Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a ‘holding trap’ rather than a ‘folding trap’ mechanism.DOI:http://dx.doi.org/10.7554/eLife.16177.001 Human, yeast and other eukaryotic cells contain compartments called mitochondria that perform several vital tasks, including supplying the cell with energy. Each mitochondrion is surrounded by an inner and an outer membrane, which are separated by an intermembrane space that contains a host of molecules, including proteins.Intermembrane space proteins are made in the cytosol before being transported into the intermembrane space through pores in the mitochondrion’s outer membrane. Many of these proteins have the ability to form disulfide bonds within their structures, which help the proteins to fold and assemble correctly, but they only acquire these bonds once they have entered the intermembrane space.An enzyme called Mia40 sits inside the intermembrane space and helps other proteins to fold correctly. This Mia40-induced folding had been suggested to help proteins to move into the intermembrane space.Mia40 contains two important regions: one region acts as an enzyme and adds disulfide bonds to other proteins, and the other region binds to the intermembrane space proteins. Peleh et al. have now generated versions of Mia40 that lack one or the other of these regions in yeast cells, and then tested to see if these mutants could drive proteins across the outer membrane of mitochondria. The results show that it is the ability of Mia40 to bind proteins – and not its enzyme activity – that is essential for importing proteins into the intermembrane space.As disulfide bond formation is not critical for importing proteins into the intermembrane space, future studies could test whether Mia40 also helps to transport proteins that cannot form disulfide bonds. Presumably, Mia40 has a much broader relevance for importing mitochondrial proteins than was previously thought.DOI:http://dx.doi.org/10.7554/eLife.16177.002 In most cellular compartments, cysteine residues are predominantly present in the reduced state. In contrast, the bacterial periplasm, the endoplasmic reticulum (ER) and the IMS of mitochondria contain dedicated oxidation machineries (disulfide relays) to introduce disulfide bonds into a broad range of substrate proteins . At leasMitochondria consist of about 600 (yeast) to 1500 (humans) nuclear encoded proteins . Followi2 bprotein of Saccharomyces cerevisiae is the best studied example for this stop-transfer targeting  play a role as chaperones for carrier proteins in the IMS  or the substrate-binding pocket (Mia40-FE and Mia40-STOP). Interestingly, the Mia40-SPS mutant still mediates protein import of Mia40 substrates with high efficiency. It also allows the accumulation of Mia40 substrates in mitochondria, albeit at reduced levels. Our observations suggest that trapping activity of Mia40 is essential since Mia40 serves as a ‘trans-site receptor’ whose hydrophobic binding to incoming polypeptides drives protein import into the mitochondrial IMS. Hence, protein oxidation in the IMS is not directly linked to translocation, and mechanistically is independent of the transport reaction, similar to the situation in the ER and the periplasm.9C domain) was replaced by a hemagglutinin tag , precipitated proteins with trichloroacetic acid (TCA) to denature them and to preserve their redox state, and incubated them with mmPEG24  required to allow efficient substrate oxidation. We therefore tested whether the addition of a chemical oxidizer can support the growth of the Mia40-SPS mutant. Diamide is a thiol-specific chemical oxidant that induces disulfide bond formation in proteins  and whice plates . Interese plates . When GA9C domain of this protein), or it might act downstream in oxidizing proteins that were initially imported by Mia40-SPS. To test whether diamide directly supports the functionality of the Mia40-SPS protein, we isolated mitochondria containing either Mia40-SPS or Mia40-FE, pretreated them with increasing amounts of diamide and then added a radiolabeled cysteine-less model substrate of Mia40, 10CS. This protein represents a mutant version of the IMS protease Atp23 in which all ten cysteine residues were replaced by serine residues  and we assume that the up- and down-regulation of Mia40 regulates the levels of cytochrome 2b indirectly. Thus, the strong rate-limiting function of Mia40 on the import of its substrates is obviously highly relevant to the protein composition of the IMS in general and critical even for proteins which are imported in a Mia40-independent fashion.In contrast, upregulation of Mia40 did not increase, but rather reduced, the import of cytochrome b2, a well-b2. Depletihrome b2 . ImmunopMia40 is a highly conserved IMS protein which consists of two functionally and structurally distinct elements. Here we show that both the redox-active cysteine pair and the substrate-binding region of Mia40 are essential and mutants lacking individual elements cannot cross-complement each other. However, we found that cysteines in the CPC motif of Mia40 are dispensable for the import reaction of all Mia40 substrates we tested. This was unexpected because it was suggested that the mitochondrial disulfide relay operates as a folding trap  Lutz et. This moThe oxidoreductase activity of Mia40 is obviously important, but it apparently plays a role after translocation. This is consistent with a recent study which showed that mutants of small Tim proteins, in which all cysteine residues were replaced by serine residues, accumulated to wild type levels in the IMS as soon as the iAAA protease Yme1 was deleted .Surprisingly, the presence of diamide can partially suppress the growth defect of Mia40-SPS cells suggesting that the substrate-specificity of the mitochondrial disulfide relay is not absolutely necessary. The observation of oxidized Tim10 in the Mia40-SPS mutant indicates that the CPC of Mia40 is not absolutely essential for protein oxidation in the IMS. Whether Erv1 can directly oxidize IMS proteins or whether other factors might serve as oxidoreductases here is not known, but it will be interesting to test the relevance of the recently identified thioredoxins and glutaredoxins in the IMS in this process . Howeverin organello competition import experiments  or in YP  medium containing 2% galactose, glucose or glycerol  the agenesis . The res24, mmPEG12 and AMS were used to modify reduced thiols. The pulse-chase labeling of Cox19 was performed as described  and resuspended in lysis buffer I . The extract was diluted tenfold in lysis buffer II . After incubation for 10 min at 4°C, the extract was cleared by a clarifying spin . Antibodies against Mia40, Erv1, Atp23, Tim9, Cmc1, Cox19 or hemagglutinin were coupled to protein A-sepharose beads. The beads were incubated with the mitochondrial extract at 4°C, washed three times with the lysis buffer II , resuspended in SDS-sample buffer and boiled for 5 min at 96°C. Samples were analyzed by SDS-PAGE and autoradiography. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Vivek Malhotra (Senior editor) and three reviewers, one of whom, Nikolaus Pfanner, is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "An essential trans-site receptor activity of Mia40 drives protein import into the intermembrane space of mitochondria" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this manuscript Peleh et al. have analyzed the function of Mia40 in protein transport into mitochondria. Using a set of MIA40 mutant strains, they define how the proposed substrate binding domain and the redox active CPC motif promote protein translocation into the mitochondrial intermembrane space. They find that the redox active CPC motif is dispensable for the translocation process and that the substrate-binding domain can promote vectorial movement of the polypeptide independent. Thus, a predominant mechanism applied by Mia40 for substrate recognition is a trapping mode, for which disulfide bond formation is not necessary.The results presented here provide new and unexpected mechanistic insight into the transport of intermembrane space proteins The surprising finding that transport is not linked to oxidative folding but rather facilitated by substrate binding to Mia40 's binding domain changes the current concept of the Mia40 transport pathway. The manuscript provides a new mechanistic model on the import driving forces. The findings are interesting not only for protein translocation specialists but also for a broader audience since they provide a conceptual framework to explain the involvement of Mia40 in the biogenesis of several non-redox dependent proteins.The data are of high technical quality and the manuscript is very well written. The findings presented in this manuscript clearly change our view of intermembrane space-protein transport.Essential revisions:1) The point mutant FE that disrupts the hydrophobic substrate binding patch may not be redox-active since there is no oxidized protein detectable at steady state, in contrast to the STOP variant that lacks the entire substrate binding domain . The aut2) The authors should discuss their data and the resulting novel conclusions for mitochondrial protein import more accurately. Statements such as "Mia40 serves as a trans-receptor" have been published in earlier papers. Based on earlier papers the critical point in initiating the oxidative folding pathway in mitochondria is the trapping by Mia40. The important novelty of the current work lies in the demonstration that trapping does not have to be mediated by a redox reaction, i.e. the formation of a disulfide bond, but can be facilitated by hydrophobic interactions of Mia40 with the incoming protein substrates. Several parts of the manuscript, including the title, abstract and model, should be rewritten to precisely specify these important conclusions.3) It has been a long standing question in the redox field why Mia40-bound substrates could easily be detected under wild-type conditions whereas the substrate interactions of ER and bacterial oxidoreductases  were highly transient. The identification of two different activities in Mia40 elegantly solves this problem. It will thus be good to discuss these general implications for the fields of protein translocation and oxidative protein folding in more detail, e.g. by addressing this open question (difference between Mia40 and PDI/DsbA) already in the Introduction.4) Several papers have specified the cysteine residues of substrates in close proximity of the MISS/ITS signal for Mia40 recognition and showed the import deficiencies of substrates lacking this Cys residue. The in vivo accumulation of these proteins indicates, however, that this "recognition" defect may be bypassed by different means (either by use of other Cys residues or by folding that is not fully dependent on oxidation).It would be good to have a WB to see the levels of Erv1 overexpression. Is it possible that substrate proteins form some other intermolecular disulfide bonds that do not involve the CPC motif of Mia40, i.e. with Erv1? Additionally, the possibility of substrate binding by Mia40-SPS via a disulfide bond should be tested in the presence of chemical reductant.5) The authors did not provide a sufficient explanation concerning the action of diamide. One can assume that diamide should in fact improve the overall oxidative folding/mitochondrial accumulation of substrates; is this the case? Diamide may or may not improve the substrate binding to Mia40-wt or Mia40-SPS? The answer to this question would be important to specify the stage diamide works. Finally, does diamide influence Mia40 binding to Erv1 or substrate binding to Erv1? The mechanism for diamide should be proposed based on the experimental data.6) The import experiments presented in What is the purpose of The text does not mention the number of replicates used for the quantitations in  Essential revisions:1) The point mutant FE that disrupts the hydrophobic substrate binding patch may not be redox-active since there is no oxidized protein detectable at steady state, in contrast to the STOP variant that lacks the entire substrate binding domain . The aut9C motif, our shift experiments do not allow to make clear conclusions on the redox state of the CPC motif. Therefore, we followed your suggestion and added novel experiments using the STOP mutant which is partially oxidized. First, we performed import experiments into Mia40-STOP mitochondria with Atp23, Cmc1 and Tim9. All three proteins were imported well in the presence of Mia40 but no import was observed into Mia40-STOP mitochondria. These novel experiments are shown in Indeed, the redox state of the FE mutant is not clear. In the background of the four reduced cysteines in the Cxmia40- 3 background in the presence of the STOP mutant whereas co-expression of the Mia40-SPS mutant largely restored the accumulation of these proteins. This supports our conclusion that the hydrophobic bind cleft is both essential and (to a certain degree) sufficient to import IMS proteins.Moreover, as requested we analyzed protein levels in mitochondria of the STOP mutant by Western blotting. In the novel 2) The authors should discuss their data and the resulting novel conclusions for mitochondrial protein import more accurately. Statements such as "Mia40 serves as a trans-receptor" have been published in earlier papers. Based on earlier papers the critical point in initiating the oxidative folding pathway in mitochondria is the trapping by Mia40. The important novelty of the current work lies in the demonstration that trapping does not have to be mediated by a redox reaction, i.e. the formation of a disulfide bond, but can be facilitated by hydrophobic interactions of Mia40 with the incoming protein substrates. Several parts of the manuscript, including the title, abstract and model, should be rewritten to precisely specify these important conclusions.We agree with the referee in this point. A trapping function of Mia40 via disulfide bonds was proposed before and the specific recognition of MISS/ITS signals was of major importance to understand the role of Mia40 as a trans-site receptor. We now adapted the model in 3) It has been a long standing question in the redox field why Mia40-bound substrates could easily be detected under wild-type conditions whereas the substrate interactions of ER and bacterial oxidoreductases  were highly transient. The identification of two different activities in Mia40 elegantly solves this problem. It will thus be good to discuss these general implications for the fields of protein translocation and oxidative protein folding in more detail, e.g. by addressing this open question (difference between Mia40 and PDI/DsbA) already in the Introduction.We discuss this aspect in more detail in the Abstract and in the Discussion.In the Abstract we added the sentence: “The oxidoreductases of the periplasm and the ER, DsbA and protein disulfide isomerase, are members of the thioredoxin superfamily which are structurally and phylogenetically distinct from the components of the mitochondrial disulfide relay.”In the Discussion, the last paragraph reads: “The bacterial periplasm, the ER and the mitochondrial IMS are the three compartments in which dedicated machineries mediate the oxidative folding of a broad range of proteins . […] It therefore appears likely that the ability of Mia40 to trap incoming preproteins efficiently at the trans-site of the TOM complex made Mia40 the superior oxidoreductase to mediate the translocation and oxidation of IMS proteins.”4) Several papers have specified the cysteine residues of substrates in close proximity of the MISS/ITS signal for Mia40 recognition and showed the import deficiencies of substrates lacking this Cys residue. The in vivo accumulation of these proteins indicates, however, that this "recognition" defect may be bypassed by different means (either by use of other Cys residues or by folding that is not fully dependent on oxidation).It would be good to have a WB to see the levels of Erv1 overexpression. Is it possible that substrate proteins form some other intermolecular disulfide bonds that do not involve the CPC motif of Mia40, i.e. with Erv1? Additionally, the possibility of substrate binding by Mia40-SPS via a disulfide bond should be tested in the presence of chemical reductant.We now added the requested Western blot data as novel Furthermore, we performed Western blotting experiments with mitochondrial extracts of the Mia40-SPS mutant in the absence or presence of diamide with or without overexpression of Erv1. As shown in the novel In addition, we added a novel set of pull down experiments in which we tested a potential disulfide-mediated interaction of Cmc1 and Tim9 with Mia40 and Erv1 after import into wild type, Mia40-SPS and Mia40-STOP mitochondria. Whereas both proteins are efficiently precipitated with Mia40, no interactions were observed with Mia40-SPS, Mia40-STOP or Erv1. Even if Erv1 was overexpressed in the Mia40-SPS mutant, only very small amounts of Cmc1 and Tim9 were recovered with Erv1-specific antibodies. These experiments are now shown as novel Moreover, we performed immunoprecipitation experiments with Mia40-SPS-containing mitochondria that were pretreated with 5 mM DTT. As shown in the novel 5) The authors did not provide a sufficient explanation concerning the action of diamide. One can assume that diamide should in fact improve the overall oxidative folding/mitochondrial accumulation of substrates; is this the case? Diamide may or may not improve the substrate binding to Mia40-wt or Mia40-SPS? The answer to this question would be important to specify the stage diamide works. Finally, does diamide influence Mia40 binding to Erv1 or substrate binding to Erv1? The mechanism for diamide should be proposed based on the experimental data.Diamide is a chemical oxidizer that induces the formation of disulfide bonds. As shown in the novel mia40-4 cells. This also shows that the Mia40- SPS does not improve the oxidation rate of Cox19-HA. This provides additional evidence that Mia40-SPS does not serve as oxidoreductase here.We also tested in more depth the redox state of proteins imported into the Mia40-SPS mutant. As shown in the novel 6) The import experiments presented in What is the purpose of The text does not mention the number of replicates used for the quantitations in We followed the suggestion of the referee and removed the former panels 4F and 4H. For the quantification shown in the study  and 7C,


CDH1 germline mutations. In Brazil, gastrointestinal tumors are among the most prevalent tumor types and constitute a serious public health problem, especially in the northern and northeastern regions. This study aimed to investigate germline mutations, methylation pattern and genomic rearrangements in the CDH1 gene and quantitative changes in the DNA of HDGC patients in northern and northeastern Brazil.Hereditary diffuse gastric cancer (HDGC) is a hereditary autosomal inherited syndrome associated with Twenty-seven DNA samples from the members of four families affected by HDGC were analyzed using array comparative genomic hybridization (aCGH), DNA sequencing and methylation pattern.No evidence of gain and loss events or any rearrangements were found in any of the samples tested using aCGH. No promoter region hypermethylation was observed either. Two of the four families presented different types of germline mutations. The 185G > T and 1018A > G germline mutations detected in this study have been described in Asian and European families, respectively. The ancestors of the two families carrying these mutations had originated from those continents.CDH1 gene germline mutations in Brazilian families with HDGC. In our study, 50% of the families showed no CDH1 gene alterations, and it is possible that in regions with a high incidence of gastric cancer, such as northern and northeastern Brazil, environmental factors might have induced the different genetic alterations analyzed in this study.This is the first study to evaluate  In northern and northeastern Brazil, gastric cancer (GC) is the second most common type of cancer and is considered a serious public health problem because it is usually diagnosed at an advanced stage. In addiIn order for a family to qualify for a diagnosis of HDGC, the following criteria must be met: two or more documented cases of diffuse gastric cancer in first- or second-degree relatives with at least one diagnosed before the age of 50 years or three or more cases of documented diffuse gastric cancer in first- or second-degree relatives independent of the age of onset,5.CDH1 inactivation) and the predisposition to diffuse gastric cancer was first identified in a large family in New Zealand[The correlation between an E-cadherin gene germline mutation  signed consent forms that had been approved by the Research Ethics Committee of University Hospital João de Barros Barreto  under protocol number 274/12. Assurance was provided that the use of biological materials and participation in the study would not cause any harm to nor have any negative influence on patient treatment.All biological materials except one sample  was blood DNA. And all genetic screening analysis - mutation, deletion and methylation - were all looking for germline changes, not somatic.Peripheral blood samples were collected in EDTA-containing tubes and extracted with the QIAamp® DNA Blood Mini Kit  according to the manufacturer’s instructions.Paraffin block sample was obtained from one patient, and extracted with the QIAamp DNA® FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions.screening of the CDH1 gene. Gene fragments containing the analyzed polymorphisms were amplified by polymerase chain reaction (PCR) using gene-specific reaction conditions and primers . The sequences were obtained as electropherograms and analyzed with the software package provided with the equipment. The generated sequences were analyzed with the BIQ Analyzer software package.High-density probe microarray analyses were performed to determine the copy number variation (CNV). The complete genomes of all patients were evaluated with the goal of identifying genes related to tumor development.Affymetrix® CytoScan™HD Array  was used; this system features a total of approximately 1.9 million probes for detecting CNV and 750,000 SNP molecular markers. The standard protocol incorporates the following eight procedures before scanning the chip: genomic DNA digestion, NSP adapter ligation, fragment amplification by PCR (Polymerase Chain Reaction), PCR product purification, PCR product fragmentation, end-labeling, hybridization and washing.The Chromosome Analysis Suite software v1.2.1 (Affymetrix®) was used for the chip analysis.The CDH1 promoter region was amplified using a nested PCR strategy[For methylation analyses, the samples were subjected to DNA modification using sodium bisulfite. A fragmstrategy. Fragmenstrategy. Methylastrategy softwareCDH1 gene (HDGC). Streptavidin-biotin-peroxidase staining as described by Hsu et al.[An anti-cadherin  commercial primary antibody was used to detect the protein product of the u et al. was adopu et al..The detailed patient data are shown in Table Of these 27 patients, nine (33.4%) had been previously diagnosed with diffuse gastric cancer, whereas the remaining patients did not have any type of gastric tumor. Among the patients diagnosed with DGC, the age at diagnosis ranged from 27–67 years.After the retrospective study and using information collected from the analyzed patients, it was possible to identify relatives with a history of DGC and other types of tumors Figure .CDH1 gene, namely family A from northern Brazil and family B from northeastern Brazil .CDH1 protein detection was performed only for patient AM05, a carrier of the germline mutation 185 G > T and member of family A. This patient died before the start of the study, and a genetic analysis was performed on a paraffin-embedded tumor tissue. The other study participants with this syndrome donated peripheral blood for genetic analysis, and consequently, there was no need to request paraffin blocks of the resected tumor tissues. The tumor cells of patient AM05, revealed negative immunoreactivity to the E-cadherin protein.CDH1 gene promoter region fragment in one CpG island containing 22 CpG dinucleotides, to find possible heritable epimutations. No promoter region hypermethylation was observed in all 27 tested individuals, from the four families, and also in 10 healthy controls.We analyzed the methylation pattern of a In addition to sporadic manifestation, gastric cancer can be associated with various syndromes that predispose the carrier to cancer. Among the familial forms of gastric cancer, HDGC is the only syndrome with a well-defined genetic cause,3.CDH1 inactivation) and the predisposition to HDGC was first identified in a family in New Zealand[The relationship between a genetic germline mutation in the E-cadherin-encoding gene ; this mutation causes a change in the protein structure from the amino acid glycine to valine. This missense mutation was first described by Shinmura et al.[In this study, two individuals in family A, a father and son Figure A, were cra et al. in a famCDH1 gene germline mutation was also identified in two brothers from family B who had developed an early form of gastric cancer (before 45 years of age)[CDH1 locations to mutations.A  of age). The mut of age),37. BecaCDH1 gene in HDGC[A literature review revealed the occurrence of a total of 122 germline mutations in the  in HDGC. Althoug in HDGC,13; thisCDH1 mutation-associated HDGC might share a common ancestry, as was suggested for families A and B in this study[Of the mutations reported in the literature, approximately 15% are shared by many families worldwide, suggesting that is study,39.CDH1 gene mutations, there were considerable numbers of HDGC-affected members in different generations . Based on this, it may be possible in the future to test families C and D, for allele-specific loss on CDH1.In the other two families (C and D), in which the evaluated subjects carried no CDH1 gene germline mutations. This finding might be consequent to exposure to environmental risk factors or the susceptibility of individuals to genetic alterations in low-penetrance genes[In other populations in which the incidence of gastric cancer is high, there are also reports of HDGC-carrier families with large numbers of affected individuals who do not carry ce genes,41.CDH1 gene sequencing was not conducted to identify possible mutations, the immunohistochemical analysis revealed the involvement of CDH1 in the early development of gastric cancer in a Brazilian population[In a study conducted in southeastern Brazil on 88 patients with early gastric cancer, 16 of whom had familial histories of gastric cancer, it was possible to detect changes in the E-cadherin protein expression in 41 individuals via immunohistochemical analysis. Although pulation.CDH1 gene acts as a tumor suppressor, other gene inactivation mechanisms of this gene besides germline mutations in one allele should be investigated to identify changes to the second allele in somatic cells (second event of the Knudson hypothesis[CDH1 gene deletion[Because the pothesis), which deletion and promdeletion, were inCDH1 promoter region induces transcriptional silencing[CDH1 gene promoter region hypermethylation in patients with HDGC have not been conclusive. For example, studies by Li et al.[et al.[CDH1 promoter hypermethylation-mediated gene silencing in 53–57% of patients with HDGC. Conversely, Wu et al.[Cytosine hypermethylation in the CpG dinucleotides present within the ilencing and mighi et al. and Conc.[et al. demonstru et al. reportedCDH1 gene promoter region hypermethylation in the families with HDGC syndrome. Our results reinforce the theory put forth by Yamada et al.[CDH1 promoter hypermethylation was not a predisposing factor of gastric cancer.The analyses conducted in the present study did not detect da et al. who, aftCDH1 gene might also suffer deletions even in the absence of point mutations in the germlines of patients with HDGC[et al.[In addition to epigenetic alterations in the promoter region, the ith HDGC. Kim et [et al. used mulet al.[CDH1 gene deletions occurred in the peripheral blood in approximately 4% of families affected by this syndrome. The same authors also observed that all families with CDH1 deletions originated from countries with low gastric cancer incidence rates. A large number of patients with HDGC, in northern and northeastern from Brazil, needs to be analyzed to verify if the absence of CDH1 gene deletions and other quantitative genomic alterations, as found in patients of this work, corroborate this observation because these regions have high gastric cancer incidence rates.Oliveira et al. geneticaHelicobacter pylori (H. pylori) infection during childhood as well as the high consumption of salt-preserved foods, infrequent use of refrigeration and low consumption rates of cereals, fresh fruits and vegetables are considered risk factors for gastrointestinal tumors[It is widely known that the most common forms of cancer develop as a result of interactions between endogenous and environmental factors such as the diet. Environl tumors-55.CDH1 gene germline mutations and quantitative gene alterations in Brazilian families with HDGC. CDH1 gene germline mutations were found in 50% of the families evaluated. The detected mutations appeared to originate from Asia and Europe. This migratory flow does not rule out the possibility that environmental factors might have caused these same mutations in the affected members of the analyzed families. No quantitative changes were observed in the genomes of any of the analyzed families. It is possible that in regions with high gastric cancer incidence rates such as northern and northeastern Brazil, environmental factors or other molecular mechanisms might induce different genetic alterations from those analyzed in this study.This is the first study to evaluate The authors declare that they have no competing interests.Conceived and designed the experiments: CFAMN, MBLB, BNB, LML, RMRB. Performed the experiments: CFAMN, MBLB, BNB, LML, ABB. Analyzed the data: CFAMN MBLB, BNB, HFR, RMRB. Wrote the paper: CFAMN, PPA, JAR, GRP, RMRB. All authors read and approved the final manuscript.


Portable, low-cost, objective and reproducible assessment of muscle strength in the lower limbs is important as it allows clinicians to precisly track progression of patients undergoing rehabilitation. The Nintendo Wii Balance Board (WBB) is portable, inexpensive, durable, available worldwide, and may serve the above function.The purpose of the study was to evaluate (1) reproducibility and (2) concurrent validity of the WBB for measuring isometric muscle strength in the lower limb.A custom hardware and software was developed to utilize the WBB for assessment of isometric muscle strength. Thirty older adults (69.0±4.2 years of age) were studied on two separate occasions on both the WBB and a stationary isometric dynamometer (SID). On each occasion, three recordings were obtained from each device. For the first recording, means and maximum values were used for further analysis. The test-retest reproducibility was examined using intraclass correlation coefficients (ICC), Standard Error of Measurement (SEM), and limits of agreement (LOA). Bland-Altman plots (BAP) and ICC’s were used to explore concurrent validity.No systematic difference between test-retest was detected for the WBB. ICC within-device were between 0.90 and 0.96 and between-devices were from 0.80 to 0.84. SEM ranged for the WBB from 9.7 to 13.9%, and for the SID from 11.9 to 13.1%. LOA ranged for the WBB from 20.3 to 28.7% and for the SID from 24.2 to 26.6%. The BAP showed no relationship between the difference and the mean.A high relative and an acceptable absolute reproducibility combined with a good validity was found for the novel method using the WBB for measuring isometric lower limb strength in older adults. Further research using the WBB for assessing lower limb strength should be conducted in different study-populations. The succesful completion of daily activities such as standing up from a chair or getting out of bed requires an individual to possess sufficient muscle strength. ReducedMuscle strength or performance is measured in one of four ways: (a) as the maximum force which can be produced during an isometric contraction, (b) as the maximal load, which can be performed once during weight-lifting (eg. squat), (c) as the peak torque during a concentric or eccentric isokinetic contraction, or (d) as a function test (eg. chair-stand test). Isokinetic and isometric dynamometry are considered the gold standard methods for strength assessment, 10]. H. H10]. HTherefore, a need exists to find alternative, reliable, easy to use and accessible methods for measuring muscle strength, which can be applied across different settings. This alternative could potentially be the Nintendo Wii Balance Board (WBB) as it is portable, inexpensive (approximately 80 $), durable and wideThe reproducibility of the WBB was tested with an intra-rater inter-day design with seven days between the two test sessions. Concurrent validity was also explored by comparing the WBB against a stationary isometric dynamometer ((SID)—Leg Force, Newtest, Finland). The study followed guidelines for reporting reliability and agreement studies (GRRAS).Participants were identified and recruited via member lists from senior citizen clubs and organizations in the Aalborg area. Thirty older adults were screened for eligibility and enrolled into the study by using telephone interview. Participants were included in the study if they were 65 years or more, willing and capable of coming to the hospital by themselves twice within a 7 day span, and could pass a small custom cognitive impairment screening . In addition, participants were excluded if they had acute illness within the previous 3 weeks, neurological disease , or had orthopedic surgery within the previous 6 months on either upper or lower extremities. The study was approved by the Danish North Jutland regional ethics committee and all participants gave written informed consent.We loaded both the WBB and the SID with 12 known weights ranging from 25 to 300 kg (adding 25 kg each time) in order to calibrate the measured force values in both evaluation methods. Each mass was centered on both the WBB and the SID “All participants preformed a standardized warm-up for 5 minutes of sub-maximal cycling with a cadence of 60 rmp with a one kg load. After the initial warm-up participants were randomly (coin toss) assigned to begin on either the WBB or the SID. In the assigned apparatus, participants then performed 3–5 submaximal presses, which served as both warm-up and habituation to the actual strength tests. All tests were performed in a clinical examination room at the university hospital during the same time-of-day and without shoes. Prior to actual testing participants received standardized instructions, which were to “press the feet on the WBB or SID as hard and as fast as possible until being told to stop” in order to ensure that maximal force was achieved. Both the WBB and SID test was initiated verbally with a countdown “3,2,1 Push, push, push”. During both tests (WBB and SID) participants were allowed visual feedback on the force produced as this has been shown to effect the outcome. Betweenthorder Butterworth filter (cut-off frequency 20 Hz). The custom software recorded the isometric force-time curve from the lower extremities during a 20-second period and kilogram was the unit of measurement. Participants were fitted with a standard kite harness  around their hips, seated in a standard chair (seat height 45 cm), and connected to the WBB via custom seatbelt straps. The seatbelt straps were adjusted in length corresponding to a knee angle of 120 degree. The WBB itself was held at a 57-degree angle from the ground in a custom designed Aluminum-mount. This particular angle was chosen as pilot studies indicated that the 57-degree angle was the angle where most individuals could apply the highest pressure. Finally, participants were allowed to grab on to the side of the chair in order to stabilize themselves “The WBB consists of a rigid platform with four uni-axial vertical stain gauge transducers located in the corners of the board. In order to collect data from the WBB data was streamed to a laptop computer  using Bluetooth HID wireless protocol and custom programs written in C#. Sensor values were reported as four channels of 16-bit digital data sample at approximately 100 Hz. The signal was subsequent digitally filtered using a 4th order Butterworth filter (cut-off frequency 20 Hz) .Participants were seated in a laboratory-grade stationary isometric SID  with a knee angle of 120 degree and with their feet against a fixed strain-gauge instrumented footplate, while holding on to handlebars (with their hand) for stabilization “t test was used to explore systematic bias between sessions. Relative reproducibility was assessed through an ICC 3.1 two-way mixed model using absolute agreement and the results of a single measurement. The ICC values were reported with a corresponding 95% confidence interval (95% CI), , . The curThe present protocol for measuring lower limb muscle strength had limitations. Firstly, the seamless adjustment of the belt between the harness and the aluminum-board meant that the tester had to measure the angle of the knee (120 degrees) each time and adjust the belt accordingly. This factor  may be one of the largest modifiable contributors to within-subject variation, as the tester needed to be cautious on the exact length of the belt between test and retest. It may be more appropriate to work with three given lengths of the belt, i.e. long, middle and short, adjusted based on the subject’s leg length (trochanter major to the floor). This modified assessment protocol would limit the influence of belt length as a source of within- subject variation. Secondly, the study lacked between-tester data on the method. These data could be relevant for further validation. On the other hand, we would not expect any significant difference between testers as the present method is objective, straightforward and well described. Finally, the present method did not dictate another recording if the last obtained strength value was the highest in the present session. Continuing to record additional values until a “ceiling” had been reached could have decreased the within-subject variation. A strength of the present study was the use of both relative and several absolute statistics. This transparency in reporting should help the reader to better judge the reproducibility and provide basis for comparisons with other studies.In summary, the novel approach using the WBB for measuring isometric lower limb strength in older adults showed a high relative reproducibility, an acceptable absolute reproducibility and a good concurrent validity. The results encourage further research using the WBB for muscle strength testing in different study-populations.S1 Dataset(XLSX)Click here for additional data file.


Accurate real-time monitoring systems of influenza outbreaks help public health officials make informed decisions that may help save lives. We show that information extracted from cloud-based electronic health records databases, in combination with machine learning techniques and historical epidemiological information, have the potential to accurately and reliably provide near real-time regional estimates of flu outbreaks in the United States. Influenza is a leading cause of death in the United States (US), where up to 50,000 are killed each year by influenza-like illnesses (ILI)The US Centers for Disease Control and Prevention (CDC) monitors ILI in the US by gathering information from physicians’ reports about patients with ILI seeking medical attentionMany attempts have been made to design methods capable of providing real-time estimates of ILI activity in the US by leveraging Internet-based data sources that could potentially measure ILI in an indirect manner45678910131415161716170, and such estimates tend to differ from region to region in the USNational-level flu estimates are hard to translate into actionable information that enable local health officials to make better decisions during a surge in clinical visits, for exampleretrospectively the high correlation between aggregated data obtained from electronic health records (EHR) and flu syndromic surveillance systems2425Diverse studies have shown Here, we demonstrate EHR data collected and distributed in near real-time by an electronic health records and cloud services company, athenahealth, combined with historical patterns of flu activity using a suitable machine learning algorithm, can accurately track real-time influenza activity (as reported by the CDC), at the regional scale in the United States. Additionally, we show that the signal to noise ratio in this data source is high. EHR data provides us with an “early count” of ILI activity in much the same way as exit polls enable us to forecast election results. We build a machine learning model that optimally exploits the data by building a system as timely as GFT used to be, yet as stable and reliable as CDC validated data sources. Although our model is capable of forecasting influenza levels weeks into the future, we decided to present here the real-time monitoring of ILI. We name our model ARES, which stands for AutoRegressive Electronic health record Support vector machine.http://www.athenahealth.com). Its electronic health records, medical billing, and care coordination services are organized around a single cloud network, allowing for the collection of unique insights related to patient-provider encounter data for more than 72,000 healthcare providers in medical practices and health systems nationwide. This database includes claims data for over 64 million lives and electronic health records for over 23 million lives.Athenahealth is a provider of cloud-based services and mobile applications for medical groups and health systems , though they also see patients in the following settings: inpatient hospital (11.3%), outpatient hospital (6.6%), nursing facility (1.5%), emergency room (1.4%), and other (5.8%). The age statistics for the athenahealth network are as follows: younger than 15 years old (15%), 15 to 24 years (6%), 25 to 44 years (14%), 45 to 64 years (29%), and 65 years or older (36%). The place of service and age distribution statistics are close to the statistics reported by the CDC’s National Ambulatory Medical Care Survey (NAMCS). This suggests that medical visits to athenahealth’s medical provider network provide a good representation of ambulatory care, including flu activity, in the US.There are two main differences to note between the athenahealth and NAMCS statistics. First, providers in the athenahealth network see patients that are slightly older than average, though re-weighting the data change the athenahealth ILI rates very little. Second, emergency departments are underrepresented in the athenahealth network, causing the raw athenahealth %ILI estimates to be consistently lower than the values reported by the CDC. This indicates that the athenahealth data could be adapted to build specialized models that mimic ILI rates.Flu Vaccine Visit Count, which accounts for the number of visits where a flu vaccine was administered; Flu Visit Count, the number of visits where the patient had a flu diagnosis; ILI Visit Count, the number of visits where the patient had either a flu diagnosis or a fever diagnosis with an accompanying sore throat or cough diagnosis; and Unspecified Viral or ILI Visit Count, the number of visits where the patient had either an unspecified viral diagnosis, a flu diagnosis, or a fever diagnosis with an accompanying sore throat or cough diagnosis. It is important to note that Unspecified Viral or ILI Visit Count contains ILI Visit Count, which itself contains Flu Visit Count. InILI Visit Count was used as an independent variable.To develop these models, we used the following list of athenahealth variables as input: gis.cdc.gov/grasp/fluview/fluportaldashboard.html), for the same time period, to use as both a comparator as well as to provide historical independent variables for our models. We note that using weekly information from reports published by the CDC as a gold standard for US national and regional influenza activity may have limitations. An evaluation of the CDC’s data collection approach has been published and improvements suggested inWe also obtained the national and regional ILI weekly values from the CDC (http://www.google.org/flutrends). All of the data used was downloaded on Oct 15, 2015 and all of the experiments were conducted in Python 2.7.We obtained historical GFT data for the same time period to use as a comparison from the Google Flu Trends website , (t-1) and (t). We also incorporated (autoregressive) historical information from the unweighted percentage ILI estimates obtained from the CDC for weeks (t-2) and (t-1). The variable capturing “flu vaccine visit counts” was not used in the models since it showed no significant correlation with the historical CDC-reported %ILI.We built a collection of regional models that dynamically recalibrate every week in order to optimally include all of the available data up to the week of flu estimation. This dynamic recalibration is inspired by data assimilation techniques used in industrial and financial time series forecastingOur ARES models map the variables described above into a %ILI real-time estimate, using a support vector machine (SVM) model(t-2) and (t-1), and (b) for the dynamically trained linear model: the value of athenahealth’s ILI value at time t.For comparison purposes, we produced real-time estimates using two baseline methodologies: (a) a dynamically-trained autoregressive model that only used historical CDC information, called AR(2) throughout the paper; and (b) a dynamically-trained linear model that used athenahealth’s %ILI information onto CDC’s ILI, as introduced inThe training period for our first flu estimate consisted of data from 6/28/2009 through 1/1/2012 for all the implemented models. This choice was made so that our training period was at least two years in length as inOur first out-of-sample real-time estimate of ILI was produced for the week of 1/8/2012. Time series of real-time (out-of-sample) estimates using ARES, the AR(2) model, and the linear model, were generated up to and including the week of June 28, 2015. In order to understand the predictive power of each of the independent variables to estimate CDC’s ILI, we plotted the values of the coefficient associated with each variable in the  linear models as a function of time. These values are presented in the multiple heatmaps of In this study we have shown that EHR data in combination with historical patterns of flu activity and a robust dynamical machine learning algorithm, are capable of accurately predicting real-time influenza activity at the national and regional scales in the US.nationally was established using a dynamically-trained linear model inviral visits during the week of the estimates typically improve results.While the ability of athenahealth’s ILI data to predict CDC’s ILI Flu estimates using ARES lead to 2–3 fold error reductions in the national and regional point estimates, when compared to the dynamic linear model introduced inModels that use only historical information to predict future ILI typically show high Pearson correlation and not very large (RMSE) error values when compared with CDC’s ILI; however, they consistently show lags of 1 to 2 weeks with respect to the observed CDC’s ILI values, making them systematically inaccurate. This happens to our baseline AR(2) model implementation across regions, as shown in Specifically, using ARES improved Pearson correlation (from 0.958 to 0.996) and reduced the error (RMSE) three-fold for the national level when compared to the autoregressive, AR(2), model. Pearson correlation across regions was generally improved, with the largest improvement in region 3 (from 0.932 to 0.987) and the mildest taking place in region 2 (from 0.96 to 0.978). The average error (RMSE) generally improved, with the greatest performance in region 4, where more than a two-fold reduction was achieved, and the mildest reduction in region 9, where a 20% reduction in error was achieved.The only region where the combination of historical CDC data and EHR data did not lead to improvements when compared to the AR(2) model was region 7, where correlations went from 0.958 to 0.938, and the average error (RMSE) went from 0.4 to 0.51 (%ILI). This may be explained by the fact that athenahealth manages very few facilities in the Midwestern United States.ARES correctly estimated the timing and magnitude of the national peak week of the 3 flu seasons. For 6 out of the 10 regions, ARES correctly estimated the timing of the peak week at least 2 (out of the 3) flu seasons; for regions 2 and 9 ARES only estimated the timing of 1 peak correctly, and for regions 7 and 10, ARES did not capture this feature. When ARES did not get the timing correctly, the estimated peak happened 1.4 weeks off, on average. The peak values were estimated with 0.148% error on the national level, and with 0.445% error on average for the 10 regions.While the historical predictions of the now discontinued Google Flu Trends system have been shown to contain multiple inconsistencies1314151617revised version of historical CDC ILI reports to dynamically train all of our models. While this revised information was not technically available at the time the flu estimates were produced (it was probably available a week or two later), our main point in this work is to show the methodological advantages of combining historical data with EHR data to improve flu estimates. This goal is achieved since all models (including the baseline ones) were trained with the same revised data. However, our comparison with the historical GFT values may not be strictly fair. It is important to highlight that this decision was not taken lightly. Our experience training near real-time flu prediction models17revised historical CDC reports to train them, as opposed to using only the available ones (revised and unrevised) at the time. See the differences between Table 1 and We would like to note that in this study we used the http://www.healthmap.org/flutrends/. In this live implementation, the hyperparameters of the SVM linear model are chosen dynamically with 10 fold cross validation. The predictions with dynamic hyperparameters have historically led to similar performance to the flu estimates presented in this study, perhaps with slight improvements.The discrepancies between the flu estimates using ARES and the observed CDC values, as captured by Pearson correlation and RMSE values, are comparable to those shown inFinally, ARES avoids many of the limitations faced by most non-traditional digital disease surveillance systems, such as a lack of specificity, while providing the main advantage of non-traditional systems, timeliness. The most limiting aspect of ARES is that it relies on electronic health records, data that are not generally publically available. At the time of the writing of this report, athenahealth data is provided to several groups of flu researchers around the country but is not publically available. Future work includes integrating ARES into a network model and testing the accuracy of ARES at the state and city levels, in other countries, as well as on other communicable diseases.We have shown that EHR data in combination with historical patterns of flu activity and a robust dynamical machine learning algorithm provide a novel and promising way of monitoring infectious diseases at the national and local level. Our methodology provides timely flu estimates with the accuracy and specificity of sentinel systems like the CDC’s ILI surveillance network. This demonstrates the value of cloud-based electronic health records databases for public health surveillance at the local level.How to cite this article: Santillana, M. et al. Cloud-based Electronic Health Records for Real-time, Region-specific Influenza Surveillance. Sci. Rep.6, 25732; doi: 10.1038/srep25732 (2016).


Odds of survival were greatest when first Ebola virus–positive blood sample collected had low viral load. t), a surrogate for viral load, in first Ebola virus–positive blood sample tested. Of these patients, 151 were alive when detected and had reported healthcare facility admission dates and Ct values available. Time from symptom onset to healthcare facility admission was not associated with survival, but viral load in the first Ebola virus–positive blood sample was inversely associated with survival: 52 (87%) of 60 patients with a Ct of >24 survived and 20 (22%) of 91 with a Ct of <24 survived. Ct values may be useful for clinicians making treatment decisions or managing patient or family expectations.To determine whether 2 readily available indicators predicted survival among patients with Ebola virus disease in Sierra Leone, we evaluated information for 216 of the 227 patients in Bo District during a 4-month period. The indicators were time from symptom onset to healthcare facility admission and quantitative real-time reverse transcription PCR cycle threshold (C Zaire ebolavirus) disease (EVD) in West Africa began in eastern Guinea in December 2013 (The epidemic of Ebola virus (t), which can serve as an approximation of viral load, at the time of EVD diagnosis. Early treatment, which is made possible by early admission, is thought to improve chances of survival  the total elapsed time from reported symptom onset to healthcare facility admission and 2) cycle threshold . During the first 2 months of the study period, the isolation unit did not consistently provide supportive care, but during the second 2 months, after the unit was reorganized by a new management team, the unit did provide such care. Throughout the 4-month study period, patient blood samples were transferred from this isolation unit to the field diagnostic laboratory, located a few kilometers away.Patients with confirmed EVD were transferred to an ETU managed by Médecins Sans Frontières. In the ETU, located in the same compound as the field laboratory, the patients received care for EVD , as well as presumptive care for other diseases, nutritional support, and psychosocial counseling  demographic information and symptom onset dates from the case investigation forms; 2) admission dates and death reports from the government hospital isolation unit; 3) ETU admission dates and patient outcomes ; 4) EVD diagnostic test results; and 5) confirmation of deaths from the district burial team, which buried the bodies of deceased EVD-positive patients.https://epiinfovhf.codeplex.com/). In the event of missing or conflicting information, we requested verification or additional information from the original sources. For each infected person, we compiled symptom onset date; healthcare facility admission dates; outcome type and date; patient age, sex, and place of residence; and laboratory test results. To ensure a complete linkage and to identify persons with duplicate records, we reviewed all information for errors.These sources routinely reported this information to the Bo District surveillance team, which maintained a database by using the Epi Info Viral Hemorrhagic Fever application  all EVD patients admitted to any healthcare facility (primary cohort), 2) only patients admitted to the ETU (ETU subgroup), and 3) patients admitted to the isolation unit during the last 2 months of the assessment (November 16–January 10) when patients were consistently receiving care in the unit .t is defined as the number of cycles of RNA replication that have occurred when the Ebola virus–specific RNA signal is detected. A total of 40 cycles of replication are run for a given specimen; if no RNA signal is detected and the β-2-microglobin control result is positive, the test result is negative. Therefore, the lower the Ct for a positive specimen, the higher the relative quantity of virus.The field laboratory, operated by the US Centers for Disease Control and Prevention (CDC), tested persons suspected of having EVD by using nucleoprotein (NP) and viral protein (VP) 40 quantitative real-time reverse transcription PCRs and a β-2-microglobin control. These tests detect Ebola viral RNA in blood specimens . The association between Ct and 50% tissue culture infective doses per milliliter (TCID50/mL) is provided in the CDC document Ebola Virus VP40 Real-Time RT-PCR Assay . We also excluded patients for whom Ct values or admission dates were not available.Because it is unknown whether Ct at first test, and average number of days from symptom onset to healthcare facility admission.We calculated the case-fatality proportion for all patients in Bo District for whom outcome was known, who were admitted to a healthcare facility, and who were admitted to the ETU. We stratified the primary cohort and the 2 subgroups by patient outcome and described those who survived and those who died in terms of sex, average age, average Ct values for the Ebola patients and created a scatterplot with a LOESS  curve to serve as a graphical representation of patient survival by Ct. We did this with the LOESS function in R (https://www.r-project.org/) by using the default span of 0.75 and degree of 2. We determined the Ct that was most accurate when used as a dichotomous predictor for survival. We also categorized Ct into 3 levels according to visual inspection of the relationship between Ct and survival.We examined the distribution of Ct , and days from symptom onset to admission to a healthcare facility . We ran 3 multivariable logistic regression models in R (1 for each group) that included sex, age (continuous variable), Ct , and days from reported symptom onset to admission to a healthcare facility. We used the Pearson correlation coefficient to examine collinearity between time from symptom onset to healthcare facility admission and Ct.We ran unadjusted logistic regression analyses in R to determine if the following covariates are associated with patient survival: sex, age , Ct values were missing for 2, and admission dates were missing for 5. The primary cohort comprised the remaining 151 patients. Although 123 patients were admitted to the ETU, admission dates were known for only 99; the ETU subgroup comprised these 99 patients. Dates of admission to the isolation ward during the final 2 months of the study period (when treatment was consistently provided) were known for another 68 patients; the final 2 months subgroup comprised these 68 patients  could be confirmed for 216 patients, but outcome information was missing for 11. Of the 216 patients with outcome data, 164 were detected and admitted to a healthcare facility, but the other 52 died in the community before being detected. Of the 164 patients, 6 died before blood could be collected for confirmatory testing, Cpatients .Outcome was known for 216 persons, of whom 142 (66%) died. Among the 164 persons admitted to a healthcare facility, 90 (55%) died. Among the 123 persons admitted to the ETU, 49 (40%) died . All survivors were ultimately discharged from the ETU.t for the survivors in the 3 groups was in the upper 20s and the mean Ct for the deceased was in the low 20s. The mean number of days from symptom onset to healthcare facility admission was nearly same for those who survived and those who died , the ETU subgroup (49%), and the final 2 months subgroup (50%) died. Of the 151 patients in the primary cohort, 90 (60%) were female; of these, 47 (52%) died. The 2 subgroups had similar proportions. The mean age (in years) of survivors in each of the 3 groups was low- to mid-20s, and the mean age for those who died was low- to mid-30s. The mean Cwho died .t values in the low- to mid-20s  of 91 with a Ct of <24 survived. Of all 72 survivors, Ct was >24 for 52 (72%); of the 79 who died, Ct was <24 for 71 (90%).The average chance of survival among patients in the primary cohort showed a sharp increase for those with C mid-20s . Of notet —as both a continuous and a categorical variable—is strongly associated with survival . Analysis results for the 2 subgroups were similar  (<10 days (145 of 151 patients), the magnitude of the association was greatly diminished . The Pearson correlation test between the time from symptom onset to admission to a healthcare facility and Ct yielded a small but statistically significant relationship . Ct values were slightly higher for patients for whom time from symptom onset to admission was longer than for those for whom this time was shorter   and prod shorter .The overall observed case-fatality proportion of 66% in this assessment is higher than some other case-fatality estimates for this epidemic but less than those reported for many previous outbreaks of EVD  at time of detection were more likely to die than were those with higher Ct values. This finding was consistent across patients regardless of time from symptom onset to admission to any healthcare facility (Similar to the results of some analyses of ETU patients (facility , suggestt might also be useful as a prognostic tool is increasing. This finding was first reported >10 years ago after an outbreak of Sudan virus infection (To date, quantitative real-time reverse transcription PCR has been used in the Ebola response to distinguish EVD cases from non-EVD cases and to determine when a convalescing patient can be released from an ETU. However, evidence that Ct as a prognostic indicator could have several benefits for clinicians. First, it could guide patient triage and help clinical staff determine the best treatment course for the gravely ill, particularly when intravenous fluids or advanced supportive therapies are in short supply. Such therapies might help improve patient outcomes when used on those who have higher viral loads at the time of initial care and those who are more ill than their peers. Second, if a patient has mild symptoms but a high viral load, clinicians can be prepared for the patient’s condition to deteriorate quickly. Third, healthcare staff also can use this information to manage patient and family expectations regarding probable outcomes.Using Ct values used for this assessment are specific to the CDC laboratory equipment and VP40 assay used. Use of other laboratory equipment, procedures, or assays could yield different results, thus affecting the optimal Ct. Standardization of equipment, procedures, and assays would facilitate the use of Ct as a prognostic indicator. Last, determining an optimal Ct for prognosis probably depends on specific patient characteristics and the effectiveness of care provided; the strength of the association between Ct and survival could be confounded by these or other unmeasured factors.Limitations of this study include probable misreporting of patient age and symptom onset date, as well as the relatively small number of cases. Although we included all known deaths among persons with EVD to calculate the overall case-fatality proportion, the 58 persons who died before or shortly after detection were excluded from the analysis because comparable blood samples or reliable symptom onset dates were not available. This exclusion is potentially a source of bias if the time from symptom onset to death for these 58 persons is longer than the time from onset to admission for the remaining patients . In addit of >24 in the first Ebola virus–positive sample was a strong predictor of survival among persons who were alive when detected by the surveillance system. In this population, the time from reported symptom onset to healthcare facility admission was not associated with survival. Additional studies are needed to validate these findings and to continue to explore how Ct values can be combined with other biomarkers (In summary, the case-fatality proportions found by this study were higher than estimates that do not include all deaths of known infected persons or that are limited to ETU patients only. A CTechnical Appendix. Notes about persons excluded from analyses of prognostic indicators for Ebola virus disease survival, Sierra Leone.


Cartilage remodeling is currently among the most popular topics in osteoarthritis research. Remodeling includes removal of the existing cartilage and replacement by neo-cartilage. As a loss of balance between removal and replacement of articular cartilage develops , joints will begin to degrade. In the last few years, significant progress in molecular understanding of the cartilage remodeling process has been made. In this brief review, we focus on the discussion of some current “controversial” observations in articular cartilage degeneration: (1) the biological effect of transforming growth factor-beta 1 on developing and mature articular cartilages, (2) the question of whether aggrecanase 1 (ADAMTS4) and aggrecanase 2 (ADAMTS5) are key enzymes in articular cartilage destruction, and (3) chondrocytes versus chondron in the development of osteoarthritis. It is hoped that continued discussion and investigation will follow to better clarify these topics. Clarification will be critical for those in search of novel therapeutic targets for the treatment of osteoarthritis. Cartilage remodeling is a continuous process in which the existing cartilage is removed (or degraded) and replaced by new cartilage (regenerated). The balance between degradation and regeneration has been considered so critical that tipping the scale toward degradation results in osteoarthritis (OA). Therefore, an understanding of the molecular basis of the degradation and regeneration processes will undoubtedly provide valuable information for those in search of novel therapeutic protocols for the treatment of OA. In this review article, we focus on several recent discoveries on the topic of articular cartilage degeneration.et al. indicates that the half-life of collagens in human mature cartilages is 117 years1. The long half-life of the collagens indicates a slow turnover of the collagens in the cartilages. However, the half-life of the aggrecan (the large monomer) turnover is about 3.4 years2. This suggests that the rate of the turnover may be different in the different parts of the extracellular matrix. For example, the rate of the turnover is high in the aggrecan-rich pericellular matrix of chondrocytes and the rate of the turnover is low in the collagen-rich interterritorial and territorial matrices in articular cartilages3.With regard to the remodeling of the extracellular matrix, mature articular cartilages are, in general, considered relatively quiescent tissues. For example, a study by Verzijl4.Although this review focuses on articular cartilages, we have to point out that OA is currently considered the consequence of the whole joint failure. In addition to articular cartilages, the subchondral bone, peri-articular cartilage, synovial membrane, ligaments, and menisci contribute to the development of OAIs transforming growth factor-beta 1 (TGF-β1) a culprit or protector in the development of OA? Currently, numerous pharmaceutical companies are considering the use of TGF-β1 as a stimulant to repair damaged articular cartilage for the treatment of OA. Is this the correct choice?in vitro andex vivo experiments in which TGF-β1 can stimulate chondrocytes to synthesize and release proteoglycans and type II collagens6. In addition, three independent mouse genetic studies9 demonstrate that Tgf-β1 is required for the formation of articular cartilage at early stages of development in mice. Without Tgf-β1, articular cartilage is not formed properly, eventually an immature joint becomes an OA-like joint in mice. Moreover, a human genetic study reports that a two-nucleotide deletion, 741-742del AT (nonsense mutation), inSMAD-3 causes early-onset OA in a human family10. All of the aforementioned results support the argument that TGF-β1 is a protector against the development of OA. Unfortunately, the situation is not that simple. Numerous other independent investigations suggest that TGF-β1 may, in fact, be a factor in joint destruction. First, studies with animal models, by Itayemet al., suggest that intra-articular injections of TGF-β1 into adult rat knee joints cause early onset of OA12. Second, a human genetic study reports that a nucleotide change, 859C>T or 782C>T inSMAD-3, increases the level of TGF-β1 and activity of the TGF-β1 signaling pathway in human families is associated with early-onset OA10. This is in agreement with the observation from two other studies indicating that the level of TGF-β1 is significantly higher in human OA tissues than in healthy articular cartilages14. Third, we found increases in the expression of Tgf-β1 and of p-Smad2/3 in articular chondrocytes of knee joints in mouse models of OA15. The increased expression of p-Smad2/3 was associated with elevated expression of a serine protease, high-temperature requirement A1 (HtrA1), in the chondrocyte. HTRA1 is capable of degrading extracellular matrix molecules, particularly most pericellular components of articular chondrocytes16. Another independent research group also demonstrates that TGF-β1 induces HTRA1 in human primary chondrocytes17. Fourth, we determined whether the removal of Tgf-β type II receptor (Tgfbr2) from the articular cartilage of adult knee joints could attenuate the OA progression. We deletedTgfbr2 in the articular cartilage of adult mouse knee joints and then subjected the mice to destabilization of the medial meniscus (DMM). We found a significant disparity in the progression of articular cartilage degeneration in knee joints between mice with or withoutTgfbr2 at 8 and 16 weeks following the surgery. The progression toward OA was significantly (P <0.05) delayed inTgfbr2−/− mice.TGF-β1 has been considered an anabolic factor to articular chondrocytes, based largely on results fromet al. suggests that a high level of TGF-β1 does more harm than good to the tendon18. One study by Bakkeret al. reports that the constitutive overexpression of active TGF-β1 in adult mouse knee joints results in OA associated with an increase in the production of proteoglycans in articular cartilage, hyperplasia of synovium, and chondro-osteophyte formation19. A study by Zhenet al. demonstrates that inhibition of TGF-β1 signaling in mesenchymal stem cells of subchondral bone delays the development of OA in adult mice20.Several studies also indicate that the increase in the amount of TGF-β1 in other joint tissues has detrimental effects on adult joints. A study by MaedaTgfbr2 suggest that the TGF-β1 dose-dependent manner may not be the case. Another plausible explanation is that effective TGF-β1 signaling acts in a developmental stage-dependent manner. In this scenario, TGF-β1 is required for the development of articular cartilage; however, once a joint is formed, TGF-β1 is no longer needed. In any case, induction of TGF-β1 in an adult joint causes articular cartilage degeneration. Therefore, inhibition activity of TGF-β1, not application of TGF-β1, may be considered in treatment of OA in mature joints.How can this “conflicting” role of TGF-β1 in the pathogenesis of OA be explained? One plausible explanation is that effective TGF-β1 signaling acts in a dose-dependent manner. In this scenario, an appropriate level of TGF-β1 is required for the development and maintenance of articular cartilages. Therefore, TGF-β1 below or above this level results in articular cartilage degeneration. However, results from our study with mice without22. Both of these aggrecanases degrade aggrecans (proteoglycans). This indicates that both aggrecanases may be ideal therapeutic targets in the development of disease-modifying OA drugs. In fact, two independent research groups used mouse gene-targeting techniques to delete ADAMTS-4 or ADAMTS-5. One group found that the deletion of ADAMTS-5 could protect aggrecan from being degraded in a mouse model of inflammatory arthritis. With regard to the development of OA, it is not clear whether both aggrecanases are involved in cartilage destruction23. The results from another group demonstrated that the removal of ADAMTS-5 in mice could significantly delay the progressive process of articular cartilage degeneration at 4–8 weeks following the DMM surgery24. This suggests that ADAMTS-5 may play a role in early stages of OA development. However, lack of evidence indicating elevated expression of ADAMTS-5 at early stages of articular cartilage degeneration in any one of the existing mouse models of OA raises a question as to how important a role this enzyme has in the development of OA.Proteoglycans are the basic elements of articular cartilage and are indispensable in the ability of articular cartilage to resist compressive pressure. Thus, much of the effort in the OA research field is focused on the search for an enzyme, or enzymes, that degrades proteoglycan. In 1999, two enzymes, ADAMTS-4 and ADAMTS-5, were clonedSox925. The elevated expression of ADAMTS-5 is associated with the disappearance of aggrecans. Surprisingly, there is no progression of articular cartilage degeneration in this model. This is contrary to our current understanding that aggrecans are indispensable for articular cartilage health. Consistent with this observation, another independent investigation indicates that an increase in the expression of bone morphogenetic protein 2 (Bmp2) elevates levels of the neo-epitope, VDIPEN341, of aggrecan in articular cartilage without inducing an acceleration of cartilage degeneration in mice. Furthermore, Davidsonet al. find that the increased expression ofBmp2 does not exacerbate the degenerative condition of articular cartilage that has been induced by the DMM in mouse knee joints26.More importantly, a recent study indicates that the expression of ADAMTS-5 is increased in the articular cartilage of knee joints in adult mice because of inactivation ofet al. demonstrates that articular cartilage degradation is completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation but irreversible after induction of metalloprotenase (MMP)-mediated aggrecan and collagen type II degradation27. This study suggests that the aggrecanases may be involved with the reversible processes of cartilage degradation (or extracellular matrix turnover) but MMPs cause the irreversible degeneration of articular cartilages.One plausible explanation for the aforementioned observation is that the loss of proteoglycans alone may not be sufficient to initiate or accelerate articular cartilage degeneration. Instead, the degradation of both proteoglycans and type II collagen may be required in the development of OA. Interestingly, a study by Karsdal28. Thus, it will be important to understand whether aggrecanases play roles in the development of OA through other joint tissues.There is evidence that the removal of ADAMTS-5 may protect joints against OA by stabilizing subchondral boneWe discuss the aggrecanases in this brief review. However, other enzymes, such as MMPs, elastase, and cathepsins, also play important roles in the pathogenesis of OA.in vitro experiments in cartilage research and repair. They have allowed a wealth of information to be obtained about the genetic regulation of chondrocyte function, activation of signaling pathways, and gene expression profiles in chondrocyte response to chemical or mechanical stimulation. Many investigators use chondrocytesin vitro to study physiological and pathophysiological events while mimickingin vivo biological conditions. In particular, researchers almost exclusively use primary chondrocytes as a resource to regenerate functional articular cartilage. Regarding this method, however, a question remains: are primary chondrocytes alone adequate for investigating the role of chondrocytes in OA development and articular cartilage repair?Primary chondrocytes and chondrocyte cell lines are the primary tools for35. This concept was proposed by Benninghoff in 1925. About 40 years later, Szirmai further evaluated the structure of the chondron by a more systematic analysis. At that time, however, the chondron was not widely recognized as a functional unit. Some 20 years later, C.A. Poole’s research group completed additional experiments to physically isolate chondrons from cartilage and showed that chondrons are true anatomic and functional entities. Chondrons consist of chondrocytes, the pericellular matrix, and a capsule surrounding the pericellular matrix. The pericellular matrix contains laminin, fibronectin, biglycan, decrin, fibromodulin, matrilin 3, and cartilage oligo matrix protein (COMP). The pericellular capsule is composed mostly of type VI and IX collagen and proteoglycans. The capsule and the pericellular matrix separate chondrocytes from the adjacent interterritorial or territorial matrices containing type II collagen. Clearly, under normal conditions, type II collagen is not exposed to chondrocytes. It is conceivable that disruption of the pericellular matrix exposes chondrocytes to type II collagen and can alter the metabolic events in chondrocytes, eventually leading to cartilage destruction. In fact, results from human and mouse genetic studies indicate the significant role of the pericellular matrix in protecting articular cartilage against the development of OA. For example, the deficiency of one or a combination of two components of the pericellular matrix, such as type VI collagen, type IX collagen, matrilin 3, decrin, biglycan, and fibromodulin, results in early onset of OA in mice40. In human genetic studies, mutations in type IX collagen and COMP are associated with OA46.Chondrocytes and their pericellular matrix are considered to be the primary structural and functional units, termed chondrons, of articular cartilage47. Very interestingly, the significant role of the pericellular matrix of a cell is also demonstrated in neural tissue. A study by Gogollaet al. points out that the perineuronal net (pericellular matrix of neurons) in the amygdala plays a significant role in protecting the neurons from the loss of “fear memory”48. The authors also find that functional and structural changes of sensory systems are caused by the absence of a perineuronal net in the visual cortex. The significant role of the pericellular matrix of a neural cell in the brain coincides with its perceived role within the chondron. If that is the case, maintaining the integrity of the pericellular matrix will be one of the key issues in protection against OA. We believe that more attention should be directed toward the pericellular matrix of chondrocytes for the identification of novel biomarkers and therapeutic targets for OA.In 1998, a study by Lee and Loeser provided evidence that the pericellular matrix of chondrocytes could play critical roles in the maintenance of normal metabolic activities of chondrocytes in articular cartilage and that the disruption of the pericellular matrix is associated with articular cartilage degeneration49.Data from a very recent investigation provide more evidence that chondrons are not only the functional unit in the maintenance of articular cartilage homeostasis but also the basic elements in the regeneration of articular cartilage. Chondrons derived from adult articular cartilage are more efficient than chondrocytes in the regeneration of articular cartilage. This information is particularly critical for the articular cartilage repair fieldin vitro50. It is well known that chondrocyte dedifferentiation is one of the major obstacles in cartilage tissue repair. A study by Zelenskiet al. shows that the deletion of type VI collagen alters the mechanical properties of the pericellular matrix of chondrocytes51. This, in turn, increases the extent of cell swelling and osmotically induced transient receptor potential cation channel subfamily V member 4 (TRPV4) signaling in an age-dependent manner. These findings suggest that alterations in pericellular matrix properties can influence mechanotransduction via TRPV4 or other ion channels, which eventually leads to articular cartilage destruction.Collagen type VI is one of the major components of the capsule of the pericellular matrix. Results from one very recent study indicate that soluble collagen type VI can be a stimulant for chondrocyte proliferation. The soluble collagen type VI may also prevent proliferating chondrocytes from being dedifferentiatedIn summary, chondrons, instead of primary chondrocytes or chondrocyte cell lines, may be a more appropriate choice for investigating the biological functions and effects of chondrocytes in the development of OA and cartilage repair. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


We found a statistically significant interaction between distance to nearest power line and domestic radon regarding risk of childhood leukemia (p = 0.01) when using the median radon level as cut-off point but not when using the 75th percentile (p = 0.90). We found no evidence of an interaction between distance to nearest power line and traffic-related air pollution (p = 0.73). We found almost no change in the estimated association between distance to power line and risk of childhood leukemia when adjusting for socioeconomic status of the municipality, urbanization, maternal age, birth order, domestic radon and traffic-related air pollution. The statistically significant interaction between distance to nearest power line and domestic radon was based on few exposed cases and controls and sensitive to the choice of exposure categorization and might, therefore, be due to chance.We investigated whether there is an interaction between distance from residence at birth to nearest power line and domestic radon and traffic-related air pollution, respectively, in relation to childhood leukemia risk. Further, we investigated whether adjusting for potential confounders alters the association between distance to nearest power line and childhood leukemia. We included 1024 cases aged <15, diagnosed with leukemia during 1968–1991, from the Danish Cancer Registry and 2048 controls randomly selected from the Danish childhood population and individually matched by gender and year of birth. We used geographical information systems to determine the distance between residence at birth and the nearest 132–400 kV overhead power line. Concentrations of domestic radon and traffic-related air pollution (NO In 2001 a working group at the International Agency for Research on Cancer (IARC) classified exposure to extremely low-frequency magnetic fields (ELF-MF) as ‘possibly carcinogenic to humans’ and exposure to extremely low-frequency electric fields were grouped as ‘not classifiable as to its carcinogenicity to humans’ In 2005 a large-scale case-control study from Great Britain The lack of an accepted biological explanation for the observed association between ELF-MF and childhood leukemia have raised doubt about the causality of the association. Several mechanisms of how extremely low-frequency electric and magnetic fields can cause cancer have, however, been proposed The major objective of our study was, therefore, to investigate possible interactions between distance from residence to nearest power line and domestic radon and traffic-related air pollution, respectively, in relation to childhood leukemia risk; to our knowledge this has not previously been investigated. Additionally, we investigated whether adjusting for potential confounding factors alters the association between distance from residence at birth to nearest power line and childhood leukemia. We adjusted for factors observed to be associated with childhood leukemia risk in other studies, specifically socioeconomic status of the municipality, urbanization, maternal age, birth order, domestic radon and traffic-related air pollution The Danish Data Protection Agency (2007-41-0239) approved the study. In accordance with Danish law written consent was not obtained as the study was entirely register-based and did not involve biological samples from, or contact with study participants.We identified all children in Denmark diagnosed with leukemia  before the age of 15 years during the period 1968–1991 (inclusively) through the virtually complete nationwide Danish Cancer Registry We identified 1024 cases diagnosed with leukemia and selected 2048 matched controls, constituting a population of 3072 children .The addresses of cases and controls at birth were obtained from the Danish Civil Registration System and we identified geographical coordinates by linkage to the Danish Address Database. The geographical coordinates refer to the front door of each residence (for apartments the main entrance door) and are precise within a few meters. We collected data on existing and historical 132–400 kV overhead power lines with alternating current from the seven Danish transmission companies. The mapped grid includes 4336 km of current and historical power lines . In addition information on the date when the line was put into and out of operation was collected. For the lines for which we only had information on the year of operation (77.5%), we set the date of operation to 31 December and the date when the line was put out of operation was set to 1 January. This restrictive approach was applied to avoid assigning exposure to unexposed individuals. The distance from residence at birth to the nearest power line that existed at the date of birth was calculated in ArcGIS 9.3. We successfully calculated distance for 2797 (91.0%) of the addresses at birth. Distance could not be calculated due to missing information in the address for 100 (9.8%) cases and 175 (8.5%) controls . DistancDomestic radon concentration at address at birth was estimated using a validated regression model constructed to predict radon concentrations in Danish dwellings on the basis of register data including geographical region, soil type, and house characteristics such as type of house, floor, basement, and building materials. Details on the model are given in previous papers Exposure to domestic radon at address at birth was successfully estimated for 2904 (94.5%) addresses. We could not estimate domestic radon exposure for 62 (6.1%) cases and 106 (5.2%) controls .x (nitrogen monoxide (NO) + nitrogen dioxide (NO2)), as an indicator for air pollution from traffic because NOx correlates strongly with other traffic-related pollutants in Danish streets, especially ultrafine PM (Particulate matter): r = 0.93 for total particle number concentration  and r = 0.70 for PM10 (mass of particles with an aerodynamic diameter less than 10 µm) x at the front door of the address at birth was estimated by use of the Operational Street Pollution Model taking into account both the street-level pollution as well as the background pollution We used the sum of nitrogen oxide gasses, NOExposure to traffic-related air pollution at the birth address was successfully estimated for 2942 (95.8%) addresses and could not be estimated for 45 (4.4%) cases and 85 (4.2%) controls .Socioeconomic status of the municipality  has been described in detail previously We had no information on socio-economic status of the municipality for 6 cases and 12 controls, on urbanization for 100 cases and 175 controls, on maternal age for 43 cases and 77 controls and on birth order for 43 cases and 78 controls .Because of the nested case-control design of the study, with equal follow-up time for a case and the matched controls, the rate ratios We analyzed the association between distance to nearest power line and risk of childhood leukemia both with and without adjustment for potential confounders. The potential confounders included socio-economic status of the municipality , urbanization , maternal age at time of birth , birth order , domestic radon  and traffic-related air pollution .x, we dichotomized domestic radon and traffic-related air pollution using the median value as cut-off; in sensitivity analyses we used the 75th percentile as cut-off point. In an additional sensitivity analysis we divided radon and NOx into tertiles in order to investigate a potential dose-response association. All interaction analyses were adjusted for the potential confounders: socioeconomic status of the municipality, urbanization, maternal age at time of birth, birth order and domestic radon and traffic-related air pollution, respectively. From the former study on radon We analyzed possible interactions between distance to power lines and domestic radon and traffic-related air pollution, respectively. Since there is no known biological relevant cut-off point for radon or NODue to small samples in some of the categories in the analysis of the interaction between distance to power line and radon, exact methods for logistic regression were also applied using LogXact. It was only possible to compute models without adjustment for confounding factors.We excluded 139 (13.6%) cases and 248 (12.1%) controls due to missing data on distance to power line or any of the potential confounding factors: domestic radon, traffic-related air pollution, socioeconomic status of the municipality, urbanization, maternal age or birth order. Further, we excluded 6 cases and 179 controls since they no longer had a matched control or case, leaving 879 (85.8%) cases and 1621 (79.2%) controls for the analyses .In 3 was 2.88 (95% CI: 1.01–8.27) compared to children living ≥600 meters from a power line and exposed to domestic radon <42 Bq/m3. Children living 200–599 meters from a power line and being exposed to domestic radon <42 Bq/m3 had a lower risk with an RR of 0.24 (95% CI: 0.07–0.83) compared to children living ≥600 meters from a power lines and exposed to domestic radon <42 Bq/m3. There was no evidence of interaction between distance to nearest power line and traffic-related air pollution (p = 0.73). The sensitivity analysis including domestic radon and traffic-related air pollution with a cut-off at the 75th percentile showed no significant interaction with domestic radon (p = 0.90) or air pollution (p = 0.59) )   and ther = 0.59) . The int7–7.07)) . When ra7–7.07)) . The RR th percentile of radon exposure as cut-off point. We found no evidence of an interaction between distance to nearest power line and traffic-related air pollution. We found almost no change in the estimated association between distance to power lines and risk of childhood leukemia when adjusting for socioeconomic status of the municipality, urbanization, maternal age, birth order, domestic radon and traffic-related air pollution.We found a statistically significant interaction between distance to nearest power line and domestic radon regarding risk of childhood leukemia when using the median radon level as cut-off point but not when using the 75Our study was a register-based case-control study covering the entire population of Denmark. Cases were identified in a virtually complete national cancer registry, and the Central Population Registry provided an excellent basis for an unbiased sampling of controls. The potential for selection bias in our study is, therefore, minimal. We identified around 90% of current as well as historical power lines with ≥132 kV and according to two of the transmission companies  the data on power lines had a precision of 3–5 meters. The geographical coordinates used to identify the front door at the address at birth had a precision of few meters. For apartments the main entrance door was identified, which for the majority of apartments is within 5 meters, however, for some types of apartments a larger deviation may be expected.Distance to nearest power line, domestic radon and air pollution was estimated for the address at birth, although exposure accumulated over all childhood addresses might be more relevant It is a limitation of the study that the analyses of interaction were based on few cases and controls for some of the cells of 3) were correctly classified and 60% of the highest exposures (>100 Bq/m3) were correctly classified x concentration at the front door has been validated by comparing the calculated nitrogen dioxide (NO2) values with measured values at 204 locations in Denmark with an R2 value of 0.82 Exposure to domestic radon and traffic-related air pollution at the birth address were both modeled using successfully validated prediction models which have been applied in previous studies In a previous study, where we included all children diagnosed with leukemia before the age of 15 years during the period 1968–2006, we report a OR of 0.76 (95% CI: 0.40–1.45) for children who lived 0–199 meters from the nearest power line compared with children who lived ≥600 meters away We found a statistically significant interaction between distance from residence to nearest power line and domestic radon but no interaction between distance and traffic-related air pollution. The interaction with radon showed a higher risk of childhood leukemia if living close to power lines and being exposed to high radon concentrations. This is in line with the hypothesis by Fews et al. th percentile was used as cut-off also suggests that the interaction found when using the median as cut-off might be due to chance. Even if the interaction between power lines and radon in relation to childhood leukemia is true, the potential effect in terms of absolute risk would be very small.The interpretation of the observed interaction is not straightforward since it is not only based on a statistically significant higher risk among those exposed to high radon and living close to a power line but also a statistically significant lower risk among those being exposed to low domestic radon and living 200–599 meters from a power line, which is not easy to explain. The observed interaction might be due to chance because of few cases and controls in several cells. The finding of no statistically significant interaction when the 75We found almost no change in the estimated association between distance to power lines and risk of childhood leukemia when adjusting for socioeconomic status of the municipality, urbanization, maternal age, birth order, domestic radon and traffic-related air pollution. This is in line with other studies investigating the association between ELF-MF and childhood leukemia, where the inclusion of potential confounders had no substantial effect on the estimates The statistically significant interaction observed between power lines and radon might be due to chance since numbers were small and the interaction was no longer significant when changing the cut-point for radon exposure. We found no support for an interaction between power lines and traffic-related air pollution and no change in the estimated association between distance to power lines and the risk of childhood leukemia by adjustment for potential confounding factors.Figure S1Number of cases and controls in the study.(TIF)Click here for additional data file.Table S1The joint effects of distance to nearest power line and domestic radon and air pollution, respectively, on leukemia.(DOCX)Click here for additional data file.Table S2The joint effects of distance to nearest power line and domestic radon and air pollution, respectively, on leukemia.(DOCX)Click here for additional data file.Table S3The joint effects of distance to nearest power line and domestic radon on leukemia for the period 1968–1994.(DOCX)Click here for additional data file.Table S4The joint effects of distance to nearest power line and domestic radon on leukemia risk with exact methods and with asymptotic methods.(DOCX)Click here for additional data file.