributes to changes in synaptic efficacy and local connectivity, ultimately affecting learning and memory (for review see ##REF##12676795##[19]##). Owing to its diverse functions, TrkB expression is regulated at multiple steps in response to various cellular conditions ##REF##11529494##[22]##, ##REF##7559588##[23]##, ##REF##12055187##[24]##. For example, ischemia stimulates TrkB protein synthesis in order to promote cell survival ##REF##9590552##[25]##. Interestingly, ischemia also inhibits cap-dependent translation ##REF##1731336##[26]## suggesting that the upregulation of TrkB occurs in a cap-independent manner. In accordance with this observation, our lab has demonstrated that the 5′ leader of the mRNA encoding for human TrkB contains an IRES ##REF##15908588##[16]##.</p>", "<p>We hypothesized that if IRES-dependent translation was a critical mechanism for the synthesis of TrkB protein, then both the presence of an IRES and its mechanism would be evolutionarily conserved. The human TrkB 5′ leader is derived from alternative transcriptional start sites and alternative splicing of five exons ##REF##11798182##[27]##. Interestingly, the IRES was localized to exon 5, which is present in all 5′ leader variants ##REF##15908588##[16]##. Unlike the human 5′ leader, the mouse TrkB 5′ leader is transcribed from two promoters and is comprised of two exons ##REF##10395916##[28]##. However, similar to the human TrkB 5′ leader, all of the mouse TrkB 5′ leaders contain a 3′ common exon, exon 2. The human exon 5 and the mouse exon 2 sequences share a 63% identity with regions of high similarity on the extreme ends of the exons. Consequently, we predicted that the mouse TrkB 5′ leader would mediate internal initiation and that the IRES would be located within Exon 2.</p>", "<p>In the present report we establish that the mouse TrkB Exon 2 does contain an IRES. But surprisingly, an IRES is also located within Exon 1 demonstrating the presence of two IRESes initiating translation of the same open reading frame. The two IRESes are differentially utilized based on the differentiation state of a neuronal cell line. This phenomenon is explained in part by the observation that PTB1 differentially affects translation mediated by the two IRESes, despite the fact that PTB1 binds both IRESes with similar affinity.</p>" ]

[ "<title>Materials and Methods</title>", "<title>Constructs</title>", "<p>The mouse TrkB 5′ leaders (##FIG##0##Fig 1a##), with the exception of L1, were cloned from a mouse brain cDNA library (Clontech) with EcoRI and NcoI restriction sites. L1 was created by PCR amplifying exon 1 and exon 2 separately. Exon 1 was created with an EcoRI site at the 5′ end and a blunt 3′ end. Exon 2 had a blunt 5′ end and an NcoI site at its 3′ end. The central blunt ends were ligated together and the combined exons were the inserted into the RF vector using the EcoRI and NcoI sites at the insert ends. The RF vector was a generous gift from Dr. Anne Willis, University of Nottingham.</p>", "<p>In order to create the dual monocistronic vector, the RF constructs were then digested with EcoRI and HpaI to isolate the 5′ leader, the <italic>Photinus</italic> luciferase gene, and the SV40 3′ UTR. This portion was then ligated into the pGL3 vector (Promega). To isolate the 5′ leader and <italic>Photinus</italic> luciferase gene from this vector, it was digested with SnaBI and ApaLI. The fragment was then ligated into a backbone that already contained a monocistronic <italic>Renilla</italic> luciferase gene, creating a vector that contains both luciferase genes in a monocistronic context. The mono- and dicistronic constructs used for <italic>in vitro</italic> transcription were made as described in ##REF##15908588##[16]##.</p>", "<p>To create the promoterless constructs, the β-globin RP vector was digested sequentially with SmaI and EcoRV to release the upstream intron and promoter. The blunt ends were then ligated together to create the promoterless vector. The 5′ leaders were exchanged using the EcoRI and NcoI restriction sites.</p>", "<p>DNA containing the cricket paralysis virus (CrPV) IRES was PCR amplified from plasmid DNA designed as described in ##REF##10497018##[52]## using M13 primers. The original CrPV IRES DNA was a generous gift from Dr. Peter Sarnow, Stanford University.</p>", "<p>Capped RNA used for <italic>in vitro</italic> translation and RNA transfections was <italic>in vitro</italic> transcribed using the mMessage Machine kit (Ambion) per manufacturer's instructions as described in ##REF##15908588##[16]##. Uncapped RNA used for <italic>in vitro</italic> binding assays was <italic>in vitro</italic> transcribed using the MEGAScript kit (Ambion) per manufacturer's instructions.</p>", "<title>Cell Culture/Transfections</title>", "<p>C6 and N2a cell lines were purchased from ATCC and cultured in DMEM, 10% fetal bovine serum, and 200 mM L-glutamine. Two micrograms of DNA was transfected into the cells using FuGene6 reagent (Roche). The cells were harvested 24 hours later and lysed using passive cell lysis buffer (Promega). The lysate was then assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) measured using a Luminoskan luminometer.</p>", "<p>Co-transfections were performed as described above using 1.8 µg of RP DNA and 0.2 µg of either the Pactag null vector (based on pACTAG-2) or DNA encoding for a hypophosphorylated version of 4E-BP1 ##REF##8521827##[33]##. The 4E-BP1 and pACTAG-2 constructs were a generous gift from Dr. Nahum Sonenberg, McGill University.</p>", "<p>RNA transfections using 2 µg of RNA were performed using the RNA Transmessenger kit (Qiagen) per manufacturer's instructions. The cells were incubated for seven hours after transfection, harvested, and assayed for luciferase activity.</p>", "<p>SH-SY5Y cells were purchased from ATCC and cultured in DMEM, 10% fetal bovine serum, and 200 mM L-glutamine. Cells were plated four days prior to transfection and differentiated by treating with 2 µM retinoic acid. Upon transfection, the media was replaced with DMEM. Three hours into the transfection, the media was exchanged and retinoic acid or the carrier DMSO was added to the cells for the remainder of the incubation.</p>", "<title>PTB1 Purification</title>", "<p>Rosetta cells (Novagen) were transformed with the plasmid PGEX-2TK-PTB1, encoding GST-tagged full-length human PTB1. The PTB1 construct was a generous gift from M. A. Garcia-Blanco. Bacteria were grown in 2XYT media to a density 0.35 ODU_595nm at which time expression of GST-1 was induced with 0.2 mM IPTG. After 18 hrs of expression at 25°C, the cells were collected by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 0.25 M NaCl, 1 mM TCEP, 1 mM EDTA). After lysis by sonication, the NaCl concentration was raised to 1 M and the lysate clarified by centrifugation. The resulting supernatant was applied to glutathione-coupled agarose (GE-Healthcare), and the column was washed with lysis buffer to remove the excess NaCl. To liberate PTB1, thrombin was added and the column was incubated at 25°C for 3 hours with gentle agitation followed by elution of the PTB1. Glycerol was added to the PTB1 containing fractions to a final concentration of ∼10% (v/v). Afterwards, the fractions were concentrated. The PTB1 was further purified by size exclusion chromatography on a Sup200 column (GE Healthcare) in lysis buffer. To remove any bound nucleic acids from PTB1, the protein was bound to a heparin column (GE Healthcare) in lysis buffer, and eluted with a 0.25–2 M NaCl linear gradient. The PTB1 containing fractions were concentrated, dialyzed against the storage buffer (50 mM Tris-HCl pH 8.0, 0.25 M NaCl, 1 mM TCEP, 1 mM EDTA, 20% glycerol (v/v)), and stored at 4°C.</p>", "<title>\n<italic>In Vitro</italic> Translation</title>", "<p>One microgram of <italic>Photinus</italic> luciferase monocistronic mRNA was added to rabbit reticulosyte lysate (RRL) (Red Nova, Novagen) in the presence of cap analog (Ambion). Following a 1 hour incubation at 30°C, the sample was assayed for <italic>Photinus</italic> luciferase activity.</p>", "<p>For the <italic>in vitro</italic> translation assays involving PTB1, 1 µg of dicistronic mRNA was added to RRL (Promega) in the presence of 0.4 µg of PTB1 protein. The lysates were incubated for 1 hour at 30°C and then the samples were assayed for luciferase activity as described above.</p>", "<title>PTB1 Binding Assay</title>", "<p>Approximately 2 pmol of radiolabeled RNA (500 cpm) was combined with increasing amounts of recombinant PTB1 protein in protein binding buffer (10 mM Tris-HCl pH 8, 100 mM KCl, 2.5 mM MgCl2, 5% glycerol (v/v)) ##REF##9214659##[53]## in triplicate. The reaction was incubated for 10 minutes at 37°C and then passed through a dot blot apparatus containing a sandwich of nitrocellulose and charged nylon membranes. A ratio of the fraction of bound RNA to the total RNA was calculated and plotted against the PTB1 concentration. The curve was fit using the Langmuir formula [(m0*m1/(M0+m2)+m3);m1 = .9;m2 = 1e−9;m3 = .001] to determine the dissociation constant.</p>", "<title>siRNA Transfection</title>", "<p>SH-SY5Y cells were treated for two days with either DMSO (mock) or 2 µM retinoic acid to induce differentiation. The cells were incubated with 300 µM siRNA (Dharmacon) and Dharmafect 1 reagent (Dharmacon). After 24 hours, the siRNA and media were aspirated off and fresh media was applied to the cells for an additional 24 hours. The cells were treated with DMSO or 2 µM retinoic acid for an additional 48 hours. At this stage, the cells were either harvested for Western blot analysis or transfected with RNA as described above.</p>" ]

[ "<title>Results</title>", "<title>The Mouse TrkB 5′ Leaders Contain a Potential IRES Element</title>", "<p>To determine if the full-length 5′ leaders generated from the first promoter, Leader 1 (L1, 1.428 kb), from the second promoter, Leader 2 (L2, 448 nt), and Exon 2 (Ex2, 344 nt) exhibited IRES activity, they were inserted into the intercistronic region of the dicistronic luciferase DNA vector pRF (##FIG##0##Fig 1a and 1b##)##REF##9467968##[29]##. The 5′ leader of the human β-globin mRNA (50 nt) and the leader of the encephlomyocarditis virus (EMCV) (608 nt) were also inserted as negative and positive controls, respectively ##REF##2538648##[30]##. The individual constructs were transfected into two neural cell lines, C6 and N2a, and after 24 hours the cells were harvested and assayed for luciferase activity. A ratio of the <italic>Photinus</italic> luciferase to the <italic>Renilla</italic> luciferase was calculated following a luciferase assay of the cell lysates. The ratio obtained for the negative control, β-globin, was set to one, and the ratios obtained from the other constructs were normalized to that value.</p>", "<p>As expected, the positive control EMCV exhibited an increased <italic>Photinus</italic>:<italic>Renilla</italic> (P∶R) luciferase ratio when compared to the β-globin 5′ leader in both cell lines. EMCV generated a ratio of 28 in C6 cells and 16 in N2a cells (##FIG##1##Fig 2##). All three mouse TrkB 5′ leaders also exhibited higher P:R ratios than the negative control. Ex2 exhibited a P∶R ratio of 9 in C6 cells and 15 in N2a cells, while the full-length L2 had a ratio of 7 in both cell lines. Interestingly, L1 yielded a ratio ranging from 200–300 in the cell lines. These results suggest that all three mouse TrkB 5′ leaders can internally initiate translation, however the presence of other regulatory elements could also lead to an increased P∶R ratio from the DNA construct.</p>", "<title>A Cryptic Promoter is Present in the Mouse TrkB 5′ Leader</title>", "<p>The dicistronic DNA construct has been used as the major assay to test for IRES activity. However, additional mechanisms can account for translation of the second cistron. The dicistronic DNA construct used in these assays contains an intron upstream of the <italic>Renilla</italic> luciferase gene to increase transcription and translation efficiency. However, the presence of a cryptic splice site located in the 5′ leader of interest would lead to the splicing of the <italic>Renilla</italic> luciferase gene, leaving only the <italic>Photinus</italic> luciferase gene to be translated and effectively increasing the P∶R ratio. In addition, the presence of a cryptic promoter in the 5′ leader would generate a monocistronic mRNA encoding only the <italic>Photinus</italic> luciferase gene. This process would create an mRNA consisting only of the <italic>Photinus</italic> luciferase gene and again, would lead to an increase in the P∶R ratio.</p>", "<p>To determine if a cryptic promoter is present in the TrkB 5′ leaders, we inserted the TrkB and the β-globin 5′ leaders into the intercistronic region of a promoterless dicistronic luciferase construct (a generous gift from Dr. Anne Willis ##REF##15998809##[31]##). Transfection of the promoterless dicistronic DNA into C6 cells yielded robust <italic>Photinus</italic> luciferase activity from the TrkB L1 5′ leader (##FIG##2##Fig. 3##); the P∶R ratio was 76 fold higher than that obtained from the dicistronic construct containing the β-globin 5′ leader. In addition, both Ex2 and L2 showed a ten-fold increase in the P∶R ratio. It was perhaps surprising that L2 and Ex2 exhibited cryptic promoter activity because a Northern blot analysis did not reveal additional RNA species (data not shown), although this incongruity has been observed previously ##REF##12370285##[32]##. These results indicate that all three TrkB 5′ leaders exhibit some level of cryptic promoter activity.</p>", "<title>The Mouse TrkB 5′ Leader Internally Initiates Translation from Dicistronic RNA</title>", "<p>To overcome the limitations of cryptic promoter activity (and cryptic splicing which we did not examine), we <italic>in vitro</italic> transcribed the dicistronic DNA. The resulting dicistronic mRNA was transfected into C6 cells. All three TrkB 5′ leaders exhibited a P∶R ratio higher than that observed from the dicistronic mRNA containing the β-globin 5′ leader (##FIG##3##Fig. 4##). The largest ratio of approximately six was seen with L2. Ex2 generated a P∶R ratio of four, and L1 showed the lowest ratio of approximately 2.5. This result demonstrates that all three mouse TrkB 5′ leaders can mediate IRES-dependent translation. The result also indicates that the presence of a cryptic promoter in a 5′ leader does not preclude its ability to internally initiate translation.</p>", "<title>Mouse TrkB 5′ Leaders Initiate Translation When Cap-Dependent Translation is Inhibited</title>", "<p>With a capped monocistronic RNA, it cannot be determined whether translation initiation is occurring in a cap-dependent manner or through an IRES. To dissociate between the two mechanisms, cap-dependent translation was inhibited <italic>ex vivo</italic>. We inserted the 5′ leaders upstream of the <italic>Photinus</italic> luciferase gene. The <italic>Renilla</italic> luciferase gene was contained on the same plasmid (pRM) under an independent promoter and served as an internal transfection control (##FIG##0##Fig 1b##). The multiple cloning site from the pGL3 plasmid (83 nt) was used as a negative control in this experiment because the Renilla gene on the pRM plasmid also utilizes this multiple cloning site as its 5′ leader. We have previously shown that the pGL3 multiple cloning site does not contain an IRES and exhibits a P∶R ratio equivalent to that obtained from a dicistronic DNA construct containing the β-globin 5′ leader ##REF##15908588##[16]##. The mouse TrkB and pGL3 DNA constructs were co-transfected into C6 cells with either a hypophosphorylated form of 4EBP ##REF##8521827##[33]## or a null vector. When expressed, the hypophosphorylated 4EBP sequesters eIF4E, inhibiting cap-dependent translation ##REF##15690031##[34]##. The percentage of luciferase activity remaining in the presence of the hypophosphorylated form of 4EBP when compared to that observed in the presence of the null vector was calculated. Translation of <italic>Renilla</italic> luciferase mRNA containing the pGL3 multiple cloning site served as the internal control to monitor cap-dependent translation.</p>", "<p>In the presence of hypophosphorylated 4EBP, the <italic>Renilla</italic> luciferase activity decreased to approximately 23–30% of the control level for all of the constructs. This result demonstrated that cap-dependent translation was being inhibited (##FIG##4##Fig 5a##). In addition, the level of <italic>Photinus</italic> luciferase activity generated from the mRNA containing the multiple cloning site from the vector pGL3 decreased to 28% of the control confirming that its translation was also cap-dependent. On the other hand, the <italic>Photinus</italic> luciferase mRNA containing the EMCV IRES decreased to 51% of the control transfection when cap-dependent translation was inhibited confirming that it does initiate translation through a cap-independent mechanism. The relatively large decrease in the overall level of translation from the mRNA containing the EMCV IRES when cap-dependent translation is shut down is believed to be due to the addition of a cap structure to the mRNA. This creates an artificial context for the EMCV 5′ leader and when uninhibited, allows for the cap structure to compete with the IRES for translational machinery. All three mRNAs containing the different mouse TrkB 5′ leaders showed a smaller reduction in activity compared to that observed from the pGL3 mRNA. Ex2 and L2 exhibited similar reductions of approximately 50%. L1 exhibited only a 15% decrease in <italic>Photinus</italic> luciferase activity in the presence of hypophosphorylated 4EBP. This result supports the conclusion that all three TrkB 5′ leaders can initiate translation independent of the cap. It also suggests that the contribution of cap- and IRES-dependent translation may vary for each leader.</p>", "<p>To further demonstrate the ability of the TrkB 5′ leaders to initiate cap-independent translation, <italic>in vitro</italic> transcribed mRNA containing the three mouse TrkB 5′ leaders or the β-globin 5′ leader upstream of the <italic>Photinus</italic> luciferase open reading frame was translated in rabbit reticulocyte lysate (RRL). Increasing concentrations of cap analog, which binds and sequesters eIF4E inhibiting cap-dependent translation, was added to the lysate. Indeed, the level of <italic>Photinus</italic> luciferase activity derived from the mRNA containing the β-globin 5′ leader was inversely proportional to the concentration of cap analog (##FIG##4##Fig 5b##). However, the level of translation from the mRNAs containing the mouse TrkB 5′ leaders was relatively stable irrespective of the cap analog concentration. These results demonstrate that the mRNA containing the TrkB 5′ leader can be translated in a cap-independent manner.</p>", "<title>The Mouse TrkB 5′ Leader Contains Multiple Contains Multiple IRESes</title>", "<p>Our results indicate that an IRES element is located within exon 2, which is present in both mouse TrkB 5′ leaders. To determine whether the unique upstream regions in each 5′ leader can also internally initiate translation, these regions were inserted into dicistronic RNA constructs and transfected into C6 cells. The unique region from Leader 2, L2U (104 nt, ##FIG##0##Fig 1a##), generated a P∶R ratio similar to that observed with the negative control β-globin (##FIG##5##Fig 6a##). However, the upstream sequence from Leader 1, Ex1 (1.084 kb), generated a P∶R ratio comparable to that observed from Ex2 indicating that Ex1 contains an IRES.</p>", "<p>To confirm the <italic>ex vivo</italic> results, we inserted Ex1 and Pr2U into monocistronic <italic>Photinus</italic> constructs and <italic>in vitro</italic> translated the mRNA (##FIG##5##Fig 6b##). As expected, translation from the L2U construct decreased in the presence of increasing amounts of cap analog similar to that observed from the mRNA containing the β-globin 5′ leader. On the other hand, translation from the Ex1 construct remained relatively constant. Taken together, the <italic>ex vivo</italic> and <italic>in vitro</italic> data demonstrate the ability of Ex1, in addition to Ex2, to internally initiate translation.</p>", "<p>Exon 1 of mouse TrkB can be alternatively spliced at three sites creating three different leaders, which all contain the first 259 nt of the exon (##FIG##0##Fig 1a##). To identify the region within Exon 1 that contains the IRES we created dicistronic constructs containing the individual segments, Ex1a, Ex1b, and Ex1c, or in contiguous pairs, Ex1ab and Ex1bc. Transfection of C6 cells with the dicistronic RNA showed that all 5′ leaders containing Ex1a generated a P∶R ratio higher than β-globin (##FIG##6##Fig 7##). However, Ex1b, Ex1c, and Ex1bc exhibited a P∶R ratio equivalent to that obtained from β-globin. These results indicate that the IRES element in Ex1 is located within Ex1a.</p>", "<title>PTB1 Protein Binds both Mouse TrkB IRESes</title>", "<p>PTB activates or enhances IRES activity from a number of cellular mRNAs ##REF##11063741##[6]##, ##REF##11313462##[9]##, ##REF##18631133##[35]##. As mentioned previously, unr and PTB bind to the Apaf-1 IRES, inducing conformational changes and allowing for internal initiation to occur ##REF##12667457##[5]##. PTB binds to the Apaf-1 IRES at two polypyrimidine tracts located 74 and 118 nucleotides from the initiator codon, respectively. Sequence comparison revealed that the mouse TrkB 5′ leader has two polypyrimidine tracts in similar locations of 75 and 124 nucleotides from the initiaton codon suggesting that PTB may also influence RNA secondary structure, and ultimately the IRES activity, of the mouse TrkB 5′ leader. To determine the ability of PTB1 to directly bind the Ex1a and Ex2 IRESes, we performed a filter binding assay. The cricket paralysis virus (CrPV) IRES was used as a negative control since it has been established that the CrPV IRES does not require protein factors to initiate translation ##REF##12470947##[36]##. Radiolabeled RNA consisting of Ex1a, Ex2, or CrPV was incubated in the presence of increasing amounts of recombinant PTB1 protein and passed through a dot blot apparatus. The resulting binding curve was fit using the Langmuir equation. As expected, the CrPV IRES did not bind to PTB1 with a significant affinity (##FIG##7##Fig 8##). However, Ex1a and Ex2 bound to PTB1 with K_d values equaling 85 nM and 46 nM, respectively. Although PTB1 binding to both IRESes falls within the same order of magnitude, the two-fold difference may reflect a biologically significant difference.</p>", "<title>The Two TrkB IRESes Are Differentially Regulated</title>", "<p>To address whether the binding of PTB1 plays an important role in Ex1a and Ex2 IRES activity, dicistronic RNA was <italic>in vitro</italic> translated in the presence of 0.4 µg of PTB1 protein isoform ##REF##2533575##[37]##. Somewhat surprisingly, no change in the P∶R ratio from Ex1a was observed when PTB1 was present despite the ability of PTB1 to bind to Ex1a (##FIG##8##Fig 9a##). Conversely, the P∶R ratio from the dicistronic mRNA containing Ex2 increased by 40% in the presence of PTB1. This result suggests that PTB1 stimulates Ex2 IRES activity and that PTB1 interacts with the mouse TrkB IRESes differently.</p>", "<p>Changes in cell state, such as mitosis ##REF##10882097##[12]##, inhibit cap-dependent translation and promote ITAF synthesis and IRES activity. Since the TrkB receptor contributes to different cellular functions in neural stem cells and neurons, we were interested in determining whether differentiation also affected IRES activity. To examine the effects of differentiation on the TrkB IRESes, we chose SH-SY5Y cells, a neuroblastoma that generates a differentiated neuronal phenotype when exposed to retinoic acid ##REF##11313462##[9]##. Dicistronic RNA containing Ex1a, Ex2, or the β-globin 5′ leader was transfected into SH-SY5Y cells that were either treated with DMSO (undifferentiated) or retinoic acid (differentiated) for four days (##FIG##8##Fig. 9b##). The dicistronic mRNA containing Ex1a exhibited a P∶R ratio approximately three-fold higher than β-globin in both the undifferentiated and differentiated cells. Surprisingly, in undifferentiated cells Ex2 did not demonstrate IRES activity, yielding a P∶R ratio similar to that of β-globin. However, in the differentiated cells the P∶R ratio of Ex2 increased to approximately two and a half fold that of β-globin. This result suggests that the Ex1a IRES is constitutively active is SH-SY5Y cells, while the Ex2 IRES is only active in differentiated SH-SY5Y cells.</p>", "<p>The ability of Ex2 to internally initiate translation in differentiated but not undifferentiated SH-SY5Y cells indicates the presence of a factor that is induced upon differentiation. Since we have already established that PTB1 affects the IRES activity from the mouse TrkB IRESes differentially, it suggests that PTB1 may be the factor responsible for the differential regulation observed in the SH-SY5Y cells. Indeed, differentiation of SH-SY5Y cells by retinoic acid leads to the induction of PTB1 ##REF##11313462##[9]##, but not the neural isoform, nPTB (data not shown). To investigate whether the presence of PTB1 affects TrkB IRES activity, PTB1 protein was knocked down in differentiated SH-SY5Y cells using siRNA. Western blot analysis revealed that the siRNA reduced PTB1 expression level by greater than 80% from that observed in untreated differentiated cells (##FIG##9##Fig. 10a##). Additionally, transfection of dicistronic RNA into the PTB1 depleted differentiated SH-SY5Y cells showed a decrease in Ex2 IRES activity, suggesting that PTB1 is required for internal initiation from the Ex2 IRES (##FIG##9##Fig. 10b##). The Ex1a IRES activity was not affected by the reduction in PTB1, confirming the differential regulation seen above.</p>" ]

[ "<title>Discussion</title>", "<p>In the present report, we demonstrate that the mouse TrkB 5′ leader contains two IRESes. One IRES is located within Ex2, a region similar to the region within the human TrkB 5′ leader found to exhibit IRES activity ##REF##15908588##[16]##. The other IRES is unique to the mouse TrkB 5′ leader and is found in the 5′ end of Exon 1. Moreover, we found that the two IRESes exhibit different characteristics. The IRES in Ex2 is active in differentiated SH-SY5Y cells, binds PTB1, and its activity is enhanced in the presence of PTB1. On the other hand, the IRES within Ex1a is active in both differentiated and undifferentiated SH-SY5Y cells, binds PTB1, but its activity is unaffected in the presence of PTB1. We also noted that the TrkB 5′ leaders contain a cryptic promoter, indicating that 5′ leaders can contain both a cryptic promoter, as well as an IRES.</p>", "<title>Limitations of Using Dicistronic DNA Constructs to Demonstrate IRES Activity</title>", "<p>Historically, dicistronic constructs have been used to examine 5′ leaders for the ability to internally initiate translation. However, the increased P∶R ratios that have been interpreted as the presence of an IRES can also be due to the presence of cryptic splice sites or cryptic promoters ##REF##12370285##[32]##. Removal of the promoter and intron from the dicistronic construct demonstrated that all three TrkB 5′ leaders exhibit some level of cryptic promoter activity. This result does not rule out the possibility that cryptic splicing occurs in the original dicistronic vector, and therefore, it is still possible that both processing events can occur and contribute to the second RNA species.</p>", "<p>Our results indicate that the presence of a cryptic promoter in a eukaryotic 5′ leader does not preclude the presence of an IRES. Indeed, it was previously reported that both an IRES and a cryptic promoter are present in the 5′ leader of hepatitis C virus (HCV) ##REF##12582247##[38]##. To more conclusively demonstrate IRES activity in cells, we transfected cells directly with RNA. This approach should reveal IRES activity exhibited by mRNA without subjecting it to nuclear processes. Indeed, aberrant RNA species following transfection of DNA constructs have not been observed when the corresponding RNA was transfected ##REF##15037781##[39]##. Using RNA transfections, all three mouse TrkB 5′ leaders demonstrated IRES activity. In addition, the TrkB 5′ leaders were able to initiate translation <italic>in vitro</italic> when cap-dependent translation was inhibited. Together, these data suggest that despite the presence of a cryptic promoter, the mouse TrkB 5′ leaders can internally initiate translation.</p>", "<title>Multiple IRESes Present in a Single mRNA</title>", "<p>The mouse TrkB mRNA is one of only a few eukaryotic mRNAs to contain two IRESes within the same mRNA. Transcription of the mouse TrkB gene from promoter 1 yields the 5′ leader 1 with IRESes located within Ex1 and Ex2. The IRES in Ex1 was further localized to the 5′ end, a region that is within all alternatively spliced variants. The rationale for multiple IRESes on the same 5′ leader is not known. It is possible that multiple IRESes work cooperatively to increase IRES activity. If this was occurring with the Ex1a and Ex2 IRESes, we predict the presence of both IRESes (L1) would generate higher IRES activity than either IRES alone. However, L1 exhibited a lower P∶R ratio than either Ex1a or Ex2 alone (##FIG##3##Fig 4##).</p>", "<p>A second possibility is that the two IRESes initiate at different initiator codons. This situation occurs with the two IRESes located within the c-myc mRNA. The upstream element, IRES 1, initiates translation of the MYCHEX1 open reading frame (ORF) while the downstream IRES 2 initiates the c-myc 1/ c-myc 2 ORF ##REF##11464293##[40]##. Additionally, two IRESes exist in the 5′ leader of the mRNA coding for endothelial growth factor (VEGF). They control initiation at two alternative start sites yielding a protein with a different N-terminus ##REF##11731620##[41]##, ##REF##9774635##[42]##. Interestingly, an upstream ORF (uORF) located between the two IRESes determines which IRES is utilized ##REF##18304943##[43]##. The mouse TrkB Exon 1 contains 15 potential uORFs that could also regulate the usage of the two TrkB IRESes. Only one uORF (−1048 nt in Ex1b) is in a moderately favorable Kozak context. It would therefore, be of interest to determine if the predicted 4.2 kD product encoded in Exon 1 is synthesized. We cannot identify at present, whether one or both IRESes is utilized in the full length L1 5′ leader. However, since all 5′ leaders containing one or two IRESes (regardless of the presence of uORFs) yield equivalent levels of luciferase protein it indicates that the major ORF is the <italic>Photinus</italic> luciferase ORF. This observation is in agreement with the identification of a single initiation start site in both the mouse and human TrkB genes ##REF##11798182##[27]##, ##REF##10395916##[28]##, and would be the first example of two unique IRESes being used to produce the identical protein.</p>", "<p>A third explanation is that the two IRESes are differentially employed. Multiple mechanisms may exist to ensure the presence and/or use of one IRES. For example, neural activity increases transcription mediated by promoter 2 generating an mRNA with only one IRES ##REF##12900419##[44]##. Second, the presence of RNA upstream of an IRES may inhibit its function ##REF##12757712##[45]##. In this case, the Ex1a IRES would be active in the full-length leader if the presence of the upstream sequence inhibits the Ex2 IRES. Finally, when both IRESes are present, IRES selection may be regulated by the presence of ITAFs. It has been well documented that IRESes are neither constitutively nor ubiquitously active. IRESes, including those in the mouse TrkB 5′ leaders, as well as human TrkB ##REF##15908588##[16]##, c-IAP1 ##REF##14970392##[46]##, Apaf-1 ##REF##12458215##[47]## and c-myc ##REF##10637319##[48]##, demonstrate varying activities in different cell lines, presumably due to differential expression of ITAFs. Indeed, we have shown that the two IRESes within the TrkB 5′ leader are differentially regulated within a cell line. This level of regulation is likely functional within primary neurons. For example, Ex2 IRES is only active in differentiated SH-SY5Y cells, a model for post-mitotic neurons that are capable of neural activity ##REF##9463428##[49]##. Neural activity in turn promotes the use of promoter 2 generating a TrkB 5′ leader containing only the Ex2 IRES ##REF##12900419##[44]##. This observation provides a link between the differential usage of the TrkB promoters and TrkB translation.</p>", "<p>At the molecular level, differential usage of the two TrkB IRESes may be regulated in part by PTB1. IRES activity <italic>in vitro</italic> mediated by the Ex2 IRES was increased in the presence of PTB1. In addition, Ex2 IRES activity correlates with the expression pattern of PTB1 within SH-SY5Y cells ##REF##11313462##[9]## and is decreased when PTB1 levels are significantly reduced. IRES activity mediated by Ex2 mirrors that of the Apaf-1 IRES in that it is also only active in differentiated SH-SY5Y cells and is regulated by PTB1 ##REF##11313462##[9]##.</p>", "<p>Binding of an accessory protein to an mRNA does not implicate the protein as an ITAF. <italic>In vitro</italic> translation assays and RNA transfections into SH-SY5Y cells demonstrate that although PTB1 binds to both Ex1a and Ex2, it only affects Ex2 IRES activity. Ex1a does not require PTB1 to internally initiate translation and the changes in the level of PTB1 does not alter Ex1a IRES activity, an observation seen previously for one of the IRESes in the VEGF 5′ leader ##REF##9774635##[42]##. It remains possible that PTB1 plays a role in enhancing Ex1a IRES activity in conjunction with additional factors that are absent from RRL and SH-SY5Y cells. The Apaf-1 and BAG-1 IRESes require the presence of unr and poly(rC) binding protein 1 respectively, in addition to PTB1, for IRES activity ##REF##12667457##[5]##, ##REF##12527772##[50]##.</p>", "<p>The position of the PTB1 site within the mRNA secondary structure can determine whether PTB1 binding can affect IRES activity. Therefore, the inability of PTB1 to enhance Ex1a IRES activity may be due to the context of the PTB1 binding sites. The Willis lab demonstrated that the PTB1 binding site, a CCU repeat, can only internally initiate translation when present within a stem structure and not as single stranded RNA ##REF##15998809##[31]##. Consequently, it would be predicted that potential PTB1 binding sites within Ex2 are in a double stranded conformation, while those within Ex1a would exist in a single stranded state.</p>", "<p>It is also possible that PTB1 binding to the TrkB mRNA occurs to mediate another mRNA processing event. PTB1, in addition to regulating internal initiation, also affects RNA splicing ##REF##2533575##[37]##. Since Exon 1 is alternatively spliced, it is possible that PTB1 is binding for that purpose. In addition, the assay was performed in isolation of other polypyrimidine binding proteins that may normally compete with PTB1 for the binding sites within TrkB and prevent the binding of PTB1.</p>", "<p>Differential use of the TrkB IRESes may be a mechanism to regulate protein expression levels. For instance, the selection of which IRES is utilized will alter the sequence and distance over which the ribosome must scan, in a similar manner to that predicted for ribosomes recruited to the cap structure. For example, a long G/C rich 5′ leader will impede ribosomal scanning and decrease the overall level of protein synthesis ##REF##15145049##[51]##. In the case of TrkB, the distance from the Ex1a IRES is over 1kb long, is G/C rich, and contains multiple upstream AUGs. Therefore, when less TrkB is required, initiation could occur from the Ex1a IRES. However, when TrkB upregulation is required to affect synaptic plasticity in response to neural activity, initiation could occur from the shorter, and now more prevalent Ex2 IRES. Consequently, the presence of multiple IRESes on a single 5′ leader may provide an additional mechanism to regulate protein synthesis.</p>" ]

[]

[ "<p>Conceived and designed the experiments: SLT LK. Performed the experiments: SLT JSP. Analyzed the data: SLT JSP JSK LK. Contributed reagents/materials/analysis tools: SLT JSP JSK. Wrote the paper: SLT LK.</p>", "<p>Current address: Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, United States of America</p>", "<p>A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5′ leaders (1428 nt and 448 nt), both of which include the common 3′ exon (Ex2, 344 nt). Dicistronic RNA transfections and <italic>in vitro</italic> translation of monocistronic RNA demonstrated that both full-length 5′ leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity <italic>in vitro</italic> and <italic>ex vivo</italic>, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5′ leader are differentially regulated, in part by PTB1.</p>" ]

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[ "<p>We would like to thank Tara Dobson for her technical assistance. We would like to acknowledge Dr. Vincent Poirel for his work on developing and optimizing transfection protocols used in this manuscript. We would also like to thank David Costantino for his technical assistance with the CrPV plasmid. Additionally, the authors would like to thank the University of Colorado Denver School of Medicine translation group for comments on the data and experimental design.</p>" ]

[ "<fig id=\"pone-0003242-g001\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g001</object-id><label>Figure 1</label><caption><title>The mouse TrkB 5′ leaders.</title><p>(A) Schematic representation of the gene structure of the mouse TrkB 5′ leader ##REF##10395916##[28]## and variations used for luciferase assays (B) as well as the two controls, the β-globin 5′ leader and EMCV IRES. The boxes represent exons, lines represent introns, and the blue arrows indicate the transcriptional start site of the two promoters.</p></caption></fig>", "<fig id=\"pone-0003242-g002\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g002</object-id><label>Figure 2</label><caption><title>The mouse TrkB 5′ leaders exhibit an increased <italic>Photinus</italic> to <italic>Renilla</italic> luciferase (P∶R) ratio.</title><p>Dicistronic luciferase constructs containing the β-globin, EMCV, and TrkB 5′ leaders were transfected into the C6 and N2a neural cell lines. The P∶R ratio from each construct was normalized to that obtained from the β-globin construct, whose P∶R ratio was set to one.</p></caption></fig>", "<fig id=\"pone-0003242-g003\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g003</object-id><label>Figure 3</label><caption><title>The mouse TrkB 5′ leaders exhibit cryptic promoter activity.</title><p>Promoterless dicistronic luciferase constructs containing the β-globin or the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio was normalized to that obtained from construct containing the β-globin 5′ leader.</p></caption></fig>", "<fig id=\"pone-0003242-g004\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g004</object-id><label>Figure 4</label><caption><title>The mouse TrkB 5′ leaders exhibit IRES activity when expressed in dicistronic RNA constructs.</title><p>Dicistronic luciferase mRNA containing the β-globin and the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio for each construct was normalized to that of the negative control, β-globin.</p></caption></fig>", "<fig id=\"pone-0003242-g005\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g005</object-id><label>Figure 5</label><caption><title>The mouse TrkB 5′ leaders are able to initiate translation when cap-dependent translation is inhibited.</title><p>A) A dual monocistronic construct containing the pGL3 multiple cloning site upstream of the <italic>Renilla</italic> luciferase and the pGL3 multiple cloning site, EMCV, or the TrkB 5′ leaders upstream of the <italic>Photinus</italic> luciferase gene was co-transfected into C6 cells with a construct encoding for a hypophosphorylated 4EBP construct or a null vector. The level of <italic>Photinus</italic> luciferase activity from each mRNA, when co-transfected with the null vector, was normalized to 100 percent. The <italic>Renilla</italic> and <italic>Photinus</italic> luciferase activity obtained in cells co-transfected with hypophosphorylated 4EBP is represented as a percentage of the activity obtained in cells co-transfected with the null plasmid. B) Monocistronic <italic>Photinus</italic> luciferase mRNA containing the β-globin or TrkB 5′ leaders was <italic>in vitro</italic> translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. <italic>Photinus</italic> luciferase activity for each mRNA in the absence of cap analog was set to 100 percent.</p></caption></fig>", "<fig id=\"pone-0003242-g006\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g006</object-id><label>Figure 6</label><caption><title>The mouse TrkB mRNA contains two independent IRESes.</title><p>A) The additional sequence upstream of Exon 2 from both full-length leaders (Ex1 and L2U) and Exon 2 alone (see ##FIG##0##Fig 1a##) were individually inserted into dicistronic RNA vectors and transfected into C6 cells. The resulting P∶R ratios were normalized to the ratio observed from the mRNA containing the β-globin 5′ leader. B) The unique regions of the full-length TrkB 5′ leaders, as well as the β-globin 5′ leader were inserted upstream of a monocistronic <italic>Photinus</italic> luciferase open reading frame and <italic>in vitro</italic> translated in the presence of increasing concentrations of cap analog. The initial level of <italic>Photinus</italic> luciferase activity was set to 100 percent for each mRNA.</p></caption></fig>", "<fig id=\"pone-0003242-g007\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g007</object-id><label>Figure 7</label><caption><title>The upstream IRES in the mouse TrkB 5′ leader is located within Ex1a.</title><p>The exon 1 splice variants (shown in the schematic, see also ##FIG##0##Fig 1A##) were inserted into dicistronic RNA vectors and transfected into C6 cells. The resulting P∶R ratios were normalized to the ratio observed from β-globin 5′ leader.</p></caption></fig>", "<fig id=\"pone-0003242-g008\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g008</object-id><label>Figure 8</label><caption><title>The mouse TrkB 5′ leaders bind PTB1 protein.</title><p>Purified PTB1 was added in increasing amounts to radiolabeled RNA containing Ex1a, Ex2, or the negative control CrPV IRES. A Langmuir plot was created using the calculated fraction bound. Disassociation constants of 85 nM and 46 nM were determined for the PTB1 interaction with Ex1a and Ex2, respectively.</p></caption></fig>", "<fig id=\"pone-0003242-g009\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g009</object-id><label>Figure 9</label><caption><title>PTB increases IRES activity from the Ex2 IRES, but does not affect Ex1a IRES activity.</title><p>A) Dicistronic luciferase mRNA containing the Ex1a, Ex2, or β-globin 5′ leader was <italic>in vitro</italic> translated in RRL that was either untreated or supplemented with 0.4 mg of PTB1. The change in the P∶R ratio for the samples when in the presence of PTB1 relative to the untreated sample are shown. B) The two mouse TrkB IRESes demonstrate differential regulation. SH-SY5Y cells treated with 2 mM retinoic acid or with DMSO (mock) for four days were transfected with dicistronic mRNA containing the β-globin, Ex2, or Ex1a 5′ leaders. The P∶R ratios were normalized to that from the mRNA containing the β-globin 5′ leader.</p></caption></fig>", "<fig id=\"pone-0003242-g010\" position=\"float\"><object-id pub-id-type=\"doi\">10.1371/journal.pone.0003242.g010</object-id><label>Figure 10</label><caption><title>PTB expression is required for IRES activity mediated by Ex2.</title><p>A) Western blot analysis of PTB and GAPDH (as a loading control) from lysates obtained from differentiated and undifferentiated SH-SY5Y cells transfected with siRNA directed against PTB or mock transfected for 72 hours. B) Dicistronic RNA containing the two mouse TrkB IRESes were transfected into differentiated SH-SY5Y cells depleted of PTB1 by siRNA. The P∶R ratios were normalized to that from the mRNA containing the β-globin 5′ leader in control undifferentiated SH-SY5Y cells.</p></caption></fig>" ]

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[ "<fn-group><fn fn-type=\"COI-statement\"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type=\"financial-disclosure\"><p><bold>Funding: </bold>NIH grant AG028156</p></fn></fn-group>" ]

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[ "<title>Introduction</title>", "<p>Myosin filaments (also known as thick filaments), which interact with actin filaments in muscle to produce force and movement, are assemblies of myosin molecules and accessory proteins. Much is known about their 3D structure, but high resolution information is hard to obtain. Mutations in cardiac muscle myosin and its associated proteins (e.g. C-protein; myosin binding protein C (MyBP-C)) are known to be associated with a number of myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) (<xref rid=\"bib58 bib59 bib41 bib52\" ref-type=\"bibr\">Watkins et al., 1992, 1995; Seidman and Seidman, 2001; Tajsharghi et al., 2003</xref>). To understand how heart muscle normally works and how the known mutations affect contractility it is essential to understand the structure and properties of normal mammalian cardiac myosin filaments.</p>", "<p>Myosin molecules comprise two heavy chains and four light chains. Parts of the heavy chains twist together to form a 1500-Å long coiled-coil α-helical rod-shaped tail domain on one end of which are two elongated globular myosin heads (crossbridges) formed by the rest of the heavy chains together with the light chains. The way in which myosin molecules and accessory proteins associate to form a myosin filament can vary depending on the type of muscle. Generally the tails pack together, often in a helical arrangement, to form the filament backbone from which the heads project. The heads are ATPases that interact with actin filaments to produce force and movement (##REF##14064165##Huxley, 1963##). In vertebrate striated muscle the myosin molecules pack together into bipolar myosin filaments with the rods forming a roughly cylindrical filament backbone and the myosin heads arranged in a quasi-helical array on the filament surface. Halfway along each filament, where the rod packing is anti-parallel, there is a head-free region known as the bare-zone or M-region (<xref rid=\"bib19 bib42 bib43\" ref-type=\"bibr\">Huxley, 1963; Sjostrom and Squire, 1977a, 1977b</xref>). The first myosin heads in each half A-band are at the edges of the M-region. The backbone also has on its surface additional non-myosin protein components including C-protein (MyBP-C; ##REF##4269687##Offer et al., 1973##); or possibly its analogue X-protein (##REF##3543050##Bennett et al., 1986##) and the A-band part of titin (<xref rid=\"bib31 bib53\" ref-type=\"bibr\">Labeit and Kolmerer, 1995; Trinick, 1996</xref>).</p>", "<p>The head arrangement on vertebrate striated muscle myosin filaments approximates to a 3-stranded right-handed helix where each strand possesses nine subunits (pairs of myosin heads) per turn and a pitch of 1287 Å. Since the filament is 3-stranded, the axial repeat is reduced from 1287 Å to 430 Å. The pairs of heads from three myosin molecules project from the backbone at regular intervals to form a so-called ‘crown’ of heads, with successive crowns separated axially by roughly 143 Å (##REF##4567398##Squire, 1972##). The angle between each of the three head pairs on one crown is 120°. Each 430 Å repeat contains three such crowns (<xref rid=\"bib45 bib46 bib47 bib16 bib50 bib9 bib28\" ref-type=\"bibr\">Squire, 1972, 1973, 1974; Harford and Squire, 1986; Stewart and Kensler, 1986; Cantino and Squire, 1986; Kensler and Stewart, 1989</xref>). Vertebrate striated muscle myosin filaments are characterised by a systematic departure from perfect helical symmetry, known as a perturbation. This was originally identified by the presence of ‘forbidden’ meridional reflections (i.e. X-ray meridional reflections seen at orders of the 430 Å repeat other than multiples of 3) in the X-ray diffraction patterns from relaxed vertebrate skeletal muscles (<xref rid=\"bib20 bib48 bib16\" ref-type=\"bibr\">Huxley and Brown, 1967; Squire et al., 1982; Harford and Squire, 1986</xref>).</p>", "<p>Electron microscopic analysis of vertebrate striated muscle myosin filaments has included 2D analysis of images of negatively stained myosin filaments from fish, frog, chicken, and rabbit skeletal muscles (<xref rid=\"bib27 bib28 bib29 bib50 bib30\" ref-type=\"bibr\">Kensler and Stewart, 1983, 1989, 1993; Stewart and Kensler, 1986; Kensler and Woodhead, 1995</xref>), shadowed, freeze-fractured myosin filaments from frog skeletal muscle (##REF##3484742##Cantino and Squire, 1986##), and myosin filaments from freeze-substituted frog skeletal muscle (##REF##1453458##Craig et al., 1992##). These studies have shown the presence of the perturbation in 3-stranded crossbridge arrangement in all these species, consistent with the perturbation first described by ##REF##5586931##Huxley and Brown (1967)##, but could not definitively demonstrate the nature of the perturbation. Myosin filaments in vertebrate skeletal muscles have also been studied in 3D (<xref rid=\"bib50 bib15 bib4 bib5\" ref-type=\"bibr\">Stewart and Kensler, 1986; Eakins et al., 2002; AL-Khayat and Squire, 2006; AL-Khayat et al., 2006</xref>), but only recently has single particle analysis been used (<xref rid=\"bib3 bib5\" ref-type=\"bibr\">AL-Khayat et al., 2004, 2006</xref>).</p>", "<p>Myosin filament 3D structure has also been extensively studied in several well-ordered invertebrate striated muscle tissues such as insect flight muscle, tarantula, <italic>Limulus</italic>, scorpion and scallop (<xref rid=\"bib12 bib51 bib35 bib57 bib37 bib2\" ref-type=\"bibr\">Crowther et al., 1985; Stewart et al., 1985; Morris et al., 1991; Vibert, 1992; Offer et al., 2000; AL-Khayat et al., 2003</xref>). In these cases helical reconstruction by the Fourier-Bessel approach has usually been applied, since the myosin head arrays in these muscles appear to be helical. The exception is the ground-breaking study by ##REF##16121187##Woodhead et al. (2005)## who applied single particle analysis and helical averaging to cryo-EM images of tarantula myosin filaments and achieved a very detailed 3D reconstruction. We have also applied the single particle approach to other helically ordered myosin filaments from both insect and scallop invertebrate striated muscles (<xref rid=\"bib6 bib7\" ref-type=\"bibr\">AL-Khayat et al., 2008a, b</xref>).</p>", "<p>Recent 3D-information about the structure of the vertebrate striated muscle myosin filament from fish skeletal muscle has been achieved both by modelling of the X-ray diffraction data (<xref rid=\"bib18 bib4\" ref-type=\"bibr\">Hudson et al., 1997; AL-Khayat and Squire, 2006</xref>) and by single particle analysis of EM images (##REF##16731006##AL-Khayat et al., 2006##). Both these studies have provided better structural detail than had been obtained previously (##REF##12064942##Eakins et al., 2002##) and they started to quantify for the first time the perturbation of the crossbridge array in the fish myosin filament. This work, together with the earlier 3D reconstruction of isolated frog skeletal muscle myosin filaments (##REF##3495665##Stewart and Kensler, 1986##), has provided significant information about the crossbridge arrangement in the vertebrate skeletal muscle myosin filament. However, less is known about the structure of myosin filaments in vertebrate heart muscles. Although 2D-analysis of isolated cardiac thick filaments has been performed (<xref rid=\"bib23 bib24 bib25 bib26\" ref-type=\"bibr\">Kensler, 2002, 2005a, 2005b; Kensler and Harris, 2008</xref>), a 3D reconstruction of the structure of the cardiac muscle myosin filament has not been performed.</p>", "<p>In the present work, the 3D structure of myosin filaments isolated from normal mammalian rabbit cardiac muscle has been studied. Using the technique of single particle analysis of non-helical filamentous systems (<xref rid=\"bib39 bib3 bib5\" ref-type=\"bibr\">Paul et al., 2004; AL-Khayat et al., 2004; AL-Khayat et al., 2006</xref>) we produced a 3D reconstruction at about 40 Å resolution of the head arrangement in the C-zone region of relaxed rabbit cardiac myosin filaments. The results are very similar to the published structure of the vertebrate skeletal (fish) muscle myosin filament from EM 3D reconstruction (##REF##16731006##AL-Khayat et al., 2006##) and from X-ray diffraction modelling (##REF##16884926##AL-Khayat and Squire, 2006##), possibly suggesting a common structural theme for vertebrate striated muscle myosin filaments (cardiac and skeletal). However, for the first time, we show evidence that at low radius from the filament axis, the myosin heads origins are closer to being on a perfect helix than at high radius and that especially the azimuthal perturbation becomes more marked as the radius increases. This new structure serves as a starting point from which to understand the effects of the mutations in myosin and C-protein (MyBP-C) associated with different cardiomyopathies.</p>" ]

[ "<title>Materials and methods</title>", "<title>Electron microscopy</title>", "<p>Myosin filaments were isolated from rabbit ventricular muscle in relaxing solution as described by <xref rid=\"bib23 bib24\" ref-type=\"bibr\">Kensler (2002, 2005a)</xref>. The isolated myosin filaments were applied to a thin carbon film support on holey carbon grids and negatively stained. Only filaments lying over holes were used in the current analysis. Electron micrographs were collected under minimal dose conditions on a JEOL 1200 electron microscope at 40 K magnification.</p>", "<title>Preparation of images for single particle analysis</title>", "<p>A total of 52 electron micrographs were digitised using a Nikon Super Coolscan 8000 16-bits per colour pixel scanner at a step size of 6.35 μm/pixel (equivalent to 1.58 Å sampling in the specimen). Digitised images for the whole micrographs were saved on the PC in TIFF format and were then transferred to a Linux UNIX operating system PC running the IMAGIC suite of programs (##UREF##8##van Heel et al., 2000##) to analyse them. Initially, they were converted to floating point IMAGIC format using EM2EM. The resulting images were then binned by a factor of 4 in both the <italic>x</italic> and <italic>y</italic> directions, resulting in a final step size of 6.35 Å/pixel and converted to MRC format for pre-processing using the MRC suite of programs (##REF##8742717##Crowther et al., 1996##) and also using locally developed software. Regions were selected which contained intact half-filaments which were relatively straight, not overlapped by other actin and myosin filaments, and had readily identifiable bare-zones (##FIG##0##Fig. 1##(A)). Location of the bare-zone was essential to properly deduce the location of the C-protein stripes. The area selected on either side of each half-filament was also required to have as little background as possible (##FIG##0##Fig. 1##(B)), so as to reduce noise in the calculated Fourier transforms (##FIG##0##Fig. 1##(C)). Images of whole myosin filaments were cut into two halves with the whole bare-zone (M-band) included in each half-filament. In order to preserve polarity in the processing, half-filaments (i.e. from the M-band to the pointed end of the myosin filament) were then rotated to make each filament image vertical and oriented with its bare-zone (M-band) region at the bottom (##FIG##0##Fig. 1##(B)).</p>", "<p>From the 52 available micrographs and using the above selection criteria, 153 half-filaments were identified. Half-filament images were floated in 2048 square arrays and their Fourier transforms computed (##FIG##0##Fig. 1##(C)). The sixth order of the 430 Å repeat, the 71.5 Å meridional reflection, which was strong in most computed Fourier transforms, was used to calibrate the magnification and to adjust the sampling of each half-filament from all the different micrographs to be exactly 7.54 Å/pixel. The majority of the Fourier transforms for the filaments showed up to the 11th order of the 430 Å repeat corresponding to 39 Å resolution (the titin sub-repeat; ##FIG##0##Fig. 1##(C)). The correctly scaled half-filament images, in MRC format and with the pixel size accurately scaled to 7.54 Å/pixel, were then read again into IMAGIC and converted back to IMAGIC format using the EM2EM command.</p>", "<p>All the further single particle image analysis was carried out within IMAGIC. The modified exact filter method for back-projection described in ##REF##15477103##Paul et al. (2004)## was used for calculating the 3D reconstruction. This allows the thickness of the central section to be adjusted taking into account the fact that the diameter of the filament is less than the size of the cube. 3D structures were visualised with both IMAGIC and PyMOL (##UREF##3##DeLano, 2002##).</p>" ]

[ "<title>Results</title>", "<title>Selection of myosin filament segments</title>", "<p>##FIG##0##Fig. 1##(A) shows a typical micrograph of negatively stained isolated rabbit cardiac myosin filaments that contain good detail and from which half length myosin filaments were selected as shown in ##FIG##0##Fig. 1##(B). As previously reported (<xref rid=\"bib23 bib24\" ref-type=\"bibr\">Kensler, 2002, 2005a</xref>), well-preserved rabbit cardiac muscle myosin filaments, which are bipolar, have regular myosin head arrays in each half-filament with clear bare-zones (M-regions) halfway along. M-band protein density was sometimes visible in the middle of the M-region. The filament Fourier transforms showed meridional peaks out to the 11th order of 430 Å at 39 Å (##FIG##0##Fig. 1##(C)). Our aim in this study was to produce a 3D reconstruction of the structure of the myosin filament from only within the C-zone area (<xref rid=\"bib42 bib43\" ref-type=\"bibr\">Sjostrom and Squire, 1977a, 1977b</xref>). This should result in a closer representation of the C-protein distribution in the final 3D structure than has been achieved before (##REF##16731006##AL-Khayat et al., 2006##). Previously particles were selected from the whole of the half-filaments and thus included data from the P-zone and D-zone regions of the A-band as well as the C-zone (##FIG##1##Fig. 2##(A)).</p>", "<title>Locating C-protein along the filaments</title>", "<p>To locate the C-zone, 1D density profiles were calculated for each of the 153 individual half-filaments examined. These half-filaments ranged in length from 6000 to 7000 Å. Their 1D profiles were aligned together by cross-correlation using a program especially developed for this purpose, Cross-Corr (Knupp, C. unpublished). The Cross-Corr program was used to align and sum these 1D profiles to give averages in order to locate precisely the positions of C-protein. Seven stripes of higher density were observed. The first particle was taken to start at a distance 2040 Å from the middle of the M-band (##FIG##1##Fig. 2##(A)) and to include at its centre the first C-protein line, C3, reported by <xref rid=\"bib42 bib43\" ref-type=\"bibr\">Sjostrom and Squire (1977a, 1977b)</xref> and ##REF##3543050##Bennett et al. (1986; they called it C5)##. Beyond this, six further strong peaks were seen separated by approximately 430 Å. These are referred to as C6, C9, C12, C15, C18 and C21 as in <xref rid=\"bib42 bib43\" ref-type=\"bibr\">Sjostrom and Squire (1977a, 1977b)</xref>.</p>", "<title>Particle selection, alignment, classification and 3D reconstruction</title>", "<p>From a dataset of 153 half-filaments, 802 boxed square segments, each of length 128 pixels (equivalent to 7.54 Å/pixel × 128 pixels = 965 Å) containing just over 2 × 430 Å repeats (just over six 143 Å-myosin crown levels) were selected by stepping the box along the half-filaments at intervals of 430 Å (##FIG##1##Fig. 2##(A)). The chosen box length was used as a compromise between the easier alignment of a longer particle and a better compensation of the effects of any filament bending or distortion obtained with a shorter particle. Up to seven particles were selected from each half-filament to ensure that they only include the C-zone area.</p>", "<p>Since the three crown levels within the 430 Å repeats were different, it was essential to make sure that the selected particles were in proper axial register. The aim was to align all the segments and classify them so that each class will be the same structure but rotated about the filament long axis. The simplifying assumption was made that in the main part of the bridge regions of vertebrate myosin filaments within the C-zone area, the 430 Å repeats are all the same. In order not to mix the different crowns within a 430 Å repeat, the alignment was checked thoroughly by calculating the 1D density profiles for the sum of the particles that came from each of the 153 individual half-filaments. A typical example of the sum of particles within a half-filament is shown in ##FIG##1##Fig. 2##(D) and its corresponding 1D profile is similar to that shown in ##FIG##2##Fig. 3##(E). The 1D density profiles for the sum of particles of each of the 153 half-filaments were then aligned together and the positions of the peaks calculated. The shift values needed to superimpose similar peaks were found for each half-filament. The sums of the shifted particles within each individual half-filament, together with their corresponding 1D profiles, were then re-calculated to check that they were all properly aligned.</p>", "<p>A circular mask was applied to the dataset of 802 images and these were then rotationally and translationally aligned to an initial reference corresponding to the average sum of all the particles. Since the selected filament images were already quite accurately rotationally and translationally aligned, the angular range in the alignment was restricted to ±15° in order to preserve the polarity of the filaments. Also, the axial shift along the filament long axis was restricted to be between −50 Å and +50 Å to prevent the crown levels getting out of step. The aligned images were then classified by Multi-variate Statistical Analysis (MSA) (##UREF##8##van Heel et al., 2000##) to group those particles corresponding to the same view.</p>", "<title>Angular assignment and class refinement</title>", "<p>Because the aligned filament segments are likely to be related by rotation about a single axis (the filament long axis) with little out-of-plane rotation normal to this axis, <italic>ab initio</italic> angular assignment by standard angular reconstitution is not possible (##REF##15477103##Paul et al., 2004##). Therefore the approach adopted here (as suggested by ##REF##15477103##Paul et al. (2004)##) was based on using a starting reference 3D model. The starting model was used to create an ‘anchor set’ of 2D re-projections as a reference from which to assign projection angles to class averages derived from the individual segments. By convention, these projection angles are defined by the three Euler angles <italic>α</italic>, <italic>β</italic> and <italic>γ</italic>. <italic>α</italic> is the rotation angle in the plane of the image (i.e. the plane of the EM grid), <italic>β</italic> is the out-of-plane tilt angle (to allow for the fact that the filaments may not lie perfectly flat on the grid or the grid itself may not be flat) and <italic>γ</italic> is the rotation angle around the filament long axis. We had already developed and implemented the same procedure in the previous study on the fish skeletal myosin filaments (##REF##16731006##AL-Khayat et al., 2006##). In that work a number of strategies were used to test the uniqueness of the final 3D reconstruction. These included using various different starting 3D models to generate the anchor sets so as to avoid any model bias, using a totally independent angular assignment method where no starting model was required (##REF##15533440##Patwardhan et al., 2004##), and using different software, EMAN (##REF##10600563##Ludtke et al., 1999##). All these different methods led to essentially the same final 3D reconstruction, giving us confidence in the methodology that we were using. In the present study, we therefore used a single first reference 3D model to generate an anchor set of 2D projections to use for angle assignment (Euler angles) for each of the class averages. This reference model was generated by calculating a 3D reconstruction of a single class average with Euler angles <italic>α</italic>, <italic>β</italic> and <italic>γ</italic> of 0°, 90°, and 0°, respectively, assigned to it as its angles of view and imposing 3-fold symmetry around the long axis. The anchor set produced from this reference 3D map was used to provide a first set of Euler angles for all the class averages. An improved 3D reconstruction could then be calculated. 2D re-projections of this new 3D map were used to make a new set of reference images with which to re-align the original raw image dataset of the 802 particles using Multi-Reference Alignment (MRA), to re-classify them and to carry out a further process of angular reconstitution/assignment. This procedure was repeated with the class images gradually improving, and refinement was stopped when no further significant change was observed and the assigned angles became stable.</p>", "<title>The final 3D reconstruction</title>", "<p>The final 3D reconstruction (##FIG##2##Fig. 3##(A–D); M-band direction downwards) was calculated with imposed C3 symmetry using the best 17 class averages containing a total of 301 particles and using the modified weighted back-projection method of ##REF##15477103##Paul et al. (2004)##. According to the Fourier Shell Correlation (##FIG##2##Fig. 3##(H)), the resolution of the reconstruction lies between 40 Å (0.5 bit criterion, ##REF##16125414##van Heel and Schatz, 2005##) and 48 Å (0.5 correlation coefficient criterion), and the 3D image was filtered to include frequencies from 300 Å up to 40 Å resolution. The contour threshold used to show these images corresponded to the theoretical volume of the myosin molecules within the length represented (9 whole molecules/430 Å repeat) plus an additional 15% to account for non-myosin proteins (titin and C-protein) present in the myosin filament. The reconstruction shows slightly more than two full 430 Å repeats of the myosin filament and the two successive 430 Å repeats are substantially similar to each other. Although these regions contain some overlapping image data, they were not averaged together during the analysis and the structural similarity of the successive 430 Å repeats can be taken as support for the validity of the reconstruction.</p>", "<p>Four views related by a 30° rotation around the filament axis are included in ##FIG##2##Fig. 3##(A–D) to illustrate the different crown structures as well as their angular perturbation from helical symmetry. Surface density features in the 3D map, presumably mainly myosin heads, can be seen following a perturbed helical path. There are three levels of myosin heads within each 430 Å repeat, but they are not identical. On levels labelled 1 and 3, the projecting masses appear to have approximately the same azimuthal positions, whereas those on levels labelled 2 are at quite a different azimuth. The overall appearance suggests that there is on average a large departure from helicity in the azimuthal positions of the crown densities from the constant 40° angular rotation expected from ideal helical symmetry.</p>", "<p>The azimuthal and axial perturbations from helical symmetry are further demonstrated in ##FIG##2##Fig. 3##(G) which shows the density in circumferential sections (like a radial net) through the 3D reconstruction at radii of 90, 110, 130 and 150 Å from the filament axis. For comparison, the circumferential section at a radius of 110 Å from the filament axis in the EM reconstruction of fish skeletal muscle myosin filaments (##REF##16731006##AL-Khayat et al., 2006##) is shown as ##FIG##2##Fig. 3##(F) where the same leftwards shift of mass on level 1 is seen.</p>", "<p>Analysis of the positions of centres of mass on each crown in ##FIG##2##Fig. 3##(G) reveals an interesting feature. ##FIG##3##Fig. 4##(A and B) show how the azimuthal and axial perturbations vary with radius. The azimuthal and axial displacements of density peaks on crowns 1 and 3 relative to those on crown 2 are plotted as a function of radius between 90 and 150 Å. ##FIG##3##Fig. 4##(A) shows that there is a very clear trend for the angular separation of peaks on crowns 1 and 3 relative to crown 2 to tend towards the 40° expected for a perfect helix as the radius reduces. In ##FIG##3##Fig. 4##(B) for the axial perturbations it is also conceivable that the axial shift is tending towards 143 Å at low radius between levels 2 and 3 as indicated by the extrapolated yellow lines. However, what happens between levels 1 and 2 is less clear cut.</p>", "<p>In the present reconstruction, as in the fish muscle myosin filament (##FIG##2##Fig. 3##(F)), additional density which may be C-protein is associated with crown 1. However, there also appears to be extra density running from crown 1 down to the adjacent crown 3 (##FIG##2##Fig. 3##(G)) which could also correspond to C-protein, titin, or density associated with the myosin rods in the backbone.</p>" ]

[ "<title>Discussion</title>", "<title>Single particle analysis of vertebrate mammalian cardiac myosin filament structure</title>", "<p>The technique of single particle analysis of non-helical filamentous systems involving the creation of particles by dividing the myosin filaments into segments has already been applied to produce a 3D reconstruction of the myosin head arrangement in relaxed vertebrate striated (fish) skeletal muscle (<xref rid=\"bib3 bib5\" ref-type=\"bibr\">AL-Khayat et al., 2004, 2006</xref>). These filaments deviate from ideal helical symmetry and their structure can not be accurately determined by traditional Fourier-based helical 3D reconstruction. Our new approach has allowed the 3D analysis of EM images of these non-helical filaments without invoking helical symmetry (<xref rid=\"bib3 bib5\" ref-type=\"bibr\">AL-Khayat et al., 2004, 2006</xref>). The method can preserve perturbations in the myosin head array within the 430 Å repeat length which are otherwise averaged out in helical reconstructions.</p>", "<p>The new approach was originally applied to the 3D structure of myosin filaments in vertebrate skeletal muscle, but until the present work it had not been applied to the medically important 3D structure of myosin filaments in vertebrate heart muscles. The current analysis not only had the benefit of extending the work from vertebrate skeletal to cardiac myosin filaments, but was also an improvement in that the analysis was carried out using only the C-zone part of the filament. Studying cardiac myosin filament structure in the normal state is an essential starting point from which to understand the mechanisms of the diseased system, in particular the effects of the mutations in myosin and its accessory proteins such as C-protein (MyBP-C) associated with different cardiomyopathies.</p>", "<p>Good evidence that our current 3D map is a reliable structure is that it helps to explain the features observed in the myosin filament images determined by 2D filtration by ##REF##15721584##Kensler (2005a)## using the same images that were used in our current single particle analysis. ##FIG##4##Fig. 5##(I–L) shows projected views of our 3D map which reproduce the features seen in the filament images of ##REF##15721584##Kensler (2005a)##. In particular what was described as the “comma-shaped” heads on level 2 (boxed density in ##FIG##2##Fig. 3##(B) in ##REF##15721584##Kensler, 2005a##) is now seen as a projecting density which is curved upwards in the 3D map (##FIG##2##Fig. 3##(A–D)). The comma-shape feature is also boxed in ##FIG##4##Fig. 5##(J and L)). The projected views of the 3D map are also consistent with the “saw-tooth” pattern of myosin head densities described by ##REF##15721584##Kensler (2005a)## (labelled by red lines in ##FIG##3##Fig. 4##(B) in ##REF##15721584##Kensler, 2005a##) and clearly demonstrated in the 2D re-projection of the current 3D map (##FIG##4##Fig. 5##(I–L)). It is now evident that this “saw-tooth” pattern observed in both fish skeletal and rabbit cardiac myosin filaments is due to both the 3-stranded arrangement of the heads as well as the azimuthal perturbation inherent in both filaments.</p>", "<title>Axial and azimuthal crown perturbations</title>", "<p>Comparison of our new map for rabbit cardiac muscle myosin filaments with that for fish skeletal muscle myosin filaments (##REF##16731006##AL-Khayat et al. 2006##; ##FIG##3##Fig. 4##) shows that the angular appearance of the single particle 3D reconstructions and the clear axial and azimuthal perturbations are common to both filament types. In both cases levels 1 and 3 have heads pointing on average in roughly the same azimuthal directions compared with level 2 which in both cases is rotated by roughly 60° compare ##FIG##2##Fig. 3##(F) with ##FIG##2##Fig. 3##(G; 110 Å). The radial perturbation is minor in both the fish and rabbit myosin filaments. Comparison between the angles and axial spacings/separations between the three levels in both fish skeletal and the current rabbit heart myosin filaments are shown in ##TAB##0##Table 1##. It can be seen that the angular perturbation in rabbit is slightly larger than in fish. It can also be seen that the angular separations are closer to 60° in rabbit compared to the corresponding ones in fish. This could be due to the current C-zone analysis allowing better resolution of the angles than those in fish. Moreover, the axial spacing between levels 2 and 1 is lower in rabbit (−134.4 Å) compared to that in fish (−149.0 Å) whereas the spacing is larger between levels 2 and 3 in rabbit (154.0 Å) compared to fish (135.0 Å).</p>", "<p>The axial perturbations measured for rabbit cardiac myosin filaments by ##REF##15721584##Kensler (2005a)## between what we define as levels 2 and 1, and 2 and 3 were −132.9 Å and 153.0 Å, respectively, as measured from six filtered images. [Note that Fig. 4(E) in ##REF##15721584##Kensler (2005a)## referred to our levels 1, 2 and 3 as 2, 3 and 1, respectively. His level 0 in Fig. 4(E) (##REF##15721584##Kensler, 2005a##) is our level 2.] Our new values based on the average of data plotted in ##FIG##3##Fig. 4## are very similar at −134.4 Å and 154.0 Å, respectively (##TAB##0##Table 1##).</p>", "<title>Location of non-myosin protein densities</title>", "<p>There is evidence from the earlier work on fish skeletal myosin filaments that the main C- (or X-) protein density is located at level 1. A similar conclusion can be reached for the rabbit cardiac myosin filaments (##FIG##2##Fig. 3##(E)) where the strongest projected density is also at level 1. A further important observation in ##REF##16731006##AL-Khayat et al. (2006)## was that the azimuthal perturbation between the three crowns probably adjusts the head locations to fit perfectly within the hexagonal filament lattice common to all vertebrate striated muscles so that the myosin heads do not clash with neighbouring actin filaments. The same conclusion can be reached for the cardiac myosin filaments studied here. That the myosin filaments from different species and from skeletal and cardiac muscles show similar perturbations and a similar location for C- (X-) protein argues that there may be a single basic three-dimensional plan common to all vertebrate striated muscle myosin filaments, presumably with minor variations.</p>", "<p>A feature seen in the fish myosin filament reconstruction is that there is axial density linking levels 3 and 1 (see ##FIG##2##Fig. 3##(F)). Similar density is absent between levels 1 and 2 and 2 and 3. However, this linking density between levels 3 and 1 is rather more marked in the cardiac reconstruction (arrowed in ##FIG##2##Fig. 3##(B), ##FIG##2##Fig. 3##(G; 110 Å), arrowed in ##FIG##4##Fig. 5##(J)) which could be due to the selection of particles in this case from only the C-zone. We therefore tentatively attribute this “extra” density to C-protein in the cardiac thick filament and infer that similar connecting C-protein density also occurs in fish skeletal muscle myosin filaments. In the X-ray diffraction modelling (see ##FIG##4##Fig. 5##(b) and (d) in ##REF##16884926##AL-Khayat and Squire, 2006##) a similar region of longitudinal connecting density between levels 3 and 1 was also present. Since C-protein and other accessory proteins were not included in the analysis, the best fit model had the myosin heads on level 1 dipping down and almost touching the heads on level 3 to account for this connecting density. The current 3D-EM reconstruction suggests the alternative possibility that this axial connecting density may be due to the presence of C-protein at this location.</p>", "<p>Skeletal muscle C-protein is known to be a modular structure composed of ten fibronectin 3-like (Fn-3) and immunoglobulin I-like (Ig-I) domains (termed C1–C10), with an N-terminal Pro-Ala-rich domain; (Pro-Ala)-C1(IgI)-C2(IgI)-C3(IgI)-C4(IgI)-C5(IgI)-C6(Fn3)-C7(Fn3)-C8(IgI)-C9(Fn3)-C10(Ig) (<xref rid=\"bib36 bib8 bib56\" ref-type=\"bibr\">Offer et al., 1973; Bennett et al., 1986; Vaughan et al., 1993</xref>). In the case of cardiac C-protein there is an additional N-terminal domain C0(Ig) before the Pro-Ala domain and there is an insertion between the first two Ig domains (C1 and C2) (##REF##8576942##Yasuda et al., 1995##). The connectivity of mass in our reconstruction running between the projected myosin head masses of levels 1 and 3 (arrowed in <xref rid=\"fig3 fig5\" ref-type=\"fig\">Figs. 3(B) and 5</xref>(J)) could be titin or C-protein. If the density corresponds to C-protein, this may be consistent with the C-terminal part of the C-protein (domains C6 to C10) running axially along the axis of the myosin filament as proposed by ##REF##12899839##Squire et al. (2003)##. It could be this longitudinal connecting density that causes the myosin head masses on levels 3 and 1 to be kept azimuthally aligned. The C0 to C5 part of C-protein is thought to be able to project out from the myosin filament and to bind to actin (##REF##12899839##Squire et al., 2003##).</p>", "<p>A weak feature of uncertain origin in our new map and seen in the circumferential sections of the current 3D reconstruction at low radius (e.g. dashed red lines in ##FIG##2##Fig. 3##(G; 90 and 110 Å radius), is a continuous circumferential density halfway between levels 1 and 2. Although this feature was less continuous in the 3D map of fish skeletal muscle (##FIG##2##Fig. 3##(F)), a similar circumferential band of density at a radius of 50 Å was present in the earlier 3D map of the isolated frog skeletal muscle thick filament (##REF##3495665##Stewart and Kensler, 1986##; Fig. (11a); note that the numbering of the crossbridge levels in the frog myosin filament map is different from the current rabbit myosin filament map). It could be argued that this continuous density in the circumferential section of the rabbit cardiac myosin supports the model for the structure of C-protein proposed by ##REF##12386147##Moolman-Smook et al. (2002)## with a trimeric collar arrangement around the myosin filament. At present it is unclear whether the difference in the density between the rabbit cardiac and fish skeletal myosin maps is due to a genuine difference between the filaments or is a difference due to the present more selective analysis where only the C-zone area was included.</p>", "<p>We have further investigated the locations of non-myosin densities by investigating the circumferential sections at a radius closer to the backbone of the filament where the myosin heads do not contribute as much and therefore any densities seen may be attributed to myosin rods, C-protein or titin. This is shown in ##FIG##2##Fig. 3##(I) which is a circumferential section at a radius of 75 Å from the filament axis in the current rabbit EM reconstruction. The red box in ##FIG##2##Fig. 3##(I) highlights the circumferential density above crown 1, discussed earlier (##FIG##2##Fig. 3##(G; 90 and 110 Å dashed red lines)) that may conceivably be attributed to a C-protein collar. In addition to this there is a weak meshwork of density, possibly attributable to titin or other features of the filament backbone, in the regions between what are taken to be the myosin head masses on crowns 1 and 3 and 2 and 3 as shown arrowed in ##FIG##2##Fig. 3##(I). These densities were not seen in the reconstruction of the fish skeletal myosin filaments of ##REF##16731006##AL-Khayat et al. (2006)## and may correlate with the strong meridional reflections on the 10th and 11th layer lines in the transforms of the rabbit cardiac myosin filaments (<xref rid=\"bib24 bib25\" ref-type=\"bibr\">Kensler, 2005a, 2005b</xref>). The reflection on the 11th layer line corresponds to the 39 Å spacing expected for the domains of titin. This weak meshwork of density may correspond to the pattern of beaded striations at this spacing on the backbone reported by ##REF##15721585##Kensler (2005b)##.</p>", "<title>The arrangement of myosin heads in cardiac muscle myosin filaments</title>", "<p>We have investigated the possible organisation of the myosin head pairs in our new 3D map by asking whether the observed densities are consistent with the 2D crystal structure of vertebrate smooth muscle two-headed myosin determined by ##REF##11287639##Wendt et al. (2001)##. In order to obtain the best fit and due to the fact that the crowns were not identical, the two-headed Wendt structure was modelled in different orientations in each crown (##FIG##4##Fig. 5##(A–H). The fitting was done for each crown individually within PyMOL by orienting the Wendt structure by hand within one of the three projecting mass densities on that crown. The other two 3-fold-symmetry-related Wendt structures on each crown were then positioned using the 3-fold transformation command in the CCP4 software package (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ccp4.ac.uk/\">http://www.ccp4.ac.uk/</ext-link>). It appears that crowns 1 and 3 can both accommodate the Wendt model quite well, but that crown 2 does not fit. This agrees with the conclusions for fish skeletal myosin filament (##REF##16731006##AL-Khayat et al., 2006##) in that the Wendt structure fitted reasonably well into two crowns (levels 1 and 3) but not the third (level 2). We conclude that the ##REF##11287639##Wendt et al. (2001)## structure is not a totally universal myosin head arrangement even though it fits well to the helically averaged crowns in tarantula invertebrate striated muscle myosin filaments (##REF##16121187##Woodhead et al., 2005##). Instead it appears to occur on two of the three levels of fish skeletal and rabbit cardiac muscle myosin filaments, but not on the third.</p>", "<p>Moreover, the Wendt model fits much better within crown 1 in the current rabbit cardiac myosin filament map than it did on crown 1 in the fish skeletal myosin. In addition, in the current reconstruction, the Wendt model fits somewhat less well within crown 3 than it did on the same crown in fish skeletal myosin. The projected myosin head density on level 2 in the current 3D map is more curved upwards (##FIG##2##Fig. 3##(A–D), ##FIG##4##Fig. 5##(A–D)) than observed on the same level in the fish skeletal myosin filament (##REF##16731006##AL-Khayat et al., 2006##). The difference in the fitting between the current map and the previous fish skeletal could be due to the fact that myosin filaments are different in cardiac and skeletal muscles. Obtaining a map at higher resolution would be beneficial in order to assess this difference in more detail.</p>", "<p>There could be several possible reasons for the difference observed in the fit of Wendt model to the third level of fish skeletal and rabbit cardiac myosin filament compared to the very good fit in tarantula myosin filaments (##REF##16121187##Woodhead et al., 2005##). One of these possibilities could be that these four species have different regulatory systems in that that both vertebrate smooth (Wendt model) and tarantula striated muscles are myosin-regulated by phosphorylation of their regulatory light chains (<xref rid=\"bib1 bib44 bib10\" ref-type=\"bibr\">Aksoy et al., 1976; Sobieszek, 1977; Craig et al., 1987</xref>), whereas both fish skeletal and rabbit cardiac muscles are regulated by calcium binding to their thin filaments (<xref rid=\"bib22 bib32\" ref-type=\"bibr\">Kendrick-Jones et al., 1970; Lehman and Szent-Gyorgyi, 1975</xref>).</p>", "<p>Recent work by ##UREF##4##Jung and Craig (2007)## reported preliminary results that show that myosin molecules isolated from tarantula and <italic>Limulus</italic> striated muscles (both phosphorylation-regulated) can have similar head–head interactions to those seen in vertebrate smooth and scallop striated myosin. Unlike these myosin-regulated muscles, head–head interaction was not observed in vertebrate skeletal and cardiac muscle myosins, in agreement with previous shadowing observations. However, their preliminary data showed that these unregulated myosin molecules can also exhibit head–head interactions if blebbistatin is bound to the heads (slowing phosphate release by promoting the ‘closed’ conformation of switch 2 in the motor domain of the head). Therefore the ##UREF##4##Jung and Craig (2007)## results support the idea that myosin head–head interaction may be quite a common motif in the inhibited state of myosin II molecules. Nevertheless, vertebrate and invertebrate systems are evolutionarily widely separated. In addition to the fact that the various species have different regulation systems in that smooth, tarantula, <italic>Limulus</italic> and scallop myosins are all myosin-regulated whereas both skeletal and cardiac are thin filament-regulated, the tarantula, <italic>Limulus</italic> and scallop myosin filaments are all perfect/true helical systems. This true helical symmetry requires that there are the same orientations, configurations and interactions between the two-heads on each crown. On the other hand, vertebrate myosin filaments (skeletal and cardiac) are not true helical systems. The perturbation from helical symmetry could be reflecting differences in the configurations, interactions and functions of the two-headed myosin molecules on different crowns. It is therefore probably not surprising, as we have shown in the current rabbit cardiac myosin and the previous fish skeletal myosin (##REF##16731006##AL-Khayat et al., 2006##), that the pairs of heads at the three crowns do not all share exactly the same arrangement (the Wendt model). The third possibility, along with the differences in regulation and helicity, is that tarantula, <italic>Limulus</italic> and scallop myosin filaments all have different compositions of accessory proteins (paramyosin core, etc.) compared to fish skeletal and rabbit cardiac (which have surface-located titin and C-protein). It could be that the extra surface proteins in vertebrate myosin filaments disrupt the fitting of the Wendt structure in one of the three levels, although it still fits well into the other two crowns. Fourthly, the differences in lattice arrangements between these species and the need to fit the underlying 9-fold structure of vertebrate myosin filaments into the 6-fold filament array, presumably requires there to be systematic differences in the head arrangements on different crowns.</p>", "<p>We therefore conclude that, even if the vertebrate myosin heads do sometimes tend to take up the Wendt configuration in isolation, as per the work of ##UREF##4##Jung and Craig (2007)## on isolated myosin molecules, the vertebrate myosin filaments with their perturbed helical myosin head arrangement, the presence of surface accessory proteins, the special lattice organisation in vertebrates, and their different regulatory system may all reflect the need for heads to depart from this arrangement on one crown.</p>", "<title>The origin of the perturbations in vertebrate striated muscle myosin filaments</title>", "<p>A method to test the effect of C-protein on the myosin filament structure is to study a system which does not have C-protein. ##REF##11909824##Harris et al. (2002)## reported the development of a knockout mouse for cardiac C-protein which shows hypertrophy and significant contractile defects, but retains normal sarcomeric structure. In a recent study, ##REF##17993479##Kensler and Harris (2008)## examined the structure of myosin filaments isolated from the C-protein knockout mouse heart muscle. They reported that myosin filaments isolated from the knockout mouse hearts appeared similar in length and diameter to the wildtype filaments, but the “forbidden” meridional reflections, thought to derive from a perturbation from helical symmetry in the wildtype filament, were weaker or absent in the Fourier transforms of the cMyBP-C^−/− myocardial myosin filaments. In addition, the crossbridge array in the absence of cMyBP-C appeared to be more easily disordered and sensitive to the negative staining conditions. These studies provide support for the idea that C-protein interactions with myosin may affect the myosin head arrangement as proposed by ##REF##16731006##AL-Khayat et al. (2006)##. As noted by ##REF##17993479##Kensler and Harris (2008)##, 2D studies of the filaments do not allow a determination of whether an azimuthal perturbation is present in the filaments. The large azimuthal perturbation observed for cardiac myosin filaments in this study would not give rise to the observed forbidden meridional reflections which arise from an axial crossbridge perturbation and/or the presence of additional mass on a 430 Å repeat. It is only 3D analysis, as in the present study, that allows a detailed and separate study of the axial and azimuthal components. This highlights the advantage of the single particle method in showing the azimuthal perturbation which is not evident from the observed forbidden meridionals in X-ray diffraction patterns and FFTs from 2D electron micrograph images.</p>", "<p>The 3D reconstructions of the vertebrate thick filament (both the current rabbit cardiac and the previously published fish skeletal myosin filaments (##REF##16731006##AL-Khayat et al., 2006##)) have clearly shown the presence of a perturbation in the crossbridge arrangement, but until now have not addressed the question of whether the perturbation in the crossbridges extends to the packing of the myosin rods in the backbone. We have shown here that the rabbit cardiac 3D map contained perturbations that were pronounced at large radius and reducing at smaller radius. We also note that our current rabbit cardiac myosin filament map complements the reported abstract of the wildtype mouse cardiac myosin filament of ##UREF##6##Perez-Zoghbi et al. (2007)## who have stated that the three crowns in their map were not identical. However, our evidence suggesting the greater observed helical perturbation of the heads at higher radius compared to at lower radius from the filament axis is distinct to our current study. Future higher resolution 3D analysis work in both wildtype and C-protein knockout mouse heart muscle myosin filaments, as well as in our current studies of the isolated rabbit cardiac myosin filament, should help to select between the alternatives: EITHER that the azimuthal and axial perturbations within the myosin filament are an inherent feature of the packing of the myosin rods in a non-helical way within the filament backbone, OR that the myosin head origins lie on a perfect helix and that the conformational perturbations are confined to the myosin heads only and are due to the presence of titin, which does not have a 143 Å repeat, and the effect of titin in locating C-protein on every third crown level. Our evidence so far from the present analysis is that the second of these options is more likely.</p>" ]

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[ "<p>A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2 × 430 Å long, each of which was treated as an independent ‘particle’. The resulting 40 Å resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 Å repeat, with successive crown rotations of approximately 60°, 60° and 0°, rather than the regular 40° for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac).</p>", "<title>Keywords</title>" ]

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