Body fat mass distribution and deposition are determined by multiple environmental and genetic factors. Obesity is associated with insulin resistance, hyperinsulinemia, and type 2 diabetes. We previously identified evidence for genotype-by-diabetes interaction on obesity traits in Strong Heart Family Study (SHFS) participants. To localize these genetic effects, we conducted genome-wide linkage scans of obesity traits in individuals with and without type 2 diabetes, and in the combined sample while modeling interaction with diabetes using maximum likelihood methods (SOLAR 2.1.4).SHFS recruited American Indians from Arizona, North and South Dakota, and Oklahoma. Anthropometric measures and diabetes status were obtained during a clinic visit. Marker allele frequencies were derived using maximum likelihood methods estimated from all individuals and multipoint identity by descent sharing was estimated using Loki. We used variance component linkage analysis to localize quantitative trait loci (QTLs) influencing obesity traits. We tested for evidence of additive and QTL-specific genotype-by-diabetes interactions using the regions identified in the diabetes-stratified analyses.P < 0.02). In analysis accounting for QTL-specific interaction (P < 0.001), we detected a QTL for weight on chromosome 1 at 242 cM (LOD = 3.7). This chromosome region harbors the adiponectin receptor 1 gene, which has been previously associated with obesity.Among 245 diabetic and 704 non-diabetic American Indian individuals, we detected significant additive gene-by-diabetes interaction for weight and BMI (These results suggest distinct genetic effects on body mass in individuals with diabetes compared to those without diabetes, and a possible role for one or more genes on chromosome 1 in the pathogenesis of obesity. Body fat mass distribution and deposition are determined by multiple environmental and genetic factors. Obesity is associated with insulin resistance, hyperinsulinemia, and incident type 2 diabetes ,2. MoreoDiabetes and obesity are associated with increased morbidity and mortality in the general population  and amonThe genetic component of obesity-related traits among American Indians has been previously described -11. In o2). WHR (in cm) was also recorded. Percentage body fat was measured using Quantum II bioelectric impedance  [The SHFS recruitment and genotyping methods have been described elsewhere ,13. Briehip, MI) . The perhip, MI) . The SHFParticipants, who are members of extended families, were genotyped for approximately 400 microsatellite markers spaced at average 10 cM intervals across the autosomal chromosomes. Pedigree relationships were verified using the Pedigree Relationship Statistical Tests program , which e2, sex, age-by-sex interaction and study center using linear regression models and an alpha = 0.05. The WHR trait was scaled for analysis by multiplying by 10.We conducted a genome scan of obesity traits while modeling interaction with diabetes status using maximum likelihood methods implemented in SOLAR 2.1.4. We studied 976 individuals from extended families enrolled between 1998 and 1999 (SHS phase 3). We examined the distribution of the obesity-related quantitative traits of body weight, BMI, WHR and percentage of body fat, and excluded observations greater than 2.5 standard deviations above or below the mean (< 5 observations excluded). The traits were adjusted for the effects of age, ageG) and the genetic standard deviations for diabetic and non-diabetic subjects. To test for differential additive genetic effects among diabetic and non-diabetic subjects, we compared a model in which ρG  =1 to a model without constraints. To test for differences in the magnitude of the genetic effects among diabetic and non-diabetic subjects, we compared a model in which the genetic standard deviations of the two groups are constrained to be equal to a model without these constraints. We considered evidence for genotype-by-environment interaction if the genetic correlation between diabetics and non-diabetics was different than 1 and/or the genetic variance for obesity traits in diabetics was significantly different than in non-diabetics.We examined the evidence for additive genotype-by-diabetes interaction on obesity traits using maximum likelihood variance decomposition methods extended to allow for the genetic covariance between diabetic and non-diabetic subjects ,20. We tG = 1.0), the test statistic is asymptotically distributed as a 50:50 mixture of a chi-square and a point mass at 0 [We also tested for QTL-specific diabetes-by-obesity interaction. For these analyses, we compared a model in which the QTL standard deviations are constrained to be equal, to a model without constraints, using the likelihood ratio statistics. When comparing models with variances constrained to be equal, interpretation of significant differences were based on the assumption of an asymptotic chi-square distribution for the likelihood test statistic. For models that restricted the genetic correlation to one , we performed genome scans accounting for the diabetes interaction. We adjusted the obtained LOD scores for the difference in the number of parameters between the polygenic and linkage versions of the interaction model, taking into account that each of the QTL standard deviations has a boundary at the null hypothesis.For the obesity traits showing significant additive genotype-by-diabetes interaction at alpha = 0.05  and 26% had diabetes , BMI (p = 0.0002) and WHR (p = 0.02), suggesting that at least some of the genes influencing these obesity traits differed in diabetic compared to non-diabetic subjects  .We then evaluated the evidence for genotype-by-diabetes interaction at the QTL level, using the combined samples of diabetic and non-diabetic individuals and the maximum LOD score QTL identified on the diabetes-stratified analysis. The QTL- specific genotype-by-diabetes interaction was significant for weight on chromosome 1 at 242 cM  mice show increased adiposity and decreased glucose tolerance, associated with decreased locomotor activity and energy expenditure [ADIPOR1 variants have been associated with type 2 diabetes, pre-diabetes states and measures of obesity although not in all studies [2) with impaired glucose tolerance, ADIPOR1 SNPs were associated with measures of body size including weight, waist, hip circumference and BMI [ADIPOR1 and ADIPONECTIN was observed in increased overall and abdominal adiposity and higher substrate oxidation [ADIPOR1 is a potential candidate gene for pleiotropic effects on obesity and diabetes susceptibility in humans.Within the 1-LOD support region lays the ceptor-α . ADIPOR1enditure . ADIPOR1 studies -33. For  and BMI . In addixidation . TherefoIt is difficult to gauge the LOD score evidence reported herein in the face of the number of comparisons made in conducting gene-by-diabetes genome scans. In total, we conducted three genome scans . If we were to use a too conservative Bonferoni correction, we would need to observe a P = 0.05/3 * 380 markers: P = 0.00004 or a corresponding LOD score of 3.7. However, given the correlation and non-independence of the three genome scans of each obesity trait, a systematic correction would be extremely difficult.Multiple interacting genes may underlie this QTL on chromosome 1, and therefore, densely genotyping this region is the next logical step to be taken to identify these genes influencing obesity in the context of diabetes. Our findings are limited to American Indian populations and should be validated in replication samples of other populations.ADIPOR1 gene is located. This gene has been associated with multiple measures of body size and diabetes-related traits in humans and experimental models and, therefore, it is likely a good candidate for susceptibility to fat deposition in diabetic individuals. Importantly, our findings support that this region of the genome likely harbors susceptibility variants for obesity that are importantly different in type 2 diabetes individuals and those with normal glucose metabolic profile. Thus, future studies should more comprehensively evaluate the ADIPOR1 gene, especially with respect to the complex relationship between body weight and type 2 diabetes.In summary, we identified a diabetes-specific QTL on chromosome 1 at 242 cM for weight in American Indians, in the region where the The authors declare that they have no competing interests.NF, JWM and KN participated in the design of the study and drafted the manuscript. NF and HHHG performed the statistical analysis. SAC and SL carried out the molecular genetic studies. VPD, BVH, ETL, LGB and RRF contributed with acquisition of data and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Strong Heart Family Study additional QTLs.Click here for file


We hypothesized that sedation with dexmedetomidine (an α2 adrenoceptor agonist), as compared with lorazepam (a benzodiazepine), would provide greater improvements in clinical outcomes among septic patients than among non-septic patients.Benzodiazepines and αa priori-determined subgroup analysis of septic vs non-septic patients from the MENDS double-blind randomized controlled trial, adult medical/surgical mechanically ventilated patients were randomized to receive dexmedetomidine-based or lorazepam-based sedation for up to 5 days. Delirium and other clinical outcomes were analyzed comparing sedation groups, adjusting for clinically relevant covariates as well as assessing interactions between sedation group and sepsis.In this Of the 103 patients randomized, 63  were admitted with sepsis and 40  without sepsis. Baseline characteristics were similar between treatment groups for both septic and non-septic patients. Compared with septic patients who received lorazepam, the dexmedetomidine septic patients had 3.2 more delirium/coma-free days (DCFD) on average , 1.5  more delirium-free days (DFD) and 6  more ventilator-free days (VFD). The beneficial effects of dexmedetomidine were more pronounced in septic patients than in non-septic patients for both DCFDs and VFDs . Additionally, sedation with dexmedetomidine, compared with lorazepam, reduced the daily risk of delirium  in both septic and non-septic patients . Risk of dying at 28 days was reduced by 70% [hazard ratio 0.3 ] in dexmedetomidine patients with sepsis as compared to the lorazepam patients; this reduction in death was not seen in non-septic patients .In this subgroup analysis, septic patients receiving dexmedetomidine had more days free of brain dysfunction and mechanical ventilation and were less likely to die than those that received a lorazepam-based sedation regimen. These results were more pronounced in septic patients than in non-septic patients. Prospective clinical studies and further preclinical mechanistic studies are needed to confirm these results.NCT00095251. Recent advances in critical care medicine have identified acute brain dysfunction (delirium and coma) as a highly prevalent manifestation of organ failure in critically ill patients that is associated with increased morbidity and mortality -6. AccumSedative and analgesic medications, routinely administered to mechanically ventilated (MV) patients , contrib2 (α2) adrenoceptor agonist, provided safe and efficacious sedation in critically ill MV patients, with significant improvement in brain organ dysfunction (delirium and coma) compared with the benzodiazepine, lorazepam (LZ). The principal findings from the MENDS trial were recently corroborated by the Safety and Efficacy of Dexmedetomidine Compared With Midazolam (SEDCOM) trial of 366 critically ill patients, which showed a reduction in the prevalence of delirium in patients sedated with DEX compared with midazolam; patients on DEX also showed a reduction in the duration of MV [The Maximizing Efficacy of Targeted Sedation and Reducing Neurological Dysfunction (MENDS) trial  demonstron of MV . In the 2 adrenoceptor agonists and benzodiazepines have different molecular targets (α2 adrenoceptors and gamma-aminobutyric acid type A (GABAA) receptors, respectively) and neural substrates for their hypnotic effects that may play a critical role in maintaining sleep architecture in critically ill patients [2 adrenoceptor agonists exert opposing effects on innate immunity, apoptotic injury and mortality in preclinical models of infection [A receptor antagonists are under investigation as anti-infective agents [2 adrenoceptor agonists enhance macrophage phagocytosis and bacterial clearance [per se exerts superior anti-inflammatory and organ-protective properties compared with other sedatives [The αpatients ,24; imprpatients -27. In anfection -30 likelnfection  and macrnfection , wherease agents . Contraslearance -36, whillearance , and arelearance . DEX peredatives ,39,40 anedatives  and apopedatives , and thuedatives  and may edatives . Sympathedatives ; in lineedatives , presumpedatives  and SEDCedatives  studies.a priori-planned subgroup analysis among patients from the MENDS trial to determine if sedation with DEX compared with LZ in septic and non-septic patients affected clinical outcomes, including duration and prevalence of acute brain dysfunction and 28-day mortality.Multiple levels of evidence thus converge to support our hypothesis that sedation with DEX may lead to better outcomes for patients with sepsis than benzodiazepine sedation. We therefore conducted an The MENDS study , conducted between August 2004 and May 2006,  was apprThe primary outcome of interest was delirium/coma-free days, defined as the days alive without delirium or coma during the 12-day study period . SecondaDelirium was measured daily until hospital discharge or for 12 days using the Confusion Assessment Method for the ICU (CAM-ICU) . EfficacFor other outcomes, patients were followed in the hospital from enrollment for 28 days, or until discharge or death if earlier.Data were analyzed using an intention-to-treat approach. Continuous data were described using medians and interquartile ranges or means and standard deviations, and categorical data using frequencies and proportions. We used Pearson chi-squared tests for categorical variables and Wilcoxon rank-sum tests for continuous variables to test for baseline differences between the two study groups, stratifying by the presence or absence of sepsis.P value of less than 0.15 to be significant, indicating that the treatment group affected the outcome in question differently among septic and non-septic patients.We used multivariable regression to examine associations between treatment group and outcomes, assessing for interactions between sepsis and the effect of treatment group on each outcome . All regression models included sepsis, treatment group, and a treatment group by sepsis interaction term as independent variables, in addition to the following covariates: age, severity of illness according to the acute physiology component of the Acute Physiology and Chronic Health Evaluation (APACHE) II score at enrollment, and use of drotrecogin alfa (activated) within 48 hours of enrollment. Because the trial was not powered to detect interactions, we considered an interaction term For the primary outcome, we used bootstrap multiple linear regression to calculate a non-parametric 95% confidence interval (CI) for the adjusted difference in mean delirium/coma-free days between the two treatment groups, because of the skewed distribution of this outcome variable. Specifically, we fitted a multiple linear regression model (which included the independent variables described above) in each of 2,000 datasets randomly generated from the original data using the bootstrap method  and determined the 95% CI of the adjusted difference in mean delirium/coma-free days using the 2.5 and 97.5 percentiles of the 2,000 regression coefficients of these models. The same approach was used to analyze delirium-free days, coma-free days, and ventilator-free days.For time-to-event outcomes (time to ICU discharge and death), Cox proportional hazards models were used. Kaplan-Meier survival curves were created for graphical representation of these time-to-event outcomes. When examining 28-day mortality, patients were censored at the time of last contact alive or at 28 days from enrollment, whichever was first. Censoring for ICU or hospital discharge analyses occurred at time of death or, rarely, at study withdrawal.To examine the effect of treatment group on the probability of being delirious each day during the study drug period , we used Markov logistic regression. These models, with an outcome of daily mental status, adjust for the previous day's mental status as well as the relevant covariates described above. Due to the multiple assessments included for each patient, generalized estimating equations were applied to this regression model to account for the correlation of these observations within each patient.P values of 0.05 or less were considered to indicate statistical significance. We used R (version 2.10) for all statistical analyses.For all results except for interaction terms, two-sided P = 0.37), percentage of patients receiving antibiotics on study day 1 , and percentage treated with corticosteroids . Although not statistically significant, drotrecogin alfa (activated) administration may have been less common among DEX septic patients than LZ septic patients  despite a similar severity of illness according to APACHE II scores (Table Sixty-three patients in the MENDS study  met the P for interaction = 0.09) and for ventilator-free days ; among all patients (regardless of sepsis), DEX-treated patients had 70% lower odds, compared with LZ-treated patients, of being delirious on any given day (odds ratio (OR) = 0.3, 95% CI = 0.1 to 0.7; Figure P = 0.005) and disorganized thinking  , and not as much by level of arousal.Septic patients sedated with DEX had a mean (95% CI) of 3.2 1.1 to 4.9) more delirium/coma-free days, 1.5 (-0.1 to 2.8) more delirium-free days, and 6 (0.3 to 11.0) more ventilator-free days than patients receiving LZ, after adjusting for relevant covariates. However, no substantial difference was seen in these outcomes between non-septic patients treated with DEX and LZ . The proportional hazards assumption for time to death within 28 days was validated graphically and via examining model residuals [Septic patients sedated with DEX additionally had a lower risk of death at 28 days as compared with those sedated with LZ (hazard ratio (HR) = 0.3, 95% CI = 0.1 to 0.9; Figure esiduals .P = 0.01); however, efficacy of sedation among the non-septic patients was similar for both treatment groups (67% of days (50 to 86%) vs 60% of days (27 to 75%), P = 0.27). Median (interquartile range) DEX dose was 0.8 mcg/kg/hour (0.3 to 1.1) and LZ dose was 3.6 mg/hr (2.2 to 7.1) in the septic patients. In the non-septic group, median infusion rate were 0.6 mcg/kg/hr for DEX and 2.7 mg/hr for LZ. Septic patients sedated with DEX received more fentanyl per day  vs 117 (0 to 1460), P = 0.01) than septic patients sedated with LZ, while fentanyl use was similar in the non-septic DEX and LZ groups (520 mcg/day (133 to 1778) vs 262 (10 to 775), P = 0.20).Among the septic patients, those sedated with DEX achieved sedation within one point of their ordered RASS target more often than those sedated with LZ (accurately sedated on 67% of days (50 to 83%) vs 52% of days (0 to 67%), P > 0.10). Development of new secondary infections beyond the first 48 hours after enrollment was similar in the originally non-septic group in the DEX and LZ study arms (17% vs 15%).Incidence of hypotension, vasopressor use and cardiac arrhythmias monitored during the study are shown in Table This subgroup analysis presents data indicating that the choice of a sedative may be important for sepsis patients in determining clinical outcome. Septic patients treated with DEX had shorter duration of acute brain dysfunction (delirium and coma), lower daily probability of delirium, shorter time on the ventilator, and improved 28-day survival as compared with septic patients treated with LZ. Our results further suggest that sedation regimens incorporating DEX have a greater impact on these important outcomes in patients with sepsis than in patients without sepsis. These findings suggest that choice of sedative is vitally important in the vulnerable septic patient population and, along with other strategies , needs t2 adrenoceptor agonists appear to promote macrophage phagocytosis and bactericidal killing [Our findings could be the result of either a beneficial effect of DEX in the setting of sepsis, a deleterious effect of LZ in this setting, or both . Benzodi killing -36. Give killing , althougNonetheless the mortality benefit that was provided by DEX over LZ in our patients with sepsis may be due to several factors. These include differences in the effects of these sedative regimens on both innate immunity and inflammation  and alsoWe did not observe any adverse events in the septic DEX group (with the possible exception of bradycardia), and there were no differences in liver, renal, cardiac, or endocrine safety outcomes  in septic patients treated with DEX vs LZ, attesting to its safety in critically ill septic patients. DEX has been reported to cause hypotension and bradycardia in patients, due to the inhibition of central norepinephrine release, peripheral vasodilation and a vagomimetic action . AlthougAlthough fentanyl doses were significantly greater in septic DEX-treated patients than in LZ-treated patients -- likely because supplemental analgosedation may be needed to achieve heavy sedation for a DEX-treated patient -- it is unlikely that the benefits observed in the DEX group were attributable to the use of fentanyl. Indeed, available evidence indicates that opioids have immunosuppressive effects and are capable of increasing mortality in animal models of infection ,56. Addi2 adrenoceptor agonist based sedation compared with GABAergic sedation in septic patients, we did not observe all the benefit in the non-septic group. DEX-treated patients did have lower odds of development of delirium, whether septic or non septic; however, the improvements in duration of brain dysfunction were predominantly seen in the septic patients on DEX. This may be because the non-septic group was smaller than the septic group and thus had limited statistical power to identify any beneficial or detrimental effect of either treatment. Additionally differences in pathogenesis of delirium may account for the greater benefit seen in septic patients. Furthermore septic shock is associated with neuronal apoptosis in the brain, including the locus ceruleus [2 adrenoceptors. Given that DEX prevents central neuroapoptosis via activation of α2 adrenoceptors [Interestingly, although we observed significant benefits of αceruleus , where toceptors , these nA receptor. Indeed, LZ is a significant risk factor for delirium [There are several limitations to this investigation. First, we categorized patients as septic and non-septic based on the presence of at least two SIRS criteria and suspected infection, in accordance with the consensus definition . As in cdelirium  and may a priori-identified subgroup analysis, sedation with DEX reduced the duration of brain organ dysfunction, lowered the probability of delirium, increased time-off mechanical ventilation, and reduced 28-day mortality as compared with LZ in septic patients; the benefit of DEX sedation was greater for septic patients than for non-septic patients in terms of duration of acute brain dysfunction (delirium or coma), time on mechanical ventilation, and mortality. Prospective multicenter, randomized controlled trials are needed to confirm these results and examine the mechanisms underlying the effect of DEX on outcomes, including mortality, in sepsis.In this a priori designed subgroup analysis of the MENDS study, septic patients receiving DEX had more days free of brain dysfunction and MV and were less likely to die than those that received a LZ-based sedation regimen. Patients on DEX had lower odds of developing delirium whether septic or non-septic as compared with those on LZ.• In this • The majority of benefits conferred by DEX sedation were more prominent in septic patients than in non-septic patients.2 adrenoceptor rather than the GABAA receptor.• Further prospective clinical and preclinical study is warranted into the potential benefits of sedation with drugs targeting the αAPACHE: Acute Physiology and Chronic Health Evaluation; CAM-ICU: Confusion Assessment Method for the ICU; CI: confidence interval; DEX: dexmedetomidine; HR: hazard ratio; LZ: lorazepam; MV: mechanical ventilation; OR: odds ratio; RASS: Richmond Agitation-Sedation Scale; SIRS: systemic inflammatory response syndrome.PPP, DLH, MM and TDG have received research grants or honoraria from Hospira Inc. EWE has received research grants and honoraria from Hospira, Inc, Pfizer, and Eli Lilly, and a research grant from Aspect Medical Systems. All other authors report that they have no competing interests.RDS developed the hypothesis with PPP, MM and EWE. All authors were involved in the study design and interpretation. The analysis was performed by PPP, TDG, SM, AKS, JLT and EWE. All authors contributed to data interpretation. Primary responsibility for drafting the manuscript lay with PPP and RDS who contributed equally to the paper.


Mouse and rat models are mainstays in pharmacology, toxicology and drug development – but differences between strains and between species complicate data interpretation and application to human health. Dioxin-like polyhalogenated aromatic hydrocarbons represent a major class of environmentally and economically relevant toxicants. In mammals dioxin exposure leads to a broad spectrum of adverse affects, including hepatotoxicity of varying severity. Several studies have shown that dioxins extensively alter hepatic mRNA levels. Surprisingly, though, analysis of a limited portion of the transcriptome revealed that rat and mouse responses diverge greatly .We employed oligonucleotide arrays to compare the response of 8,125 rat and mouse orthologs. We confirmed that there is limited inter-species overlap in dioxin-responsive genes. Rat-specific and mouse-specific genes are enriched for specific functional groups which differ between species, conceivably accounting for species-specificities in liver histopathology. While no evidence for the involvement of copy-number variation was found, extensive inter-species variation in the transcriptional-regulatory network was identified; Nr2f1 and Fos emerged as candidates to explain species-specific and species-independent responses, respectively.Our results suggest that a small core of genes is responsible for mediating the similar features of dioxin hepatotoxicity in rats and mice but non-overlapping pathways are simultaneously at play to result in distinctive histopathological outcomes. The extreme divergence between mouse and rat transcriptomic responses appears to reflect divergent transcriptional-regulatory networks. Taken together, these data suggest that both rat and mouse models should be used to screen the acute hepatotoxic effects of drugs and toxic compounds. Our arrays contained 8,125 orthologous genes, allowing us to analyze a much larger portion of the transcriptome than the previous study. We confirmed that a very small fraction of TCDD-responsive genes show conserved patterns in mouse and rat liver, and that these represent specific GO categories. Further, we performed library-based and de novo transcription-factor binding-site analyses to rationalize the patterns of expression. Finally we validated our results with a gene-by-gene comparison to a public dataset.This has major implications for the use of mouse and rat as model organisms to study toxic responses, making it important to evaluate and extend this finding. We analyzed the transcriptional response to TCDD in TCDD-sensitive strains of mouse (C57BL/6) and rat . At the 19-hour exposure time, we expect that most responses are likely to represent primary events although some early morphological manifestations of hepatic toxicity emerge within one day, at least in mice [We used mRNA expression microarray analysis to study the transcriptional response to TCDD in young adult male C57BL/6 mice and L-E rats. Eleven mice were used, five treated with corn-oil vehicle and six treated with 1000 μg/kg TCDD for 19 hours . Eight r in mice . RNA extWe first analyzed the rat and mouse data separately. The arrays from each species were pre-processed independently and subjected to ProbeSet-wise linear modeling. Following a multiple-testing correction, we identified extensive responses to TCDD in both species Figure . The MOEadjusted < 10-3) both species exhibited primarily inductive responses. At less stringent thresholds, however, the rat showed similar amounts of induction and repression whereas the mouse response remained predominately induction  changes in response to TCDD in both species , suggesting that orthologs respond similarly in the two species. This result is independent of the padjusted threshold of 0.01 used  in either species. To normalize signal across species we employed gene-wise median-centering and root-mean-square-scaling. The normalized matrices were then merged gene-wise and subjected to divisive hierarchical clustering  indicating that genes that respond to TCDD in one species are less likely to respond in the other than predicted by chance alone. Again, this finding is independent of the padjusted parameter selected . This could result from at least three mechanisms. First, aspects of hepatotoxicity common to both species may be mediated by the small "core" of species-independent responses. Second, toxicity might not be mediated by any of the mRNA changes in the liver. Third, toxicity might be mediated by different genes in each species, but these genes may ultimately lead to dysregulation of a common set of pathways, thereby leading to phenotypic similarities.To test this third possibility, we studied the functional enrichment of genes that respond to TCDD in mice-only, in rats-only, or in both species using gene ontology analysis . We idenHaving demonstrated large-scale differences between the rat and mouse transcriptional responses to TCDD and having shown that these differences lead to alterations in specific pathways, we next considered mechanism. One obvious mechanism for differences in mRNA expression is changes in gene dosage. Indeed many mRNA expression studies, including those of untreated rat liver, have found localized domains or "islands" of differential expression . We assessed the genomic localization of species-specific and species-independent responses to TCDD in both species. No large regions or islands are apparent amongst the common (black), the rat-specific (red), or the mouse-specific genes (blue) in either the rat Figure  or the mAnother mechanism that might underlie differences in the transcriptional response to TCDD between mouse and rat is the differential activity of one or more regulatory genes. Alterations in the activities of a small number of regulatory genes, such as transcription factors, could lead to the large number of mRNA differences observed between species. One way to find evidence for differential transcription factor activities is to use transcription-factor binding-site (TFBS) enrichment analysis . We searSixty separate TFBS motifs were enriched in at least one of the six datasets searched  rats [Cyp1a1-/- mice [Testing the first two hypotheses will require the use of tissue-specific transgenic animals and are thus beyond the scope of this study. To test the idea that toxicity results from the dysregulation of a pathway common to mouse and rat we used gene-ontology enrichment analysis. We searched for dysregulated functional groups in the lists of rat-specific and mouse-specific genes and found only one pathway dysregulated in both species – cellular lipid metabolism. Because this minimal overlap could occur by chance alone, and with caveats regarding tissue-specificity and transcriptome-coverage, it appears likely that the genes mediating TCDD-induced toxicity are amongst those reported in Table io) rats . CYP1A1 -/- mice , but indIt would be of interest to repeat our analysis using expression profiles collected 96 to 240 hours after TCDD exposure, when overt toxicities are manifest. At these later time-points it would no longer be possible to discern primary from secondary events, but instead there would be an increased ability to contrast the final toxic pathways perturbed in each species.While about 15% of TCDD-responsive genes are species-independent and thus potentially mediate TCDD toxicities, the roles of the remaining 85% of genes are intriguing to consider. There are two key questions. First, does altered expression of these genes have functional consequences? Second, what mechanisms mediate species-specific responses?If the species-independent genes had no functional consequence we might expect them to be randomly distributed across different functional classes. Instead, gene-ontology enrichment analysis Table  shows th50 values for each species to ensure that a similar ultimate phenotype was being considered. Nevertheless it is possible that the type of hepatic injury is species-specific and that this leads to the observed differences in transcript profiles. Several lines of evidence suggest against this possibility, including the evolutionary proximity of rat and mouse, the similar responses of known TCDD-responsive genes, and the inter-species similarity of the vast majority of morphological changes. Further work will be needed to clarify between these two situations.The vast majority of TCDD toxicities are common to both species, but some species-specific differences do exist. For example, multinucleated giant hepatocytes occur only in rats, while hydropic degeneration, fat accumulation, inflammatory cell infiltration as well as apoptosis and necrosis are more pronounced in mice ,41,46-48Many different mechanisms can be proposed to explain the large species-specific responses observed, including copy-number variation, differences in transcription-factor binding affinities, and differential transcription-factor activities. We did not find evidence that copy-number variation plays a role Figure , so we fIt has been established that transcription-factor binding is poorly correlated with changes in mRNA expression . FurtherOur library-based analysis identified numerous motifs enriched in both species-dependent and species-independent manners, some clearly associated to the biology of dioxin-toxicity. For example, the enrichment of p53 motifs in mouse promoters suggests that a p53-dependent apoptotic response was initiated in mouse, but not in rat. Apoptosis is a common sequela to TCDD exposure in mouse liver ,47 and, MotifSampler is a variant of Gibbs sampling that introduces a higher-order Markov background model and incorporates a Bayesian mechanism to estimate the missing value: the number of motifs occurring in each sequence . MotifSaWe directly compared our results to those of Boverhof and coworkers at the gene- level Figure . This isThus we have shown that most TCDD-responsive genes are species-specific and that the small core of species-independent genes shares both common functions and common transcriptional-regulatory elements. Taken together these data suggest that the vast majority of dioxin-responsive genes play no direct role in the toxic responses. Rather, it may be that a small number of regulatory genes under direct AHR control lead to dramatic inter-species differences. It would be highly advantageous to further refine the list of genes associated with dioxin-toxicities by profiling the transcriptional response of additional strains of mouse and rat, or by exploiting additional rodent species.This study has significant implications for the use of rodent models to understand mechanisms of pharmacological or toxicological compounds. If transcriptional responses have been extensively remodelled in rat and mouse since their last common ancestor then hepatic expression studies may yield contradictory and species-specific results. It may be necessary to test both species and focus on the subset of overlapping genes. An optimal study design might include several strains of mice and several strains of rat, thereby focusing the list of transcriptional responses to a small number that exhibit both intra- and inter-species homogeneity and are thus highly likely to be directly involved in the phenotypic effects of the compound under investigation.Turku/AB) (LE) strain of rat was selected for these analyses because it harbours a wild-type Aryl Hydrocarbon Receptor (AHR) and is the most TCDD-sensitive rat strain reported, with an acute LD50 of 9.6  or 17.7  μg/kg [ad libitum. The temperature in the animal room was 21 ± 1°C, relative humidity 50 ± 10%, and lighting cycle 12/12 hours light/dark. The study plans were approved by the Animal Experiment Committee of the University of Kuopio and the Kuopio Provincial Government. Liver was harvested between 8:30 and 11:00 from rats treated by gavage with a single 100 μg/kg dose of TCDD or with corn oil vehicle for 19 hours. Four control (corn-oil treated) and four experimental (TCDD-treated) rats were used in this study.The Long-Evans (+/+) C57BL/6 mice harbouring wild-type AHR alleles were bred at the National Public Health Institute, Kuopio, Finland from stock originally obtained from The Jackson Laboratory. At 15 weeks of age mice were given a single dose of 1000 μg/kg TCDD or corn oil vehicle by gavage. TCDD initially was dissolved in ether and added to corn oil; the ether subsequently was evaporated off. Liver was harvested 19 hours after treatment, sliced, snap-frozen and stored in liquid nitrogen until homogenization. Five control (corn-oil treated) and six experimental (TCDD-treated) mice were used in this study.Wildtype  and was >99% pure as determined by gas chromatography-mass spectrometry.Total RNA was extracted from both rat and mouse livers using Qiagen RNeasy kits according to the manufacturer's instructions . Total RNA yield was quantified by UV spectrophotometry and RNA integrity was verified using an Agilent 2100 Bioanalyzer .Affymetrix RAE230A and MOE430-2 arrays were run according to manufacturer's protocols at The Centre for Applied Genomics at the Hospital for Sick Children . Four independent biological replicates  were analyzed for each condition , while five control and six treated mice were analyzed . The raw array data have been deposited into the GEO repository with accessions GSE10769 (mouse) and GSE10770 (rat).-/- animals described elsewhere [-/- mice in the quantile-normalization step of pre-processing. The data for control rats have been analyzed separately elsewhere , while the data from TCDD-treated rats have not. Again, pre-processing was performed separately for this study to avoid introducing bias.Microarray data were loaded into the R statistical environment (v2.6.2) using the affy package (v1.16.0) . These dlsewhere , but werAll clustering analyses employed divisive hierarchical clustering using the DIANA algorithm as implemented in the cluster package (v1.11.10) and with Pearson's correlation as a similarity metric. Heatmaps were visualized using the lattice (v0.17-6) and latticeExtra (v0.3-1) packages. Subsets for clustering were selected using global variance thresholds. When both species were clustered together, scaling was performed separately for the mouse and the rat arrays to ensure that probe, normalization or loading differences did not bias the analysis.adjusted < 0.01 for most downstream analyses. At this level we would expect no more than 1% of our hits to be false-positives. We also considered the p-value sensitivity of the overall analysis by varying the threshold from 10-1 to 10-7 in log steps and calculating the number of differentially-expressed ProbeSets at each threshold.To identify TCDD-induced changes in mRNA abundance model-based t-tests were fit using the limma software package (v2.12.0) and subjected to an empirical Bayes moderation of the standard error . P-value. Genomic localization of TCDD-responsive genes was performed using the geneplotter (v1.16.0), annotate (v1.16.1), mouse4302 (v2.0.1) and rae230a (v2.0.1) packages, all in version 2.6.2 of the R statistical environment.Genes were annotated with version na24 of the Affymetrix NetAffx annotation Homologs were mapped between the two arrays using the Homologene database (build 58). Genes were mapped according to Entrez Gene ID to Homologene IDs and only genes present in both species were retained for downstream analyses. When multiple ProbeSets were present for a single Entrez Gene ID they were collapsed by selecting the ProbeSet with the minimum p-value. We compared this approach to selecting the ProbeSet with the maximum coefficient or the one with the maximum average signal, and only minor differences were observed amongst the three strategies. We also contrasted these three approaches with a sequence-based annotations provided by the custom CDF annotations mapping individual Probes to Entrez Gene IDs, as provided in BioConductor (v1.9) .We used a library-based method to search for transcription-factor binding-sites enriched or depleted in specific gene sets . Using tThese same comparisons were also made using a pattern-discovery algorithm, MotifSampler . First, Unsupervised machine-learning was performed as described above, using divisive hierarchical clustering through the DIANA algorithm as implemented in the cluster package (v1.11.10) and with Pearson's correlation as a similarity metric. Heatmaps were visualized using the lattice (v0.17-6) and latticeExtra (v0.3-1) packages.Functional enrichment analysis was performed using the GOMiner tool . AnalysiPCB performed most bioinformatic and all statistical analyses and wrote the first draft of the paper. RY performed the motif-discovery analyses. ABO and RP guided the study design. All authors contributed to the study design and provided input into the final manuscript.Figures demonstrating supplementary findings. Demonstrations of array quality, parameter insensitivity of analyses, and the importance of appropriate scaling in cross-species clustering.Click here for fileMicroarray Data for All Mouse ProbeSets. Annotation and linear-modeling fits for all ProbeSets on the MOE430-2 array.Click here for fileMicroarray Data for All Rat ProbeSets. Annotation and linear-modeling fits for all ProbeSets on the RAE230A array.Click here for fileHomolog Response Analysis. Comparison of four separate methods of mapping homologs between rat and mouse.Click here for fileMicroarray Data for All Ortholog Pairs. Annotation and linear-model fits for ortholog pairs significantly affected by TCDD in both rat and mouse.Click here for fileGene Ontology Analysis. Complete results of gene ontology analyses.Click here for fileMotif Enrichment Analysis Using CLOVER. Complete results of promoter analysis using the CLOVER program and the JASPAR library.Click here for filede novo Motif Discovery Using MotifSampler. Top hits from MotifSample de novo motif discovery analysis.Click here for file


At present, there are no clinically reliable disease markers for chronic fatigue syndrome. DNA chip microarray technology provides a method for examining the differential expression of mRNA from a large number of genes. Our hypothesis was that a gene expression signature, generated by microarray assays, could help identify genes which are dysregulated in patients with post-infectious CFS and so help identify biomarkers for the condition.Human genome-wide Affymetrix GeneChip arrays  were used to compare the levels of gene expression in the peripheral blood mononuclear cells of male patients with post-infectious chronic fatigue (n = 8) and male healthy control subjects (n = 7).Patients and healthy subjects differed significantly in the level of expression of 366 genes. Analysis of the differentially expressed genes indicated functional implications in immune modulation, oxidative stress and apoptosis. Prototype biomarkers were identified on the basis of differential levels of gene expression and possible biological significanceDifferential expression of key genes identified in this study offer an insight into the possible mechanism of chronic fatigue following infection. The representative biomarkers identified in this research appear promising as potential biomarkers for diagnosis and treatment. Persistent fatigue after infection is recognised and forms part of a clinically-defined syndrome (chronic fatigue syndrome or CFS). CFS is a highly heterogeneous illness which is characterised by the presence of new-onset, persistent or relapsing fatigue of sufficient severity that interferes with normal activity. Patients also report impaired short-term memory and concentration, muscle pain and post-exertional malaise . The cliIn recent years, numerous microarray studies have been undertaken in order to distinguish patients with chronic fatigue from healthy controls. This subject has also been covered extensively in a special 2006 issue of the journal Pharmacogenomics. In this issue, several papers utilised numerous multivariate projection methods, bioinformatics, algorithms and computational analyses of microarray data, in order to provide better discrimination of subjects with unexplained chronic fatigue and CFS, from healthy controls -7.Although chronic fatigue is well recognised after certain infections , CFS is not commonly considered to be due to persistent infection. A preceding history of non-infection is frequently reported and recent studies indicate other factors may be responsible for the symptoms of this condition ,9. As a We hypothesised that patients with persistent fatigue developing after community-acquired infections are biologically different from healthy subjects and this would be reflected in a differential gene signature. Recent advances in genome sequencing and automated chip manufacture have made DNA chip or microarray technology readily available . This teIn our study, we aimed to obtain a complete "gene signature" for non-psychiatric patients with post-infectious persistent chronic fatigue. We excluded female subjects in order to avoid confounding factors from the monthly reproductive cycle. By using an Affymetrix GeneChip Human Genome U133 double-chip set that contains nearly 45,000 probe sets, representing 39,000 transcripts derived from more than 33,000 human gene sequences, most of the known human genome was encompassed in this work.All patients underwent full medical and neurological evaluation clinically and had appropriate investigations to exclude alternative explanations for their symptoms. All patients with CFS fulfilled the international research criteria for diagnosis . CFS pat5 cells) were stored under liquid nitrogen.Male patients with post-infectious CFS and normal healthy controls underwent venous blood sample collection (prior to 11 am) in 2 × 5 ml EDTA tubes. PBMC were isolated immediately and stored within 2 hours of sampling. EDTA-treated whole blood was diluted 1:1 with phosphate buffered saline (PBS). Two volumes of blood were overlaid onto one volume Histopaque-1077 (Sigma) and centrifuged at 20°C, 500 g for 30 min. The PBMC interfaces were washed twice with PBS and the pellets were re-suspended in PBS. Aliquots were counted and dry pellets . All 15 samples were then hybridised to Affymetrix GeneChip HG-U133A and HG-U133B arrays. The arrays were washed and stained using Affymetrix protocols on the Fluidics Station 400 and scanned on the Gene Array Scanner 2500.GCOS1.1.1 software (Affymetrix) was used to generate raw data from scanned images. Data analysis was performed using FunAlyse, an in-house built automated pipeline in Sir Henry Wellcome Functional Genomics Facility (SHWFGF), University of Glasgow.This analysis consists of the Robust Multichip Average (RMA) normalisation , followe® Systems, ) in order to identify possible biological processes associated with differentially expressed genes. The initial set of differentially expressed, redundant Affymetrix probe-sets was first mapped to Entrez Gene identifiers [The above RP-generated expression profile was then analysed with the Ingenuity Pathway Analysis (IPA) software  and western blot assays, in order to verify the microarray data for one of the putative biomarkers, defensin-α1.cDNA synthesis and PCR were performed as described previously ,24 and psense 5'-CAAGAGCTGATGAGGTTGCT-3'antisense 5'-GAAGGTACAGGAGTAATAGC-3'.5 PBMC. PAGE gels were assessed for equal protein loading by coomassie blue staining. The gels were then electrophoretically transferred onto PVDF membranes (BioRad), for 2 hours. Blots were blocked with 10% normal goat serum for 30 min, and then incubated for 2 hours at room temperature with a monoclonal antibody to human neutrophil defensin α1–3 , diluted in TBS/0.05% Tween-20. Proteins were detected with a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG  and protein bands were detected using SIGMA FAST BCIP/NBT.PBMC pellets were re-suspended in sample-reducing buffer  and boiled for 5 min. The cell lysates were loaded onto 10% PAGE gels, each track equivalent to 2 × 10A number of genes were significantly over- or under-expressed in CFS patients, as compared to healthy controls, in genome-wide microarray analysis. The Rank Products analysis revealed that 413 Affymetrix probe-sets were significantly up-regulated and 189 significantly down-regulated  in the CFS samples. Of the significantly up-regulated probe-sets, 141 showed the expression changed at least 2-fold and 20 at least 3-fold. The corresponding numbers for the down regulated probe-sets are 57 and 2, respectively.Annotating the above list of 602 differentially expressed probe-sets with EntrezGene IDs and subsequent submission to the IPA server resulted in successful mapping of the above probe-sets to 366 non-redundant "focus" genes with records in the IPA database. Of these, 286 genes were significantly up-regulated and 80 significantly down-regulated in the CFS samples. Of the significantly up-regulated genes, 105 genes showed that expression changed at least 2-fold and 16 genes at least 3-fold. The corresponding numbers for the down-regulated genes are 25 and 3, respectively. The ten most highly up-regulated genes and the three most highly down-regulated genes are presented in Table To investigate the biological context of genes differentially expressed in CFS patients, the IPA software was used, as described in Methods. Figure The gene signature of CFS highlighted changes in gene expression in three main areas: oxidative stress, apoptosis and viral-like immune dysfunction. These genes are summarised in Table et al. [There is a large body of evidence from researchers in CFS indicating that oxidative stress contributes to disease progression and resultant symptoms -29. Our et al.  showed tThere is active apoptosis in the cells of patients with CFS and consistent with this is the finding that many apoptosis-associated genes are up-regulated, including Caspase 1 Table . Severalet al. [Immune evasion is a well-documented mechanism by which viruses, bacteria, parasites and cancer cells avoid destruction by the host. Immune modulations can be varied and complex. They include targeted disruption of T-cell function, suppression of T-cell cytotoxic death receptors, induction of apoptosis proteins, inhibition of innate immune systems such as dendritic cell and natural killer (NK) receptors. From this study, several genes associated with the immune system are shown to be up-regulated in patients with CFS, thus indicating possible functional alterations Table . Among tet al.  indicateet al. [In addition to the MHC, within the top up-regulated genes there are several genes encoding immune proteins which belong to the innate immune system. Patients with CFS have been suspected to have subtle immune dysfunction. Altered NK cell function in CFS patients has been reported previously by Morrison et al. , and is et al.  and is ket al. -37.The immune modulation in post-Lyme disease fatigue is remarkably similar to the gene signature presented here for patients with CFS . It is kUp-regulated immune-related genes in patients include lactotransferrin, defensin-α1 and integrins. Lactotransferrin is an iron-binding protein responsible for immunological reactions, while defensins are a family of microbicidal and cytotoxic peptides released in response to viral and bacterial infections. Defensins are also involved in the anti-microbial defence of epithelial surfaces . Patients with CFS are known to have increased susceptibility for infections and candidiasis is frequently reported .Integrins are extracellular matrix proteins involved in cell adhesion and also activate cytosolic signal cascades for cell growth and regulation. Arginase is a key enzyme considered to be active in immune modulation and is also involved in NK cell function. Other notable up-regulated genes include haemoglobins, especially foetal haemoglobin, and this may suggest a response to increased oxidative stress. Another family of up-regulated genes relate to cellular apoptosis and serine-threonine kinase is a member of brain kinase family of protein implicated in neural apoptosis and formation of corpora amylacea. Of the down-regulated genes, ribosomal protein gene and zinc finger protein genes are involved in cell cycle and cellular growth.Two members of the defensin gene family were found to be amongst the most highly over-expressed genes in the microarray; defensin-α1 and -α4. Defensins are a family of cytotoxic peptides found in the microbicidal granules of neutrophils and macrophages, and are released in response to viral and bacterial infections. These proteins are thus likely to play a role in phagocyte-mediated host defence. As a result of the high defensin levels identified in the microarray data, defensin-α1 was chosen for further analysis. We aimed to confirm this data at the RNA and protein level, using another cohort of blood samples from 10 CFS patients and 10 normal healthy controls. Oligonucleotides and antibodies specific to defensin-α1 were used to detect and compare changes at the RNA and protein level using RT-PCR and western blotting, respectively. Figure et al. [et al. , CXCR4 was also up-regulated.A comprehensive gene signature of non-psychiatric patients with CFS has been generated by a whole-genome microarray assay. The microarray data presented here illustrates differences in gene expression across the human genome between post-infectious, non-psychiatric male patients with CFS and healthy controls. Analysis of the gene signature of CFS suggests that three significant pathways are altered in patients: oxidative stress, apoptosis and viral-like immune modulation. Previously, Gu et al.  performeet al.  demonstret al. . As a reThe overall picture emerging from these results point to a significant, albeit phenotypically subtle, perturbation of function in relation to microbial defence, viral immuno-surveillance and cell growth amongst patients with post-infectious chronic fatigue. A possible criticism of the work is that female patients were excluded. As already explained, the primary reason for exclusion was to avoid any confounding endocrine influence on gene signature. Previous research has found few gender-related differences in terms of symptom severity. There was no gender effect in a prospective cohort study of patients with post-infectious fatigue attending primary care . In geneRecent studies have attempted to determine the molecular basis of CFS -49. TablThe data presented here clearly illustrate important differences in gene expression between patients with post-infectious CFS and healthy controls. While these differences in gene signatures may offer a rational explanation for the symptoms, and confirm that CFS is a true and biological illness, gene microarray results do not imply that CFS is primarily a genetic disorder. In addition, the research data do not prove if differentially expressed genes are the predisposing cause or downstream effect of CFS. We believe, however, that the data presented here is an important first step forward towards the goal of identifying putative biomarkers to support the clinical diagnosis of CFS. Furthermore, the biological pathways identified by the over-expressed genes offer a rational understanding and possible explanation of the complex and often multi-systemic nature of CFS, which is common and remains a potentially disabling illness in search of an effective cure.CFS: Chronic Fatigue Syndrome; DAF: Decay Acceleration Factor; FC: Fold Change; FDR: False Discovery Rate; GMN: Global Molecular Network; IPA: Ingenuity Pathway Analysis; MHC: Major Histocompatibility complex; NFκB: Nuclear Factor kappa B; PBMC: Peripheral Blood Mononuclear Cells; RT-PCR: Reverse Transcription-Polymerase Chain Reaction; RMA: Robust Multichip Analysis; TLR: Toll-Like Receptor.The Court of the University of Glasgow has filed a patent entitled "Materials and Methods for Diagnosis and Treatment of Chronic Fatigue Syndrome" Patent File Number GB0502042.5.AC, CC and JG participated in the design and coordination of the study, carried out the molecular genetic studies, participated in the sequence alignment and helped to draft the manuscript. CC carried out the immunoassays. PH carried out the IPA analysis and performed the statistical analysis. SH carried out the analysis and interpretation of IPA analysis and drafted the manuscript. PB participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


E. coli K12. Next, we demonstrate one way in which network connectivity measures and global and local function distribution can be exploited to predict protein function for previously uncharacterized proteins.Protein-protein interactions networks are most often generated from physical protein-protein interaction data. Co-conservation, also known as phylogenetic profiles, is an alternative source of information for generating protein interaction networks. Co-conservation methods generate interaction networks among proteins that are gained or lost together through evolution. Co-conservation is a particularly useful technique in the compact bacteria genomes. Prior studies in yeast suggest that the topology of protein-protein interaction networks generated from physical interaction assays can offer important insight into protein function. Here, we hypothesize that in bacteria, the topology of protein interaction networks derived via co-conservation information could similarly improve methods for predicting protein function. Since the topology of bacteria co-conservation protein-protein interaction networks has not previously been studied in depth, we first perform such an analysis for co-conservation networks in Our results showed, like most biological networks, our bacteria co-conserved protein-protein interaction networks had scale-free topologies. Our results indicated that some properties of the physical yeast interaction network hold in our bacteria co-conservation networks, such as high connectivity for essential proteins. However, the high connectivity among protein complexes in the yeast physical network was not seen in the co-conservation network which uses all bacteria as the reference set. We found that the distribution of node connectivity varied by functional category and could be informative for function prediction. By integrating of functional information from different annotation sources and using the network topology, we were able to infer function for uncharacterized proteins.Interactions networks based on co-conservation can contain information distinct from networks based on physical or other interaction types. Our study has shown co-conservation based networks to exhibit a scale free topology, as expected for biological networks. We also revealed ways that connectivity in our networks can be informative for the functional characterization of proteins. Co-conservation, a measure of the degree to which proteins are gained and lost together through evolution , has deTopological analysis of other types of biological networks, including protein-protein interactions, regulatory interactions, and metabolic networks, has demonstrated that structural features of network subgraphs can provide quantitative insight into biological function -33. For Due to the availability of genome-wide data, nearly all previous investigations of network topology have been in yeast, and the majority has been based on high-throughput assays of protein-protein interactions (PPI). In contrast, this paper examines co-conservation networks in bacteria using different reference genomes, the first in-depth study to our knowledge, of the topological characteristics of such networks. This characterization can be used for current and future comparison to like studies in other organism and network types. Co-conservation networks are distinct from physical interaction networks as they capture putative functional relationships which are not necessarily dependent on direct protein binding. We find that bacterial co-conservation networks show both biologically important similarities and differences with yeast PPI networks. For example, similar to reports of significance in yeast PPI networks ,34,35, nSuch differences call into question the broad applicability of the yeast methods for predicting function based on network topology. Here, the topological properties of bacterial co-conservation networks and their relationship to function are examined. Based on this assessment, we demonstrate the use of co-conservation network topological properties to predict the function of uncharacterized proteins.E. coli K12 and 267 other completely sequenced bacteria (referred to as the All reference set) is shown in Figure E. coli K12 protein, which other proteins it is co-conserved with in a large reference group. We have previously described how such interaction networks can vary with the selection of reference group ; Information storage and processing ; Cellular processes ; Metabolism .Click here for fileAverage connectivity of each functional category in networks using different reference sets and using TIGR annotation. The average connectivity of each functional category in networks with and without removing proteins appearing in more than 90% or less than 10% of organisms using different reference sets and using TIGR annotation.Click here for fileFunctional classification of hub versus non-hub proteins using COG. Functional classification of hub versus non-hub proteins. Hub proteins are highly connected. a) COG connectivity versus log number of proteins. b) COG connectivity versus percentage of proteins, normalized within given connectivity.Click here for fileFunctional classification of hub versus non-hub proteins using TIGR. Functional classification of hub versus non-hub proteins. Hub proteins are highly connected. a) TIGR connectivity versus log number of proteins. b) TIGR connectivity versus percentage of proteins, normalized within given connectivity.Click here for fileFunction prediction in clusters containing at least 1 and maximum 10 proteins using different source of annotations. Function prediction in clusters containing at least 1 and maximum 10 proteins with at least one unclassified proteins across all sources and at least one source of annotation. KEGG version downloaded 1/5/2007, COG version downloaded 1/5/2007, TIGR version downloaded 1/5/2007. Predicted function column lists putative function based on annotated cluster members. As partial validation of the prediction, more recent annotations from EcoCyc version downloaded 1/5/2007 and a later version of COG downloaded 1/7/2007 (New COG) are shown.Click here for fileTopological analysis of the networks using different reference sets. Topological analysis of the networks without removing proteins appearing in more than 90% or less than 10% of organisms using different reference sets.Click here for file


Studies on HPV infection in pregnant women and HPV transmission to the child have yielded inconsistent results.To estimate mother-to-child HPV transmission we carried out a prospective cohort study that included 66 HPV-positive and 77 HPV-negative pregnant women and their offspring attending a maternity hospital in Barcelona. To estimate HPV prevalence and genotype distribution in pregnancy we also carried out a related screening survey of cervical HPV-DNA detection among 828 pregnant women. Cervical cells from the mother were collected at pregnancy (mean of 31 weeks) and at the 6-week post-partum visit. Exfoliated cells from the mouth and external genitalia of the infants were collected around birth, at the 6-week post-partum visit, and around 3, 6, 12, and 24 months of age. All samples were tested for HPV using PCR. Associations between potential determinants of HPV infection in pregnant women and of HPV positivity in infants were also explored by logistic regression modelling.Overall cervical HPV-DNA detection in pregnant women recruited in the HPV screening survey was 6.5% (54/828). Sexual behavior-related variables, previous histories of genital warts or sexually transmitted infections, and presence of cytological abnormalities were statistically significantly and positively associated with HPV DNA detection in pregnant women recruited in the cohort. At 418 infant visits and a mean follow-up time of 14 months, 19.7% of infants born to HPV-positive mothers and 16.9% of those born to HPV-negative mothers tested HPV positive at some point during infants' follow-up. The most frequently detected genotype both in infants and mothers was HPV-16, after excluding untyped HPV infections. We found a strong and statistically significant association between mother's and child's HPV status at the 6-week post-partum visit. Thus, children of mothers' who were HPV-positive at the post-partum visit were about 5 times more likely to test HPV-positive than children of corresponding HPV-negative mothers (p = 0.02).This study confirms that the risk of vertical transmission of HPV genotypes is relatively low. HPV persistence in infants is a rare event. These data also indicate that vertical transmission may not be the sole source of HPV infections in infants and provides partial evidence for horizontal mother-to-child HPV transmission. Despite the overwhelming evidence for a sexual transmission of high-risk HPVs, other routes of transmission have been proposed. Several studies have explored whether HPVs can be vertically transmitted from mother to child by direct contact during labor, or horizontally through manipulation of the child with infected hands, bathing, towels and fomites . StudTo assess in a low-risk country the prevalence and determinants of HPV infection in pregnant women as well as perinatal transmission and persistence of HPV types, we carried out an HPV screening survey in pregnant women and a prospective study of HPV-positive and HPV-negative mothers and their infants.The project was initiated in 1995 by carrying out a prospective cohort study of pregnant women attending a public maternity hospital for prenatal care in urban Barcelona, Spain. The hospital covers mostly general, non-selected population in its catchment area. Pregnant women were selected according to their potential risk of HPV exposure as based on their history of high-risk sexual behavior, genital warts, abnormal colposcopy, and HPV-related cervical lesions by cytology or histology. Emphasis was placed in recruiting high-risk women to procure a high yield of HPV-positive mothers and thus increase statistical power to properly study HPV transmission. A total of 115 consenting pregnant women were included and classified according to their risk of HPV exposure into high- (n = 73) and low-risk (n = 42) groups. At their first or second obstetric visit , consenting women were tested for cervical HPV-DNA status using a screening consensus primer PCR (see below). Since the yield of HPV-positive pregnant women was too low  to adequately assess mother-to-child HPV transmission, we extended the study by carrying out an HPV screening survey among unselected pregnant women to identify and recruit additional HPV-positive subjects for the prospective cohort study.Subjects for this HPV screening survey included 858 pregnant women consecutively attending the prenatal care clinic of the maternity hospital between 1997 and 2000. Consenting women were tested for cervical HPV-DNA status at their obstetric visit  using the same consensus primer PCR (see below) as that used in the initial cohort study. Women were included in the prospective cohort study only when a positive result was found (i.e. HPV-negative pregnant women from this HPV screening survey were not included in the prospective cohort study). Participation rates were not estimated and refusals were replaced by the next consenting women. This limitation should not bias our results as the purpose of the HPV screening survey was mainly the recruitment of additional HPV positive pregnant women to the prospective cohort study. Thirty women were excluded because the cervical samples collected were invalid for PCR testing leaving a total number of 828 women for the analysis of HPV prevalence among unselected pregnant women. Women from this survey that were not recruited into the prospective cohort study (those testing HPV-negative) did not receive the risk-factor questionnaire (see below).Women in the final prospective cohort study included those from the initial cohort (n = 115) and all HPV-positive women from the HPV screening survey (n = 54) along with their offspring. At the enrollment visit, performed between 2 and 12 weeks after the HPV screening visit, all 169 women signed a written informed consent and were administered a full standardised questionnaire by personal interview that included socioeconomic, reproductive, sexual behavior, and risk factor variables. A standardised clinical form was also used to systematically collect all relevant clinical parameters during pregnancy, labor, and follow up visits. The questionnaires and clinical forms were only collected for the women participating in the prospective cohort study but not for the group of women that tested HPV-negative in the HPV screening survey.® collection swab, Spain), at two time points: at the pregnancy visit , and at the 6-week postpartum visit. All samples were screened for cytological abnormalities.Cervical samples from each woman were collected using a dry swab , at or around 6 weeks, and at or around 3, 6, 12, and 24 months of age. Genital samples from boys were obtained by gently rubbing the penis glans and the inner mucosal part of the prepuce. The penile shaft was not sampled. Genital samples from girls were obtained by gently rubbing throughout the mucosal part of the vulva. Mouth samples were obtained by firmly rubbing the dors of the tongue, mucosal part of the cheeks and the hard palate, using a single swab. Particular care was taken with sample collection procedures to prevent cross-contamination between subjects and different anatomical sites by using disposable equipment and changing bed lining and gloves for each woman. All the samples from recruited infants were mostly collected by the same pediatrician (JMP) throughout the study. Since the initial anal specimens did not yield sufficient DNA for adequate HPV testing, collection of anal samples was discontinued early in the follow-up and the few results obtained for this site were not considered in our analyses. All samples were sent to the laboratory, suspended in 1 mL of 0.1 M PBS , transferred to clean sterile eppendorf tubes and kept frozen at -20°C until processed.At each visit a physical examination of the child that included also a clinical inspection of the oral cavity and the external anogenital area was performed.DNA extraction was performed using 200 μL of cervical or oral cells suspensions and 400 μL from genital cells using the Qiamp Viral DNA kit  in accordance with the manufacturer's instructions.Amplification was performed using 5 μL extracted DNA with the consensus primers MY09/MY11, which amplify a fragment from the L1 HPV gene . PCR wasAll specimens were processed blindly to child or mother HPV-risk or HPV DNA initial status. Each PCR run included DNA extraction and HPV negative controls. Particular care was taken to prevent carry-over contamination by separating pre- and post-PCR areas.HPV prevalence was estimated among women at the pregnancy visit and at the 6-week postpartum visit. HPV prevalence estimates in pregnant women did not include those in the initial cohort study as these women were selected according to their risk of HPV exposure and would yield an overestimated HPV prevalence. Determinants of both HPV positivity in pregnant women and in their offspring were explored by multivariate unconditional logistic regression models to estimate prevalence odds ratios (ORs) and 95 percent confidence limits. ORs where adjusted by age in tertiles . Even though these women were selected based on their HPV status and thus biased towards HPV positivity, this should not affect the validity of exploring potential risk factors for HPV infection in pregnant women.HPV prevalence in infants and mother to child transmission rates were estimated at the perinatal period (0 to 6 days of life), at the 6-week postpartum visit and, since few HPV positive samples were detected after the 6-week visit, all childrens' HPV results from successive visits (3 months throughout 24 months) were combined in one single prevalence estimate. Overall and type-specific HPV transmission rates were also estimated at any point during follow-up. Estimates are given according to the HPV status of the mother at pregnancy and at the 6-week postpartum visit. HPV type-specific concordance among paired mother-child samples was estimated among the HPV-positive samples that could be genotyped. Paired samples with HPV X were not counted as concordant.Two parameters were used to explore horizontal transmission: HPV positivity rates in infants born to HPV-negative mothers, and the correlation between HPV status in the mother and HPV status in the child in paired mother-child samples simultaneously collected at the 6-week visit.All women participating in the cohort study signed a written informed consent. Protocols were approved by the local ethical committees of the Maternity Hospital, where the study was carried out, and the Ciutat Sanitaria i Universitaria de Bellvitge, where the coordinating center of the study was located.Of the initial 169 recruited pregnant women , 82  were HPV positive at the pregnancy visit. The genotype distribution in these HPV-positive samples from pregnant women was in descending order of frequency: HPV 16 (32.9%), HPV X (28.0%), HPV 6/11 (25.6%), HPV 31 (11.0%), HPV 39 (9.8%), HPV 18 (7.3%), HPV 33 (3.7%). Multiple types were present in 14 women (17.1%). The most frequent combinations among these were co-infection of HPV 16 with other types (50.0%) and co-infection of HPV 31 with HPV 39 (35.7%). Cervical HPV prevalence in the unselected group of pregnant women from the HPV screening survey was 6.5% .Of the initial 169 recruited women with a known HPV-DNA status, 26 mother-infant pairs  were excluded from the prospective cohort study because no adequate sample from the child could be obtained for HPV testing neither at birth nor at any of the subsequent follow-up visits. Of the 26 excluded pairs, 16  were HPV positive. The distribution of HPV types in these 16 excluded HPV-positive women did not statistically differ from the 66 HPV-positive women finally included in the cohort (p = 0.23). Thus, the final prospective cohort study included 143 mother-infant pairs with a valid PCR result.A total of 418 study visits were performed with a mean follow-up time of 14 months. All pregnancies ended on single births.The characteristics of the 143 women and infants enrolled in the prospective cohort study along with the obstetric clinical details are detailed in Table As shown in Table The age-adjusted analyses to identify determinants of cervical HPV infection in pregnant women yielded the expected pattern of established risk factors for HPV infection Table . No assoAs expected we found a strong association between HPV positivity and presence of cervical lesions (LSIL or worse). All HSIL cases tested HPV positive being HPVs 16, 18, and HPV X the most frequently detected types among women with HSIL.At 418 infant visits and a mean follow-up time of 14 months, overall prevalence of HPV in infants at any visit was 18.2% . New infections at the post-partum visit among HPV-negative women at pregnancy occurred in only one woman . Thus, there was a strong association between HPV status at pregnancy and HPV status in the mother at the post-partum visit (p < 0.0001). Type-specific concordance among HPV-positive women between pregnancy and post-partum visit was 100% (17/17 after excluding 7 women with HPV X at either visit). The overall type-specific distribution of HPV-positive mothers at pregnancy was not statistically significantly different from that at the post-partum visit (p = 0.83). Of the 19 mothers that tested HPV X at pregnancy only 2 were HPV X persistent at the post-partum visit, 9 became HPV negative, 3 became HPV-16 positive, and in the remaining 5, no PCR result was available at the post-partum visit.Of the 26 children that tested HPV positive at some point during follow-up, 18 had at least two consecutive samples to assess HPV persistence. Three (16.7%) had two consecutive HPV-positive samples and in two of them (11.1%) there was genotype-specific persistence. In the remaining 15 infants (83.3%) their first HPV-positive sample was followed by a subsequent HPV-negative sample. There was no statistical evidence that the distribution of HPV types detected from the infants changed over time (data not shown). At the end of the study none of the infants developed oral, anogenital or cutaneous, macroscopically identifiable HPV-related lesions.Our data from both the HPV screening survey of unselected pregnant women and the prospective cohort study provide for the first time estimates for HPV prevalence, type-specific distribution, mother-to-child transmission rates as well as HPV persistence in pregnant women and in infants in Spain.Consistent with previous reports , Although HPV-DNA detection rates in samples of newborns and infants vary widely in the literature, well conducted prospective studies suggest that the risk of perinatal transmission, although existent, is relatively low. Several studies have tested infants for HPV-DNA or antibodies . Secondly, we found an association between HPV status in the mother at the 6-week postpartum visit and the HPV status in children at the same visit or thereafter. Thus, at the 6-week post-partum visit, children of mothers' who were HPV-positive at the post-partum visit were about 5 times more likely to test HPV-positive than children of corresponding HPV-negative mothers . In contrast, no association was found between mothers' HPV status at pregnancy and children's HPV status at any of the visits combined. Thus, all together, the data in Table In assessing HPV positivity in children born to HPV-negative mothers we can not rule out that these mothers were false HPV negatives at pregnancy. We need to take into account that 52% of the HPV-negative mothers came from the high-risk group of women included in the initial cohort study. Thus, increased HPV exposure may increase risk of false negative results which might somehow partially explain HPV transmission among HPV-negative mothers.Our results do not support a high prevalence of HPV during pregnancy: 6.5% of the unselected group of women was positive for HPV-DNA by consensus PCR. This relatively low HPV prevalence in pregnant women correlates with the low prevalence of HPV infection in the female general population (between 1.3% and 2.4%) ,15 and tAs expected, and consistent with the sexual mode of transmission of HPV infections, we found that an early age at first sexual intercourse, a high number of sexual partners, particularly before the age of 20, a previous history of genital warts or STDs as well as a current squamous intraepithelial lesion in the cervix as diagnosed by cytology, were the strongest determinants of HPV positivity in pregnant women Table . These eAs shown in Table In interpreting these results it should be considered that our reported HPV type-specific distribution is probably biased towards an overestimated detection of HPV X, as the samples that tested positive with the generic probe were tested with only seven type-specific probes . Thus the high percentage of samples classified as HPV X could be true rare genital HPV types, cutaneous types (unlikely because of the poor efficiency of the primer system in detecting cutaneous types) or still untyped HPVs. Furthermore the MY09/MY11 primer system has been improved by the PGMY primer system in terms of higher reproducibility of primer synthesis as well as detection of mucosal HPVs. Unfortunately, very few samples remained available for re-testing with the newer PGMY system. This limitation may also have resulted in an underestimation of the true underlying type-specific concordance.In conclusion, our study, conducted in a population at low risk for HPV and cervical cancer, confirms that high-risk HPV genotypes can be vertically transmitted to the child, although the risk of vertical transmission is relatively low. In this study, if we exclude untyped (HPV X) infections, HPV 16 has been found to be the most frequent type detected both in mothers and infants. Infants of women who tested HPV positive at six weeks after delivery are nearly five times more likely to test HPV positive than infants of HPV-negative mothers. HPV persistence in infants is a rare event. Given the substantial HPV positivity observed in children born to HPV-negative mothers, these data suggest that vertical transmission may not be the sole source of HPV infections in children and that horizontal mother-to-child transmission may play also a role. It remains to be seen whether this alternative mode of HPV transmission and acquisition may have an impact in several areas, including vaccination strategies, epidemiological studies, and the clinical management of children with HPV-associated diseases.XC: Research Grants ; Speakers Bureau ; Steering Committee . GA: Travel grants . SdS: Research Grants . FXB: Advisory Board ; Speakers Bureau (GlaxoSmithKline); Research Grants .XC, TD, FXB, SdS were the principal epidemiological investigators in the various phases of this long study, conceived the study, wrote the protocols, assured funding, identified clinical investigators and study personnel, supervised statistical analyses and performance of laboratory assays, and wrote the manuscript. MPC, TD wrote the HPV assays protocols, performed the HPV DNA detection and genotyping of the samples validating the techniques with other referent HPV laboratories, created the HPV data base, wrote part of the methods section and made substantial comments to the manuscript. AG, RR, JMP were the clinical investigators (two ObGyn and one pediatrician), trained and supervised study clinical staff  and sample collection throughout the study, and made substantial contributions to the manuscript. MJQ, JM, GA coordinated the field work in terms of study implementation and data collection, designed the study data collection forms, created clinical and laboratory databases, implemented quality assurance procedures, made the statistical analyses, produced the working and final tables and made substantial comments to the manuscript.The work was partially supported by Spanish public grants from the Instituto de Salud Carlos III , from the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR 2005SGR 00695), and from the Marató de TV3 Foundation (051530), who had no role in the data collection, analysis or interpretation of the results.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/74/prepub


There is no consensus on the role of routine follow-up imaging during nonoperative management of blunt renal trauma. We reviewed our experience with nonoperative management of blunt renal injuries in order to evaluate the utility of routine early follow-up imaging.We reviewed all cases of blunt renal injury admitted for nonoperative management at our institution between 1/2002 and 1/2006. Data were compiled from chart review, and clinical outcomes were correlated with CT imaging results.207 patients were identified . American Association for the Surgery of Trauma (AAST) grades I, II, III, IV, and V were assigned to 35 (16%), 66 (31%), 81 (39%), 26 (13%), and 2 (1%) renal units, respectively. 177 (84%) renal units underwent routine follow-up imaging 24–48 hours after admission. In three cases of grade IV renal injury, a ureteral stent was placed after serial imaging demonstrated persistent extravasation. In no other cases did follow-up imaging independently alter clinical management. There were no urologic complications among cases for which follow-up imaging was not obtained.Routine follow-up imaging is unnecessary for blunt renal injuries of grades I-III. Grade IV renovascular injuries can be followed clinically without routine early follow-up imaging, but urine extravasation necessitates serial imaging to guide management decisions. The volume of grade V renal injuries in this study is not sufficient to support or contest the need for routine follow-up imaging. Nonoperative management has become the rule for the majority of blunt renal injuries, with higher rates of renal salvage and decreased morbidity compared to primary surgical management . The non2), Glascow coma score (GCS), mechanism of injury, side of injury, grade of injury, vital signs, serial hematocrit measurements, results of follow-up imaging, complications, and delayed interventions.After receiving approval from the Institutional Review Board of the University of Tennessee Health Science Center, Memphis, Tennessee, we performed a retrospective chart review of all patients admitted with blunt renal injury and primary nonoperative management at the Elvis Presley Memorial Trauma Center between 1/2002 and 1/2006. Data collected from chart review included patient age and gender, race, body mass index  and high-grade  injuries.Patient demographics are shown in Table We noted low rates of altered injury grading after follow-up imaging. After early re-imaging, renal injuries were downgraded in 4 (21%), 5 (9%), 9 (12%), 1 (4%), and 0 cases of grade I, II, III, IV, and V injury, respectively. Grade I injuries were downgraded when subcapsular hematoma was not evident on follow-up imaging; higher grade injuries were downgraded when lacerations appeared smaller or fewer in number on follow-up imaging compared to initial imaging. Renal injuries were upgraded in 0, 2 (4%), 2 (3%), 0, and 0 cases of grade I, II, III, IV, and V injury, respectively. Overall, the rate of injury downgrading was 12% for low-grade injury and 4% for high-grade injury (p = 0.32). The rate of injury upgrading was 3% for low-grade injuries and 0% for high-grade injuries (p = 1.00). There was no significant difference in the rates of altered injury grading on follow-up imaging between low and high-grade injuries.Of note, two cases of grade III injury were upgraded to grade IV on follow-up imaging. In the first case, the initial CT was performed with suboptimal delayed excretory phase imaging; urinary extravasation that was not apparent on the initial CT was demonstrated on the follow-up CT with appropriately timed delayed excretory phase imaging. The second case of grade III injury upgrade involved a patient with two devascularized segments on follow-up imaging in addition to a stable 1.5 cm laceration noted on the initial CT scan. This patient was managed without surgical intervention, and there were no delayed urologic complications.Complications and delayed interventions were uncommon in this series. In three cases of grade IV renal injury with collecting system insult, a ureteral stent was placed after serial imaging demonstrated persistent extravasation; endoscopic management proved definitive in these patients. One patient with a grade III renal injury developed a febrile urinary tract infection that was successfully managed with IV antibiotics. There were no cases in which repeat imaging results independently prompted urologic intervention. There were no urologic complications among cases for which follow-up imaging was not obtained.The incidence of traumatic renal injuries in the United States is approximately 5 per 100,000 persons , or 15,0Our institution previously reported a retrospective review of 48 cases of blunt renal injury and primary nonoperative management, noting that one in ten patients with a grade II or higher blunt renal injury had a delayed urologic complication detected by follow-up CT scan that ultimately required invasive intervention . FollowiOur contemporary retrospective review includes 175 patients  who underwent routine early follow-up imaging during nonoperative management of a blunt renal injury. The majority of these renal units 151/85%) suffered a grade I, II, or III injury. It is noteworthy that the proportion of grade I injuries was significantly smaller than other published blunt renal trauma series  includes 32 patients  who did not undergo repeat imaging. The majority of patients in this subset had a grade I or grade II injury that was managed by the trauma surgery service without consultation by the urology service. Excluding one patient with a grade V renal injury , there were no urologic complications among these patients. Admittedly, this group has limited statistical significance given its diminutive power.We are prospectively evaluating a revised management strategy Figure , and futWeaknesses of this study include its retrospective design, with the inherent limitations and biases of a retrospective analysis. Furthermore, we do not have long-term follow-up data for the majority of the patients in this cohort, so we are unable to evaluate the impact of routine re-imaging on long term renal functional outcomes, development of hypertension, or other renal injury sequellae. We suspect that the impact of routine re-imaging on such parameters is minimal. Additionally, we have reviewed the use of routine re-imaging 24–48 hours after blunt renal injury. It has been shown that many of the delayed complications from blunt renal trauma  occur at least 1–3 weeks after the injury occurs , so it iRoutine follow-up imaging is unnecessary in the nonoperative management of blunt renal injuries of grades I-III. Grade IV renovascular injuries can be followed clinically without routine follow-up imaging, but urine extravasation necessitates serial imaging to guide management decisions. The volume of grade V renal injuries in this study is not sufficient to support or contest the need for routine follow-up imaging, however we maintain a practice of routine follow-up imaging of nonoperatively managed grade V renal injuries. Ongoing prospective study will test these conclusions.The authors declare that they have no competing interests.JM and ID conceived of the study, participated in design and coordination, and drafted the manuscript. RM and DV participated in study design, data acquisition and analysis. SJ and RG oversaw interpretation of radiographic information. CD and RW helped with manuscript drafting and critical revision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Both the peptide and receptor have been detected in a wide variety of cancers, and OGF is currently used clinically as a biotherapy for some non-thyroid neoplasias. This study addressed the question of whether the OGF-OGFr axis is present and functional in human thyroid follicular cell - derived cancer.Carcinoma of the thyroid gland is an uncommon cancer, but the most frequent malignancy of the endocrine system. Most thyroid cancers are derived from the follicular cell. Follicular carcinoma (FTC) is considered more malignant than papillary thyroid carcinoma (PTC), and anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Opioid Growth Factor , PTC (KTC-1), and FTC (WRO 82-1) cell lines, immunohistochemistry was employed to ascertain the presence and location of OGF and OGFr. The growth characteristics in the presence of OGF or the opioid antagonist naltrexone (NTX), and the specificity of opioid peptides for proliferation of ATC, were established in KAT-18 cells. Dependence on peptide and receptor were investigated using neutralization studies with antibodies and siRNA experiments, respectively. The mechanism of peptide action on DNA synthesis and cell survival was ascertained. The ubiquity of the OGF-OGFr axis in thyroid follicular cell-derived cancer was assessed in KTC-1 (PTC) and WRO 82-1 (FTC) tumor cells.-6 M OGF inhibited cell replication up to 30%, whereas NTX increased cell growth up to 35% relative to cultures treated with sterile water. OGF treatment reduced cell number by as much as 38% in KAT-18 ATC in a dose-dependent and receptor-mediated manner. OGF antibodies neutralized the inhibitory effects of OGF, and siRNA knockdown of OGFr negated growth inhibition by OGF. Cell survival was not altered by OGF, but DNA synthesis as recorded by BrdU incorporation was depressed by 28% in OGF-treated cultures compared to those exposed to sterile water. The OGF-OGFr axis was detected and functional in PTC (KTC-1) and FTC (WRO 82-1) cell lines.OGF and OGFr were present in KAT-18 cells. Concentrations of 10These data suggest that OGF and OGFr are present in follicular-derived thyroid cancers, and that OGF serves in a tonically active inhibitory manner to maintain homeostasis of cell proliferation. These results may provide a biotherapeutic strategy in the treatment of these cancers. Thyroid cancer accounts for over 37,000 persons in the U.S. annually, but only 1,600 deaths . While m5]-enkephalin, is an endogenous opioid peptide that is an important regulator in the onset and progression of a variety of human cancers -enkephalin IgG (CO172) - [Met5]-enkephalin . Independent assays were performed 2 to 4 times in duplicate.Binding assays for OGFr were performed with procedures described earlier ,27. In bTo determine the specificity of endogenous OGF, log-phase KAT-18 cells were exposed to a polyclonal antibody to OGF   or to pr4 KAT-18 cells per well were seeded in 6-well plates containing 1 ml of serum-containing media without antibiotics. In each well, 20 nM OGFr-siRNA or control siRNA solutions in serum-free media were added. Cells were incubated for 4 h at 37°C prior to the addition of OGF or NTX (10-6 M). Cultures were incubated an additional 20 h, and then 1 ml fresh complete media either lacking or containing OGF or NTX was added. At 72 h cells were collected for computing growth. Two independent experiments were conducted. The scrambled siRNAs were purchased from Ambion.The OGFr-targeted siRNAs  were designed and obtained as ready-annealed, purified duplex probes from Ambion  . For tra32P-dCTP- OGFr cDNA. To determine equal loading, blots were stripped and reprobed with radiolabeled GAPDH and optical density of each band was determined by densitometry and analyzed by QuickOne (Bio-Rad Laboratories). Each value was normalized to GAPDH from the same blot.Some cultures were transfected with siRNAs and then harvested 72 h later for Northern analysis. Total RNA was extracted using the Paris Kit (Ambion), separated on an agarose gel, and mRNA transferred to nylon membrane . Membranes were probed with KAT-18 cells were seeded onto coverglasses, transiently transfected with OGFr-siRNA, and 72 h later stained with polyclonal antibodies to OGFr in order to detect the level of protein knockdown. OGFr protein levels were assessed by semi-quantitative immunocytochemistry whereby images were taken at the same exposure time with special care not to photobleach samples. The mean intensity of staining was determined for approximately 50 cells/group.5]-enkephalin, [Leu5]-enkephalin, -enkephalin (DPDPE), -enkephalin (DAMGO), -endorphin, naltrexone (NTX), naloxone, dynorphin A1-8 (Dynorphin), morphine sulfate, endomorphin 1 (Endo 1), endomorphin 2 (Endo 2), Sigma ; U69,583, Upjohn Diagnostics .The following compounds were obtained from the indicated sources: -enkephalin and this opioid peptide is known to bind to classical opioid receptors such as μ, δ, and κ as well [An extensive literature shows that OGF interacts with OGFr to regulate cell the proliferation of human and animal cells, normal and cancer ,15,17-29 as well . In orde as well , indicat as well . Thus, NA decrease in cell number as seen by treatment with OGF could be due to a decrease in cell survival because of programmed cell death, necrosis, and/or a reduction in DNA synthesis and subsequent cell replication. Our data show that neither apoptosis nor necrosis are involved with OGF activity in ATC cells, a result consistent with a previous publication documenting a similar finding in a variety of cancer cells growing in tissue culture . Howeverin vivo. Preclinical experiments are needed to establish whether this agent can alter the course of these lethal neoplasias. If successful, then the role of OGF in the etiology and pathogenesis of these cancers will be an exciting avenue to pursue and studies using OGF as an antitumor agent in the clinic are warranted.A critical question that needs to be addressed in future studies is whether OGF has efficacy in modulating the incidence and progression of human ATC, PTC, and FTC These data demonstrate that the OGF-OGFr axis is present in follicular-derived thyroid cancers, and that OGF is a tonically active inhibitory peptide that serves in a receptor-mediated manner to repress proliferation of these thyroid cancer cells. These results support further consideration on the use of OGF as a biotherapeutic strategy in the treatment of follicular-derived thyroid cancers.OGF: opioid growth factor; OGFr: opioid growth factor receptor; ATC: anaplastic thyroid cancer; PTC: papillary thyroid cancer; FTC: follicular thyroid cancerPJM and ISZ hold patents  on both OGFr and use of OGF to regulate growth of pancreatic and colorectal cancer.DG, SSP, RND, and ABC have no competing interests.PJM, DG, and ISZ participated in the conception of the project, and overall analysis and interpretation of data. In addition, PJM and ISZ were involved in the design and drafting of the manuscript. SSP, ABC, RND, and PJM carried out acquisition of the data and statistical analyses. All authors have approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/369/prepub