The seventh sentence of the sixth paragraph in the Discussion section should have cited references 41 and 52 instead of 5 and 52.The correct sentence should read: Another promising discovery has been the finding that curcumin, derived from the dietary spice turmeric, is an effective inhibitor of Aβ aggregation [41], [52].


Resistance in virus-infected stem cell transplant recipients illustrates the need for surveillance. >4 days of oseltamivir therapy. Three of these 4 patients were critically ill. Oseltamivir resistance in 4 (13.3%) of 30 patients who were administered oseltamivir highlights the need for ongoing surveillance of such resistance and further research on optimal antiviral therapy in the immunocompromised.We describe laboratory-confirmed influenza A pandemic (H1N1) 2009 in 17 hospitalized recipients of a hematopoietic stem cell transplant (HSCT)  and in 15 patients with malignancy treated at 6 Australian tertiary centers during winter 2009. Ten (31.3%) patients were admitted to intensive care, and 9 of them were HSCT recipients. All recipients of allogeneic HSCT with infection <100 days posttransplantation or severe graft-versus-host disease were admitted to an intensive care unit. In-hospital mortality rate was 21.9% (7/32). The H275Y neuraminidase mutation, which confers oseltamivir resistance developed in 4 of 7 patients with PCR positive for influenza after  The 5 centers in Melbourne provide most specialist adult hematology services to the state of Victoria (population 5.4 million), and 2 of these centers perform all allogeneic HSCTs for the state. The participating Sydney center is the largest of 2 centers that perform adult allogeneic HSCT for the state of NSW (population 7 million). Approval for this study was obtained from human research ethics committees of each center.>18 years of age hospitalized at 1 of the 5 Melbourne study centers during May 1–August 30, 2009, or hospitalized at the Sydney center during May 1–September 15, 2009; 2) recipient of an HSCT or had an underlying malignancy (hematologic or solid tumor); and 3) had laboratory-confirmed pandemic (H1N1) 2009 virus infection identified by nucleic acid testing (NAT) during their hospital stay. At each center, investigators were directly involved in active surveillance and management of patients with pandemic (H1N1) 2009, allowing cases to be identified. Data were retrospectively abstracted onto standardized case record forms.Patients were included in the study if they had the following characteristics: 1) ,All cases were confirmed in laboratories whose performance is accredited by the National Association of Testing Authorities in Australia. For the Victorian cases, all but 1 case-patient had laboratory confirmation of pandemic (H1N1) 2009 at the Victorian Infectious Diseases Reference Laboratory (VIDRL) with pandemic (H1N1) 2009–specific NAT by using reverse transcriptase–PCR (RT-PCR) as described , and descriptive statistics were summarized with proportional outcomes. Nosocomial acquisition was defined as the development of symptoms attributable to pandemic influenza after 48 hours in the hospital that were not present on admission. Steroid-refractory or grade III–IV graft-versus-host-disease (GVHD) was defined as severe GVHD.,Length of stay was calculated as length of hospital admission or period of confinement after onset of symptoms for those with nosocomial acquisition. Epidemic curves for the 2 states were aligned with epidemic curves of community pandemic influenza activity ascertained from surveillance data  2009, except patient 11, who had multiple myeloma that had been diagnosed 3 years previously. All autologous HSCT recipients had a relapse of malignancy after transplantation, and 7 of these patients were continuing to receive active treatment for malignancy.At the time they sought treatment, all 8 recipients of autologous HSCT were Two patients (26 and 29) who had had recent a diagnosis of malignancy were in remission and continuing on the primary chemotherapy treatment plan. Seven nontransplant recipients had either not shown a response or were deemed by their treating clinicians to be refractory to their current treatments. Patient 17 had an influenza-like illness (ILI) before admission for chemotherapy and autologous HSCT (further details below). Sixteen patients  were receiving corticosteroids before the onset of pandemic (H1N1) 2009 infection. The mean dose in prednisolone equivalents was 40.9 mg/d (range 5.0–156 mg/d).Clinical features at patients’ initial visit coincided with peak activity of pandemic (H1N1) 2009 in the Victorian and NSW communities . PatientMost patients exhibited fever (94%) and cough (91%). Dyspnea, sore throat, and rhinorrhea were reported in 53%, 50%, and 50% of patients, respectively. Sixteen patients (50%) sought treatment within 48 h of symptom onset. Four patients visited the hospital after experiencing prolonged symptoms: for 12 d (patient 4), 14 d (patients 22 and 25), and 21 d (patient 14). Three of these patients had biphasic illnesses with initial prolonged mild upper respiratory tract illnesses and, on examination, had consolidation shown on chest radiograph. Patient 25 had 2 weeks of dyspnea associated with an exacerbation of chronic obstructive pulmonary disease. Patient 16 sought treatment for shock and respiratory failure.<200 cells/mL, and 18 (56%) patients had pneumonia shown on baseline chest radiograph.Diagnosis of pandemic (H1N1) 2009 was confirmed by NAT of bronchial washings of 3 patients . One of them, patient 17, had symptoms of a viral respiratory tract infection for 20 d with negative results for pandemic (H1N1) 2009 virus by RT-PCR of nasopharyngeal swab specimens taken 2 and 16 d before bronchoscopy. All other patients had the diagnosis confirmed on NAT of nasopharyngeal swab samples. Six patients (19%) had a baseline peripheral blood lymphocyte count Thirty patients (94%) were administered oseltamivir therapy. Of these patients, 13 (41%) were administered therapy within 48 hours, and 16 began therapy >48 hours after symptom onset. Duration of illness before receiving oseltamivir was unknown for patient 2. Nine patients received NA inhibitors for >5 days. Three of these patients  had a positive NAT result >5 days after oseltamivir treatment began. Antimicrobial drug treatment was changed to zanamir for patients 1 and 8 after they received initial therapy with oseltamivir (see below). The longest duration of antiviral therapy was 43 d (patient 1). Patients 15 and 31 had uncomplicated infections and were not administered oseltamivir therapy.Staphylococcus aureus bacteremia from an unrelated source. Patient 17, who came to transplant with an ILI, had bronchial washings that were positive for pandemic (H1N1) 2009 by NAT, galactomannan antigen, and Aspergillus spp. by NAT 5 days after transplant. Pulmonary nodules consistent with invasive fungal infection were seen on a high-resolution computed tomography scan. Patient 13 was infected with respiratory syncytial virus, which was detected by multiplex NAT on a nasopharyngeal swab.Four patients had notable co-pathogens identified. Patient 2 had 4 concurrent herpesviridae  detected by NAT in respiratory specimens and blood. Treatment for patient 2 included liposomal amphotericin B and foscarnet. Patient 7 had Ten (31.3%) patients were admitted to intensive care . In eachAll allogeneic HSCT recipients with the following features required admission to intensive care for mechanical ventilation: transplantation within 100 d, severe GVHD, and nosocomial acquisition of pandemic (H1N1) 2009. However, onset of symptoms for patient 1 was day 119 after allogeneic transplantation.Eight of 10 patients admitted to intensive care had evidence of pneumonia on baseline chest radiograph. Patient 1 initially had normal chest radiograph results, despite the acute onset of hypoxia. Patient 24 was transferred to intensive care after 3 days in the hospital, at which point bilateral infiltrates were seen on chest radiograph, and oseltamivir therapy was begun.>1 further positive NAT  after receiving oseltamivir. Five of these patients had a positive NAT after >5 d of oseltamivir therapy. The longest recorded duration of viral shedding during oseltamivir therapy was 28 d (patient 1).Ten patients had repeat NAT testing to determine clearance of viral shedding. Eight had >4 d of oseltamivir therapy. These 4 patients comprised 13.3% of the 30 patients who received oseltamivir. The findings for the 4 patients who were infected with oseltamivir-resistant influenza virus are summarized in The H275Y NA mutation, a substitution known to confer a high level of oseltamivir resistance, was detected in 4 (57%) of 7 patients who had detectable nucleic acid after Patient 12, who survived despite shedding oseltamivir-resistant pandemic (H1N1) 2009 virus, had a biphasic clinical course. She initially stabilized while she was treated with oseltamivir; progressive respiratory failure then developed, which coincided with her recovery from neutropenia before later improvement. The other patient who survived despite shedding oseltamivir-resistant 2009 pandemic (H1N1) virus (patient 20) had an ILI without pneumonia. Of the 2 patients with resistant isolates who died, patient 5 first experienced the apparent resolution of pneumonia but later succumbed to recurrent pneumonia, and patient 1 had persisting pneumonitis despite 36 days of oseltamivir therapy.Patient 24 had multiple positive NATs, none with the H275Y mutant detected. His NAT was positive 4 d after he began oseltamivir therapy, and he died 1 d after his last specimen, a nasopharyngeal swab, was collected.We report a case series of hospitalized cancer patients with influenza A pandemic (H1N1) 2009 virus infection and their outcomes. Patients with hematologic malignancies accounted for 50% of deaths of persons with pandemic influenza in Victoria during the first 3 months of the pandemic  and chronic lymphocytic leukemia (n = 4). There was also a paucity of patients with solid tumors (n = 2). The severity of illness from influenza in patients who have underlying multiple myeloma and chronic lymphocytic leukemia has not previously been widely appreciated, although a high rate of pneumonic illness from influenza has previously been demonstrated at 1 center  2009 has been described in another immunocompromised patient, a renal transplant recipient  2009–specific RT-PCR result after Only 1 of the patients who was in intensive care and had oseltamivir-resistant pandemic (H1N1) 2009 virus survived. The effects of oseltamivir-resistant influenza virus in this patient are uncertain. The deterioration of her condition after initial stabilization may have been related to development of oseltamivir-resistance and persistent influenza viral replication similar to that seen in a recent case report  2009 infection in immunocompromised patients.A high rate of oseltamivir resistance developed in critically ill HSCT recipients, particularly in those who continued to shed pandemic (H1N1) 2009 virus after 4 d of oseltamivir treatment. Consequently, surveillance, infection control measures, and early treatment of those at greatest risk of pandemic (H1N1) 2009 infection may prove more useful than universal chemoprophylaxis during an outbreak with an influenza strain that is not contained in the available influenza vaccine. Continued surveillance for oseltamivir-resistant influenza virus strains is needed, particularly in the immunocompromised.Oseltamivir Resistance in Adult Oncology and Hematology Patients Infected with Pandemic (H1N1) 2009 Virus, Australia


Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that  A critical stage in vertebrate sex determination is entry of the bipotential gonadal primordium into a developmental pathway leading to ovary or testis  and RA-degrading enzymes (i.e. members of the Cyp26 P450-cytochrome family) determines the precise spatial-temporal distribution of RA [Cyp26b1 expression leads to degradation of RA and protects germ cells from entering into meiosis in developing testes, while female-specific down-regulation of Cyp26b1 expression allows RA to induce germ cells to enter into meiosis in embryonic ovaries [Cyp26b1 expression in embryonic mouse testes, or addition of CYP inhibitors to wild-type embryonic testes, induced germ cells to express the pre-meiotic marker Stra8 (stimulated by retinoic acid-8), which is required for the initiation of meiosis in mammals, followed by the expression of the early meiotic marker Sycp3 , and the down-regulation of the pluripotent stem cell marker Pou5f1  [Recent studies showed that in mouse, retinoic acid (RA) in females or its absence in males -- regulated by the RA-destroying Cyp26 Cytochrome P450 enzyme family -- reinforces germ cell fate by controlling the sex-specific initiation of meiosis  ,24,35–45Aldh1a2 paralog Aldh1a1 has been reported in somatic cells of embryonic mouse testes [In mice, expression of the RA-synthesizing enzyme Aldh1a2 in the embryonic mesonephros (but not in the gonads) as females initiate meiosis led to the suggestion of a source-sink regulatory system. According to this model, RA synthesized in the mesonephros enters the neighboring gonad and causes germ cells to enter meiosis in embryonic ovaries, which lack the RA-degrading enzyme Cyp26  have been lost in zebrafish and other teleosts, and that this gene loss has altered the functional evolution of the surviving aldh1a paralogs [RA plays a role in the onset of meiosis not only in mammals, but also in other tetrapods, including birds and amphibians –49. Whetparalogs –57. WhetTo address these questions and to test the hypothesis that meiotic control mechanisms in mouse apply to zebrafish, we performed a comprehensive genomic and expression analysis of the genetic machinery that regulates the synthesis and degradation of RA during gonadogenesis, and studied genetic markers for meiosis and somatic cells of the gonad to investigate the role of RA in the onset of meiosis and sexual fate determination in zebrafish. Results revealed shared underlying regulatory themes between zebrafish and mammals but important genomic and developmental differences in the mechanisms of RA-regulated gonadogenesis and sex determination.Cyp26 paralogs: Cyp26a1, Cyp26b1, and Cyp26c1. Recent studies in mouse [cyp26b1 occurs during gonad development in zebrafish as it does in tetrapods.Tetrapods and zebrafish have three in mouse ,24, chicin mouse ,49 and uin mouse  implicatyp26b1, as well as the other two gene family members cyp26a1 and cyp26c1, in adjacent histological sections of developing zebrafish gonads by in situ hybridization (ISH)  [cyp26b1 was detected only in a few scattered cells in undifferentiated gonads at 15 dpf  and in the retina of 15 dpf juveniles. These results revealed that, in contrast to tetrapods, in which cyp26b1 is the major regulator of RA availability during early gonad development, cyp26a1 is the only paralog up-regulated in developing zebrafish gonads during the time of sexual differentiation of the gonads.To address this question, we investigated expression patterns of con (ISH) . Zebrafimination , became mination , and wasmination . This re- 31dpf) ,58–60. Ut 15 dpf , and in analyzed , althougCyp26 gene before the two rounds of vertebrate genome duplications ; alternatively, it might be that zebrafish gene orthologies had been incorrectly assigned. To rule out the possibility of error in orthology assignments, we examined gene orthologies by first investigating the phylogenetic relationships between zebrafish Cyp26a1 [The apparent convergence of the roles of Cyp26a1 in zebrafish and Cyp26b1 in mouse could have either of two explanations: first, it might represent a case of independent subfunctionalization of gene roles possessed by the ancestral  Cyp26a1 , Cyp26b1 Cyp26a1  and Cyp2 Cyp26a1  (origina Cyp26a1 ) and the Cyp26a1 –67. Phyl Cyp26a1 .Cyp26 genes would have been produced in VGD1 and VGD2 [Cyp26 genes, raising the possibility that different VGD paralogs were lost in the teleost and tetrapod lineages.Phylogenetic analyses can sometimes give erroneous orthology assignments in cases of reciprocal gene loss after ancient gene duplications –70; for and VGD2 , but zebCyp26 genes in zebrafish and mouse , and Cyp26a1 and Cyp26c1 are adjacent and transcribed in the same orientation on Mmu19, consistent with the origin of Cyp26a1 and Cyp26c1 by tandem duplication rather than by genome duplication. In teleosts, however, the three Cyp26 paralogs are on three different chromosomes (i.e. cyp26a1 on zebrafish (Danio rerio) linkage group 12 (Dre12) and on medaka (Oryzias latipes) chromosome 19 (Ola19); cyp26b1 on Dre7 and Ola18; and cyp26c1 on Dre17 and Ola15). Genomic analysis at the chromosomal level using circleplots [Cyp26b1 genomic neighborhood connected to teleost cyp26b1 neighborhoods; and the mouse Cyp26a1/Cyp26c1 neighborhood connected to teleost neighborhoods that harbor cyp26a or cyp26c1, respectively  surrounding nd mouse  and medand mouse . In mouscleplots  revealedectively . Local aith Dysf , cyp26a1th Pde6c , and cyp Tbc1d12 . These rcyp26a1 in Dre12 and cyp26c1 in Dre17 revealed that these two regions are indeed paralogons  event , which encode RA synthesizing enzymes. Because no robust genetic marker for sex is yet available for zebrafish [vasa [in situ hybridization analyses on adjacent gonadal sections from animals representing three key stages of gonad development: i) bipotential gonads (20 days post-fertilization (dpf)) when gonads are sexually undifferentiated and can become either ovary or testis  trar testis ; and iiialdh1a2 was expressed throughout most cells of the ovary-like gonad of juveniles, but was not expressed in the largest germ cells, which were probably developing oocytes judging by their morphology , which is the only other remaining aldh1a paralog in zebrafish due to the loss of aldh1a1 early in the evolution of the teleost lineage [amh was already expressed, but in only a few scattered somatic cells . Analysis by double fluorescent in situ hybridization in sections of bipotential gonads resolved this question, showing that cyp26a1 was expressed in somatic cells but not in germ cells labeled by vasa  showed broad expression of th sexes . Sex assxes ; green arrowhead in eIB); yellow arrowhead in cyp26a1 in late stage IB is compatible with a potential role of Cyp26a1 in arresting oocytes during meiosis. Expression of cyp26b1 and cyp26c1 was not detected at this crucial stage for gonad fate decision in ovaries or testis  continued to express th sexes . In maleis cysts , judgingli cells , A’. In  oocytes . These rcyp26a1  against dead end (dnd), a gene essential for germ cell survival [vasa verified total depletion of germ cells in dnd-MO-injected animals  cannot be discarded  [Pou5f1(Oct4) in germ cells entering meiosis and the up-regulation of the early meiotic marker Sycp3  [To learn whether the sexually dimorphic expression pattern of zebrafish  gene 8) –80, whic gene 8) ,44,45,81otein 3) , which iotein 3) .in silico screening of the zv9 version of the zebrafish genome database [Tetraodon, fugu, medaka) as well as the basally diverging spotted gar genome did not identify Stra8. We did, however, find an ortholog for Stra8  in the genome of the basally diverging chordate amphioxus Branchiostomafloridae, and in the elephant shark [stra8 was present in stem chordates and stem gnathostomes, but was lost secondarily in the ray fin fish lineage [Stra8 in teleosts suggests that if RA plays a role in the regulation of the initiation of meiosis in teleosts, it does not act through Stra8 as it does in mammals [To our surprise, database  by BLASTnt shark , support lineage  at least lineage . Future  mammals .Sycp3 by in situ hybridization in adjacent sections showed that all zebrafish analyzed at the bipotential stage  showed germ cells expressing sycp3 throughout the gonad, while cells expressing cyp26a1 were mostly restricted to the dorsal surface , Cyp26b1 expression up-regulates in males but down-regulates in females [Cyp26b1 in gonadal somatic cells is at a time and place appropriate to protect germ cells from RA, and thereby to prevent oogenesis in males by retarding Stra8 expression and postponing the onset of meiosis until postnatal stages [Stra8 in germ cells, and promotes the onset of meiosis and oocyte development, thereby reinforcing the female pathway  and are entering meiotic arrest in diplotene. This observation, together with the fact that cyp26a1 expression disappears late in oocyte development at stage IV when meiosis resumes, is compatible with the Cyp26a1-mediated degradation of RA in oocytes that prevents the progression of meiosis and maintains meiotic arrest. This finding is consistent with results in mouse, in which RA prevents meiotic arrest in testes of 13.5 dpc embryos [cyp26a1 and the pluripotent marker pou5f1 (oct4). The convergence of cyp26a1 and pou5f1 expression in zebrafish oocytes at meiotic arrest is compatible with the proposed roles of Pou5f1 and Cyp26b1 in mouse germ cells in promoting survival and preventing apoptosis [Interestingly, we observed that zebrafish express ogenesis . In zebrg factor . In fema embryos . Interespoptosis ,86, mechpoptosis .fgf9 gene in zebrafish and other teleosts [wnt4 genes [Overall, this work provides the knowledge base on RA metabolic gene expression necessary for the design of experiments that alter RA signaling in zebrafish developing gonads to test the model stemming from our results for the role of RA signaling in the progress of meiosis. In addition, further work is required to understand the relationship of RA signaling to the Fgf9/Wnt4 seesaw that helps regulate gonad fate in mouse ,94–98. Eteleosts ,100 and t4 genes , genomicAB strain zebrafish were used in all experiments. Animals were reared and collected under standard conditions and were handled in accordance with good animal practice. The University of Oregon Institutional Animal Care and Use Committee approved all animal work .dead end as described [dnd MO-injected embryos were fixed at 24 hours post-fertilization to confirm the presence or absence of germ cells by whole-mount in situ hybridization using vasa probe as described [To obtain animals lacking germ cells, wild-type zebrafish embryos were injected at the 1-2 cell stage with antisense morpholino oligonucleotide  directed against escribed . Siblingescribed .In situ hybridization experiments on zebrafish cryosections were performed as described [aldh1a2 and aldh1a3 were made as described [amh probe was made as described and used as a somatic cell marker to distinguish differentiating testes and ovaries when morphological features were not clear [vasa was made from its 3’ end as described [cyp26a1 (nucleotides 507-1848 of NM_131146), cyp26b1 (nucleotides 518-1548 of NM_212666), cyp26c1 (nucleotides 198-1533 of NM_001029951), pou5f1 (oct4) (nucleotides 705-1482 of NM_131112), and sycp3 (nucleotides 265-884 of NM_001040350) were cloned in TOPO vector (Invitrogen) and used to synthesize DIG-labeled riboprobes (Boehringer Mannheim). The protocol for the three-color fluorescent in situ hybridization is described in wiki.zfin.org/display/prot/3+color+Fluorescent+in+situ+on+sections.escribed . Adjacenescribed ; amh proot clear ; and proescribed . FragmenTetrapods: Homo sapiens ; Mus musculus ; Gallus gallus ; Teleosts: Danio rerio ; Takifugurubripes ; Gasterosteusaculeatus ; Oryzias latipes ; Cephalochordates: Branchiostomafloridae ; CYP26A1 and CYP26C1 in Mmu19 ; cyp26a1 in Ola19 ; cyp26b1 in Ola18 ; and cyp26c1 in Ola15 . Gene loci that are close to each other may appear to overlap as a single connecting line in circle-plots due to the selected graph resolution, and lines based on best reciprocal blast hits that were not significantly different were not considered. Clusters of conserved synteny were created by coupling results from the reciprocal best hit BLAST pipeline with the use of a 100-gene sliding-window analysis that links chromosome segments with conserved synteny . Circleerred by ). Each gFigure S1Cyp26 orthologies between zebrafish and medaka were supported by clusters of conserved synteny, which extends conclusions from zebrafish to other teleost models.A: cyp26a1 in Dre12 and Ola19; B: cyp26b1 in Dre7 and Ola18; and cyp26c1 in Dre17 and Ola15. Cyp26 orthologs have been labeled with larger fonts, and names of gene neighbors can be surfed in the high-resolution pdf electronic files.(PDF)Click here for additional data file.Figure S2cyp26a1 and tetrapod cyp26b1 are not orthologs.Comparative genomic analysis of synteny conservation between mouse and medaka supports conclusions from comparison of zebrafish and mouse , ruling out the possibility of reciprocal gene loss in different lineages, and supports the notion that teleost B: cyp26b1 in Mmu6 and Ola18; B: Cyp26a1 in Mmu19 and Ola19; and Cyp26c1 in Mmu19 and Ola15. Large fonts label cyp26 orthologs and names of gene neighbors are legible in the high-resolution pdf electronic files.(PDF)Click here for additional data file.


SLC2A9 gene having the largest effect identified so far. Gene-gene interactions (epistasis) are largely unexplored in these GWA studies. We performed a full pair-wise genome scan in the Italian MICROS population (n = 1201) to characterise epistasis signals in SUA levels. In the resultant epistasis profile, no SNP pairs reached the Bonferroni adjusted threshold for the pair-wise genome-wide significance. However, SLC2A9 was found interacting with multiple loci across the genome, with NFIA - SLC2A9 and SLC2A9 - ESRRAP2 being significant based on a threshold derived for interactions between GWA significant SNPs and the genome and jointly explaining 8.0% of the phenotypic variance in SUA levels (3.4% by interaction components). Epistasis signal replication in a CROATIAN population (n = 1772) was limited at the SNP level but improved dramatically at the gene ontology level. In addition, gene ontology terms enriched by the epistasis signals in each population support links between SUA levels and neurological disorders. We conclude that GWA epistasis analysis is useful despite relatively low power in small isolated populations.Genome-wide association (GWA) studies have identified a number of loci underlying variation in human serum uric acid (SUA) levels with the  Serum uric acid is the final oxidation product of purine metabolism in humans. High serum uric acid (SUA) levels can lead to gout and is associated with cardiovascular diseases and diabetes SLC2A9 gene are the most significant genetic risk factors associating with low fractional excretion of SUA and explain 5.3% of the SUA level variance in women and 1.7% of the SUA level variance in men in the VIS population SUA level is a complex trait that is affected by environmental (e.g. diet and excessive body weight) and genetic factors with heritability estimates of 60–87% One possible source of the unexplained variation in SUA levels is gene-gene interaction (epistasis) The Italian MICROS cohort was recruited from the villages in South Tyrol DNA samples were genotyped with Illumina Infinium HumanHap300v1/v2 or HumanCNV370v1 SNP bead microarrays and analyzed using the BeadStudio software. Quality control of the genotype data was performed for each cohort using the R/GenABEL package (Version 1.4.3) rntransform function that is implemented in the GenABEL package performing quantile normalisation of residuals from a generalized linear model analysis. The normalised SUA levels were then analysed using a linear mixed model to correct for polygenic effects and relatedness using the polygenic function in the GenABEL package and the resultant environmental residuals  were used as the trait to test for association In each individual cohort the raw SUA levels were corrected for age, sex and BMI and normalised using the mmscore function in the GenABEL package. The consensus GWA threshold of 7.3 (−log10(5.0E-08)) was applied to identify GWA significant SNPs 1SNP and 2SNP, the following genetic models are used to detect epistasis where genotypes of each SNP  were fitted as fixed factors y is the trait of interest, μ is the model constant, 1SNP (or 2SNP) is a fixed factor with three levels, 1*SNP2SNP is the interaction term, e is the random error term. The F ratio test of Model 1 against Model 3 is for the whole pair effect including interaction . The F ratio test of Model 1 against Model 2 is for the interaction between the two SNPs . SNP pairs with missing joint genotype classes  were not evaluated to reduced the risk of inflation of the type I error rate. P values were calculated based on the F distribution with relevant degrees of freedom and transformed in the −log10 scale . We applied the same −log10 scaled thresholds for both the pairF and intF tests to control the type I error rate A single SNP based GWA scan was performed in each population using a score test method (based on the additive model) implemented in the 10 scale) were derived based on Bonferroni correction for multiple tests, i.e. the 5% nominal P value corrected by the number of pair-wise tests. Considering 300,000 SNPs, a SNP-genome scan and a full pair-wise genome scan perform 3.0E+05 and 4.5E+10 association tests respectively, thus the genome-wide threshold is 11.95 (−log10(0.05/4.5E+10)) for the pair-wise genome scan. SNP-genome scans have been used to test epistasis for genome-wide significant signals specifically to increase the power of detection 10(0.05/3.0E+05/N) if there are N GWA significant SNPs.Genome-wide significance thresholds  were retained. The retained results were evaluated using the predefined thresholds to identify genome-wide significant epistatic signals. Each SNP in the retained results was annotated to the nearest gene within a window of 20 kilobases flanking the SNP based on the physical distances to either the start or end of transcription of genes (the distance is set to zero if the SNP is within a gene) without considering linkage disequilibrium (LD). A full pair-wise genome scan was also performed in the CROATIAN population as above to prepare input for GO enrichment analyses (see below).A full pair-wise genome scan was performed in the MICROS population and SNP pairs with a certain interaction signal  was used to claim significant replication for an epistatic pair (i.e. both replicated SNPs were exactly the same as the epistatic SNPs) because only one test was performed. When an epistatic pair was not replicated (e.g. due to missing genotype classes), we also tested the interactions between each of the adjacent SNPs of the first epistatic SNP and that of the second. In that case, the nominal threshold corrected by the actual number of tests was used to claim significant replication.polygenic function was also used to calculate the proportion of the phenotypic variance in SUA levels explained by epistatic pairs in the MICROS population. Because the quantile normalised SUA levels were based on the quantiles of the raw SUA levels, we used standardized SUA levels for variance calculation ). The standardized SUA levels were fitted into the full mixed model including polygenic effects and the identified SNP pairs. The difference of residual variance from a value of 1 is the proportion of phenotypic variance explained by the SNP pairs included.The 10Ppair>6.5 and −log10Pint>6.5) in each population and used the epistatic genes annotated from them as the target for the GO enrichment analysis to mine biological meanings from epistatic signals that were less significant. The GO terms enriched (P<1.0E-03) by the epistatic genes in each population were compared to identify replicated GO terms and then epistatic genes shared by each pair of replicated GO terms. The shared epistatic genes in the replicated GO terms were investigated further for a) associated biological functions or diseases and b) epistatic SNP pairs involved and their replication.A GO enrichment analysis was conducted for each of the MICROS and CROATIAN populations using the running mode of “Two unranked lists of genes” in GOrilla SLC2A9 gene. The GWA scan for the CROATIAN population also identified seven genome-wide significant SNPs which were exactly the same as those in MICROS. Thus the genome-wide threshold of 7.62 (−log10(0.05/3.0E+05/7) was used for SNP-genome scans. The inflation factor λ (computed by regression in a quantile-quantile (QQ) plot, The mean SUA level and phenotypic correlation between SUA level and BMI were similar across the MICROS, VIS and KORCULA cohorts . The polpairF and intF tests performed on chromosomes 3 and 4  to illustrate why the two tests are needed (pairF test (e.g. −log10Ppair>11.95 may pick up SNP pairs with very weak interactions (e.g. −log10Pint<3), whereas using only the intF test (e.g. −log10Pint>7.62) could pick up those with weak whole pair effects when both SNPs had small marginal effects (e.g. −log10Ppair<6)  had missing joint genotype classes and hence were ignored in this study. We plotted the P values of all the e needed . Using oPpair<6) . The QQ ir tests  showed ant tests  showed a10Ppair>4.7 and −log10Pint>3.2, 212933 in total) retained from the full pair-wise genome scan . However, we found a big cluster of SNP pairs with strong whole pair effects  but weak to high interactions . This cluster of SNP pairs all involved the SLC2A9 gene as one would expect. Two pairs: rs12130085 (NFIA) – rs737267 (SLC2A9) and rs737267 (SLC2A9) – rs9316212 (ESRRAP2) were significant based on the threshold of 7.62 for SNP-genome scans , in total 1326 SNP pairs involved 2063 unique SNPs of which 1148 (55.6%) were annotated to 910 unique genes, i.e. 1.5 pairs per gene. In contrast, 17 out of the 1326 SNP pairs involved the SLC2A9 gene including 4 GWA significant SNPs rs733175, rs737267, rs13131257 and rs13129697  and rs737267 – rs17064136 (adjacent to rs9316212) were 1.35 and 1.32 respectively. Combining the MICROS and CROATIAN data did not make the two significant pairs stronger (−log10Pint was 2.32 and 2.19 respectively) which is in line with the replication results. The two less significant SLC2A9 epistatic pairs also missed the SNP level replication in the CROATIAN population  in MICROS (910 genes from 1326 pairs) and CROATIAN (984 genes from 1260 pairs) were tested for GO enrichment. The GO terms enriched by epistatic genes in MICROS showed a complicated relationship among biological functions  and highlighted the importance of glutamate receptor activity  (SLC2A9 – LRRC16A (interaction between two SUA candidate genes) (SLC2A9) – rs4085921 (GPC6) and rs737267 (SLC2A9) – rs2302558 (CLEC16A). All the 49 epistatic gene pairs involved SLC2A9 and of these 5 involved interactions with other shared epistatic genes: ERC2, GPC6, CTNND2, CNTNAP2 and PTPRD. However, most of the 49 replicated gene pairs had a relatively weak interaction signal (−log10Pint<5).Epistatic genes annotated from the less significant SNP pairs  . Surprise genes) . Two genSLC2A9 gene may be an important epistatic locus interacting with multiple loci across the genome more frequently than expected by chance (SLC2A9 epistatic pairs (NFIA – SLC2A9 and SLC2A9 – ESRRAP2) were significant in SNP-genome scans and jointly explained 8.0% of the phenotypic variance in SUA levels where 3.4% was explained by their interaction components. However, caution should be taken in light of the billions of tests performed, the potential overestimation of the variance explained and the limited replication of the two pairs. The NFIA (nuclear factor I/A) gene is known as a cellular transcription DNA replication factor and its haploinsufficiency is associated with a central nervous system malformation syndrome and ureteral and renal defects NFIA was responsible for celiac disease ESRRAP2  is not yet known to have any related functions.Using a full pair-wise genome-wide association scan we generated a profile of epistasis in SUA levels in the Italian MICROS population. No epistatic SNP pairs reached the Bonferroni adjusted threshold for the pair-wise genome scan, which was not a surprise because of the relatively small population size. Nonetheless, we found that the y chance  and S1. SLC2A9 gene is known to interact with sex, age and dietary patterns SLC2A9 is a class II glucose/fructose transporter as well as high-capacity/low-affinity urate transporter with two isoforms expressed in basolateral and apical membranes of proximal renal tubular cells respectively SLC2A9 polymorphism was demonstrated further in two recent studies linking SUA levels with human cognitive aging SLC2A9 were involved in epistatic interactions, which adds another dimension of studying the SLC2A9 polymorphism and provides new clues of the genetic mechanism underlying SUA levels.The SLC2A9 interacting genes had clear neuronal influence  and/or membrane functions (SLC2A9 and genes that interacted with it (8 out of 910 unique genes in MICROS). The epistatic genes encoding glutamate receptors , schizophrenia and bipolar disorder , Alzheimer's disease , Parkinson's disease (DLG2), sclerosis , and diabetes .The impact of SUA levels on human cognitive dysfunction has been investigated in recent years unctions , S2, S3.Statistically detecting and replicating an epistatic pair of SNPs is far more challenging than for a single SNP signal for a number of reasons. Whereas a single SNP association is dependent on strong LD between the marker SNP and causative variant, detection of epistasis requires strong LD for both loci. Small allele frequency changes (e.g. 10%) between detection and replication populations can lead to a dramatic loss of power in replication. Such small allele frequency changes are not uncommon across the small and/or isolated populations. The issue of missing joint genotype class in replication populations  increases the difficulty of replication, especially when the epistatic pair for test has a rare genotype class  – rs4085921 (GPC6) was replicated at all three levels, rs737267 (SLC2A9) – rs2302558 (CLEC16A) at the SNP and gene levels but not the GO level as CLEC16A is not associated with an enriched GO term. Both GPC6 (glypican 6) and CLEC16A  are associated with multiple sclerosis The lack of statistical replication at the SNP level may be not too surprising considering that the epistatic signals detected in one isolated sample were to replicate in another isolated sample (CROATIAN) or a less isolated but smaller sample (i.e. SOCCS). Even when all the retained SNP pairs were considered regardless, we just found two SNP pairs (out of 212933) were replicated at the SNP level. When testing for replication at the GO level, we found 13 GO enriched terms (>50%) were replicated. Clearly replication improved dramatically as the level changes from SNP to GO annotation. A high level replication (i.e. gene or GO/pathway) does not guarantee a SNP level replication but is valuable to understand the biology of interest. Therefore, we recommend testing for replication of epistasis signals at all three levels. For example, rs737267 (10Pint of 6.5 used here) could be chosen as long as the big cluster of SLC2A9 pairs was mostly excluded Click here for additional data file.Figure S2The joint genotype – phenotype maps for two genome-wide significant epistatic pairs.(TIF)Click here for additional data file.Figure S3Gene component ontology terms enriched by epistatic genes in the MICROS population.(TIF)Click here for additional data file.Figure S4Gene component ontology terms enriched by epistatic genes in the CROATIAN populations.(TIF)Click here for additional data file.Table S1SLC2A910Ppair>6.5 and −log10Pint>6.5) in MICROS. involved epistatic pairs (−log(PDF)Click here for additional data file.Table S2Epistatic genes in the replicated GO terms shared by the MICROS and CROATIAN populations.(PDF)Click here for additional data file.Table S3Epistatic pairs with at least one shared GO gene and replicated in MICROS and CROATIAN.(PDF)Click here for additional data file.


Women with low grade squamous intraepithelial lesions (LSIL) at cervical cancer screening are currently referred for further diagnostic work up despite 80% having no precancerous lesion. The primary purpose of this study is to measure the test characteristics of 3q26 chromosome gain (3q26 gain) as a host marker of carcinogenesis in women with LSIL. A negative triage test may allow these women to be followed by cytology alone without immediate referral to colposcopy.A historical prospective study was designed to measure 3q26 gain from the archived liquid cytology specimens diagnosed as LSIL among women attending colposcopy between 2007 and 2009. 3q26 gain was assessed on the index liquid sample; and sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were measured at immediate triage and at 6–16 months after colposcopic biopsy. The sensitivity of 3q26 gain measured at immediate triage from automated and manually reviewed tests in 65 non-pregnant unique women was 70%  with a NPV of 89% . The sensitivity and NPV increased to 80%  and 98% , respectively, when only the automated method of detecting 3q26 gain was used.3q26 gain demonstrates high sensitivity and NPV as a negative triage test for women with LSIL, allowing possible guideline changes to routine surveillance instead of immediate colposcopy. Prospective studies are ongoing to establish the sensitivity, specificity, PPV and NPV of 3q26 gain for LSIL over time. Human papillomavirus (HPV) and telomerase overexpression have been recognized as independent carcinogenic factors for which the Nobel Prize in Physiology or Medicine was awarded in 2008 and 2009, respectively. Harald zur Hausen defined the link between human papillomavirus (HPV) infection and cervical cancer; and Elizabeth Blackburn, Carol Greider and Jack Szostak detailed the function of telomeres and the enzyme telomerase. Cells that are becoming cancerous or are cancerous express telomerase over-abundantly, allowing gains in the chromosomes’ telomere length. Testing for gains in human chromosome length of HPV infected cells may identify a potential biomarker for cervical cancer screening.The primary screening method for cervical cancer has been the morphologic characteristics of HPV infection in the epithelial cells of the uterine cervix. The Bethesda System, (TBS-2001) Cocktail high risk (HR) HPV testing, such as Hybrid Capture 2 (HC2) is positive in 76–80% of LSIL cytology samples The purpose of this research is to establish the test characteristics of sensitivity, specificity, positive and negative predictive values of the gain of the chromosome 3q26 region (3q26 gain) for predicting which women with LSIL cytology do not have CIN 2/3 at colposcopy.We designed a historical prospective study of women who had presented to the colposcopy clinics at Truman Medical Center, the teaching hospitals of the University of Missouri Kansas City School of Medicine, Kansas City Missouri, USA. To be eligible for inclusion, we reviewed the electronic medical record (EMR) for those women whose index liquid cytology LSIL sample had been archived, who had a colposcopically directed biopsy and who was not pregnant at the time of cytology or biopsy sampling. We were granted ethics approval, and consent waiver, from both the University of Missouri-Kansas City School of Medicine Institutional Review Board (#09-70X) and the Truman Medical Centers’ Privacy Board to use the de-identified data.Clinicians collected the index samples in the SurePath™ system  as part of routine cervical surveillance. The specimens were pelleted and resuspended for cytology processing for immediate clinical diagnosis. No other sampling was done on the specimen. The remainder of the resuspended sample was refrigerated at 2–4°C until it was used for 3q26 gain testing. The Bethesda System was used to report the cytology diagnoses , atypical squamous cells of undetermined significance (ASCUS), LSIL, high grade squamous intraepithelial lesion (HSIL)). CIN nomenclature was used to report the histologic diagnosis from the biopsy specimen .The cytology specimens were mailed at ambient temperature to the Ikonisys laboratory where each was transferred to a 15 mL tube and centrifuged for 6 minutes at 1200 RPM. The supernatant was decanted and the pellets resuspended in 5 mL of KCl for 15 minutes at 37°C. Two milliliters Carnoy’s Fixative was added and the samples centrifuged for 6 minutes at 1200 RPM. The supernatant was discarded and the pellet resuspended in 10 mL of Carnoy’s fixative and incubated overnight. Samples were centrifuged for 6 minutes at 1200 RPM, the supernatant discarded and the pellet resuspended in an appropriate volume of Carnoy’s Fixative. Slides were prepared manually and incubated at 55°C for 10 minutes. Prior to hybridization, slides were immersed in 2X SSC for 2 minutes at 73°C and treated with 1% pepsin for 10–15 minutes at 37°C. Slides were then incubated in 1X PBS for 2 minutes and fixed in 2% formalin for 5 minutes, at room temperature. Slides were washed in 2X SSC for 2 minutes at room temperature and dehydrated in an ethanol series .The slides were hybridized using the oncoFISH cervical probe kit . Briefly, 1 µL of 3q26 probe, 1 µL of CEP 7 probe, 1 µL distilled water and 7 µL of hybridization buffer were mixed and applied to the slide. A 22 mm round glass cover slip was placed over the probe and air bubbles were removed. Slides were transferred to a ThermoBrite , denatured at 76°C for 5 minutes and hybridized overnight at 37°C. Following hybridization, slides were washed in 2X SSC, 0.3% NP-40 at 73°C for 2 minutes then in 2X SSC, 0.1% NP-40 at room temperature for an additional 2 minutes. Slides were counterstained with DAPI  for 3–4 minutes at room temperature and dehydrated in an ethanol series. Ten microliters of antifade and a 22×50 mm cover slip were applied and slides were placed at −20°C for at least 15 minutes prior to analysis.We analyzed 65 slides using an automated system developed specifically for rare-cell detection and analysis . The entire sample was scanned using a 20× objective and both the total number of nuclei and number of nuclei with >2 signals for 3q26 were determined. The nuclei were ordered based on the number of 3q26 FISH signals, and up to 800 nuclei with the highest number of 3q26 FISH signals were imaged using a 40× objective. Nuclei were enumerated for both 3q26 and control centromeric 7 FISH probe signals to determine 3q26 gain. The test was positive if two or more cells with more than four 3q26 FISH signals were detected .Fourteen cytology samples were unable to be read by automated scanning for the 3q26 gain signal. These slides had less than 50 nuclei imaged at high magnification (40×), and they had an increase in background autofluorescence that could confound the signal to noise ratio. Therefore, following scanning, these fourteen slides were reviewed for 3q26 gain manually, while maintaining blinding to the subjects’ clinical information.The power analysis was calculated with Statistica software 81 LSIL cervical specimens from unique non-pregnant women were collected between May 2007 and January 2009 and stored at 2–4°C. Of these 81, 73 women had colposcopically directed biopsies on average 36 days from their index cytology (standard deviation (SD): 18). Of these 73 specimens, 65 provided sufficient archived material to run the 3q26 gain test resulting in 65 women with complete index cytology, colposcopically directed biopsy and index 3q26 gain status . Of thesThe average age of the study population was 32 years (SD 9.6 yrs) ranging from 17–59 years. Most of the women in the study were African-American (51%) or Caucasian (40%). The women had a mean gravidity and parity of 2.6 (SD 1.9) and 2.0 (SD 1.6), respectively, and 40% of the women had had a prior abnormal Pap. Ten of the 65 women (15%) had CIN 2/3 at colposcopy with the remaining having CIN 1 or normal tissue .Test characteristics were calculated for 3q26 gain status determined by both automated slide scanning and manual slide review. Seven of the 10 women with CIN 2/3 were positive for 3q26 gain on the index cytology for a sensitivity of 70% . The specificity of the 3q26 gain test was 91% ; the positive predictive value (PPV) was 44%  and the negative predictive value (NPV) was 89%  .Recalculation of the 3q26 gain test characteristics after exclusion of the manually reviewed slides does not substantially change the four test characteristics of 3q26 gain . There wOf secondary interest, sixteen women whose colposcopically directed biopsy was CIN 1 returned for repeat cytology 6–16 months  from their index cytology. None of the women had progressed to HSIL in that time frame for a non-evaluable sensitivity and a 0% positive predictive value. Twelve of the women regressed to normal cytology, one of whom had had a gain in the 3q26 chromosome on her index cytology, representing a false positive 3q26 gain test. None of the four women whose LSIL persisted had a 3q26 gain on her index cytology, making the specificity of the 3q26 gain test for regression or persistence at a 10 month average follow up 94%  and the negative predictive value 100% .In addition, four of the women with biopsy confirmed CIN 2/3 had follow up cytology 6 months (mean 203 SD 16) after their LEEP. All LEEP specimens had negative resection margins. All 6 month follow up cytologies were reported as NILM. Two of the women who showed a 3q26 gain at index cytology showed no 3q26 gain at the post-LEEP follow up cytology.HPV infections create the morphologic effects seen to codify a LSIL entity. Cells must exhibit nuclear enlargement as well as a defined, perinuclear clear area demarcated by a dense cytoplasmic ring (koilocytes); there may be nuclear smudging and binucleation. The nuclear contour of a LSIL cell may be smudged or granular or slightly irregular with hyperchromasia but the chromatin is evenly distributed. The nuclear enlargement seen in LSIL is at least three times that of the normal intermediate cell along with an increased nuclear to cytoplasmic ratio. These changes are most often seen in the superficial and intermediate cells, in sheets or individually While only 2.5% of all women screened for cervical cancer will have LSIL cytology, these women constitute nearly 50% of all the referrals to colposcopy The natural history of LSIL cytology supports an observational management guideline if the sensitivity and negative predictive value of a triage test is high enough to provide reassurance that the LSIL lesion will not progress to CIN 2/3 before the next screening interval. LSIL lesions caused by oncogenic HPV types regress, on average, in 14 months, ranging from 9–19 months, while those caused by low risk, benign, types regress, on average, in half that time Positive triage tests to improve the diagnostic accuracy of cytology have included testing for oncogenic HPV types, quantitating viral load, measuring integration and quantifying markers of cell cycle aberration, such as p16ink4a. These triage tests need high specificity and positive predictive value for identifying women whose abnormal cytology hides a true cancer precursor, balanced with an acceptable rate of false positive tests. The results of these positive triage tests would change clinical management from passive surveillance to requiring further diagnostic work up such as referral to colposcopy. Randomized controlled trials show high risk HPV testing, as a triage test after LSIL, to have specificities less than 50% for predicting those women with CIN 2+ disease where CIN 2+ is defined as CIN 2 or 3 or cervical cancer A negative triage test would be one whose negative/normal results would modify current aggressive clinical guidelines to allow observation without clinical intervention until the next screening interval without missing a significant portion of diseased women. A negative triage test is one with a high sensitivity for cancer and cancer precursors in addition to a negative predictive value exceeding 97% A promising new negative triage test for women with LSIL cytology has been the gain of chromosome length of 3q26. Prior studies report greater than 80% sensitivity for the detection of CIN 2/3 from a LSIL population Prior studies using gains in 3q26 amplification showed increasing proportions of 3q26 gain among women with increasingly more severe cervical cytology and histology fulfilling the biological plausibility and temporal association characteristics needed for this triage test to be clinically relevant in cervical cancer screening Our results show a sensitivity of 80% and a NPV of 98% for automated scanning for 3q26 gain among women with LSIL cytology at immediate colposcopy; and 100% NPV at the 6–16 month follow up visit after CIN1 biopsy. These excellent negative triage test properties are balanced by reassurance of not missing diseased women as seen from the high specificity of 90% and the PPV of 44%, nearly three times the prevalence rate of CIN 2/3 disease in our study population. These test characteristics are sufficiently high to warrant further prospective investigation of this test as a negative triage tool to change clinical guidelines to conservatively follow LSIL women negative for 3q26 gain instead of the current guidelines of immediate referral to colposcopy.Other clinical states that may benefit from a host-based negative triage test, such as 3q26 gain, include those women with atypical glandular cells of undetermined significance (AGC) The limitations of our study include the historical prospective trial design which relies on the integrity of archived liquid cytology material for 3q26 gain testing; and an unknown disease state in women whose LSIL smears were censored from analysis due to incomplete records of a colposcopic endpoint or interval surveillance screens.A prospective trial in larger numbers of women with LSIL from the general population of wide age ranges is ongoing. This study may determine a more robust characterization of the 3q26 gain test for reassuring women and physicians of the lack of need for immediate colposcopy after a LSIL Pap test.


Dync1h1 mutation. Dynactin heavy (DCTN1) and light (DCTN3) subunits were studied in the CNS of humans with sporadic ALS (SALS), mice with hSOD1G93A (SOD1/+), Dync1h1 (Cra1/+), and double (Cra1/SOD1) mutation at presymptomatic and symptomatic stages. In SALS subjects, in contrast to control cases, expression of DCTN1-mRNA but not DCTN3-mRNA in the motor cortex was higher than in the sensory cortex. However, the mean levels of DCTN1-mRNA and protein were lower in both SALS cortexes and in the spinal cord than in control structures. DCTN3 was unchanged in brain cortexes but decreased in the spinal cord on both mRNA and protein levels. In all SALS tissues immunohistochemical analyses revealed degeneration and loss of neuronal cells, and poor expression of dynactin subunits. In SOD1/+ mice both subunits expression was significantly lower in the frontal cortex, spinal cord and hippocampus than in wild-type controls, especially at presymptomatic stage. Fewer changes occurred in Cra1/SOD1 and Cra1/+ mice.It can be concluded that in sporadic and SOD1-related ALS the impairment of axonal retrograde transport may be due to dynactin subunits deficiency and subsequent disturbances of the whole dynein/dynactin complex structure and function. The Dync1h1 mutation itself has slight negative effect on dynactin expression and it alleviates the changes caused by SOD1G93A mutation.Dynactin is a complex motor protein involved in the retrograde axonal transport disturbances of which may lead to amyotrophic lateral sclerosis (ALS). Mice with hSOD1G93A mutation develop ALS-like symptoms and are used as a model for the disease studies. Similar symptoms demonstrate Cra1 mice, with  SOD1 gene are responsible for 20 % of FALS and up to 7 % of SALS cases .Analysis of variance showed that Dctn1-mRNA and Dctn3-mRNA expression was dependent on both the mutation and the clinical stage only in the frontal cortex [F(3.86) = 2.98, Glued) which mediates dynein–dynactin interaction is a critical component of the whole dynein/dynactin complex. It was found that a single base-pair change leading to a glycine-59-serine (p.G59S) substitution in glycine-rich domain of this subunit accelerated motor neuron degeneration in humans and mice which resembled ALS [DCTN1 exons and exon–intron boundaries in 286 samples diagnosed with neurodegenerative diseases  concluded that pathogenic mutations in DCTN1 are rare. Lately Fujiwara and Morimoto [Glued and microtubule stabilization cooperatively suppress axon degeneration.Dynein/dynactin complex is involved in axon maintenance, removal of damaged organelles, vesicles, misfolded and aggregated proteins from axons to the cell body . Many pobled ALS , 34. Vilbled ALS  after seMorimoto  discoverIn the present study we found that in individual SALS subjects, DCTN1-mRNA expression was higher in the motor than in the sensory brain cortex. In our earlier studies conducted on the same SALS subjects, we found that kinesins-mRNA expression was also higher in the motor cortex . In the The comparison of SALS and control cases indicated lower values of DCTN1 expression (on the mRNA and protein level) in both SALS cortexes and also in the spinal cord. Those results were supported by immunoexpression, which indicated that DCTN1 protein in the preserved SALS motoneurons revealed mild immunoreactivity and was less intense than in control material—the difference was especially evident in the spinal cord anterior horns. Our results are with agreement with the studies of Ikenaka et al. , who obsDifferently to DCTN1, in the brain cortex of SALS cases the expression of DCTN3 was similar in both SALS cortexes and it didn’t differ from the expression in control cortexes (on both mRNA and protein levels). In the spinal cord the expression of DCTN3 was much lower in SALS than in control tissues. In immunohistochemistry DCTN3 was hardly seen in all SALS tissues but was much better visible in controls.Mutations in the gene encoding SOD1 cause 2–3 % of all ALS cases and induce motor neuron degeneration in transgenic mice . Mutant All motor proteins transport their cargoes on tracts formed by microtubules. Microtubules assembly, stabilization and organization into bundles depend on tau protein . Tau alsDync1h1) mutations present phenotype similar but milder than SOD1G93A mice but in contrast to SOD1 mice their life span remains almost unchanged [Dync1h1 mutations in pathogenesis of motor neuron degeneration is controversial [Dync1h1 and SOD1G93A mutation [Cra1 mice with dynein heavy chain (nchanged , 15, 45.oversial , 47. Accoversial , Cra1 heoversial . There wmutation . All thoDync1h1 mutations defective retrograde transport is partially corrected and the life span of SOD1 mice extended [Dync1h1 mutation also attenuates motor neuron degeneration in SOD1G93A mice [In mice with double SOD1G93A and extended . Accordiextended  increase93A mice . Zhang e93A mice  suggeste93A mice . In the 93A mice , 45, 49 Dync1h1 mutation itself has slight negative effect on dynactin subunits expression but positively affects the changes caused by SOD1G93A mutation.It can be concluded that in SALS and SOD1-related ALS the impairment of dynein-mediated retrograde axonal transport and degeneration of motor neurons may be related to dynactin subunits deficiency and subsequent disruption of the whole dynein/dynactin complex structure and function. The


The aim was to compare two novel endotracheal tubes (ETT), Mallinckrodt TaperGuard  cuff) and KimVent Microcuff , with conventional Portex  in leakages across cuffs (microaspiration) under simulated clinical situations. It has been shown that globular PVC cuffs protect poorly against leakages due to microchannels formed from infolding of redundant cuff material . We hypo2O), normal lungs (PEEP 5 cmH2O) and acute respiratory distress syndrome (PEEP 10 cmH2O), and disconnection from the ventilator with and without spontaneous breathing effort. Spontaneous breathing was simulated with a respiratory gas exchange simulator. Suction was applied at 200 cmH2O sustained for 3 minutes at the Murphy eye. Each scenario was repeated with cuff pressures (Pcuff) 10, 20 and 30 cmH2O maintained by a Pcuff maintenance device.Each ETT was inserted into a silicone cylinder of 2 cm wide inclined at 35°. Then 20 ml water was added above the cuff and leakage measured every minute under five different simulated clinical conditions: mechanical ventilation for acute severe asthma (positive end-expiratory pressure (PEEP) 0 cmH2O in the presence of suction irrespective of Pcuff . Leakage under these scenarios can be reduced in TG and prevented in MC by Pcuff ≥20 cmH2O (Figure PT leaked grossly in all scenarios without PEEP and at PEEP 5 cmH2O whenever PEEP was lost. The effect of ETT type on the incidence of VAP warrants further investigation.Microcuff outperformed the others in preventing microaspiration, while Portex leaked grossly even at a recommended Pcuff of 20 to 30 cmH


PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) are associated with chronic pancreatitis. However, in many patients with a familial chronic pancreatitis pattern suggesting a genetic cause, no mutations in either of these genes can be found, indicating that other, still unknown, associated genes exist. In this respect ATP8B1 is an interesting candidate due to its strong expression in the pancreas, its supposed general function in membrane organization and the higher incidence of pancreatitis in patients with ATP8B1 deficiency.Mutations in genes encoding cationic trypsinogen (ATP8B1 coding exons and adjacent non-coding sequences of 507 chronic pancreatitis patients by direct sequencing. Exons that harbored possible relevant variations were subsequently sequenced in 1,027 healthy controls.We analyzed all 27 In the exonic regions, 5 novel non-synonymous alterations were detected as well as 14 previously described alterations of which some were associated with ATP8B1 deficiency. However, allele frequencies for any of these variations did not significantly differ between patients and controls. Furthermore, several non-synonymous variants were exclusively detected in control subjects and multiple variants in the non-coding sequence were identified with similar frequencies in both groups.ATP8B1 variants and chronic pancreatitis in our cohort of patients with hereditary and idiopathic chronic pancreatitis.We did not find an association between heterozygous  PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) can be identified CFTR mutations too enhance the susceptibility for idiopathic chronic pancreatitis Chronic pancreatitis (CP) is an inflammatory disease characterized by destruction of pancreatic parenchyma that can result in permanent impairment of both exocrine and endocrine pancreatic function ATP8B1 (formerly designated as FIC1) ATP8B1 mutations.ATP8B1 deficiency is an autosomal recessive disease characterized by mutations in n = 316) and France (n = 191). In the German patients, the diagnosis of CP was based on two or more of the following findings as described previously: presence of a typical history of recurrent pancreatitis, pancreatic calcifications and/or pancreatic ductal irregularities revealed by endoscopic retrograde pancreaticography or by magnetic resonance imaging of the pancreas, and pathological sonographic findings The study was approved by the local ethics committee of the Technische Universität München and the ethical review committee of the Université de Bretagne Occidentale. All study subjects gave their written informed consent for genetic analysis. For this study, 507 patients with hereditary or idiopathic chronic pancreatitis were included. The patients originated from Germany  and French (n = 285) origin. Whenever a non-synonymous variation in the coding sequence was encountered all 1,027 controls were sequenced for the exon involved.The control group consisted of 1,027 unrelated healthy individuals of German .Genomic DNA of peripheral blood leukocytes was extracted routinely. Primer pairs for PCR were designed to amplify all 27 coding exons, with flanking intron-exon boundaries of To detect nucleotide sequence changes of potential relevance to clinical phenotypes, we analyzed the sequence output of the patient cohort for variants that resulted in amino acid changes, nonsense variants or deletions/insertions. Exons with one of these alterations were subsequently analyzed in the control population by DNA sequencing.http://www.ncbi.nlm.nih.gov/entrez, reference sequence NM_005603.4). The A of the ATG start codon was used as nucleotide +1. The mutations are described according to the nomenclature recommended by the Human Genome Variation Society (http://www.hgvs.org/mutnomen).The reference sequence was derived from GenBank , all possibly associated in literature with episodic ATP8B1 deficiency. The second patient had two variants , both described in patients with the progressive form of ATP8B1 deficiency. Interestingly both these patients presented with chronic pancreatitis and had no signs of liver disease whatsoever.In our cohort of 507 CP patients we identified 19 different d change . Five (pATP8B1 variations in CP patients did not significantly differ from that in controls (p = 0.5). Furthermore, in the control population we detected 8 additional non-synonymous variations, of which 5 had not been described before (Those exons in which a non-synonymous variant was detected in CP patients 13 exons) were also sequenced in our control cohort of 1,027 subjects. No alteration was significantly overrepresented in the patient group. Also the combined frequency of these non-synonymous exonic d before . In addid before  and 3. T exons weATP8B1 seemed a plausible candidate gene for chronic pancreatitis due to its high expression in the pancreas, its supposed general function in membrane organization and the finding that 2 out of 10 individuals affected with ATP8B1 deficiency had chronic pancreatitis ATP8B1 variants and hereditary or idiopathic chronic pancreatitis when comparing 507 patients and 1,027 controls.At the outset of our investigations, ATP8B1 variants. We could not experimentally verify independent inheritance as no genomic material was available from parents or unaffected family members. However if these patients were indeed compound heterozygous, these genotypes are predicted to result in an ATP8B1 deficiency phenotype. Especially the p.E665X and p.A1208fs mutations change the structure of ATP8B1 significantly and can cause PFIC. Yet these two CP patients did not have any signs of liver disease or extrahepatic features of ATP8B1 deficiency other than pancreatitis. ATP8B1 deficiency without liver disease has been described before, suggesting that reduced penetrance of the liver phenotype can indeed be seen We did identify two CP patients with two or three non-synonymous ATP8B1 variants in health and disease. For example p.D70N was previously suggested to contribute to the etiology of intrahepatic cholestasis of pregnancy (ICP) as 3/182 ICP patients harbored this variant and none of 120 controls In addition, our data do contribute to a better understanding of the role of rare heterozygous ATP8B1 variants and hereditary or idiopathic chronic pancreatitis. However it suggests that pancreatitis might be the first or sole symptom of ATP8B1 deficiency. Furthermore earlier suggestions of an involvement of ATP8B1 variants in ICP might have been due to a chance effect.In conclusion, our investigation did not reveal an association between heterozygous


S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of  Cells are routinely challenged by changes in growth conditions that perturb protein homeostasis. The action of protein quality control (PQC) machinery is essential to maintain levels of non-native proteins within a tolerable range Hsp40s represent a large family of Hsp70 co-chaperones that are essential regulators of the ATP hydrolytic cycle of Hsp70 and target Hsp70 to specialized machineries and cellular locations To define Hsp70-dependent steps in triage decisions that lead to protein degradation in the eukaryotic cytosol we expressed in yeast a terminally misfolded and short-lived chimeric GFP fusion protein (slGFP). SlGFP has an N-terminal domain that is too short to fold into a stable conformation fused to tandem GFPs, so its fate can be monitored visually and biochemically. Therefore, study of slGFP degradation provides a valuable approach to define chaperone dependent steps in protein degradation without having to consider interpretations related to folding.We report that Hsp70 cooperates with Sis1 and the PQC E3 ligase Ubr1 to mediate proteasomal degradation of slGFP. Interestingly, attenuation of Sis1 or Ubr1 activity lead slGFP to accumulate in a Triton X-100-soluble state and be packaged into protein handling centers that are visualized as cytosolic puncta. The slGFP that accumulated in puncta when Sis1 activity was low was subsequently degraded in a proteasome dependent manner. Yet, in the absence of Ubr1, puncta localized slGFP was relatively stable. The Sis1/Hsp70 system and Ubr1 cooperate in degradation of a terminally misfolded cytosolic protein. Cells compensate for saturation of the Sis1/Ubr1 E3 machinery via storage of degradation competent protein assemblies in cytosolic puncta. Terminally misfolded proteins that accumulate in PQC puncta can subsequently be degraded in a process that requires Ubr1.Yeast strains and plasmids are listed in 2O, and boiled for 15 min in denaturing lysis buffer . Cell lysates were precleared at 3,000 rpm for 3 min and the protein concentration of supernatants were normalized. Normalized lysates were diluted in sample buffer  then analyzed by SDS-PAGE and western immunoblotting for the indicated proteins.Yeast cultures expressing the indicated proteins were treated as described in the text. To inhibit protein translation, cultures were treated with 200 µg/mL cycloheximide and aliquots were removed at indicated times. Cells were lysed by alkaline pretreatment Yeast strains expressing the indicated proteins were lysed by glass bead disruption in Buffer A . Cell extracts were precleared at 3,000× G for 3 min at 4°C. The supernatant was saved and protein concentrations assessed with a BioRad protein determination kit. Protein concentrations were normalized between samples to approximately 3 mg/mL and 300 µg of protein was incubated with the indicated antisera for 1 hour at 4°C then incubated with Protein G beads (50% slurry preblocked with BSA) for 30 min at 4°C. Beads were washed 2–3 times with Buffer A then resuspended in sample buffer and analyzed by SDS-PAGE and immunoblotting.2O (+1 mM NaN3 and 20 mM NEM) and lysed by glass bead disruption in Buffer A (+1 mM NEM). Cell extracts were precleared at 3,000 rpm for 3 min at 4°C, protein concentrations normalized, and GFP immunoprecipitated with anti-YFP antisera and protein G resin using standard methods. Protein G resin was washed three times in Buffer A supplemented with 0.1% SDS. Ubiquitinated slGFP was detected after SDS-PAGE and western immunoblotting of precipitated material for ubiquitin (Covance). Levels of ubiquitinated slGFP were quantified using laser densitometry and ImageJ software (NIH) and normalized as a ratio to the level of slGFP that was immunoprecipitated from the lysate (detected using anti-GFP).Yeast strains expressing slGFP were grown under selection to mid-log phase. Cells were washed with cold HGAL1 promoter for 4 hours before cells were processed for analysis. Cells were visualized with an Olympus IX81 Fluorescence microscope and images processed with Metamorph software. Exposure times and all other settings were standardized across individual experiments unless otherwise noted.Yeast strains expressing the indicated proteins and treated as described in the text were fixed in 3.7% formaldehyde and stored in phosphate buffered saline (pH 7.5) supplemented with 1.2 M sorbitol. Fixed cells were permeabilized and DNA visualized with DAPI as described in Cells were lysed by glass bead disruption in Buffer A (+1 mM DTT) and lysates pre-cleared at 3,000 rpm for 3 min at 4°C. The supernatant was saved and a quantity of lysate that contained 200 µg of protein was spun at 100,000× G for 30 min at 4°C. An aliquot was saved prior to the spin to represent the total input. Equivalent volumes from total, supernatant, and pellet fractions were added to 2× sample buffer and analyzed by SDS-PAGE and western immunoblotting for the indicated proteins.To analyze how protein degradation and aggregation pathways intersect in the cytosol we examined the degradation pathway of a labile and aggregation-prone cytosolic protein. This protein (herein referred to as slGFP for short-lived GFP) consists of a 126 amino acid N-terminal domain containing several unstructured regions that are enriched in hydrophobic motifs and putative Hsp70/Hsp40 chaperone binding sites  [27], [3ADH1 promoter. Importantly, slGFP expression from this promoter did not impact cell growth although we observed elevation in levels of heat-shock inducible chaperones Ssa1 and Hsp104. However, slGFP did not induce a global heat shock response since the levels of Ydj1 and Sis1 were unchanged  protein requires partner proteins (elongin B & C) and molecular chaperones to fold properly We expected that Sis1 was acting through its partner Hsp70 Ssa1 to accelerate slGFP turnover. To determine if Sis1 action in slGFP degradation is indeed Hsp70-dependent, a mutation was made in the Sis1 J-domain (H34Q) that disrupts a conserved Hsp70 binding motif SIS1 is essential we investigated slGFP degradation in Δsis1 complemented with a plasmid in which SIS1 is expressed under control of tetracycline repressible promoter. Sis1 levels in this strain were 85% lower than in an isogenic wild type strain even in the absence of doxycycline, though growth rates were unaffected  suggest that blocking Sis1 function impacts localization of slGFP in the same manner as inactivation of Ubr1 or San1. To explore this further, we evaluated the impact of reducing Sis1 levels on the solubility and degradation of slGFP. As affected . Furtheraffected .Δubr1 or Δsan1 strain, the slGFP in low Sis1 remained predominantly Triton X-100-soluble . Full-length slGFP is detected by western blot in Δydj1, so slGFP is translated in this strain, but may not be detected by fluorescence microscopy due to its aggregation prior to proper folding. Thus, in contrast to Sis1 action in degradation of slGFP, Ydj1 may be required to chaperone nascent slGFP.Accumulation of Triton X-100 soluble forms of slGFP in puncta in the low Sis1 strain did not appear to result from a general decrease in cytosolic chaperone capacity. This is the case because deletion of the non-essential  60 mins . Ydj1 cockground . We atteUBR1 or SAN1 decreased the rate of slGFP degradation and in each of these instances, slGFP accumulated in Triton X-100-soluble puncta. Therefore, we investigated whether Sis1 function in turnover of short-lived proteins is dependent upon Ubr1 or San1. To test for functional interactions between Sis1 and quality control E3 ligases we first asked if the presence of Ubr1 or San1 is required for Sis1 to accelerate slGFP degradation  to mediate proteasomal degradation of a terminally misfolded protein called slGFP. A decrease in the flux of slGFP through the Sis1/Hsp70/PQC E3 pathway results in the accumulation of slGFP in cytosolic puncta. Triton X-100-soluble forms of slGFP that accumulate in puncta are resolubilized and degraded in an Ubr1 and proteasome dependent manner. Thus, Sis1/Hsp70 and PQC E3s cooperate to clear terminally misfolded proteins from the cytosol. Saturation of this system can lead to storage of degradable protein species in puncta that serve as transient holding depots for detergent soluble assemblies of misfolded proteins When PQC pathways are saturated cells package non-native proteins into several different protein handling centers; the IPOD, JUNQ and peripheral compartment Sis1 is found to function in concert with Hsp70 and PQC E3s to mediate degradation of slGFP. In addition, Sis1 appears to cooperate with Hsp104 to accelerate rates of slGFP degradation, but Hsp104 is not required for normal rates of slGFP degradation. Thus, Sis1 seems to function via Hsp104 independent and dependent mechanisms to facilitate slGFP degradation. Sis1 and Hsp70 Ssa1 are present in complexes that contain Ubr1, and Hsp70 is known to function in substrate selection by PQC E3s To accelerate the rate of slGFP degradation Sis1 may function with Hsp104 in a manner similar to Sis1/Hsp104 function in prion propagation Δubr1 strains is different. SlGFP present in the puncta of the low Sis1 strain is resolubilized and degraded in a proteasome-dependent manner. Yet, in Δubr1 the puncta that contain slGFP are relatively stable and slGFP has a half-life of greater than 90 mins instead of 15 min. These data suggest that slGFP degradation intermediates accumulate in cytosolic puncta when the capacity of Sis1 and Hsp70 to handle non-native proteins is saturated. In addition, it appears that Ubr1 is required for degradation of misfolded proteins that are liberated from the puncta. Further studies are now required to understand the functional interplay between PQC factors that degrade non-native proteins and those that package non-native proteins for reversible sequestration into protein storage depots. A mechanistic understanding of these cellular processes will help define approaches to suppress the proteotoxicity associated with protein conformational disease.The half-life of puncta that contain slGFP in low Sis1 or Figure S1The Domain Structure Of SlGFP. A) Sequence and predicted secondary structure of the N-terminal 120aa of slGFP; C-coiled H-helical S-β-strand. B) Turnover of slGFP wild type and mutant lacking its nuclear localization sequence (NLS) and nuclear export sequence (NES). C) Fluorescence microscopy of slGFP and NLS/NES mutant.(TIF)Click here for additional data file.Figure S2Depletion Of Sis1 To Undetectable Levels Delays SlGFP Turnover. A yeast strain expressing slGFP as described in (TIF)Click here for additional data file.Table S1Genotypes Of Yeast Strains Used In Study Of The SlGFP Degradation.(DOCX)Click here for additional data file.Table S2Plasmids Utilized In The Study Of SlGFP Degradation.(DOCX)Click here for additional data file.


Sepsis and its common complication septic shock are generally induced by the action of lipopolysaccharide (LPS) and characterized by peripheral arteriolar vasodilatation that results in hypotension and inadequate tissue perfusion. During sepsis, secretion occurs of large amounts of inflammatory mediators such as nitric oxide (NO), interleukin 1 (IL-1) and TNFα that will modulate the inflammatory response. One significant finding in clinics is that men and women respond differently to sepsis, with better prognosis related to women [Male and female (ovariectomized and sham surgery) rats were injected intraperitoneally (i.p.) for three consecutive days with ECP 40 µg/kg or vehicle. On the third day, after ECP injection, rats receive i.p. injection of 10 mg/kg bacterial LPS or saline solution. Plasma was collected 2, 4 and 6 hours after LPS for NO and cytokine measurements.Administration of LPS increased the NO plasma concentration in males and females . ECP pretreatment decreased the NO concentration in sham females at 4 and 6 hours; conversely, it increased nitrate levels in ovariectomized and in males at 4 and 6 hours. IL-1 plasma concentration was increased in the three groups after LPS administration at 2 and 4 hours and in Sham at 6 hours; ECP pretreatment decreased IL-1 plasma concentration in all groups at 2 hours. LPS administration also increased TNFα plasma concentration at 2, 4 and 6 hours in the three groups; ECP pretreatment inhibited the increase of TNFα at 2 hours in three groups.Our results indicate that estradiol may have proinflammatory or anti-inflammatory actions depending on the gender and the mediator evaluated; this balance in mediator secretion may be protective and explain in part the better outcomes of woman during sepsis.


Chromogenic in situ hybridization (CISH) is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques.HER2 CISH pharmDx™ Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx™ Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory.Here it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx™ Kit to both HER2 FISH pharmDx™ Kit and PathVysion HER-2 DNA Probe Kit.The data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx™ Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to fluorescence-based methods.The concordance between results obtained using the recently FDA approved  HER2  amplification is observed in approximately 22% of human breast cancers [HER2  is an important marker for invasive breast cancer. The assessment of the HER2 expression level is routinely done by examining protein expression and/or gene expression levels in formalin-fixed and paraffin-embedded (FFPE) histological sections. Overexpression of HER2 protein and/or  cancers  and has  cancers .HER2 amplification is mostly done by in situ hybridization (ISH) techniques either fluorescence (FISH) [HER2 target sequences in the nuclei of tumor cells is done by fluorescence- or hapten-labeled sequence pairing probes. Implementation of CISH for determination of HER2 amplification in breast cancer has some advantages compared to FISH based detection [Tissue based assessment of HER2 protein expression levels is commonly achieved using immunohistochemistry (IHC), whereas tissue based analysis of e (FISH)  or chrome (FISH) . In ISH etection -9. ChromHER2 FISH techniques. In this paper data is reported from the comparison of 365 breast cancer specimens using a new dual color HER2 CISH method  with two well established and FDA approved HER2 FISH techniques; HER2 FISH pharmDx™ Kit  and PathVysion HER-2 DNA Probe Kit .To implement CISH in the anatomical pathological laboratories for determination of HER2 status in breast cancer the technique needs to be safe and reliable . One wayHER2 CISH pharmDx™ Kit (Dako Denmark A/S), HER2 FISH pharmDx™ Kit (Dako Denmark A/S) and PathVysion HER-2 DNA Probe Kit . Specimens were not individually identifiable and it was impossible to trace the identity of the patients. The study was performed in accordance with the current version of the World Medical Association Declaration of Helsinki and approval from an Institutional Review Board  was granted prior to study start. Evaluation of specimens were performed by three different technologists for the three ISH tests and subsequently reviewed by the pathologists. One pathologist reviewed test results obtained with PathVysion HER-2 DNA Probe Kit and HER2 CISH pharmDx™ Kit with several months in between and another pathologist reviewed test results obtained with HER2 FISH pharmDx™ Kit. Knowledge of test results was not shared between technologists or between pathologists.The study included 365 FFPE invasive breast cancer tissue specimens with known fixation history . The specimens were collected consecutively at a US reference laboratory and the first 304 specimens were included irrespective of HercepTest™  IHC score and additional 61 specimens were included based on a IHC HER2 2+ score as determined by HercepTest™ Serial sections (5 μm) were cut from each specimen and stained with H&E, HercepTest™ for HER2 protein expresion, HER2 CISH staining was performed according to the manufactures instructions (Dako Denmark A/S) at the US reference laboratory. In short, specimens were subjected to heat-pre-treatment (microwave oven) and pepsin digestion at 37°C to prepare the tissue for probe hybridization. Denaturation for 5 min at 82°C and over-night hybridization at 45°C were performed simultaneously for the HER2/Texas Red labeled DNA probe and the CEN-17/FITC labeled PNA probe using a Hybridizer (Dako Denmark A/S). Specimens were subjected to stringent wash at 65°C for 10 min before transfer to a CISH wash buffer. The signals from the fluorescent probes were converted to chromogenic signals in an IHC staining reaction performed on an automated platform . The immunohistochemical staining included blocking of endogeneous peroxidase activity, incubation with horseradish peroxidase conjugated anti-FITC and alkaline phosphatase conjugated anti-Texas Red antibodies followed by development of chromogenic signals using red and blue chromogens. The slides were counterstained with hematoxylin and mounted in a permanent mounting medium.HER2 CISH stained slides were interpreted using a bright field microscope with 40× and 60× objectives. The HER2/CEN-17 ratio was calculated based on the enumeration of 20 nuclei from the invasive tumor area. Based on ratio, the specimens were categorized into amplified (HER2/CEN-17 2.0) or non-amplified (HER2/CEN-17 < 2.0) categories. Specimens with a ratio between 1.8 and 2.2 (borderline cases) were subjected to additional enumeration of 20 nuclei and the ratio was then recalculated for the 40 nuclei to determine if amplification was present or not. Normal cells within the specimen served as an internal control for staining success. Normal cells should exhibit the ratio expected for normal diploid cells with a one to one relationship of red and blue signals.HER2 FISH pharmDx™ was performed according to the manufacturer's instructions at Dako Denmark A/S and FISH using PathVysion HER-2 DNA Probe Kit was performed according to an internally validated procedure at the US reference laboratory.HER2/CEN-17 ratios obtained by CISH and FISH were translated to a HER2 gene status of amplified when the HER2/CEN-17 ratio was higher than or equal to 2.0 or non-amplified when the HER2/CEN-17 ratio was below 2.0.In accordance with FDA approved guidelines for determination of HER2 status All specimens were tested at the US reference laboratory using HercepTest™ as per the manufacturer's instructions to determine the IHC HER2 score.IBM SPSS Statistics were used for statistical work . Agreement calculations were reported with 95% confidence limits based on the binomial distribution using equal tailed Jeffreys prior intervals  as calcuA total of 365 breast cancer specimens were included in this investigation. An overview of the HER2 IHC scores obtained from HercepTest™ staining is provided in Table HER2 amplified and non-amplified test results found by HER2 CISH, HER2 FISH and PathVysion FISH are presented in Table HER2 CISH, 11.4% were amplified by HER2 FISH and 11.0% were amplified by PathVysion FISH (data not shown). Figure HER2 CISH pharmDx™ Kit. In both panels tumor cells having distinctive blue dots are observed corresponding to the reference CEN-17 probe signals  have cluster amplification in which red signals are overlapping, but some single red signals are also visible in some tumor cells.Frequencies of HER2 CISH result and seven specimens lack a HER2 FISH result. Three of these specimens are overlapping and therefore, 348 specimens were eligible for comparison between HER2 CISH and HER2 FISH. Agreement calculations revealed an overall agreement of 98.3%  with positive agreement of 93.2%  and negative agreement of 99.0%  when comparing HER2 CISH and HER2 FISH . Positive agreement was 90.9%  and negative agreement was 98.7%   signals or made them difficult to see. In these cases the blue signals in the normal cells surrounding the tumor cells were clear and distinct and cases could therefore pass the quality control. Therefore, in cases with cluster amplification of blue signals additional caution should be taken during interpretation and results from other test methods such as IHC or FISH should be included before a final HER2 status is given.As indicated in Table  present . Three oHER2 CISH test result (Table HER2 CISH was 97.5% (352/361*100). The success rates for HER2 FISH and PathVysion FISH were 98.4 and 98.9%, respectively.Final success rates were determined after allowing for two staining runs. Of the 13 cases with a missing HER2/CEN-17 ratios was performed by plotting ratios obtained by HER2 CISH as a function of ratios obtained by HER2 FISH  were calculated for all three assays and tabulated for comparison for all the valid specimens  compared to 4:02 for PathVysion FISH (data not shown). This difference was statistically significant using a two-tailed, paired t-test .During the study, the evaluation time for each specimen was recorded for HER2 FISH and HER2 CISH performed in breast cancer specimens [HER2 CISH have not yet been presented. In the current investigation 365 primary breast cancer specimens were included and an overall HER2 status agreement close to 98% is reported when comparing test results obtained by HER2 CISH pharmDx™ Kit to results obtained by HER2 FISH pharmDx™ and PathVysion HER-2 FISH DNA Probe Kit. The study population was enriched for HercepTest™ IHC 2+ specimens because most testing modalities pass on such equivocal specimens to a genetic test. For overall agreement the lower 95% confidence interval limits were at or above 96% in the two comparisons, further stressing the reliability of the HER2 CISH pharmDx™ in this comparison to the two FISH analysis methods.Previously published studies have reported high concordance between pecimens ,15-19, hHER2 amplified or HER2 positive consecutively collected specimens reported in this study by the three ISH assays  and by HercepTest™ IHC (10.5%) seems to be lower than expected based on previously published data [The number of HER2 CISH compared to HER2 FISH and PathVysion FISH with no significant difference in HER2 signal counts between the three assays were observed. This resulted in significantly lower HER2/CEN-17 ratios observed for HER2 CISH. There was no clinical diagnostic impact of this change as the concordance agreement calculations revealed very fine agreement between HER2 CISH and the two FISH assays. In support of the good agreement the analysis of test results for specimens having a HER2/CEN-17 CISH ratio below 3.0 revealed that identical ratios were obtained for HER2 CISH, HER2 FISH and PathVysion FISH. Surprisingly, average copy numbers for both HER2 and CEN-17 were higher for the HER2 CISH method, whereas, HER2 CISH standard deviations seemed to be lower for the average HER2/CEN-17 ratio, HER2 and CEN-17 copy numbers compared to PathVysion FISH. This could be interpreted as the HER2 CISH assay being the most sensitive assay.In the current data analysis a significantly higher average CEN-17 signal counts in HER2/CEN-17 ratios in highly amplified specimens between HER2 CISH and FISH assays (see Figure Also, there was a tendency towards differences between HER2 CISH and PathVysion FISH and as could be expected the average evaluation time for a CISH staining was significantly shorter. This is most likely due to the easy access to the morphological information and hence a faster selection of areas for enumeration. In HER2 CISH a higher number of specimens were reported failed in comparison to staining with PathVysion FISH. This is likely to be due to CISH being a new technique implemented in the US reference laboratory and PathVysion FISH being a well established test method at this site. With respect to HER2 CISH the normal cells in the tissue surrounding the tumor area can be used as internal control securing good staining quality and preventing wrong diagnosis being based on a failed slide.As a parallel study, the slide evaluation time was compared between HER2 CISH and FISH data from 365 different primary breast cancer specimens it is confirmed that the FDA approved HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to today's FDA approved FISH techniques for HER2 gene status determination in FFPE breast carcinoma specimens.From this study based on JM, UH, SM and AS are employees at Dako Denmark A/S.The clinical trial was planned by UH, AS and JM. Study protocols were written by JM with help from UH and AS. Training of the US reference laboratory was performed by UH and SM. Data analysis was performed by JM and JM wrote the manuscript with help from UH, SM and AS. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/12/3/prepub


The mammalian Peg3 domain harbors more than 20 evolutionarily conserved regions (ECRs) that are spread over the 250-kb genomic interval. The majority of these ECRs are marked with two histone modifications, H3K4me1 and H3K27ac, suggesting potential roles as distant regulatory elements for the transcription of the nearby imprinted genes. In the current study, the chromatin conformation capture (3C) method was utilized to detect potential interactions of these ECRs with the imprinted genes. According to the results, one region, ECR18, located 200-kb upstream of Peg3 interacts with the two promoter regions of Peg3 and Zim2. The observed interaction is most prominent in brain, but was also detected in testis. Histone modification and DNA methylation on ECR18 show no allele bias, implying that this region is likely functional on both alleles. In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3. Overall, these results indicate that the promoters of several imprinted genes in the Peg3 domain interact with one evolutionarily conserved region, ECR18, and further suggest that ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element. In mammals, a small subset of autosomal genes are expressed mainly from one parental allele due to an epigenetic mechanism termed genomic imprinting . These gcis-regulatory elements similar to other imprinted domains . The converted DNA was used for PCR amplification using the two following primers: ECR18-bis-a  with the DMEM plus GlutaMAX medium containing 5% fetal bovine serum and 1% antibiotic-antimycotic (GibcoBRL). On the following day, the cells were transfected with 1 µg of each reporter construct using 2.5 µL Lipofectamine 2000 (Invitrogen). Fresh complete media was added 6 hrs post transfection, and total cell lysates were harvested in 100 µL of 1X reporter lysis buffer 48 hrs post transfection (Promega). The luciferase assay was performed for each promoter construct in triplicate according to the company’s protocol (Promega). We also performed a similar set of transfection experiments using an independent β-Geo reporter construct to monitor the transfection efficiency. Thus, the initial values from the luciferase assay were normalized with the β-Gal activity.For promoter assays, we first modified the promoterless β-Geo reporter  by replaMaterial S1Sequence information for 18 ECRs and oligonucleotides used for 3C and ChIP analyses.(DOCX)Click here for additional data file.