Numerous cross-sectional studies have reported reduced lung function among welders but limitations of exposure assessment and design preclude causal inference. The aim of this study was to investigate if long-term exposure to welding fume particulates accelerates the age-related decline in lung function.1) was analysed in relation to cumulated exposure to fume particulates among welders during the follow-up period.Lung function was measured by spirometry in 1987 and 2004 among 68 steel welders and 32 non-welding production workers. The decline in forced expiratory volume (FEV1 through follow-up period was in average 150 ml larger among welders than non-welders while the difference was negligible among non-smokers. The results did not reach statistical significance and within welders the decline in lung function was not related to the cumulated welding particulate exposure during follow-up periodAmong smokers the decline in FEV3 the average annual excess loss of FEV1 is unlikely to exceed 25 ml in smokers and 10 ml in non-smokers.Long-term exposure to welding emissions may accelerate the age-related decline of lung function but at exposure levels in the range of 1.5 to 6.5 mg/m Welding of metals may cause substantial exposure to fume particulates and gases ,2. The mAnalyses of short term exposure to welding fumes and gases have indicated an influence on lung function in cross-shift studies -12 whileOnly few longitudinal studies in welding populations have been published, and results have not been unambiguous ,23,23,24In 1987 the lung function was measured in 94 stainless steel and mild steel welders and 49 manual workers that were not exposed to welding emissions . The latWe obtained comprehensive information on demographic variables, characteristics of welding work and smoking habits for the period 1987 until 2004 by face-to face interviews in 1987 and 2004 , welding method , welding intermittence (average number of hours during a work shift), use of local exhaust ventilation (yes/no/sometimes), and welding in confined spaces (yes/no/sometimes). We reorganized the data to obtain welding characteristics by decades  in order to estimate lifelong cumulated exposure to welding fume particulates based upon a large Danish welding exposure database that provides ambient air particulate concentration by decade, type of steel, welding method, hours welding a day, exhaust ventilation and welding in confined spaces. The exposure matrix include 364 cells each providing the geometric mean exposure to welding fume particulates for a specific welding process.3). The process specific exposures in decades before 1970 were estimated by extrapolation based upon the declining trend of exposures that were observed from 1971 to 1985 for all types of welding processes. Very few welding exposure measurements have been carried out in Denmark since 1989. For purposes of the present exposure assessment we assumed that the average exposure levels have not changed from 1989 and onwards. However, in an alternative approach we also estimated exposures in 1990–1999 and 2000–2004 based upon linear extrapolation of exposures from 1971–85 and thus arriving at lower exposure estimates.The welding exposure matrix is based upon 1.106 measurements  of particulates in ambient air samples collected in filters placed in the breathing zone behind the welding helmet . The mea3 (SD) and among stainless steel welders 0.8 mg/m3 (SD). For each year in a welding shop we added bystander exposures corresponding to the numbers of hours during a work shift that welding was not performed (and therefore not included in the process specific exposure estimate). We did not accounted for the few metal workers using infrequent welding methods as welding robots and tube trod.Using the welding exposure matrix and the corresponding interview data on decade, type of steel, welding method, intermittence, exhaust ventilation, and welding in confined spaces we computed the product of number of years and particulate concentration in each decade and summed over all decades to arrive at a summary measure of total lifelong welding exposure (mg/m3*years), Table 3) while the estimated values for mild steel welding with MAG technology and stainless steel welding with TIG technology were slightly higher than the measured values  in three plants from which welders for this survey were recruited. This hygienic survey also included 12 full-shift stationary measurements of particulates in the vicinity of the welding processes to estimate bystander exposure. We validated the exposure estimate by comparing the welding process specific results of the 1987 measurements with the exposure levels predicted by the exposure assessment model taking decade and welding characteristics into account. There was a high agreement in welding fume measurements in 1987 and the predicted exposure levels based upon the welding fume exposure matrix and the interview information obtained in 2004. The geometric means for mild steel welding with MMA technology were identical 3.5 mg/m.5 mg/m3 1) and forced expiratory capacity (FVC) were recorded in accordance with American Thoracic Society guidelines (24). Lung function data were analysed in absolute figures as well as percentage predicted from gender, age, and height specific Danish reference data  greater than the decline among non-smokers. Moreover, the decline was significantly related to cigarette pack years through the follow-up period 1987–2004. Thus FEV1 declined 16 ml per pack year . Age, height and FEV1 at baseline and weight at follow-up was not related to lung function decline during follow-up, but the baseline FEV1 was significantly related to age (lower at higher age), height (higher at higher height) and smoking (reduced among smokers), but not to welding at baseline or to cumulated exposure up to baseline .Smokers in 1987 had a more pronounced decline in FEVWelders were not at increased risk of accelerated decline of lung function during follow-up in comparison with non-welding metal workers Table .1 that in average declined some 280 ml more in smokers than in non-smokers after adjustment for the effect of welding.Measurements of lung function 18 years after baseline indicated a limited accelerated loss of lung function among in particular smoking welders that were compared to a reference group of non-welding metal workers, turners, and electricians enrolled from the same plants. However, differences did not reach statistical significance and within the group of welders the decline in lung function was not related to the total welding particulate exposure during the follow-up period. As expected smoking had a strong effect on FEVcross-sectional study of 50 non-smoking manual metal arc welders and 50 non-smoking controls a significant smaller lung function among welders exposed for more than 9 year (640 ml in FEV1), suggesting an independent effect of welding fume on the lungs [Only few studies have been published that analyses effects of welding exposure on lungs in non-smokers. Meo et al found in a he lungs .longitudinal studies in welders addressing decline in lung function have been published (18–20). In a two-year follow-up (1996–98) from New Zealand lung function was measured in 54 TIG and MIG welders and 38 non-welders aged 40–41 years [1 (88.8 ml) than smoking referents (34.2 ml). Moreover, welders without respiratory protection or local exhaust ventilation had a greater annual decline in FEV1 (168.8 ml) than welders with protection (87.3 ml). The annual loss is surprisingly high, and higher than observed in the present study. Differences in severity of exposure to welding fumes, duration of exposure, welding in confined spaces, and use of protective equipment together with smoking habits may explain the apparent inconsistency. Our data are similar to the findings of Beckett et al [1 among welders of 40 ml and no overall excess decline in lung function in welders compared to control subjects during the three year of follow up. The participants were 51 MMA shipyard welders aged 32  fabricating steel alloy structures and 54 control subjects aged 39  . No exposure measurements were reported, and it was not indicated when the study was undertaken. Chinn et al studied English shipyards workers from 1980–84 to1987–1991. In all 346 subjects participated in the follow-up  with an average follow-up time of 6.7 years. Age at baseline was 17–30 years. The overall annual decline in FEV1 was 11.1 ml. Continuing smoking was associated with increased loss in FEV1 and atopy in smokers further increased the loss in FEV1. Continued shipyard work as welders or caulker/burners was associated with enhanced deterioration in FEV1. The annual decline in lung function is in the same range as data from the present study. The cohort was studied a decade before ours, and this might indicate a higher exposure to welding fumes than in the present study. Moreover, shipyards welders may have worked more in confined and poorly ventilated spaces with higher risk from impairment of lung function than welders working in more well ventilated areas [Few 41 years . No expott et al . They foed areas ,31.Less than 70% participated in the second survey. We cannot entirely exclude that high-level exposed workers with impaired lung function declined participation to a higher degree than others and thus introduce bias towards the null. However, the fact that the loss to follow-up was balanced in welders and non-welders and that the non-participants were younger with less tobacco smoking and respiratory symptoms do not support this hypothesis. It seems unlikely that weak associations between welding fume exposure and lung function is due to selective processes.Our findings have limitations due to small sample size, especially for analyses of subgroups, and the small sample size reduces the ability to generalize the results. On the other hand, the present data are aggregated from longitudinal analyses with a long follow-up time and accounting for the dropouts. Although a larger study group is definitely warranted the number of person-years at-risk is far higher in this study (some 1800 person-years) than in two earlier longitudinal studies [ declare that they have no competing interests.OO and SC conceived the study and wrote the protocol. SC organized the data collection. JPB performed data analyses. JPB and OO drafted the paper. All authors read, revised and approved the final manuscript.


There are many different types of pediculicides available OTC in Australia. In this study we compare the efficacy and safety of three topical pediculicides: a pediculicide containing melaleuca oil (tea tree oil) and lavender oil (TTO/LO); a head lice "suffocation" product; and a product containing pyrethrins and piperonyl butoxide (P/PB).This study was a randomised, assessor-blind, comparative, parallel study of 123 subjects with live head lice. The head lice products were applied according to the manufacturer's instructions  and the P/PB product was applied twice according to manufacturers instructions (on Day 0 and Day 7)). The presence or absence of live lice one day following the last treatment was determined.The percentage of subjects who were louse-free one day after the last treatment with the product containing tea tree oil and lavender oil  and the head lice "suffocation" product  was significantly higher compared to the percentage of subjects who were louse-free one day after the last treatment with the product containing pyrethrins and piperonyl butoxide .The high efficacy of the TTO/LO product and the head lice "suffocation" product offers an alternative to the pyrethrins-based product.The study was entered into the Australian/New Zealand Clinical Trial Registry, ACTRN12610000179033. The incidence of head louse infestation is high in many countries-3. This ®  and NeutraLice Advance® (a "suffocation" product).One essential oil based product containing 11.0% eucalyptus oil was reported to have an efficacy of 82.5% compared to a kill rate of 36.1% for a product containing pyrethrins and piperonyl butoxide using a study design similar to that employed in this study[To compare the efficacy and tolerance of three head lice treatment products when used according to the manufacturers instructions:® Key Pharmaceuticals Pty Ltd, Australia) presented as a clear oily solution1. Product containing melaleuca oil (tea tree oil) 10% w/v and lavender oil 1% w/v (TTO/LO)  presented as a white opaque lotion2. "suffocation" product containing benzyl alcohol, mineral oil, polysorbate 80, sorbitan monooleate, Carbopol 934, water and triethanolamine  presented as a pressurised aerosol mousse3. Product containing pyrethrins 1.65 mg/g, and piperonyl butoxide 16.5 mg/g (P/PB)  from three different schools in Queensland and their siblings with live head lice (adults or nymphs) in their hair or on their scalp. If parental written informed consent was provided, the children were screened for the presence of live head lice by visual inspection (see Appendix 1 - Definitions) and by dry-combing (see Appendix 1 - Definitions). Those subjects meeting the entry criteria were randomised and treated with one of the three head lice products. Subjects with a history of allergies, presence of scalp disease and those who were treated with a head lice product in the 4 weeks prior to participation in this trial were excluded.The TTO/LO and "suffocation" products were applied three times, at weekly intervals, as per the manufacturer's instructions . The P/PB product was applied twice, as per the manufacturer's instructions (on day 0 and day 7). The louse-combing procedure normally used in combination with these three products was not done so we could compare the efficacy of the components of each product without confounding the efficacy measurements by physically removing head lice by combing (which is a treatment in itself).The primary outcome measure was the louse free rate (see Appendix 1 - Definitions) assessed one day after the last treatment (at day 15 for TTO/LO and "suffocation" products and at day 8 after application of P/PB product) and was determined by wet-combing (see Appendix 1 - Definitions) for the Intention to Treat population and the Per Protocol population. A secondary outcome measure, louse free rate at day 1, was determined by dry-combingThis trial was conducted in compliance with the World Medical Association Declaration of Helsinki, the requirements of the National Statement on Ethical Conduct in Research Involving Humans, ICH E6 Guidance for the Industry; Good Clinical Practice: Consolidated Guidance, the National Privacy Principles and relevant State/Territory laws. The trial activities were approved by the Medical Research Ethics Committee of the University of Queensland, project No. 2003000184 and all parents/guardians provided written informed consent.Eligible subjects were randomly assigned to receive one of the three head lice treatments by a computer generated code using blocked randomisation (groups of six).This trial was assessor-blind. The person applying the treatment could not avoid being aware of the product being applied as the products are easily identifiable by their physical attributes; however, assessor-blinding was achieved by using different staff for applications on the one hand and assessment and CRF data entry on the other hand, and by physically separating these activities at the investigational site. Subjects were prevented from sighting the products being used. The parents of subjects were also blinded to the treatment applications. Analysts involved in data management were blinded to the identification of each treatment group until the final efficacy analysis for each treatment group was complete.If an enrolled subject had a primary-school aged sibling (aged 4 years to 12 years), the sibling was also examined for head lice and, if infested with live lice and available for enrolment, this sibling was enrolled into the same treatment arm as the subject. Those siblings not enrolled, because they were unavailable for the trial for any reason or had eggs only, were wet-combed-out at days 0 and 7 for siblings of subjects receiving the P/PB product or wet-combed-out (see Appendix 1 - Definitions) at days 0, 7 and 14 for siblings of subjects receiving TTO/LO and "suffocation" products.Intent-to-Treat (ITT) population is defined as all subjects receiving at least one treatment application. This was the primary population for determination of safety and efficacy.The per-protocol (PP) population and this was the secondary population for determination of efficacy. Subjects are considered to be per protocol (PP) if they satisfied the requirements listed in Appendix 2:Subjects who met all the protocol requirements are termed the The doses, method of application and number of weekly treatments were those recommended by the manufacturers. All three products were applied for 10 minutes. After the TTO/LO product was applied the hair was covered by a shower cap made of polyvinyl chloride to retain the volatile components of the formulation. The TTO/LO and "suffocation" products were then washed out with water; the P/PB product was washed out with a standard shampoo. The TTO/LO and "suffocation" products were applied at weekly intervals on days 0, 7 and 14. The P/PB product was applied on days 0 and 7.The efficacy of each product is defined as the "louse-free rate." The louse free rate was assessed at day 1 (by dry-combing), and at day 15 (by wet-combing) for those subjects treated with TTO/LO or "suffocation" products OR at day 8 (by wet-combing) for those subjects treated with the P/PB product. In the case of the day 1 examination, dry-combing was used and combing was stopped immediately if live lice were observed. A blinded assessor conducted the hair and scalp examinations.Often, visual inspection was sufficient to determine if live lice are present especially in cases of severe infestation. In such cases, dry/wet-combing was not necessary to confirm the presence of live lice. However, visual inspection alone was insufficient to declare a subject as "louse-free".Subjects were interviewed on site regarding possible adverse effects during and immediately following the application procedure as well as just before the next scheduled application by site staff. The incidence and severity of adverse events was compared between treatment groups. The ITT population was analysed for safety.Previous efficacy studies involving the P/PB product reported a cure rate of 36.1%. AssuminSubjects were enrolled between April and June 2009. The disposition of subjects is shown in Figure With regard to the primary efficacy endpoint for the ITT population   Table  one day With regard to the louse-free rates on day 1 , subjects in the ITT population in the TTO/LO product group were more likely to be louse-free on day 1 than subjects in the P/PB product group . Similarly, subjects in the ITT population in the "suffocation" product group were more likely to be louse-free on day 1 than subjects in the P/PB product group . For this population on day 1, the difference in louse-free rates for TTO/LO and "suffocation" products was not statistically significant (p = 0.1009). Subjects in the PP population in the TTO/LO product group were more likely to be louse-free on day 1 compared to subjects in the P/PB product group . For the PP population on day 1, 72.4% of the subjects in the "suffocation" product group were louse-free, whereas 57.1% of the subjects in the P/PB product group were louse-free; this difference was not statistically significant (adj. p = 0.2986).Demographic factors were not found to be confounders of the effect of treatment on outcome since they were not associated with the outcome. Using a multiple logistic regression model adjusting for gender, hair length, hair type, school attended and accounting for clustering, there was still a statistically significant higher louse-free rate at the day after the final treatment among children treated with the TTO/LO product  and among children treated with the "suffocation" product  than among children treated with the P/PB product.Of the 132 subjects enrolled, 36 adverse events were reported; 29 in relation to TTO/LO treatment; 3 in relation to "suffocation" product; and, 4 were related to P/PB product treatment. All adverse events reported were considered by the investigator to be related to study treatment. No adverse reaction was considered to be serious by definition. Some subjects reported more than one adverse event - 20 TTO/LO product-subjects (46.5%), 3 "suffocation" product-subjects (6.7%) and 4 P/PB product-subjects (9.1%) reported at least one adverse event.The adverse events for the TTO/LO, "suffocation" and P/PB products were rated as mild in severity with the exception of 3 adverse events in relation to the TTO/LO product which were rated as moderate in intensity. These 3 adverse events occurred in 3 subjects and were described as stinging of the eyes following product contact with the eyes; stinging of the neck; and, erythema of the skin. In one case following product-contact (TTO/LO) with the eyes, the application was washed out prior to the 10 minute contact time. In all other cases where an adverse event was reported no action was taken or required.The most commonly reported adverse events in relation to the TTO/LO product were stinging , flaky scalp/dry scalp  and erythema . In the case of the "suffocation" product 3 subjects (6.7%) reported flaky scalp/dry scalp. In the case of the P/PB product, 3 subjects (6.8%) reported flaky scalp/dry scalp and 1 subject (2.3%) reported erythema.The reported stinging or burning sensation associated with the TTO/LO product  lasted ®, 82.5%)[The efficacy of the P/PB product in this study was similar to that reported for the same product in a previous and similarly designed study of ours [It is likely, that the use of a shower cap to trap volatile components of essential oils such as melaleuca oil and eucalyptus oil contributes to the higher efficacy of these products compared to the same products applied without a shower cap. The use of a shower cap with essential oil products, however, also appears to be correlated to a higher incidence of transient mild to moderate stinging sensations, burning sensations, and erythema. The sensitivity of the skin of children varies. The "suffocation" product, which is highly efficacious yet caused little skin irritation in the present study, would be a good choice for children with inherently sensitive skin.Wet combing is a very accurate method to diagnose active head lice infestation. In contThe TTO/LO product and head lice "suffocation" product were both > 97% effective and were almost four times as effective as the P/PB product that we compared them with, when used according to manufacturer instructions. These results support the view that this new "suffocation" product is as effective in controlling head lice as an essential oil product applied with a shower cap.Associate Prof. Steve Barker and Dr. Phillip Altman have previously provided consultant-advice to Key Pharmaceuticals Pty. Ltd. the company that funded this study. The company had no active role in study design, study management, data analysis, interpretation of results or manuscript writing.PA and SB were responsible for study design. PA was responsible for protocol and document drafting, site staff training, study management, study monitoring, and writing the manuscript. SB conducted the trial and helped write the manuscript. Both read and approved the final manuscript.The proportion of subjects on whom no live head lice (adults or nymphs) were found when combed by a specified method at a specified time point.Visual inspection involves a brief examination of the hair assisted by parting of the hair in spots to determine whether live lice are present. It may be used in conjunction with dry-combing to determine active infestation with live lice.Combing from scalp to the hair tips using a head lice comb and not using water or conditioner to assist combing. Every part of the hair should be combed with a metal-toothed lice comb up to 6 times. The hair may be de-tangled with a wide-gap comb before dry-combing.Standard commercial conditioner is applied liberally to the hair, and the hair detangled with a regular wide-toothed comb. The hair is then combed with a metal-toothed lice comb to remove live lice. Every part of the hair is combed 6 times, scalp to hair tips. During combing, the comb is wiped onto a white tissue and the wipings are examined for lice. After combing, the conditioner is rinsed or towelled from the hair as desired by the subject. Wet-combing is a powerful detection technique to determine the final infestation status of a subject at the end of an efficacy trial, as the conditioner traps the lice making it highly unlikely that any lice in the hair will avoid detection. Thus, the likelihood that a subject will be incorrectly categorised as louse free when in fact a low grade infestation still exists is substantially reduced.The wet-combing method results in the smothering of lice in conditioner and the removal of head lice from the scalp, thus wet-combing has an irreversible effect on a subject's infestation status. Wet-combing was only used at the completion of the head lice trial."Wet-combed-out" is a therapeutic procedure which uses the wet-combining technique (above) to ensure the hair is louse free: wet-combing is continued until no live lice are found in six continuous passes of the comb. This is repeated for every section of the hair.• They comply with all inclusion and exclusion protocol requirements• Have a signed informed consent authorisation• Treatment was administered on day 0, 7 and 14 for Tea Tree/Lavender Oil or suffocation products or treatment was administered on days 0 and 7 for the Pyrethrins/piperonyl butoxide product• Are evaluated using the wet-combing procedure at one day after the final application (day 15 in the case of Tea Tree/Lavender Oil or suffocation products or day 8 in the case of Pyrethrins/piperonyl butoxide product)• The subject's Case Report Form is sufficiently complete to enable a valid assessment of efficacy and safety.• Have not used any other head lice products during the trial or in the 4 weeks preceding the trial• Have not used any other head lice products other than those specified in the protocol during the trial• Has not used a head lice comb during the trial• Have not bleached or dyed their hair during the trial• The subject's siblings were assessed and treated, if required, as per the section "Treatment of siblings"The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-5945/10/6/prepub


Toxoplasma gondii –inducible IRG proteins are a distinctive cytoplasmic GTPase family encoded by about 20 genes in the C57BL/6 mouse Specific cell-autonomous resistance mechanisms have been attributed to Irgm1 in the context of mycobacterial resistance. Irgm1 has been considered to act by associating with the mycobacterial phagosomal membrane and accelerating lysosomal fusion These mechanisms for Irgm1 function are widely accepted, perhaps partly because of the importance of the diseases that they are supposed to control, but also because, right or wrong, they are immediately appreciable, plausible, proximal, cell-autonomous effects on the pathogen. However, optimism that there may be such direct explanations for the loss of mycobacterial resistance as a result of the loss of Irgm1 has apparently obscured an important literature on Irgm1 deficiency and activity that points in an entirely different direction.Trypanosoma infection T. cruzi or Salmonella) really have something to do with the proposed cell-autonomous mechanisms, autophagocytosis or reduced vacuole acidification, or is it due to the profound and generalized immunodeficiency that these organisms induce in Irgm1-deficient hosts?Irgm1-deficient mice become strikingly leukopenic when infected with mycobacteria. Alan Sher and colleagues reported some years ago that the blood picture of young Irgm1-deficient adults is pretty normal, but collapses during infection Salmonelladirect cause of the susceptibility in either of these cases. There must be a more complex explanation.Gregory Taylor and colleagues showed recently that mice that are not only Irgm1- but also Irgm3-deficient  are no longer susceptible to in itself responsible for the hemopoietic and immune failures. Rather, it is the rest of the IFN response that is causing the problem in the absence of Irgm1. Stressing this point, Irgm1-deficient mice infected with a pathogen that stimulates only a Th2 response (Schistosoma mansoni), and therefore essentially no IFNγ production, show normal resistance and no lymphoid abnormalities Why do Irgm1-deficient animals rapidly develop a lymphomyeloid deficiency after infection or autoimmune stimulation? There seems to be reduced proliferative potential in the lymphomyeloid system that becomes acute after immune activation. It was shown recently that this affects the hematopoietic stem cell (HSC) as well as more peripheral lymphoid compartments T. gondii vacuole and initiating vacuolar disruption We showed that the IRG proteins fall into two groups, the GKS and the GMS sub-families, based on the sequence of their nucleotide binding domains It was shown some time ago that unregulated GKS proteins can interfere with cell proliferation. Douglas Carlow and colleagues attempted to generate fibroblast cell lines constitutively expressing the GKS effector IRG protein Irgb6 in the absence of IFNγ, and therefore in the absence of the three GMS proteins We can therefore propose the following preliminary scenario for the Irgm1-deficient mouse. In the absence of induced IFNγ production, the mouse appears relatively normal. However, for unclear reasons, there is constitutive expression of many IRG genes in HSCs Salmonella or mycobacteria infection Consistent with this scenario, Taylor and colleagues showed aggregates of GKS proteins (Irgb6 and Irga6) in bone marrow–derived macrophages from Irgm1 knock-out mice after in vitro stimulation with IFNγ A plausible interpretation of the Irgm1 phenomenon now runs like this. Infection, for example by mycobacteria, induces a high level of IFNγ, which in turn induces high levels of IRG proteins. In the absence of Irgm1, the GTPase cycle of the remaining IRG proteins cannot be properly controlled. This results in the formation of large IRG protein aggregates that in certain cell types of the hematopoietic and lymphoid systems are cytopathic or cytostatic, causing a generalized lymphopenia. Losing Irgm3 and Irgm1 together causes rapid clearance of the aggregates and relieves the cytopathic phenotype. Thus, in the Irgm1/Irgm3 double knock-out the immune picture essentially returns to normal. In a recent note, Feng and colleagues have also proposed that at least part of the Irgm1 deficiency phenotype is due to the loss of a regulatory function Toxoplasma is completely lost in the Irgm1 knock-out Toxoplasma does not return in the Irgm1/Irgm3 double knock-out Toxoplasma susceptibility phenotype without any lymphopenia or generalized immunodeficiency  was normal Salmonella since this appears to be normal in the absence of Irgm1 so long as Irgm3 is missing too, but these, of course, do not exhaust the universe of intracellular pathogens. There is much experimental work left to do to assess the validity and completeness of this revision of view about how the IRG proteins fulfill their function. It is a complex argument, but it hangs together reasonably well and offers a broad and satisfying explanation for most, if not all, of the properties of the Irgm1-deficient mouse. Above all, however, the IRG system must be understood as a highly regulated, highly coordinated system of proteins where the properties of single-gene knock-outs may be misleading.It is important to emphasize that while the present view can account for much of the complexity of the observations on Irgm1 deficiency, it remains possible that Irgm1 may have additional “autonomous” activities of its own, perhaps in the control of autophagy. It now seems unlikely that this will be true for immunity against mycobacteria or


Cattle that naturally do not grow horns are referred to as polled, a trait inherited in a dominant Mendelian fashion. Previous studies have localized the polled mutation (which is unknown) to the proximal end of bovine chromosome 1 in a region approximately 3 Mb in size. While a polled genetic test, Tru-Polled™, is commercially available from MetaMorphix Inc., Holsteins are not a validated breed for this test.e.g., the untranslated region, or UTR) where one is located in the 3'UTR of a gene and the other two are located in the 5'UTR and coding region (synonymous SNP) of another gene. The 3'UTR of genes have been shown to be targets of microRNAs regulating gene expression. In silico analysis indicates the 3'UTR SNP may disrupt a microRNA target site.Approximately 160 kb were sequenced within the known polled region from 12 polled and 12 horned Holsteins. Analysis of the polymorphisms identified 13 novel single nucleotide polymorphisms (SNPs) that are concordant with the horned/polled trait. Three of the 13 SNPs are located in gene coding or regulatory regions (These 13 novel SNPs concordant with the horned/polled trait in Holsteins represent a test panel for the breed and this is the first report to the authors' knowledge of SNPs within gene coding or regulatory regions concordant with the horned/polled trait in cattle. These SNPs will require further testing for verification and further study to determine if the 3'UTR SNP may have a functional effect on the polled trait in Holsteins. Cattle that naturally do not grow horns are termed polled, a trait inherited in an autosomal dominant fashion ,2. De-hoWhile a polled genetic test is commercially available from MetaMorphix Inc., Holsteins are not listed as a validated breed for the Tru-Polled™ test . The breBos taurus, which is unknown, was localized to the proximal end of bovine chromosome 1 (BTA01) with microsatellite markers [ATP5O, and most distal gene, KRTAP8, of the polled region from these cited sources corresponds to approximately 0.6 Mb and 3.9 Mb respectively on the public bovine genome assembly version 4.0 [The polled mutation in  markers . More re markers -11 and t markers . The locsion 4.0 . One stue.g., untranslated region, or UTR) will be analyzed in silico to determine if there is any potential functional effect.The objective of this work was to identify single nucleotide polymorphisms (SNPs) associated with the polled trait in Holsteins by sequencing targeted regions of the proximal end of BTA01 on a panel of 12 polled and 12 horned bulls  region. The other 10 SNPs are located in introns or are inter-genic and not present in a putative regulatory element as analyzed by WWW Promoter Scan [SYNJ1 gene, and SNPs bC2159_C-193T and bC2159_T372C are located in the 5'UTR and coding region of the C21orf59 gene, respectively. The bC2159_T372C polymorphism is a synonymous SNP, with both alleles coding for a serine amino acid.Of the 13 SNPs identified as concordant with the polled trait Table , only thter Scan . The bSYSYNJ1, and 3'UTRs have been shown to be targets of a gene expression regulatory system orchestrated by microRNAs  [SYNJ1, and specifically the location of SNP bSYNJ1_C3981T, may be a microRNA target, the current collection of bovine microRNA mature sequences (N = 117) was downloaded from the publicly available miRBase [As previously mentioned, the polled concordant SNP bSYNJ1_C3981T is located in the 3'UTR of the gene n sheep) . In orde miRBase . The micces N = 1 was downSYNJ1 and overlap the location of SNP bSYNJ1_C3981T  for polled is identified there may be multiple breed-specific mutations for the trait in that DNA element, if the trait arose separately in more than one breed. Another hypothesis is the trait was selectively introgressed from the first breed exhibiting polled and the causal mutation will be found to be the same in all breeds. The 13 SNPs reported here do represent the first described genetic markers for the polled trait specifically in Holsteins. In addition, these 13 SNPs are novel as they are not found in a search of public databases, such as NCBI's dbSNP [A total of 13 novel SNPs concordant with the polled trait in Holsteins Table  were ide's dbSNP  or the BSYNJ1 gene. The 3'UTR of genes has been shown to potentially be a target for microRNA regulation of gene expression by post-transcriptionally base-pairing to mRNAs [In silico analysis of the SYNJ1 3'UTR allelic variant created by the bSYNJ1_C3981T SNP revealed eight predicted microRNA target interactions from the 117 available bovine microRNAs  alters a microRNA target site. The effect could be predicted to be tissue specific in such a manner that only horn growth is affected  by an unknown mechanism of SYNJ1 function. If the SYNJ1 3'UTR SNP does have a functional effect on the polled trait in Holsteins it, and the SYNJ1 gene itself, becomes a candidate for further investigations of potential causal mutations for the polled trait in other breeds.Based on the i.e., not introns or inter-genic) predictive of the horned/polled trait in cattle as previous reports localized and fine-mapped the polled region utilizing inter-genic genetic markers such as microsatellites [SYNJ1 3'UTR SNP may have a functional effect on the polled trait in Holsteins.In summary, the polymorphisms reported here as concordant with the polled trait in Holsteins can readily be used as a genetic test for this breed. This is the first report, to the authors' knowledge, of SNPs within gene coding or regulatory regions  according to the manufacturer's protocol. Direct sequencing of purified PCR products, in both forward and reverse directions, was conducted using ABI BigDye  according to the manufacturer's protocol and resolved on an ABI 3730xl Automated Sequencer . Sequence trace alignment and polymorphism detection were carried out using recent versions of Phred/Phrap ,27 and CThe current collection of bovine microRNA mature sequences N = 117) was downloaded from the publicly available miRBase . The mic was downThe authors declare a provisional patent application has been filed related to the content presented in this report and this work was fully funded by Monsanto Company.e.g., polymorphism discovery, microRNA target detection), and drafted the manuscript. NJN participated in design of the study, contributed to selection of the Holstein panel, and provided review of the manuscript. MDG participated in design of the study, provided guidance for experimental analyses, and provided review of the manuscript.EJC participated in design of the study, contributed to selection of the Holstein panel, conducted all experimental analyses (


Trials in Africa indicate that medical adult male circumcision (MAMC) reduces the risk of HIV by 60%. MAMC may avert 2 to 8 million HIV infections over 20 years in sub-Saharan Africa and cost less than treating those who would have been infected. This paper estimates the financial and human resources required to roll out MAMC and the net savings due to reduced infections.We developed a model which included costing, demography and HIV epidemiology. We used it to investigate 14 countries in sub-Saharan Africa where the prevalence of male circumcision was lower than 80% and HIV prevalence among adults was higher than 5%, in addition to Uganda and the Nyanza province in Kenya. We assumed that the roll-out would take 5 years and lead to an MC prevalence among adult males of 85%. We also assumed that surgery would be done as it was in the trials. We calculated public program cost, number of full-time circumcisers and net costs or savings when adjusting for averted HIV treatments. Costs were in USD, discounted to 2007. 95% percentile intervals (95% PI) were estimated by Monte Carlo simulations.In the first 5 years the number of circumcisers needed was 2 282 (95% PI: 2 018 to 2 959), or 0.24 (95% PI: 0.21 to 0.31) per 10 000 adults. In years 6–10, the number of circumcisers needed fell to 513 (95% PI: 452 to 664). The estimated 5-year cost of rolling out MAMC in the public sector was $919 million (95% PI: 726 to 1 245). The cumulative net cost over the first 10 years was $672 million (95% PI: 437 to 1 021) and over 20 years there were net savings of $2.3 billion (95% PI: 1.4 to 3.4).A rapid roll-out of MAMC in sub-Saharan Africa requires substantial funding and a high number of circumcisers for the first five years. These investments are justified by MAMC's substantial health benefits and the savings accrued by averting future HIV infections. Lower ongoing costs and continued care savings suggest long-term sustainability. Observational studies have repeatedly shown that male circumcision (MC) offers substantial protection against HIV infection The encouraging results of the Orange Farm trial prompted wide interest in Africa towards MC as an HIV prevention strategy. Currently about a third of the African male population is circumcised but the practice is less common in southern and East Africa, where the HIV epidemic is especially severe. The circumcision of adult males was shown to be more effective and cost-effective than other general population HIV prevention strategies The acceptability of MAMC appears to be high. Before the clinical trials were completed, studies were conducted in nine African countries with a high HIV prevalence and a low MC prevalence. Most uncircumcised males were willing to become circumcised  and most women were willing to have their sons circumcised  The results of the Orange Farm trial suggest that MAMC could have a substantial impact on health, with favourable economic implications. An epidemic model estimated that full coverage of MAMC in African countries where the majority of males are uncircumcised would avert 2 to 8 million HIV infections in the next 10 years Given the favourable evidence for the impact and feasibility of MAMC and the WHO-UNAIDS recommendations to expand access to safe male circumcision services We developed a cost model of MAMC integrated with a demographic and HIV epidemic model. The technical details of the model are provided in For each location we specified a population composed of individuals matched by gender, age group (child/adult), male circumcision status and HIV status. HIV-positive individuals could receive antiretroviral therapy (ART) and died of AIDS or of other causes . HIV traWe assumed that a given number of adults were circumcised each year, independently of their HIV status, until full coverage was achieved. We estimated the cost and effect of this intervention on HIV as a function of time. We assumed that a fixed proportion of males were already circumcised before becoming adults and that this was constant over time.The demographic parameters and the HIV epidemic model were kept simple in order to allow for the calculation of anticipated resource needs and program cost with a reduced set of input parameters. Comparison with earlier models assured a realistic representation of the key factors related to demography, HIV and male circumcision status as well as the cost of the intervention.The full list of input parameters is reported in We assumed that MAMC services were intensive for the first five years, in order to achieve maximum attainable circumcision levels, and then dropped to the rate necessary to maintain these levels. It was assumed that in this “initial period” of five years a large constant annual number of MAMCs were performed, sampled randomly among all uncircumcised adult men willing to be circumcised. The prevalence of MC rose rapidly. When MC prevalence reached the specified threshold, MAMC acceptance was saturated and dropped to a lower rate. The proportion of males becoming adults who refused MAMC determined long-term MC saturation. Thus, after the initial period, the number of circumcisions performed annually was reduced to the number of male children/adolescents entering adulthood, not already adequately traditionally circumcised and willing to be circumcised. For this analysis, we assumed that current neonatal and childhood MC practices did not change. We assumed that the roll-out led to an increase in the prevalence of MC among adult males to 85% in all settings. In the sensitivity analysis, we assumed an MC prevalence increase to 55%, except for the 4 countries with an MC prevalence greater than this limit: Central African Republic, Liberia, Mozambique and Tanzania.initial and annual costs. We explored a public cost scenario assuming the use of government health infrastructures only and a private cost scenario assuming reliance on private health care providers only.Program costs were composed of public cost scenario, initial costs were per circumcision facility  and for training circumcisers. Annual costs included the oversight and promotion of MAMC, the salaries of full-time circumcisers, surgical staff and counsellors, the direct non-salary cost of each MAMC , facility overhead  and program overhead.In the The number of personnel required was based on the experience of the authors and expert opinion. In particular, we assumed that each circumciser could complete 10 circumcisions per day, as observed in recent clinical trials. For each circumciser, there was 1.0 medical assistant and 0.5 counsellor. These numbers were based on MC delivery experience in the trial in Orange Farm and were consistent with a recent study of 4 clinics in Swaziland Unit cost estimates  were derprivate cost scenario, all facility-level costs were included in the price per MAMC paid to providers. These providers were already equipped to perform MAMC and there were no initial costs. The price of each MAMC covered salaries of circumcisers, other health staff, counselling, surgical supplies, follow-up, treatment of adverse events and operating costs. This per-circumcision price could presumably correspond to a higher unit cost than in the public scenario, due to differences in private sector costs  or pricing strategies by providers. We spoke with informants in various settings, who provided us with a wide range of currently asked prices, from $25 in Kenya to $376 (Zambian 15 bed private clinic). In South Africa we obtained an average price of $72. In a first analysis we assumed that the cost of MAMC was $72 across all settings. This value was varied from −50% to +50% (36 to 99) in the sensitivity analysis. In addition to direct provider payments we assumed annual program overhead costs of 10% to cover the public promotion of MAMC.In the calculate costs and savings from HIV treatment, we assumed that 30% of HIV-infected individuals eligible for antiretroviral treatment were receiving it. The averted cost of medical treatment for HIV over time was a function of the number of HIV infections averted each year and the rate of disease progression, combined with associated medical costs. We assigned medical costs by stages of disease, based on a study in South Africa per capita GNP, since WHO CHOICE did not provide country-level details.To The analysis adopted the perspective of a government health care payer. In the private cost scenario, the cost of each MAMC was reimbursed by the government. Since all and only direct medical costs were included, governmental and societal perspectives were similar. Program costs were discounted to 2007 at 3% per year number of full-time circumcisers needed based on the productivity of circumcisers (circumcisions per day) and the duration of the initial period. We calculated two economic outputs. Program costs needed to roll out MAMC were calculated as the sum of the resources required to deliver a 5-year scale-up of MAMC to saturation and the resources required for the maintenance of MAMC over an additional 15 years, across all 16 settings. This 5-year period was changed to 7 and 9 years in the sensitivity analysis. Net costs or savings represented the costs of MAMC minus the savings due to averted medical care costs for HIV.We calculated the The uncertainty of anticipated resource needs and program costs to input uncertainty was determined using Monte Carlo simulations. For each input parameter, we matched a truncated Gaussian distribution (±2 standard deviations) to the specified uncertainty range indicated in Over the first 10 and 20 years of MAMC roll-out, the number of circumcisions per HIV infection averted was 10.1 (95% PI: 9.0 to 11.2) and 5.6 (95% PI: 5.1 to 6.2), respectively.The program cost over the first 5 years was $919 million (95% PI: 726 to 1 245) in the public scenario . The annThe required MAMC program funding over the first 10 years for the 16 settings varied only slightly, between $ 919, 1 019 and 971 for the initial periods of 5, 7 and 9 years, respectively. The lower cost for the longer period of 9 years was due in part to a lower initial investment in circumcision centres and MC training.Net costs and savings are reported in Over the first 10 and 20 years of MAMC roll-out, the cost per HIV infection averted varied from $338 to $168 . For SouWhen using the AFRO-E regional average salaries for the 14 lowest income settings (leaving South Africa and Botswana at higher levels), the program cost over 5 years was $1 279 million (95% PI: 1 074 to 1 657), slightly higher than the upper 95% PI of the main analysis. The program cost over 20 years was $1 912 million (95% PI: 1 612 to 2 438), again slightly above the main analysis upper 95% PI. The net savings over 20 years, taking into account averted HIV care, were $2 324 million (95% PI: 1 356 to 3 350). This value is similar to what was obtained in the main analysis despite a higher program cost, due to higher averted HIV care costs using the AFRO-E regional salaries.With a private cost set at $72 ($79.2 with overhead), the program cost over 5 years was $1 961 million (95% PI: 1 612 to 2 298) and $3 011 million (95% PI: 2 499 to 3 527) over 20 years. Net savings at 20 years were $610 million (265 to 1 676). With a cost per MC of $32 (excluding 10% program overhead), the program cost over 20 years in the private and public sector was the same. A net cost over 20 years of $0 was achieved at a private cost of $87 per AMC (excluding 10% program overhead).This study provides the first estimates of the cost of scaling up MAMC in sub-Saharan Africa, the number of circumcisers needed and the likely savings due to averted HIV medical care costs. After numerous observational studies, three randomised controlled studies, a modelling study and a cost-effectiveness study, this analysis provides further evidence supporting the rapid roll-out of MC in sub-Saharan Africa. Cumulative net costs at 20 years are negative for the base case and for almost all sensitivity analyses, indicating that the intervention will save money.This study has some limitations. On the costing/demand aspect, the predicted number of circumcisers required depends on the assumed period during which full MAMC scale-up is achieved. Our base case assumption that a period of 5 years will be sufficient to circumcise most of the uncircumcised males might be seen as optimistic. The sensitivity analysis showed that the required number of circumcisers would be less with a longer start-up phase; however, the associated costs to scale up MAMC were relatively insensitive to this duration. Our assumption that a high fraction of uncircumcised men would accept circumcision may be considered an upper limit, reflecting the hypothesis that education campaigns promoting MAMC might even increase demand above levels reported in acceptability studies. Finally, our assumption about circumciser productivity is based on recent experience and may turn out to be conservative as efforts are made to design and evaluate more efficient surgical methods and ways of deploying staff.Despite the consistent impacts found in the 3 RCTs, the exact impact that MAMC will have when scaled up in the field remains uncertain. For example, impact observations in these trials were all limited to the first 2 years after surgery. In addition, the indirect effect of circumcision on preventing mother-to-child transmission and associated reductions in medical costs were not taken into account in our model.The mathematical model used to calculate epidemiological impact was a simple susceptible-infected compartmental model Our analysis cannot be used to estimate precisely the difference in costs between a private and a public scenario, due to a lack of firm data on the cost of either scenario, especially with evidence of wide variation in private pricing. A pragmatic view suggests that each delivery approach has advantages and disadvantages. The main advantage of the private provider scenario is that it is immediately available: subsidies to private sector MAMC facilities by public or private donors will make this sector quickly operational. The disadvantage of the private sector approach is its likely higher long-term cost, the lack of private doctors in many rural areas threatening geographical equity in access and the insufficient number of doctors to fully cover the need for MAMC.The main advantage of the public sector approach is its potentially lower cost in the longer term and potentially better geographic equity. The main disadvantage is that the health system may take time to make MC available on a large scale. The public system may require infrastructure development and training of health workers.Summed over all the countries evaluated, the cost estimates for rolling out MAMC may appear to be high. We nevertheless think that this cost is affordable, for several reasons. First, the cost is high only for a few initial years: once most men have been circumcised, the cost will be reduced to the circumcision of men becoming adults . Second, the cost is an investment which will prevent spending far greater resources in treating persons with HIV/AIDS in future years. Compared with our ART costing assumptions, the costs of ART may even increase if treatment initiation criteria are widened (to earlier stages of infection/disease) in coming years, due to longer therapy and an increasing need for more expensive second- and third-line regimens. Furthermore, spending for MC is modest compared with overall HIV control efforts: our prediction of annual funding required for MAMC roll-out for a 10-year period in the public provider scenario is only one-quarter of the current spending of the PEPFAR program: $433 million annually in 5 countries of Southern Africa We calculated that, over the first 5 years, about 1/4 full-time circumcisers would be required per 10 000 adults. Current general practitioners may be too few  and too busy to fulfil such a need One of the main obstacles to the roll-out of MAMC is the relative technical difficulty of the surgery, which requires precise incisions, haemostasis and sutures. The roll-out of MAMC could be greatly facilitated and accelerated by the development of simplified, bloodless methods A major consideration in scaling up MAMC is whether to concentrate on a horizontal or vertical approach. The horizontal approach is exemplified by the integration of MAMC into routine clinical practice. It is best represented in this analysis by the private sector scenario, since many general practitioners are likely to do MAMC as part of their varied clinical activities. The vertical approach makes MAMC a stand-alone activity. The public sector scenario may work with a vertical or horizontal emphasis, or a mix. The vertical approach offers potential to contribute uniquely to a rapid scale-up and the horizontal approach offers more structure for sustainability. We believe that a combination is preferred and that the optimal scale-up methods will depend on the health care system settings. We have not distinguished the cost of the public sector's horizontal versus vertical approaches, which will be the subject of future analyses.The rapid implementation of MC will necessitate more than just funding. It will require broad involvement from many groups: national political leaders, activists, teachers, street leaders, churches and health workers. MAMC roll-out will also require strong and steady political support. The political involvement of South Africa will be key, as South Africa represents a high fraction of the population that could benefit the most and has a leading political role in the African region. Our hope is that the research done in the past 20 years regarding the potential of MC to reduce the spread of HIV will be recognized not merely as scientific progress, but as the foundation for an effective transition from knowledge to high-impact practice.Supporting Information S1Technical description of the modelling. Cost model of the roll-out of adult male circumcision in Africa integrated with a demographic and HIV epidemic model.(0.15 MB DOC)Click here for additional data file.Figure S1Compartment model of the modelled population(0.06 MB TIF)Click here for additional data file.


X monosomic mice  have a remarkably mild phenotype when compared to women with Turner syndrome . The generally accepted hypothesis to explain this discrepancy is that the number of genes on the mouse X chromosome which escape X inactivation, and thus are expressed at higher levels in females, is very small. However this hypothesis has never been tested and only a small number of genes have been assayed for their X-inactivation status in the mouse. We performed a global expression analysis in four somatic tissues  of adult 40,XX and 39,XO mice using the Illumina Mouse WG-6 v1_1 Expression BeadChip and an extensive validation by quantitative real time PCR, in order to identify which genes are expressed from both X chromosomes.We identified several genes on the X chromosome which are overexpressed in XX females, including those previously reported as escaping X inactivation, as well as new candidates. However, the results obtained by microarray and qPCR were not fully concordant, illustrating the difficulty in ascertaining modest fold changes, such as those expected for genes escaping X inactivation. Remarkably, considerable variation was observed between tissues, suggesting that inactivation patterns may be tissue-dependent. Our analysis also exposed several autosomal genes involved in mitochondrial metabolism and in protein translation which are differentially expressed between XX and XO mice, revealing secondary transcriptional changes to the alteration in X chromosome dosage.Our results support the prediction that the mouse inactive X chromosome is largely silent, while providing a list of the genes potentially escaping X inactivation in rodents. Although the lower expression of X-linked genes in XO mice may not be relevant in the particular tissues/systems which are affected in human X chromosome monosomy, genes deregulated in XO mice are good candidates for further study in an involvement in Turner Syndrome phenotype. The existence of dimorphic sex chromosomes poses a challenge to the balance of gene dosage between the sexes. In mammals, X chromosome inactivation is the mechanism by which the equality in gene expression between males and females is restored, through the transcriptional silencing of one of the X chromosomes in females. Dosage compensation between X-linked and autosomal genes is also achieved, through the upregulation of the active X chromosome in females and the single X chromosome in males .XIC in humans and Xic in mice), which contains the X-Inactive-Specific Transcript locus (XIST/Xist), a noncoding RNA essential for silencing, and several other genetic elements . The naiewed by ,4. Once iewed by ,5 and inin vitro models Carrel and Willard [Jarid1c, is expressed from the inactive X [Jarid1c but not in its human homologue (JARID1C), raising the hypothesis that this protein may be required to prevent the spreading of silencing epigenetic marks to Jarid1c, which is embedded in inactive chromatin on the mouse X chromosome [Although X inactivation is a very effective mechanism some genes are expressed from the inactive X chromosome . Using i Willard  performe Willard -12 and t Willard . Althougactive X . Additioromosome . Recentlromosome . The chaEif2s3x, Jarid1c, Utx and Usp9x were more highly expressed in the XX genotype, compared to XY, regardless of the gonadal phenotype [In recent reports, microarray technology was used to compare gene expression between males and females in several mouse tissues, from which indirect evidence of escape from X inactivation can be derived ,16,17. Nhenotype -23, whicmO, XpO and XmXp) using the breeding strategy of Davies et al.[The 39,XO mouse is another valuable model to assess the impact of the number of X chromosomes on the level of X-linked gene expression in a similar hormonal milieu, using a simple experimental design. The underlying assumption is that genes that are expressed from both the active and inactive X chromosomes would be expressed at higher levels in the XX females than in their XO littermates. We generated three genotypes  have a remarkably mild phenotype when compared to women with Turner Syndrome . In fact, while XO mice are fertile and do not display major physiological abnormalities, TS females present ovarian dysgenesis and other anatomical and physiological abnormalities . A spectWe present a global expression analysis in somatic tissues of XX and XO mice, using a microarray platform that allows a genome-wide survey of gene expression, followed by a detailed qPCR inspection of the candidate genes. Our analysis identified new genes potentially escaping X inactivation in the mouse and sheds some light on the transcriptional networks regulated by X-linked genes which are disturbed in X chromosome monosomy.We performed a genome-wide expression profiling in four different tissues of adult XX and XO mice , using the Illumina Mouse WG-6 v1_1 Expression BeadChip, which covers more than 45,000 transcripts and includes 1,326 probes interrogating the X chromosome transcriptome. According to our manual annotation these correspond to 970 different genes, thus achieving an extensive coverage of the mouse X chromosome - on the latest Ensembl release , approximately 1000 protein coding genes were annotated on the X chromosome. Brain was the tissue where the largest number of probes was detected , while 19,887 probes were detected in liver, 23,427 in kidney and in muscle 21,820. High values were obtained for the correlation coefficients (r) between biological replicates (0.98 to 1), indicating that the platform and methods applied are extremely robust. This high reproducibility is also likely due to the pooling of individuals of the same genotype , which minimizes the differences between individuals. After normalisation the samples were hierarchically clustered according to their global expression profiles, to verify the biological accuracy of the data. The samples clustered primarily by tissue and in general also by genotype, as expected (results not shown).http://www.ncbi.nlm.nih.gov, MGI http://www.informatics.jax.org/ and SOURCE http://smd.stanford.edu/cgi-bin/source/sourceSearch. This allowed us to determine the total number of X-linked transcripts expressed in each tissue and to perform a targeted analysis for each dataset. On the X chromosome the highest number of probes was detected in brain (725 present in at least one of the pools), while in liver only 538 were detected; 643 were expressed in kidney and 605 in muscle.In the chosen array platform 19,100 probes represent unique curated genes in the NCBI RefSeq database . We annotated all the remaining probes, by performing automated blast searches followed by a manual curation using combined information from several databases - NCBI Xist, an expected observation since this gene is only transcribed from the inactive X chromosome in XX females; a few scattered X-linked genes can be detected which also lie above the diagonal, particularly in liver.As a preliminary analysis of autosomal and X-linked expression in the two groups we plotted the median of the normalised hybridization signals of the XO mice against the XX in each of the four tissues analyzed Figure . In thesRecently, several statistical approaches to detect differential gene expression from microarray data have been compared  and it wXist was the only probe significantly different between the two groups of mice in all tissues (P < 0.0001).At the 0.05 significance level  different groups of probes were found to be significant in each tissue; Eif2s3x, which is ubiquitously expressed and known to escape X-inactivation in both human and mice [Utx, one other well known escapee [P < 0.05). Jarid1c, also shown to escape X inactivation in the mouse [P < 0.01) and qPCR , and in muscle only by qPCR . In kidney and brain the difference in Jarid1c expression between the two groups was not significant.and mice  is more  escapee ,12, werehe mouse , presentEif2s3x and Utx were significantly overexpressed in the brain of XX females by qPCR, as well as Ddx3x (formerly Dbx), a potential escapee that had been found to be more highly expressed in female compared to male brain by Northern Blot [Ddx3x was significantly more abundant in XX females in liver but it did not reach significance in any of the other tissues tested.Only a small number of genes were differentially expressed between the two genotypes in muscle and kidney and in brain none of the probes reached the significance threshold. However, the established escapees ern Blot . In our Eif2s3x, Utx, Ddx3x and Jarid1c were all highly significant (P ≤ 0.01). The fold change differences in expression between the two groups in this tissue ranged from 1.1 to 1.8 for the probes with significant p-values although some were expressed at low levels in both groups . The limma analysis we performed also produces a B-statistic, the log-odds that a gene is differentially expressed, which provides an additional measure of the strength of each candidate. The 11 probes presenting the largest p-values in liver all present a B < 0 and have therefore less than 50% chance of being differentially expressed. Pgk1, a gene that undergoes X inactivation, is amongst these, although presenting a small FC. We tested one other gene with B < 0 by qPCR (Pgrmc1) but could not confirm differential expression , advising caution in considering these 11 genes as differentially expressed.In liver a higher number of probes, corresponding to 31 different genes, were significantly overexpressed in XX mice (Table Sts (Steroid Sulfatase), a gene in the mouse pseudoautosomal region. Keitges et al. [Sts is not differentially expressed in any of the tissues tested and we were unable to design appropriate primers on exon-exon boundaries in order to validate our microarray results by quantitative PCR, since the genomic sequence of Sts was not available in any of the databases searched . A Blat search http://genome.ucsc.edu/cgi-bin/hgBlat?command=start of the probe sequence retrieved no matches, indicating that the gene is not on the current genome assembly (July 2007). Enox (AK137473), another gene recently described as escaping X inactivation [The array also includes a probe annotated as representing s et al.  have sugs et al. -33. Accotivation  is not rEif2s3x, Utx, Ddx3x, Jarid1c and 2610029G23Rik). Overall, FC estimates obtained by both methods were roughly concordant and all genes were significantly overexpressed by qPCR in at least one of the tissues tested  and therefore these genes were not tested in the other tissues. Additionally, four of the significant genes presenting FC < 1.2 were tested ; by qPCR only Pnck was significantly overexpressed .We analysed several of the significant genes by both microarrays and qPCR. At present, a standard definition of validation of microarray results by qPCR is still lacking and it is common practice to simply compare FC values. We required significant p-values in both methodologies as criteria for full concordance. This is especially important when small expression differences between groups are sought and it is difficult to define a fold change cut-off value. Five of the genes that were significant by microarray analysis were tested by qPCR in all four tissues (2610029G23Rik). The function of 2610029G23Rik and its human homologue CXorf26 have not yet been determined.Amongst the transcripts that reached the significance level in more than one tissue by both approaches, one has not been previously described as escaping X inactivation  16H female mice carrying the Searle's translocation. In these females the normal X chromosome is always inactive  and thert) Table . The secArgininosuccinate synthetase 1, Ass1 , which codes for one of the enzymes of the arginine biosynthetic pathway. Interestingly, the autosomal genes Eif2b2 and Eif2b5, which encode two of the subunits of the eukaryotic translation initiation factor 2B, were amongst the most significant probes in this genome-wide comparison . In humans, mutations in these genes have been detected in females with POF (Premature Ovarian Failure) and it has been hypothesized that EIF2B2 dysfunction in humans may be related to increased apoptosis of ovarian follicles [In view of the larger number of X-linked probes found to be more highly expressed in the liver of XX mice, we performed a second analysis including all probes in the array which were detected in this tissue, to verify if any autosomal transcripts were deregulated between the two groups of mice. In this genome-wide analysis 1402 probes  were significantly different between genotypes at the 0.05 significance level after correcting for the number of genes tested . Therefore the autosomal expression perturbations were more pervasive in the liver of monosomic mice, where the differences in X-linked expression are more pronounced.To further clarify if the alterations observed in autosomal gene expression were specific to liver, and are thus more likely downstream effects of the differential expression detected for several X-linked genes in this tissue, we extended the genome-wide statistical analysis to the remainder of the tissues. In kidney and muscle datasets only a small number of genes were detected as differentially expressed at a 0.05 significance level after FDR correction  terms corresponding to the significant genes detected at the genome-wide level  and the Crystallin Zeta quinone reductase (Cryz), and in genes encoding other mitochondrial proteins, may be due to a global alteration of the gene expression networks relevant for mitochondrial metabolism.Several terms related to metabolic and biosynthetic pathways, namely energy metabolism, were overrepresented in the set of significant genes and cytoplasmic/mitochondrial subcellular location was also more frequent Table . Severalhttp://www.cisreg.ca/oPOSSUM/. Following FDR correction the genes overexpressed in liver showed enrichment for six transcription factors .Considering that several functional categories are overrepresented in our liver dataset it is plausible that the expression of some X chromosome genes may be perturbed indirectly through the action of shared transcription factors within an expression network. To further explore this hypothesis we searched for conserved binding sequences for known transcription factors within the 5000 bp upstream and downstream the TSS of those genes that were overexpressed in XX compared to XO females and performed an analysis of TFBS enrichment using the oPOSSUM web tool Huwe1, Arhgef6 and Nsbp1, present TFBS for one or more of these transcription factors. It seems plausible that in XX females higher transcriptional output of some of the genes that escape X inactivation may lead to a secondary upregulation of other genes on the X chromosome belonging to the same transcriptional networks, which will therefore be overexpressed even though not escaping X-inactivation.Interestingly, several overexpressed genes located on the X chromosome, including In the present analysis we aimed to detect differences in X-linked expression between XX and XO mouse females in four different tissues, to fully characterize the genes escaping X inactivation in this model and to increase our understanding of the basis of the different phenotypes of X monosomic females in humans and mice.The identification of genes differentially expressed through the statistical analysis of microarray data represents a challenge and the application of this technique to the identification of genes escaping X inactivation is of added difficulty. Due to the biological nature of the differences we were aiming to detect, i.e., expression from the active X chromosome, which is inherently upregulated , and froWhile there was an overall agreement on the direction of change obtained by microarray and qPCR in our study, the concordance between the two approaches was not complete. It is widely accepted that several factors can contribute to discrepancies between microarray and qPCR data and each technique presents advantages and pitfalls. In a recent assessment it has been established that even though lower fold changes in microarray data (<1.4) are less likely to be confirmed by qPCR, the most determinant factor in obtaining a high correlation for these two approaches is the microarray data p-value . The samEif2s3x and Utx, and for a new candidate gene (2610029G23Rik). Jarid1c, also known to escape X inactivation, and Ddx3x, a potential escapee, only reached significance by both methods in liver. Variable expression from the inactive X chromosome across tissues has been reported for Jarid1c [The most consistent results across tissues, obtained by both methods, were those of two of the genes previously demonstrated to escape X inactivation,  Jarid1c ,40 and mThe majority of the genes detected as differentially expressed were only so in one of the tissues analyzed, with liver presenting a noticeably larger number of genes significantly overexpressed. Several factors may underlie these observations. Variable escape from inactivation has been observed in rodent-human somatic cell hybrids and in human cell lines ,41 as weFor several genes, more than one probe was detected and, in many cases, different probes were not concordant in defining a gene as significantly overexpressed, even in the same tissue. A careful analysis revealed that those probes with discordant results were often derived from genomic sequences that were not included in all transcripts of a given gene. Therefore, the existence of alternative splicing is contributing to some extent to the heterogeneity observed between tissues. Additionally, our analysis of enrichment of functional categories and TFBS within the group of differentially expressed genes suggests that secondary transcriptional regulation has an impact on the expression level of several X chromosome genes and regulation at this level is also likely to vary between tissues.Utx was female-biased in all tissues analyzed  and Eif2s3x, Ddx3x and 2610029G23Rik were also female-biased in 3 of the four tissues. Only those genes with a minimum of 3-fold differential expression between sexes were reported by Rinn and colleagues [Eif2s3x is significantly overexpressed in female hypothalamus; Utx, Ddx3x and 2610029G23Rik, amongst others, presented a fold change of 1.2 or higher in at least one of the tissues , although not reaching the threshold of significance. The fact that a much larger number of genes is female-biased in at least two tissues (56 genes at P < 0.05) in the comparison performed by Yang et al. [Ddx3x is indeed expressed from the inactive X chromosome in the mouse .Even though the global transcriptional output from the X chromosome is similar in males and in females , two stulleagues  but, in g et al.  is most P < 0.05 by both approaches, in at least one of the tissues (2610029G23Rik and Pnck). Although higher expression of Pja1 in the liver and kidney of XX females was also observed by qPCR, the difference did not reach significance by this method. Both Pnck and Pja1 are female-biased in the adipose tissue according to the data of Yang et al.[Ogt, Maob,Ndufa, Pgrmc1 and Nsbp1) are amongst the genes that are female-biased in at least one tissue, according to the data of Yang et al. [In our study, two new candidates tested were concordant at ng et al.. Additiog et al. . In the Huwe1, Arhgef6 and Nsbp1) are false positives generated by the statistical analyses of differential expression by microarray ; ii) overexpression of these genes in XX females may be due to a secondary transcriptional upregulation of the active X chromosome allele and does not reflect expression from both X chromosomes, as discussed above. The former hypothesis would imply a bias skewing the results in the same direction in two independent studies using different microarray platforms and a different study design, which is unlikely. On the other hand, the fact that these three genes present binding sites for common transcription factors gives strength to the second hypothesis, where overexpression of some X-linked genes would be due to transcriptional upregulation, through the same mechanism that is contributing to higher expression of several autosomal genes in XX females.Further evidence in support of the existence of uncharacterised escapees on the rodent X chromosome comes from studies in mouse ES cell lines ,44. Howein vitro and in vivo models suggest the existence of tissue- or cell type-specific patterns of X inactivation. However this issue can only be fully addressed by a thorough gene-by-gene comparison of allelic expression in single cell versus whole tissue homogenates.Our results, as well as the results from other studies using both In humans, complete or partial monosomy of the X chromosome results in Turner syndrome. The phenotype, which includes ovarian dysgenesis and infertility, is attributed to a lower dosage of X chromosome genes escaping inactivation. However, only 5% of the genes on the human X chromosome that are expressed in lymphoblastoid cell lines are significantly overexpressed in females at the population level . TherefoEif2b2 and Eif2b5 [We detected lower expression of several X-linked genes in XO mice. Moreover, a large number of autosomal genes were differently expressed between the two genotypes in liver, including two genes which are mutated in POF - d Eif2b5 . Due to The analysis of functional enrichment we performed in the liver dataset revealed other cellular processes that are differently regulated between the two groups of mice analyzed - ATP synthesis and mitochondrial metabolism. Impairment of energy metabolism is expected to lead to several alterations in cell homeostasis and is known to play a prominent role in the pathogenesis of neurological diseases. These results may therefore have a bearing on some of the observed neuropsychological deficits of TS. The presence of binding sites for shared transcription factors within the regulatory regions of the genes underexpressed in XO mice reinforced the hypothesis of a deregulation of specific pathways in this model.Xlr family [mO and XpO mice.Although X-linked imprinted genes can also contribute to some of the phenotypes observed in TS females, parental effects on expression have so far only been detected in some genes of the r family ,43. In oWe confirmed and extended previous global analysis of X chromosome gene expression in the mouse and uncovered a dosage-dependent effect on the expression levels of several X-linked and autosomal genes that cannot be attributed to sex-specific hormonal influences. Even though we found strong candidates for escaping X inactivation, the lack of direct evidence of escape for those genes tested suggests that other factors may underlie the differences observed between XX and XO mice.Several X-linked and autosomal genes are deregulated in XO mice and these are involved in a variety of cellular functions. We hypothesize that the TS phenotype is partially caused by the additive effect of regulatory perturbations downstream of the under-expressed X-linked genes, which may differ between tissues and between species. Moreover, genes escaping inactivation in humans but not in the mouse may lie in regulatory nodes at the intersection of a larger number of effectors, having a greater impact on genome-wide levels of expression.mO, XpO and XmXp). All genotypes were determined by bone marrow metaphase preparations and in some cases confirmed by PCR. All animal procedures and breeding were in accordance with the United Kingdom Animal Scientific Procedures Act 1986 and were subject to local ethical review.The mouse models used in our expression analysis were obtained as described in , except Tissues from 12 week old mice were harvested and stored at -80°C before use. Total RNA was isolated from homogenized tissues using Trizol reagent (Invitrogen) and purified using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. RNA integrity was verified by capillary electrophoresis in the Agilent 2100 Bioanalyzer.pO and eight 39,XmO mice were pooled by genotype into 9 groups, representing 3 biological replicates per genotype, as follows: 39,XpO-1 and 39,XpO-2 , 39,XpO-3 ; 39,XmO-1 and 39,XmO-2 , 39,XmO-3 ; 40,XX-1 and 40,XX-2  40,XX-3 . A total of 300 ng of RNA from each pool was then amplified following the Illumina TotalPrep RNA amplification protocol. The samples were labelled and hybridized to the Mouse WG-6 v1_1 Expression BeadChip whole genome expression array available from Illumina, following the manufacturer's protocols.The RNA samples of seven 40,XX, eight 39,XThe values for each bead in the array were imported into the Illumina software BeadStudio for preliminary quality checks, summarized according to bead type and then exported into R environment for statistical computing. Normalisation across all arrays was performed on a log scale using the quantile normalisation method  implemenhttp://www.inforsense.com. The distance matrix was calculated with Pearson correlation and clusters built by average linkage.Samples were hierarchically clustered using the tools implemented in InforSense Workflow Builder The microarray data for this study have been deposited to GEO under accession number GSE13520.2 ≥ 0.99 and with a standard curve slope typically -3.1 ≤ S ≤ -3.6 were accepted. Melt curve data were obtained to confirm amplification of the correct product. Expression was normalised by reference to Gapdh and in most cases the results were confirmed using Actb. Differences in expression between groups were calculated by means of a t-test. All primers were designed to span exon-exon boundaries, to prevent amplification of possible genomic DNA contaminants. For primer sequences see Additional file Quantitative real-time PCR reactions were carried out using 100 ng of cDNA in a reaction volume of 20 μl comprising Sybr Green PCR Master Mix (Applied Biosystems) and 0.3 μM of each primer in an Applied Biosystems 7000 detection system (Applied Biosystems). Efficiency of primers and quantity of cDNA in each well were derived from an experimentally determined standard curve; only reactions with rHuwe1, Arhgef6 and Nsbp1 by direct sequencing and, whenever possible, by single-base primer-extension (SNaPshot). Each SNaPshot assay was tested in genomic DNA of T16H-FVB F1 females heterozygous for the target SNPs and then used to differentiate expression from each X chromosome in the cDNAs from liver, kidney, muscle and brain of two animals. Typically 50 ng of DNA and 100 ng of cDNA were amplified using the HotStar HiFidelity Polymerase Kit (Qiagen) in a 10 μl reaction volume comprising 0.3 μM of each primer. PCR conditions were the following: 15 min pre-incubation step at 94°C, 35 cycles of denaturation at 94°C for 30 sec, annealing for 45 sec at the respective AT for each primer pair and extension at 72°C for 45 sec, followed by a final extension step at 72°C for 10 min. The PCR products were then purified with ExoSAP-IT (Usb) and single-base primer-extension reactions were carried out with SNaPshot Multiplex Kit (Applied Biosystems) in a 5 μl volume comprising 0.2 μM of the extension primer and up to 2 μl of the purified PCR product (depending on the expression level of each gene in the different tissues tested), for 25 cycles. SNaPshot reactions were cleaned up with Shrimp Alkaline Phosphatase (Usb) and the analysis of fluorescent products was performed in an ABI 3100 sequencer using the GeneMapper 4.0 Analysis Software. For primer sequences see Additional file Expression from each X chromosome was verified using polymorphisms within the coding region and/or the UTRs of XIC: X Inactivation Center; LTRs: Long Terminal Repeats; ES: Embryonic Stem (cells); TS: Turner Syndrome; FDR: False Discovery Rate; FC: Fold Change; POF: Premature Ovarian Failure; GO: Gene Ontology.AML conceived the study, designed and carried out all the experiments, analysed the data and wrote the manuscript; PSB and AO bred the mice and verified the genotypes; JB contributed to data analysis; NAA, CAS and PSB contributed to the experimental design, discussion of the results and with a critical review of the manuscript; AA contributed to the discussion of the results and with a critical review of the manuscript. All authors read and approved the final manuscript.Probes differentially expressed in liver of XX and XO mice. A linear model pairwise comparison between XX and XO mice was performed using the R package limma for all probes  detected in liver . After FDR correction 1402 probes were significantly different between genotypes at the 0.05 significance level.Click here for filePrimer sequences. This file contains the sequences of the primers used in qPCR and in the analysis of allelic expression by SNaPshot.Click here for file


Progress in the life sciences cannot be made without integrating biomedical knowledge on numerous genes in order to help formulate hypotheses on the genetic mechanisms behind various biological phenomena, including diseases. There is thus a strong need for a way to automatically and comprehensively search from biomedical databases for related genes, such as genes in the same families and genes encoding components of the same pathways. Here we address the extraction of related genes by searching for densely-connected subgraphs, which are modeled as cliques, in a biomedical relational graph.We constructed a graph whose nodes were gene or disease pages, and edges were the hyperlink connections between those pages in the Online Mendelian Inheritance in Man (OMIM) database. We obtained over 20,000 sets of related genes  by enumerating cliques computationally. The modules included genes in the same family, genes for proteins that form a complex, and genes for components of the same signaling pathway. The results of experiments using 'metabolic syndrome'-related gene modules show that the gene modules can be used to get a coherent holistic picture helpful for interpreting relations among genes.We presented a data mining approach extracting related genes by enumerating cliques. The extracted gene sets provide a holistic picture useful for comprehending complex disease mechanisms. Progress in the life sciences has recently been made by integrating biomedical knowledge on numerous genes and formulating hypotheses on the genetic mechanisms underlying various vital phenomena ,2. A larGraph-based approaches -9 have rHere we have constructed a biomedical relational graph whose nodes are pages of genes or diseases and whose edges are hyperlink connections between pages by using over 10,000 entities in the OMIM database . The OMIThe experimental materials were taken from the Online Mendelian Inheritance in Man (OMIM) database, a well-known catalog in which human genes and genetic disorders are assigned descriptive code numbers . Using tKEGG pathway data that contains sets of genes were obtained (November 2003) from a database produced by the Cancer Genome Anatomy Project .G consists of a set V of nodes and a set E of non-weighted edges connecting pairs of nodes. The number of edges connected to node v ∈ V is referred to as the degree of node v in G. The subgraph of G is induced by the subset V' of V. A subgraph in which every pair of nodes is connected by an edge  is called a clique, and the size of which is the number of nodes in it. A clique is called maximal if it is included in no other clique. Pseudo-cliques are the subgraphs obtained by relaxing the connectivities, and the connectivity is measured by the edge density ρ. The edge density of a pseudo-clique S is the ratio of the number of edges in S to the number of edges in a clique that has the same number of nodes that S does. It is calculated as follows:An undirected graph E(S)| is the number of edges in S and |S| is the number of nodes in S. By setting ρ(S) to a threshold value θ (0 ≤ θ ≤ 1) pseudo-cliques whose edge density is not less than θ can be enumerated [θ decreases from 1 to 0, we get pseudo-cliques whose connectivity is more and more relaxed.where |umerated . As a θ In the work presented in this paper we obtained gene modules  by enumerating pseudo-cliques in a graph whose node were genes and genetic disorders and whose connecting edges were biomedical relations.Correspondence analysis  is a metIn the work presented in this paper items and cases are assigned respectively to modules and genes/diseases contained.We will present an example of enumerating cliques from a part of an undirected graph used in experiments described later in this paper. This enables us to see clique enumeration from a biomedical relational graph and to investigate the validity in an accessible fashion.Figure The node sets for all the maximal cliques obtained from the graph in Figure V has kv connected nodes, the clustering coefficient is the ratio of the actual number of edges to the possible kv(kv-1)/2 edges. The characteristic path length and clustering coefficient [We constructed an undirected graph whose nodes were gene or disease pages in the OMIM database and whose edges were hyperlink connections between those pages. As shown by the data listed in Table fficient  were resIn the graph were 20,486 maximal cliques and the largest maximal clique contained 12 nodes. The 20 most frequent genes in the maximal cliques are listed in Table θ decreased (Table θ = 0.88 and did not increase further even when θ was decreased to 0.7 (data not shown).When pseudo-cliques were extracted by the relaxing edge connecting condition, the maximum sizes increased as ed Table . Prelimiθ = 0.88) were taken as gene modules in the following analysis. The sensitivity and specificity of the current method in extracting biologically relevant genes using cliques can not be readily assessed. One would assume that cliques with larger size are likely to be more biologically relevant. To account for this, we estimated whether a cutoff value on the clique size can be set. We examined the maximum size of cliques in randomized graphs having common node degrees to the OMIM hyperlink graph. In the experiments of enumerating pseudo-cliques (θ = 0.88) in the randomized graphs which were generated using an edge-swapping approach [For this reason, 25,642 pseudo-cliques  were compared by calculating values of the Jaccard's coefficient rJ (0 ≤ r J ≤ 1). The Jaccard's coefficient is often used as a criterion when evaluating the similarity of two sets, and is the ratio of the size of the intersection of the sets to the size of the union of the sets. Its value thus approaches 1 as the extent of coincidence increases. The 15 most relevant KEGG pathways and the corresponding gene module sizes are listed in Table To evaluate how well the gene modules correspond to known gene pathways, we compared the sets of genes in the modules with those in the KEGG pathway database . The 66 β signaling. The third is in regulation of food intake and energy monitoring. Repeated captures suggest that the pairs have various functions in biological setting.In order to evaluate how well the gene modules accord with interactions between genes, we compared the sets of genes in the modules with gene pairs in the protein-protein interaction data . The 650κB family genes (including RelA and RelB) and genes that form complexes with them (IκB). The gene module in the third row is made up of 'DNA repair'-related genes. The BRCA1-associated proteins; the BLM, MSH6, MSH2, and MLH1 proteins; and subunits of the RFC complex are involved in DNA repair [κB activity by binding to the TRAF2 gene product. The gene modules thus comprise various types of related genes including gene families, complexes, and pathways. As illustrated above, the current results have biological coherence for analyzing relations among genes.Table A repair . The genA repair . TNF recFor applying gene modules to disease mechanism analysis, we assembled gene modules associated with the metabolic syndrome  as an exGene modules associated with diabetes, hyperlipidemia, hypertension, and obesity are obtained by their containing the disease nodes, which are non-insulin-dependent diabetes mellitus (MIM 125853), familial combined hyperlipidemia (144250), essential hypertension (145500), and obesity (601665). Out of 25,642 modules, 110, 16, 34, and 28 modules are obtained, respectively. There were no overlaps among the modules. Then a total of 188 modules and 124 genes contained were identified.The 10 most frequent genes in the 188 modules are listed in Table We combined the 188 modules and 124 genes using correspondence analysis . As shown in Figure Genes related to the above-mentioned aldosterone metabolism  are at the top of the figure. Genes related to the renin-angiotensin system  are around the hypertension node indicated by the cluster of 't' marks, and apolipoprotein genes  are gathered around the hyperlipidemia node close to the 'renin-angiotensin system'-related genes. It is worth noting that 'food intake regulation'-related genes such as LEP, NPY, MC4R, AGRP and POMC genes are grouped comprehensively . Insulin resistance is defined as a status in which the action of insulin is insufficient and proper energy conversion is impaired. The TNF gene related to insulin resistance , which iWe have shown that related genes can be extracted comprehensively by enumerating pseudo-cliques in biomedical relational graph. Over 20,000 gene modules that include genes in the same family, genes encoding proteins in the same complexes, and genes encoding components of the same signaling pathway were extracted automatically. Furthermore these gene modules were utilized for visualizing relations between genes and diseases. Extraction of related genes (referred to as gene modules in this paper) would be more important since most biomedical tasks are performed not by individual genes, but by sets of functionally associated genes. The method using pseudo-cliques can generally extract related gene sets under a single computational operation although the application is restricted to genes connected by their relations in the manner of the OMIM hyperlink connections. For clarifying complex disease mechanisms, obtaining relationships among genes associated with the diseases should be crucial. Comprehensive extraction of related-gene sets by clique-based data mining may provide us with a systematic methodology for gaining insight into the genetic mechanisms underlying various biological phenomena including diseases.TM and ET conceived and designed the experiments. TM and CY performed the experiments. TM and MM analyzed the data. All authors have read and approved the final manuscript.