Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology.We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation , oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes.The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. Arachis hypogaea L.) is the second-most important legume in the world, with a total global production of 48 million tons [Cultivated peanut  as templates for probe design  was performed for all 14,352 probes present in the array using the Blast2GO search tool . Query aThe quality of the microarray was evaluated using the two comparisons: (1) two biological replicates of the same tissue-type labeled with the same dye and (2) the same tissue from two biological replicates labeled with either Cy3 or Cy5 dyes. The correlation coefficients of log transformed normalized ratios between the two replicates and two different dyes (dye-swap) were calculated by KaleidaGraph 3.6  Figures  and 2B. In recent studies, leaf, root, seed coat, and cotyledon tissues were utilized for global expression profiling in soybean ,8 and leGene expression profiles of different tissues provide information about the biological function of the genes expressed in those tissues ,26. For Due to paucity of information on peanuts in global repositories like NCBI, only half of the pod-abundant transcripts could be meaningfully annotated  number were mapped to twenty five different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Of these, 18 pathways relevant to pods were presented in Additional File Quantitative real time PCR has become a gold standard for the gene expression and generally used for validation of microarray results . To valiPeanut, being an under represented crop in terms of genome sequencing and physical mapping, needs a comprehensive tool for dissecting complex mechanisms of development and tolerance to biotic and abiotic stresses. To attain this broad objective, we have designed and characterized a high density oligonucleotide microarray suitable for transcript profiling of various peanut tissues. Analysis of pod abundant transcripts suggested the presence of distinct pathways involved in generation of secondary metabolites apart from the accumulation of transcripts for storage and desiccation-related protein. These peanut microarrays are publicly available and can be upgraded with additional oligonucleotides designed from subsequent sequencing efforts from the peanut research community. The expression profiles generated by these peanut microarrays will provide starting points for in-depth studies on candidate genes that can be utilized in reverse genetics to assign gene functions.Field grown plants of peanut cultivar FlavRunner 458 were used for tissue collection. The harvested tissue from leaves, pegs, stem, root and pods were immediately frozen in liquid nitrogen and stored at -80°C until further analysis.Total RNA from different tissue was isolated using the RNeasy Plant Minikit . Pooled frozen tissue from five plants were ground to a fine powder in liquid nitrogen and approximately 100 mg of homogenized tissue was used for total RNA isolation according to manufacturer's protocol, except the homogenized seed tissue was initially extracted in 600 μl of RLT buffer and during purification, samples were incubated in buffer RW1 for 5 min during the column washing step. RNA samples were treated with Turbo DNAfree  prior to cDNA synthesis.An aliquot of 450 ng of total RNA was used for cDNA synthesis utilizing the Low RNA Input Fluorescence Linear Amplification Kit (Agilent Technologies). Resulting cDNA was transcribed into cRNA and labeled with either cyanine 3 or cyanine 5-labeled nucleotides  using T7 RNA polymerase (Agilent Technologies). Labeled cRNA was purified with RNeasy Mini columns . The cRNA quality and quantity were determined spectrophotometrically using a NanoDrop ND-1000 spectrophotometer.in situ hybridization kit from Agilent Technologies. A total of 5 tissue samples were compared in three biological replicates with dyes swapped in the second biological replicate. Arrays were incubated at 65°C for 17 h in rotating hybridization chamber. Arrays were washed at room temperature under constant agitation for 10 minutes in 6× SSC with 0.005% Triton X-102 followed by a 5 minutes in cold, 0.1× SSC, 0.005% Triton X-102.Labeled cRNA from pod tissue was hybridized in combination with different tissues Figure  using th® 4000B microarray scanner at 5-μm resolution and images were saved as uncompressed tagged image files. For detection of significant differentially expressed genes, each slide image was processed by Agilent Feature Extraction software (version 9.1). This software measured Cy3 and Cy5 signal intensities of whole probes. Since dye bias tends to be signal intensity-dependent, probe sets for dye normalization were selected by rank consistency. Normalization was done by locally weighted linear regression (LOWESS). Ratios were log-transformed and significance values  were calculated based on a propagate error model and universal error model. In this analysis, the threshold of significant differentially expressed genes was determined with a p-value ≤ 0.05 . Low-quality spot data generated due to artifacts were eliminated prior to data analysis. Processed intensities from feature extraction analysis were imported into the TIGR Multiexperiment Viewer software (MEV 4.1) and significant genes at a p-value of ≤ 0.05 and more than two-fold difference in expression were defined as differentially expressed.Arrays were scanned using a GenePixThe Gene Ontology functional annotation tool Blast2GO  was utilTotal RNA samples were treated with Turbo DNAfree  prior to cDNA synthesis. One microgram of total RNA was used to synthesize first strand cDNA using SuperScript First Strand Synthesis system for RT-PCR . The primers for pod abundant genes and actin standard were designed using Integrated DNA Technologies primer designing tools. The efficiency of the primer pairs was determined on cDNA derived from the pod of FlavRunner 458 cultivar using a 1:2 serial dilution series. Primer efficiency reactions were performed in triplicate in volumes of 25 μL using SuperArray SYBRGreen reaction mix . Reactions were subjected to real-time qRT-PCR using the Roche LightCycler 480 Real-Time PCR System and data analyzed using the LightCycler 480 quantification software  .nd derivative maximum analysis with the arithmetic baseline adjustment. Crossing point values for each gene were normalized to the respective crossing point values for the reference gene actin. Data are presented as normalized ratios of genes along with error standard deviations estimated using the Roche Applied Science E-method [Samples were analyzed in a 25 μL volume using the Roche LightCycler 480 . Reactions were performed in triplicate using cDNA templates from five tissues samples for each gene. A master mix of SYBRGreen and primers was prepared for each primer pair. RT-PCR reactions were performed on 40 ng total RNA with 400 nM specific primers under the following conditions: one cycle of denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec (denaturation) and 60°C for 15 sec . The PCR reaction was followed by a melting curve program (60 – 95°C with a heating rate of 0.1°C per second and a continuous fluorescence measurement) and then a cooling program at 40°C. Negative controls lacking reverse transcriptase were run with all reactions. PCR products were also run on agarose gels to confirm the formation of a single product at the desired size. Crossing points for each transcript were determined using the 2E-method .PP was responsible for the conception and design of the experiment and final revisions of the manuscript. PP and KRK designed the array and performed all data analysis and interpretation. KRK carried out the tissue collection, performed RNA extractions, array hybridizations, and real-time PCR. DR and WF assisted in tissue collection and participated in data interpretation and preparation of the manuscript. BG generated cDNA libraries and contributed to array design and preparation of the manuscript. MB and NP provided seed and contributed to data analysis and manuscript preparation. MG contributed to the conception of the experiment and manuscript preparation. All authors have read and approved the final manuscript.Differentially expressed tissue specific genes. This table includes the list of statistically significant (p < 0.05) differentially expressed genes, including fold changes and functional descriptions, for leaf, stem, peg, and root tissue-specific genes.Click here for filePod abundant transcripts compared to all other tissues. The data provided represent the list of differentially expressed pod abundant transcripts. GO mapping and annotation of probe sequences was performed by Blast2go tool (version 2.2.3).Click here for filePathways catalyzed by pod specific enzymes. This figure includes eighteen different pathways catalyzed by 12 pod specific enzymes.Click here for file


Apoptosis is a cell suicide programme characterised by unique cellular events such as mitochondrial fragmentation and dysfunction, nuclear condensation, cytoplasmic shrinkage and activation of apoptotic protease caspases, and these serve as the noticeable apoptotic markers for the commitment of cell demise. Here, we show that, however, the characterised apoptotic dying cancer cells can regain their normal morphology and proliferate after removal of apoptotic inducers. In addition, we demonstrate that reversibility of apoptosis occurs in various cancer cell lines, and in different apoptotic stimuli. Our findings show that cancer cells can survive after initiation of apoptosis, thereby revealing an unexpected potential escape mechanism of cancer cells from chemotherapy. Chemotherapy is one of the major cancer treatments by promoting cancer cells into different types of cell death including apoptosis . Althoug−1 penicillin and 100 μg ml−1 streptomycin , at 37°C under an atmosphere of 5% CO2/95% air. Human prostate cancer PC3 cells were cultured in the same condition with RPMI-1640 medium. Cells were seeded on tissue culture plates until the cell density reached 70% confluency before being subjected to each experiment. Apoptotic stimuli jasplakinolide , staurosporine  and ethanol  were applied to the cells.Human cervical cancer HeLa cells, skin cancer A375, liver cancer HepG2, breast cancer MCF7 cells (from American Type Culture Collection) were cultured in DMEM  supplemented with 10% heat-inactivated FBS , 100 U mlM MitoTracker Red CMXRos (Invitrogen) and 250 ng ml−1 Hoechst 33342 (Invitrogen), respectively for 20 min, and the cells were washed two times with PBS and then cultured in suitable fresh medium (Invitrogen).Cells were grown to 70% confluence on a glass coverslip . Mitochondria and nucleuses were stained with 50 n2-independent medium (Invitrogen) on a thermo-cell culture FCS2 chamber  mounted onto the adapter in the stage of an inverted fluorescence microscope Cell Observer . Cell morphology was visualised by differential interference contrast (DIC) microscope device, and the mitochondria and nucleuses were by fluorescence with excitation 561 and 405 nm, respectively. Drugs and culture medium were introduced to the cell culture chamber through the perfusion tubes (Bioptechs) connected to the cell chamber. Cell images were captured with a monochromatic CoolSNAP FX camera  using a × 63 numerical aperture (NA) 1.4 Plan-Apochromat objective (Carl Zeiss), and analysed by using AxioVision 4.2 software (Carl Zeiss).Cells were cultured in COμm interval between each focal plane. The images were analysed by using LSM image examiner software (Carl Zeiss).Confocal cell images were captured with an inverted laser-scanning microscope LSM 5 LIVE (Carl Zeiss), with 1 One thousand cells were grown in a 96-well plate for 24 h, and then treated with different conditions. At each designed time point, cells were subjected to the corresponding assays according to the manufacturer's instructions. The activity of effector caspases was measured by using the homogeneous caspase assay kit . The activity of mitochondria was measured by 3--2,5 -diphenyltetrazolium bromide (MTT) assay (Sigma). The cell survival was detected by the cell proliferation ELISA BrdU assay kit (Roche). Results of assays were measured by SpectraMax 250 microplate reader .The morphology of the cells was observed by DIC, whereas mitochondria and nucleuses were visualised by fluorescence microscopy. At least 100 cells were examined in at least three independent cell counting.For cell proliferation, at each indicated time point, the cells were harvested by trypsinisation and thoroughly resuspended. The cells were stained with trypan-blue and counted in triplicate under a microscope with hemocytometer. Total cell number is calculated by multiplying the determined cell density with the total volume of suspension.μg protein per lane was separated on a 10% SDS-PAGE gel and transferred onto a Hybond ECL® membrane . After blocking, the membrane was incubated overnight at 4°C with 1 : 1000 anti-caspase-3 antibody  followed by another hour of incubation in the corresponding horseradish peroxidase-conjugated secondary antibody  at 1 : 5000 dilutions, and the signal was detected with the ECL western blotting detection system (Amersham Biosciences).Approximately 3 Our aim was to induce cancer cells undergoing apoptosis, and investigate whether the apoptotic dying cancer cells could survive after the removal of apoptotic reagents. We initially exposed human cervical carcinoma HeLa cells to an apoptotic inducer jasplakinolide  and 2. TInterestingly, our results indicate that HeLa cells could actually survive after the apoptotic induction. Our living cell microscopy showed that the shrunk cells regained their normal cellular morphology in 14.5 h after the wash . Our conOur discovery on reversibility of jasplakinolide-induced apoptosis in HeLa cells evoked a novel phenomenon that cancer cells could survive after initiation of apoptosis, and therefore prompted us to investigate whether this was a general phenomenon in different apoptotic inductions and in various cancer cell lines. Our results on other apoptotic stimuli to HeLa cells showed that, in response to the induction of apoptotic inducers ethanol and staurosporine . After rIn this study, we have shown that cancer cells could survive after initiation of apoptosis induced by different stimuli, and the reversibility of apoptosis was observed in various cancer cell lines. We provided evidence that cancer cells could escape from demise even after the cells undergoing critical apoptotic events such as mitochondrial fragmentation and dysfunction, nuclear condensation, cell shrinkage and activation of caspases. Importantly, the reversibility of apoptosis was abolished when the cells reached the apoptotic event of nuclear fragmentation, suggesting that this is an important cellular event indicating the point-of-no-return in apoptosis.Our discovery on the reversibility of apoptosis in cancer cells lead to several unanswered key questions: what are the components of the machinery driving the reversibility of apoptosis, and how are they linked to the regulation of apoptosis in cancer cells as a whole? To what extent does the reversibility of apoptosis contribute to the survival and repopulation of cancer cells during the cycles of anticancer treatment? Intriguingly, can inhibition on the reversibility of apoptosis in cancer cells suppress cancer relapse? Providing answers to these questions will be critical in understanding the mechanism for regulation on the reversibility of apoptosis, and provide us new potential targets for therapeutical advancement to our war of cancer. A more in-depth analysis will be required to clarify these points.


Chronic obstructive pulmonary disease (COPD) is characterized by both airway remodeling and parenchymal destruction. The identification of unique biomarker patterns associated with airway dominant versus parenchymal dominant patterns would support the existence of unique phenotypes representing independent biologic processes. A cross-sectional study was performed to examine the association of serum biomarkers with radiographic airway and parenchymal phenotypes of COPD.Serum from 234 subjects enrolled in a CT screening cohort was analyzed for 33 cytokines and growth factors using a multiplex protein array. The association of serum markers with forced expiratory volume in one second percent predicted (FEV1%) and quantitative CT measurements of airway thickening and emphysema was assessed with and without stratification for current smoking status. Significant associations were found with several serum inflammatory proteins and measurements of FEV1%, airway thickening, and parenchymal emphysema independent of smoking status. The association of select analytes with airway thickening and emphysema was independent of FEV1%. Furthermore, the relationship between other inflammatory markers and measurements of physiologic obstruction or airway thickening was dependent on current smoking status.Airway and parenchymal phenotypes of COPD are associated with unique systemic serum biomarker profiles. Serum biomarker patterns may provide a more precise classification of the COPD syndrome, provide insights into disease pathogenesis and identify targets for novel patient-specific biological therapies. While airflow obstruction is the hallmark of chronic obstructive pulmonary disease (COPD), the distinct processes of parenchymal destruction and small airway fibrosis can induce similar impairments in expiratory flow. Varying contributions of airway remodeling and emphysema can be found in any individual, leading to significant heterogeneity in disease expression Although the presence of systemic inflammation in COPD is widely accepted The following is an abridged version of the The study protocol was approved by the University of Pittsburgh Institutional Review Board. Participating subjects provided written informed consent for research use of their CT scans and blood samples.Two hundred and thirty-four participants were selected from the Pittsburgh Lung Screening Study (PLuSS) cohort. Participants were current or former smokers ages 50–79 and were selected to represent the spectrum of visual radiographic emphysema and airflow obstruction . SubjectSpirometry was performed on all subjects upon entry into the PLuSS cohort. Testing was performed using standard methodology The subjects underwent low-dose CT examinations performed on either a LightSpeed Plus 4-detector (n = 110) or LightSpeed Ultra 8-detector (n = 124) . The CT examinations were acquired using a helical technique at 120 or 140 kVp with a mean tube current-time product of 28.9 (+7.9) mAs. Images were reconstructed contiguously at 2.5 mm section thickness with a 2.5 mm interval using a GE Healthcare high-spatial frequency kernel with a range of pixel dimensions from 0.54 to 0.98 mm.The apical bronchus of the right upper lobe was manually selected from the CT images and analyzed in cross-section. Wall area as a percentage of total airway area (WA%), which has been associated with lung function Stored serum samples were analyzed for thirty-three serum chemokines and growth factors using a bead-based cytometric immunoassay system . A detailed description of the methods of the multiplex assay performed at the Core facility has been described previously by others Continuous data were summarized as either mean ± standard deviation or median and quartiles and categorical data were expressed as percentages. The association between the forced expiratory volume in the first second percent predicted (FEV1%) and WA% and F-950 was analyzed using univariate linear regression analysis. The contribution of WA% and F-950 to FEV% was then determined using multiple regression analysis. Finally, the relationship between WA% and F-950 was assessed with the Pearson's correlation coefficient.Serum biomarkers with concentrations above or below the detection threshold of the assay were respectively assigned the highest or lowest extrapolated value for that given marker . BecauseThe selected cohort consisted of 149 current and 85 former smokers . The subBoth WA% and F-950 were correlated with FEV1% in univariate analysis  and each CT parameter independently contributed to FEV1 decline in multivariate analysis (r = 0.6). Although there was a strong association of airway thickening and parenchymal emphysema CT phenotypes with FEV1%, there was no association of the CT phenotypes to each other  suggesting that each parameter represents an independent, unique phenotype .Four of the 33 markers were modestly associated with FEV1% (p = <0.05) before and after adjustment for current smoking status . EotaxinSeven of the 33 biomarkers were associated with WA%. Interleukin (IL) -6, IL-13, IL-2 receptor, Interferon gamma (IFN-γ), and c-reactive protein (CRP) were directly associated with WA% while regulated on activation normal T cell expressed and secreted (RANTES) was inversely associated with this CT parameter . NotablyThree of the 33 inflammatory markers, IL-6, MMP-7 and tumor necrosis factor alpha (TNF-α), were associated with quantitative emphysema . While IStratified analysis by smoking status revealed a relationship between specific biomarkers and FEV1%  that difLikewise, the relationship between serum biomarkers and WA% in current versus former smokers demonstrated associations dependent on current smoking status . While IRecently, the paradigm has shifted away from viewing COPD as a single disease entity to viewing it as a heterogeneous syndrome with variable contributions of peripheral airway fibrosis and emphysema. Our study illustrates this heterogeneity in a cohort of high-risk smokers and demonstrates systemic inflammatory patterns distinctive to individual anatomic phenotypes. Although the presence of a generalized systemic inflammatory response in individuals with both stable and progressive COPD has been well established The severity of airway remodeling and parenchymal emphysema can be characterized with quantitative CT analysis. Histological-radiological correlate studies have demonstrated a relationship between the extent of low attenuation areas depicted on CT images and histologic emphysema Our findings of distinct inflammatory patterns associated with airway thickening and parenchymal emphysema supports the existence of unique biological processes contributing to the syndrome of COPD. Of the markers demonstrating a significant association with airway thickening, only EGFR demonstrated an association with FEV1 and only IL-6 was simultaneously associated with parenchymal emphysema. Because our study focused on the measurement of serum biomarkers, we can not know whether the patterns of peripheral inflammation reflect a “spill-over” of the local inflammatory milieu within the lungs or represent a synchronous, systemic molecular diatheses associated with either airway or parenchymal disease. The significant but modest associations imply that a complex interplay of chemokines and growth factors, rather than one or two inflammatory mediators, is associated with the development of individual phenotypes.In a complex disease where pathogenic processes progress after cessation of cigarette smoking Although the multiple simultaneous measurements enabled by the high-throughput nature of Luminex technology increases the probability of type I error, we did not correct for multiple comparisons in our analysis. A formal adjustment would minimize the number of false positive findings but would also increase the probability of missing clinically meaningful associations. In this exploratory study, we did not want to overlook possible relationships and instead focused on those markers that demonstrated both biological plausibility and consistency in their associations with CT parameters. For instance, we found that IL-13, a cytokine associated with lung In conclusion, our study illustrates the heterogeneity of the COPD syndrome exemplified by independent variability of airway remodeling and parenchymal destruction and demonstrates an association between distinct quantitative CT phenotypes and serum inflammatory biomarker patterns. Many of these associations varied according to current cigarette use, suggesting a complex interplay of inflammation and environment leading to the variable expression of airway disease and obstruction. The traditional definition of chronic airflow obstruction fails to recognize the diversity of biologic processes represented by varying patterns of disease expression, which most likely will vary in response to molecular therapeutics. The challenge is to develop tools that precisely classify individuals based on their unique pathophysiologic phenotypes. Whereas quantitative CT indices of emphysema and airway remodeling have been shown to correlate with physiology and histology, the identification of inflammatory markers that segregate with anatomic phenotypes further validates and facilitates more robust disease sub-classification and provides further insight into molecular-cellular mechanisms, potential therapeutic targets and easily measured surrogates of disease activity.Methods S1(0.04 MB DOC)Click here for additional data file.Table S1Distribution of eligible subjects by GOLD classification and semi-quantitative emphysema score  N = 3297(0.03 MB DOC)Click here for additional data file.Table S2Distribution of subjects randomly selected from each of the 9 strata formed by cross-classification according to GOLD and emphysema score N = 260(0.03 MB DOC)Click here for additional data file.Table S3Distribution of final subjects selected for analysis by GOLD and emphysema score N = 234(0.03 MB DOC)Click here for additional data file.Table S4Number of out-of-range (OOR) values and values assigned for 33 markers(0.05 MB DOC)Click here for additional data file.Table S5Regression co-efficients for associations between log-transformed serum analyte levels and WA and F-950(0.05 MB DOC)Click here for additional data file.


TSHR intron 1.The thyroid stimulating hormone receptor (TSHR) gene is an established susceptibility locus for Graves' disease (GD), with recent studies refining association to two single nucleotide polymorphisms (SNPs), rs179247 and rs12101255, within −2–6.2×10−15, OR = 1.38–1.45) and rs12101255  exhibited strong association with GD in all three cohorts. Logistic regression suggested association of rs179247 is secondary to rs12101255 in all cohorts. Inheritance modeling suggested a co-dominant mode of inheritance in all cohorts. Genotype-phenotype correlations provided no clear evidence of association with any specific clinical characteristics.We aimed to validate association of rs179247 and rs12101255 in Polish and UK Caucasian GD case-control subjects, determine the mode of inheritance and to see if association correlates with specific GD clinical manifestations. We investigated three case-control populations; 558 GD patients and 520 controls from Warsaw, Poland, 196 GD patients and 198 controls from Gliwice, Poland and 2504 GD patients from the UK National collection and 2784 controls from the 1958 British Birth cohort. Both rs179247 (P = 1.2×10TSHR intron 1 SNPs with GD in three independent European cohorts and have demonstrated that the aetiological variant within the TSHR is likely to be in strong linkage disequilibrium with rs12101255. Fine mapping is now required to determine the exact location of the aetiological DNA variants within the TSHR.We have validated association of  TSHR region may influence TSHR structure, expression and/or post-translational processing, which in turn could initiate or exacerbate the autoimmune response against the TSHR in GD.The thyroid stimulating hormone receptor (TSHR) is primarily expressed on the thyroid follicular cell surface membrane and via its ligand, TSH, is one of the key regulators of thyroid growth and hormone production. TSHR autoantibodies with either stimulatory (TSAb) or blocking (TBAb) activity are a key feature of autoimmune thyroid disease (AITD), with TSAb having a pre-dominant effect in Graves' disease (GD) leading to hyperthyroidism TSHR genetic studies focused on several non-synonymous single nucleotide polymorphisms (nsSNPs) and despite a number of studies, no replicable GD associations emerged TSHR intron 7 in Japanese and TSHR intron 1 in UK Caucasian cohorts, which provided the first convincing evidence for TSHR association with GD TSHR nsSNP rs3783941 (Arg248Ser) with GD in a genome wide screen of 15,000 nsSNPs in 900 UK Caucasian GD cases and 1500 controls TSHR region, refined association to within 40Kb of TSHR intron 1 in 768 GD patients and 768 controls of UK Caucasian origin −8, OR = 1.53) and rs12101255 , with logistic regression suggesting these SNPs or variants in strong linkage disequilibrium (LD) may drive the association signal in the region Original All GD patients in the cohorts investigated in the current study were unrelated and gave informed written consent and the project was approved by the relevant local research committees, including; the South Birmingham Research Ethics committee (MRec/01/7/60), Medical Research Centre, Polish academy of science ethics committee and the Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Gliwice Branch ethics committee.Two independent Polish GD case-control collections were investigated in the current study. The first, from Warsaw, included 558 Caucasian GD patients recruited from the Department of Endocrinology, Medical University of Warsaw, Poland. The second was from Gliwice, and consisted of 196 Caucasian GD patients, ascertained at the Department of Nuclear Medicine and Endocrine Oncology, Centre of Oncology, Gliwice, Poland. Criteria for clinical diagnosis were the same for both collections, which were based on clinical and biochemical evidence of hyperthyroidism, confirmed by the presence of diffuse goiter, detectable TSHR autoantibodies  and/or increased radioiodine uptake, as described previously The UK cohort consisted of 2504 GD patients which make up part of the AITD UK national collection. Of these, 308 GD patients were investigated previously Genotyping of rs179247 and rs12101255 was performed using TaqMan® SNP genotyping technology  according to the manufacturer's protocol, independently in the three respective centres.2 and LD blocks were subsequently defined using the Gabriel et al algorithm http://www.broad.mit.edu/mpg/haploview) Prospective power calculations in both Polish and UK cohorts demonstrated we had >99% power (α = 0.05) to detect a significant difference in allele frequencies between cases and controls, assuming an OR = 1.50 as reported previously vs ≥31 years), severity of ophthalmopathy based on NOSPECS classification (NOSPECS 0 – 1 vs NOSPECS 2 – 6) and smoking status (smokers vs non-smokers).Association analysis of rs179247 and rs12101255 with specific clinical manifestations of GD were performed in all 3 cohorts for; age of GD diagnosis (≤30 years −5–9.0×10−3 and OR = 1.42–1.45). Similarly, the T allele of rs12101255 displayed an increased frequency in both Warsaw and Gliwice GD patients compared to controls   and rs12101255  . In the Warsaw and Gliwice cohorts allele A of rs179247 was more frequent in GD patients (P = 6.0×10ctively) . As ther4–1.84]) .−14, OR = 1.38 [95% CI = 1.27–1.49]) and rs12101255 allele T . To determine whether the strong associations could be explained by either a dominant or recessive effect, we modeled the mode of inheritance for these variants and found that neither, dominant or recessive models improved association, suggesting a co-dominant effect within the region , with three common  ≥0.11) haplotypes observed . Interestingly, if the associated allele at rs12101255 was changed to the non-associated rs1210255 (C) and rs179247 (A) remained the same (haplotype 2) there was no association in any population , suggesting rs12101255 (A) may drive the association within haplotype 1. The non-associated alleles of rs179247 (G) – rs12101255 (C) make up haplotype number 3 and display an increased frequency in controls compared to GD patients.The two SNPs demonstrated strong but incomplete LD , whereas rs179247 became non-associated after conditioning for rs12101255 . This suggests association at rs179247 maybe driven by rs12101255 or that rs12101255 is in stronger LD with the aetiological variants within the region.Since haplotype analysis suggested an important role for rs12101255 in driving association, we performed logistic regression to try and split association between the two SNPs. In both the combined Polish data set and in the UK national collection both SNPs demonstrated strong association when entered into the model (co-dominant), as expected. However, after conditioning on each SNP, rs12101255 remained associated after conditioning for rs179247  and rs12101255 (T) were increased in GD patients compared to controls with mode of inheritance suggesting a co-dominant effect. The magnitude of genetic effects observed in the current study are similar (OR≈1.50) to those observed in the original report TSHR represents a major susceptibility locus for GD in-line with other previously identified non-HLA susceptibility loci CTLA4PTPN22The present study provides convincing replication and further association mapping of TSHR introns 7 and 8 strongly associated with GD TSHR (800Kb) implementing Tag SNPs in a UK Caucasian cohort, ruled out any association within introns 7–8 in Caucasians TSHR intron 1 association in European Caucasian GD patients. Further studies are now required in Japanese and other ethnic backgrounds to independently confirm previous reports of association within TSHR intron 7. Indeed, it should be noted that Hiratani et al investigated 3 variants within intron 1 and identified some evidence of association with TSHR intron 1 SNP, rs2268474 (P = 0.026) in the Japanese Previous Japanese investigations identified numerous SNPs within TSHR intron 1 we attempted to resolve the issue of whether the two SNPs had independent effects or if the effect of one SNP was secondary to LD with the other. Logistic regression in all cohorts suggested that the effect of rs179247 could be secondary to rs12101255. This conclusion was consistent with single SNP analysis, which demonstrated a greater magnitude of genetic association with rs12101255 compared to rs179247 in all our cohorts. Furthermore, haplotype analysis demonstrated that the associated allele T of rs12101255 drives the highly associated haplotype of rs179247 (A) and rs12101255 (T). The increased size of the National AITD cohort of 2504 GD patients compared to the smaller dataset of 768 UK Caucasian GD investigated previously has allowed differentiation between these two highly associated SNPs Since rs179247 and rs12101255 are located 18.5Kb apart within TSHR intron 1 SNPs with GD in three European Caucasian data sets and for the first time association in Polish GD subjects. Detailed investigation of all genetic variants within the region to identify the aetiological variant/s is now required, which may lead to advances towards developing TSHR targeted therapeutics.In conclusion, our results confirm strong association of Table S1Clinical Characteristics of Polish GD patients from the Warsaw and Gliwice cohorts and the UK GD National Collection. Shows the number of patients and percentage in brackets where appropriate, possessing specific GD clinical phenotypes in Warsaw, Gliwice Polish cohorts and UK GD National Collection.(DOC)Click here for additional data file.Table S2Association test results for rs179247 and rs12101255 assuming either dominant or recessive modes of inheritance. The table displays association tests for rs179247 and rs12101255 in the GD case-control cohorts from Warsaw, Gliwice, Polish combined and UK GD national collection, assuming either dominance or recessive mode of inheritance. Allele counts are compared by means of the χ2 test of significance, with the χ2 P displayed. P<0.05 considered indicative of a significant difference between GD cases and controls.(DOC)Click here for additional data file.Table S3Shows the genotypes of rs179247 with specific GD clinical features. Each clinical feature is compared with genotype counts in the both two Polish and UK cohorts, using the following segregations; age of GD onset (<30 years old vs ≥30 years old), severity of ophthalmopathy determined by NOSPECS classification (NOSPECS <2 vs NOSPECS ≥2), smoking status (current or previous smoker vs non-smoker) and in the UK GD National Collection cohort only, thyroid antibody status whether positive for one of either thyroid peroxidise, thyroglobulin or TSHR (positive vs negative) and presence or abscence of a difuse, palpable goitre (presence vs abscence). The Chi-squared test P-value comparing genotype and allele counts is displayed. * = P<0.05.(DOCX)Click here for additional data file.Table S4Shows the genotypes of rs12101255 with specific GD clinical features. Each clinical feature is compared with genotype counts in the both two Polish and UK cohorts, using the following segregations; age of GD onset (<30 years old vs ≥30 years old), severity of ophthalmopathy determined by NOSPECS classification (NOSPECS <2 vs NOSPECS ≥2), smoking status (current or previous smoker vs non-smoker) and in the UK GD National Collection cohort only, thyroid antibody status whether positive for one of either thyroid peroxidise, thyroglobulin or TSHR (positive vs negative) and presence or abscence of a difuse, palpable goitre (presence vs abscence). The Chi-squared test P-value comparing genotype and allele counts is displayed. * = P<0.05.(DOCX)Click here for additional data file.


Animal studies have demonstrated that low paraoxonase-1 (PON1) status  is associated with neurotoxic effects after exposure to several organophosphate (OP) insecticides. However, few human studies have investigated associations between PON1 status and intermediate end points, such as serum cholinesterase [butyrylcholinesterase (BuChE)] inhibition, among OP-exposed individuals.We evaluated the relation between plasma PON1 status and BuChE inhibition among OP-exposed agricultural pesticide handlers.PON1 Q192R genotype. We evaluated percent change in BuChE activity from baseline level in relation to PON1 status.Agricultural pesticide handlers in Washington State were recruited during the 2006 and 2007 spray seasons when they were seen for follow-up ChE testing by collaborating medical providers as part of a statewide monitoring program. Blood samples were collected from 163 participants and tested for PON1 status based on plasma PON1 activity [arylesterase (AREase)] and p = 0.036). Lower levels of plasma PON1 activity were significantly associated with greater BuChE inhibition (p = 0.004). These associations remained after adjustment for year, days since baseline test, age, and OP exposure in the last 30 days.We observed significantly greater BuChE inhibition among QQ homozygotes relative to RR homozygotes  and low plasma PON1 activity were associated with BuChE inhibition among OP-exposed agricultural pesticide handlers. Corroborative findings from future studies with prospective collection of blood samples for PON1 testing, more sensitive markers of OP-related effects, and larger sample sizes are needed. N-methyl-carbamate (CB) insecticides inhibit cholinesterase (ChE) enzyme activity, including both serum ChE [butyrylcholinesterase (BuChE)] and erythrocyte ChE [acetylcholinesterase (AChE)]. OP/CB insecticides are widely used in agriculture in the United States and abroad. Washington State is the nation’s leading producer of apples and several other tree fruits, and OP/CBs are applied in many orchards in eastern Washington. In 2004, the Washington State Department of Labor and Industries (WADLI) initiated a ChE monitoring program for agricultural pesticide handlers who are exposed to toxicity category I or II OP/CBs (Organophosphate (OP) and I OP/CBs . In thisInhibition of AChE activity in the central and peripheral nervous systems is considered to be the main mechanism of OP/CB toxicity . In addiThe health effects of acute OP/CB poisonings have been well characterized , and a gPON1 coding region affects the catalytic efficiency of the enzyme for chlorpyrifos oxon metabolism. In a study by It is possible that some individuals may be especially susceptible to health effects related to OP/CB exposure. High-density lipoprotein–associated paraoxonase-1 (PON1) is thought to be one important determinant of an individual’s sensitivity to some OP insecticides, based primarily on evidence from studies in animal models . PON1 hya) PON1 status is most relevant for protection against direct exposure to the oxon forms of OP insecticides (b) most, if not all, OP exposures may include oxon residues (c) the safety studies for OPs such as chlorpyrifos were carried out with the highly pure parent compound  mostresidues ; and c) compound .Despite convincing evidence in animal models, few epidemiologic studies have evaluated PON1 status as a determinant of OP sensitivity. We recruited agricultural pesticide handlers from the Washington State ChE monitoring program for a study to test the hypothesis that PON1 status affects an individual’s risk of BuChE inhibition and is thus a biomarker of susceptibility.n = 25). For these individuals, the record with greater BuChE inhibition was included in the data set, and the other record was excluded . Analyses were also repeated with random selection of a single record for these individuals who participated in both years.Agricultural pesticide handlers in the statewide ChE monitoring program were recruited for this study from two collaborating clinics in eastern Washington during the 2006 and 2007 spray seasons (April–July). Blood samples for determination of PON1 status and self-reported OP/CB exposure information were collected at the time of follow-up ChE testing during the spray season. A total of 163 participants with nonhemolyzed blood specimens for PON1 testing and both baseline and follow-up ChE tests were included in this analysis. This sample represents 54% of the 304 handlers who were invited to participate. Study participants were similar to all handlers in the statewide ChE monitoring program in terms of age, race/ethnicity, and sex . We exclIn addition to collection of blood specimens for PON1 testing, participants were asked to complete a computer-based survey regarding OP/CB exposure in the preceding 30 days and demographic characteristics. Surveys were completed by 141 of the participants included in this analysis. Another 11 participants had partially complete surveys; nonmissing data from incomplete surveys were used where applicable.This study was conducted in compliance with all applicable U.S. requirements for research involving human subjects, and all study procedures were approved by the institutional review board at the University of Washington. Study participants provided written informed consent before participating in this study.PON1 coding region and level of plasma PON1 activity. Genotype at position 192 was determined using a previously established TaqMan-based genotyping assay  with genomic DNA extracted from EDTA-treated whole-blood specimens (p > 0.9).Handlers’ PON1 status was characterized based on genotype at position 192 in the pecimens . The genThe level of plasma PON1 activity was characterized by measuring arylesterase (AREase) activity in lithium heparin–treated plasma samples; AREase activity is considered to be a good surrogate for PON1 concentration in plasma , 2006. PAll study participants were tested for annual baseline BuChE activity  and at follow-up during the spray season as part of the statewide monitoring program. ChE test results were obtained from collaborating health care providers. Methods for collection and processing of blood specimens for ChE testing have been described previously . BecauseIn this study, we focused on BuChE inhibition for several reasons. First, BuChE is more sensitive than is AChE to inhibition by many OPs, including chlorpyrifos, which was the most widely used OP among study participants . Second,The primary aim of this study was to test the hypothesis that low PON1 status is a determinant of BuChE inhibition among OP-exposed pesticide handlers. For these analyses, PON1 status was characterized in terms of both observed Q192R genotype and level of plasma PON1 activity (AREase activity). The primary end point was percent change in BuChE activity comparing levels at follow-up during the OP/CB spray season with preseason baseline levels. We considered the degree of change in BuChE to be the most biologically relevant end point because of wide interindividual variability in baseline BuChE activity . We alsoWe performed several different analyses to characterize the relation between PON1 status and BuChE inhibition. First, we evaluated mean BuChE inhibition from baseline after stratifying by both Q192R genotype and tertiles of plasma PON1 activity. We compared the degree of BuChE inhibition within each stratum with the reference category of individuals who were hypothesized to have the lowest risk . Comparisons were based on linear regression with robust standard error estimates. We also performed linear regression with plasma PON1 activity modeled as a continuous predictor of BuChE inhibition after stratification by Q192R genotype.n = 19), we used birth dates from administrative records that were collected as part of the ChE monitoring program. Cumulative OP/CB exposure within the preceding 30 days was estimated quantitatively based on survey responses regarding OP/CB use, work activities, and personal protective equipment (PPE) use . Briefly, we calculated a score for OP/CB toxicity based on data from U.S. EPA cumulative risk assessments, and scores for work activities and PPE use based on algorithms developed as part of the Agricultural Health Study , time since baseline ChE test , age , and cumulative OP/CB exposure score .We performed several exploratory analyses in this study. Linear regression analyses were repeated after restricting to chlorpyrifos-exposed handlers, and after excluding handlers who reported only using CB insecticides. We also conducted linear regression analyses for BuChE inhibition in relation to Q192R genotype and level of plasma PON1 activity after stratifying by tertiles of OP/CB exposure to evaluate whether the relations between PON1 status and BuChE inhibition differed by degree of OP/CB exposure.p-values were < 0.05.All statistical analyses were performed using Intercooled Stata, version 9.2 . Findings were considered statistically significant if Characteristics of study participants are reported in n = 163; p < 0.001, paired t-test). Mean BuChE inhibition from baseline activity was 5.5% ; this is consistent with the degree of BuChE inhibition observed among all handlers in the statewide ChE monitoring program . Differences in AREase category frequencies within each Q192R genotype were not statistically significant .Overall, participants in this study had significantly lower BuChE activity at the time of follow-up during the OP/CB spray season relative to baseline  levels ( program . Relativ program . Handlern = 163). Based on this analysis, Q192R genotype appears to modify the relation between plasma PON1 activity and BuChE inhibition. A highly significant association between lower plasma PON1 activity and greater BuChE inhibition was observed among RR individuals . This suggests that even individuals with the high catalytic efficiency genotype (192R homozygotes) for chlorpyrifos oxon hydrolysis may still be at risk of BuChE inhibition if they have low plasma PON1 activity. There was a borderline significant relationship between plasma PON1 activity and BuChE inhibition among QR individuals , and we observed no association among QQ individuals . However, handlers with QQ genotype had significantly greater BuChE inhibition than did RR individuals (p = 0.036), which suggests that low catalytic efficiency of chlorpyrifos oxon hydrolysis may be a determinant of BuChE inhibition regardless of the level of plasma PON1 activity.n = 110, p = 0.03). We also observed a significant trend toward greater BuChE inhibition by number of Q alleles (p = 0.028). We observed a borderline significant association between level of plasma PON1 activity (modeled as a continuous predictor) and BuChE inhibition in the adjusted analysis (p = 0.053). Analyses stratified by year of participation indicated that the associations between Q192R genotype and BuChE inhibition within each year were similar to the results of the main adjusted analysis. Although we observed greater BuChE inhibition with decreasing AREase activity in both years, this association was stronger in 2006 than in 2007 . However, a test for interaction by year was not statistically significant (p = 0.529). Results of adjusted analyses evaluating follow-up BuChE activity were generally consistent with the results for BuChE inhibition . When we restricted the analyses to chlorpyrifos-exposed handlers, associations between BuChE inhibition and Q192R genotype and plasma PON1 activity after covariate adjustment were similar in magnitude but no longer statistically significant . Similarly, results were essentially unchanged after excluding 21 handlers who reported using only CB insecticides . When analyses were repeated after a single record from handlers who participated in both years was selected at random (rather than sampling based on BuChE inhibition), similar results were obtained (data not shown).We also evaluated the degree of BuChE inhibition in relation to Q192R genotype and plasma PON1 activity after adjusting for covariates . As in tn = 110, p = 0.036). Relative to individuals with high plasma PON1 activity, those with low plasma PON1 activity were three times as likely to have BuChE depression; this association was not statistically significant. However, when level of plasma PON1 activity was modeled as a continuous predictor, a statistically significant decrease in odds of BuChE depression was observed with increasing plasma PON1 activity (p = 0.042). There were relatively few cases of BuChE depression in the study population . Consequently, the confidence intervals for these odds ratios are fairly wide, and results should be interpreted with caution.As shown in We also performed several analyses to evaluate whether effect estimates for the association between PON1 status and BuChE inhibition differed by degree of OP/CB exposure. After stratifying by tertiles of OP/CB exposure score, we found that the magnitude of the association between QQ genotype (vs. RR genotype) and BuChE inhibition increased with greater OP/CB exposure . We observed no such trend in the relationship between plasma PON1 activity and BuChE inhibition after stratification by OP/CB exposure.PON1 Q192 alloform and low plasma PON1 activity were determinants of BuChE inhibition among OP-exposed agricultural pesticide handlers. These findings are consistent with expectations based on results from animal studies  are considered to be more highly exposed to pesticides than agricultural workers who perform other activities, this population is especially well suited for evaluating PON1-mediated susceptibility to OP exposure. Previous studies of PON1-related susceptibility among individuals with occupational OP exposure have relied on self-reported health outcomes such as chronic ill health  or symptThis study had several limitations. There is the possibility of uncontrolled confounding leading to inaccurate assignment of PON1 status because PON1 activity may be modified to some extent by certain medications , dietary habits , and environmental exposures  . HoweverAbsolute measurements of BuChE activity differed in 2006 and 2007 because of an administrative change in the laboratory conducting ChE testing for the statewide monitoring program. However, within each year baseline and follow-up ChE tests were performed by the same laboratory, so we were able to use the degree of BuChE inhibition from baseline levels as the main end point even though absolute BuChE measurements differed by year. Moreover, we included year of participation as a covariate in all adjusted models to control for differences by laboratory. Other newly developed biomarkers of OP exposure could potentially address concerns about BuChE activity measurements and may be studied in the future .The relatively low participation rate in our study (54%) may be another limitation. However, study participants were similar to all participants in the statewide ChE monitoring program in terms of demographic characteristics and the degree of BuChE inhibition observed , which sCompared with RR individuals, relatively few QQ individuals had low plasma PON1 activity. It is possible that these differences in plasma PON1 activity levels by Q192R genotype may be attributable to the healthy worker survivor effect . However, given the relatively low frequency of acute pesticide-related illnesses in this population , such diFinally, although we had a larger sample size than most previous studies of PON1-mediated OP sensitivity , this stIn this study we found that both low PON1 catalytic efficiency  and low plasma PON1 activity were associated with BuChE inhibition among OP-exposed agricultural pesticide handlers. Regulatory risk assessments should take differences in PON1-related sensitivity to OP insecticides into consideration when characterizing interindividual variability in risk related to OP exposure. At some point in the future, biologic monitoring for PON1 status among pesticide handlers may be warranted to identify individuals who are at particularly high risk of OP-related health effects. However, issues of test validity as well as the ethical and legal aspects of genetic testing in the workplace will need to be addressed before such a program could be implemented .a) collection of blood specimens for determination of plasma PON1 activity before, during, and after exposure periods; b) evaluation of both AChE and BuChE inhibition as end points; c) other newly developed biomarkers of OP exposure; and d) a larger sample size to assess interaction between the degree of OP exposure and PON1 status in terms of risk of BuChE inhibition.Findings from this study need to be confirmed in future studies, which would benefit from


The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.c. >95% concordance over 26 months for C57). Analyses of mice that had been relocated before and after maturity indicated marked, reproducible changes in fecal consortia and that occurred only in young animals. Implantation of a female BDF1 mouse with genetically distinct (C57 and Agoutie) embryos produced highly similar GTM profiles (c. 95% concordance) between mother and offspring, regardless of offspring strain, which was also reflected in urinary metabolite profiles. Marked institution-specific GTM profiles were apparent in C3H mice raised in two different research institutions.The GTM composition of six highly inbred, genetically distinct strains of mouse  was profiled using eubacterial –specific PCR-DGGE and quantitative PCR of feces. Animals exhibited strain-specific fecal eubacterial profiles that were highly stable (Strain-specific data were suggestive of genetic determination of the composition and activities of intestinal symbiotic consortia. However, relocation studies and uterine implantation demonstrated the dominance of environmental influences on the GTM. This was manifested in large variations between isogenic adult mice reared in different research institutions. The interplay between GTM and host is complex, with diverse positive and potentially negative implications for host health in the short and longer-term. Intestinal colonization-resistance for example, may reduce the incidence and severity of intestinal infections through limitation of adhesion sites and growth substrates and by the accumulation of inhibitory metabolites The gastrointestinal tract microbiota (GTM) of mammals supports a microbial ecosystem, which at up to 10Several recent investigations have demonstrated considerable integration of prokaryotic and mammalian metabolism with respect to polysaccharide metabolism Evidence is emerging to suggest a considerable degree of individuality and temporal stability within human and murine GTMs. Various studies using PCR-DGGE and cluster analyses of human Factors responsible for the development, maintenance and stability of individual-specific GTMs remain unclear. It is possible that, through the inherent stability and colonisation resistance of complex microbial climax communities, GTMs may maintain specific profiles through microbial competition, particularly during colonization versus environmental factors upon development, composition and maintenance of the GTM remains incomplete and there are relatively few reports addressing this in the literature, probably due to difficulties associated with experimentally separating environmental from genetic influences.Despite a considerable increase in interest in the function and natural history of the GTM and in strategies for its therapeutic compositional manipulation, insight into the broad contributions of host genetics et al.et al.The possible involvement of host genotype, particularly as it relates to immuno-phenotype, has been frequently postulated as a major influence on GTM composition and stability although this has been difficult to prove. For example, recent studies have investigated the possible correlation between host genetic relatedness and fecal microbial profiles in human adult twins. Zoetendal Despite the obvious usefulness of studies using human twins, they do not present a definitive means of separating genetic from environmental influences due to the frequent confounding influence of close social and familial contact. In the Stewart study for example et al.Further evidence for the importance of host genetics in the determination of host-microbe interaction is provided by a recent study by Khachatryan c. 12 months Whilst the environment represents a continuous microbial challenge to the GTM, it is believed that external influences upon the microbial composition of the GTM are greatest in nascent microbiotas, following birth. In new-borne mammals, the sterile intestine represents a readily colonisable environment, being subject to microbial immigration from sources such as the birth canal and faecal material Other environmental variables may significantly influence the composition of the GTM, including antibiotic use where it has for example been shown that oral ciprofloxacin dosing alters the abundance of approximately one third of GTM taxa, decreasing the richness and diversity with profiles returning to re-treatment states for most taxa 4 weeks after the end of treatment vs. environmental influences; i) the GTMs of age- and gender-matched mice were profiled, including isogenic murine strains maintained within separate locations and within two different research establishments; ii) relocation studies were conducted whereby immuno-competent and severely immuno-compromised mice were moved in order to assess the contributions of immunophenotype and consortial-intrinsic colonization resistance and finally, iii) the implantation of a female BDF1 mouse with embryos from two genetically distinct mouse strains effectively differentiated between the effects of genetic and environmental influences upon fecal eubacterial profiles and urinary metabolites in co-gestated animals. Better understanding of the factors that influence the stability and composition of the murine GTM is likely to have significant implications for use of experimental animal models but may also be of fundamental importance in terms of general understating of the development and composition of mammalian (and thus human) GTMs.In the current investigation, we have utilized six strains of highly inbred experimental mice, which, through considerable genetic relatedness between siblings and amenability to environmental manipulation, represent useful models for the evaluation of intrinsic and extrinsic influence upon the GTM composition. In order to study the influence of host genetics The composition and activities of the mammalian GTM significantly influences host physiology including immune function Better understanding of strain and location-dependant variation in murine GTMs has obvious implications for the use of animals as experimental models. Additionally, however fecal material from the highly inbred mouse strains can be used to represent paradigm vertebrate microbiotas which could provide fundamental insights into factors influencing the development and maintenance of GTMs.Cluster analysis of fecal denaturing gradient gel electrophoresis (DGGE)-derived microbial fingerprints was used to compare the GTMs of mature female mice from the following mouse strains: C3H, C57, GFEC, CD1, CBA nu/nu, and SCID, which were selected as paradigm genetically distinct mice. The use of CBA nu/nu and SCID mice additionally enabled specific immune-impairment to be studied since these animals lack T cells and T and B cells, respectively. GTM fingerprints from replicate (n = 3–6) animals, housed at the University of Manchester (designated environment A) clustered together, based on mouse strain with inter-strain concordance, ranging from 71 to 100% and intra-strain concordance between 50 and 55% . This obc. 4 or 8 weeks of age. This resulted in marked changes in GTM profiles that were only manifested in immature animals, as evidenced by the distinct clustering of GTM profiles in these animals  and three C57 embryos. Fecal samples were collected from the offspring and mother at weekly intervals following weaning (3 weeks of age) until 10 weeks of age. Fostered, genetically distinct progeny (Ag and C57) could not be differentiated from the BDF1 mother based on GTM fingerprints , with deIn order to examine the practical implications of the purported major influence of environment upon GTM profiles and for corroboration, feces from C3H mice obtained from Stanford University and The University of Manchester breeding houses was subjected to PCR-DGGE and cluster analysis. Data generated in this manner indicated that mice from the Manchester breeding house had substantially different GTM profiles from C3H mice at Stanford University , GFEC , BDF1 , CBA nu/nu and CD-1 nu/nu (both T-cell deficient), and SCID mice (T- and B-cell deficient). All strains are bred in-house with the exception of the C57, CD-1 nu/nu and SCID mice that were purchased at >4 weeks old from Charles River, UK.Mature Mice (n = 6) from two distinct strains  were selected for this experiment. The mice were divided into two groups (n = 3 in each group) and identified by means of ear tagging. One group of mice were relocated to environment B  upon reaching maturity (8 weeks of age), whilst the other group remained in the original breeding environment (environment A). Fecal samples were collected weekly from all individuals and subjected to 16S PCR-DGGE. Fingerprints were sorted using cluster analysis. Mice were transported and housed in individual autoclaved housing units within the quarantine area of environment B, and fed the same autoclaved food. This was repeated from immature animals whereby mice (n = 3) of two genetically distinct strains  were selected for the experiment. Upon weaning (4 weeks of age), all mice were moved to environment B. In all cases, fecal samples and urine were collected weekly from all individuals and archived at −70 for subsequent analysis.Agoutie and C57 mouse embryos at the blastocyte stage were implanted into a pseudo-pregnant BDF1 strain mother. Mice were born naturally and allowed to remain with the surrogate mother until weaning. At this stage the mother was removed and sacrificed for health screening. One fecal sample was collected from the surrogate mother before she was removed and fecal samples were collected from the offspring from weaning until nine weeks of age.Fecal samples were collected from individual mice directly into a sterile Eppendorf tubes, weekly from 4 weeks of age. Samples were then frozen within 2 hours of receipt. DNA was extracted using a Qiagen stool mini DNA extraction kit  with the added step of using a mini bead beater to homogenize the sample. Glass beads were added to the feces with sterile buffer (provided in Qiagen kit) and mixed for 90s. The amount and quality of DNA extracted was estimated by electrophoresis of 5-µl aliquots on a 0.8% agarose gel and comparison to a molecular weight standard (stained with ethidium bromide). DNA extracts were stored at −60°C prior to analysis in nuclease-free containers.5′-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3′) and HDA2 (5′-GTATTA CCG CGG CTG CTG GCA C-3′) as previously described (40). The reactions were performed in 0.2-ml tubes with a DNA thermal cycler . In all cases, reactions were carried out with Red Taq DNA polymerase ready mix , HDA primers , nanopure water (16 µl), and extracted community DNA . The thermal program was 94°C (4 min) followed by 30 thermal cycles of 94°C (30 s), 56°C (30 s), and 68°C (60 s). The final cycle incorporated a 7-min chain elongation step (68°C). Electrophoresis was carried out at 150 V and 60°C for approximately 4.5 h. Gels were stained with SYBR Gold stain  for 30 min. Gels were viewed under U.V illumination and RAW images captured using a Canon D60 DSLR camera with a 50mm macro lens with UV filter attached.The V2-V3 region of the 16S rRNA gene (corresponding to positions 339 to 539 of Escherichia coli) was amplified with eubacterium-specific primers HDA1-GC  aided by running common samples on multiple gels, to allow comparison of more than one gel. Gel images were then analysed using Bionumerics software . Lane boundaries were applied to gels and images which were optimised to reduce the background noise and smiling of lanes (where applicable). Automatically detected bands  were used to create matching profiles for lanes (in relation to each other). The matching profiles for each lane were used to produce an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram. The UPGMA algorithm weighs each lane being analysed equally and computes the average similarity or dissimilarity of each lane to an extant cluster. The dendrogram that is produced using this method can then be used to observe clustering patterns between different lanes.et al (2004) Primers sets, designed to specifically target lactobacilli, fusobacteria, enterobacteriaceae, enterococci, Clostridium perfringens, bifidobacteria and total bacteroidetes are given in Fecal samples from six strains of mice (n = 3 of each strain) were subject to Q-PCR using methods as described in Bartosch Urine samples, collected weekly and stored at −20°C were diluted with water (1∶3) and analysed by Ultra performance liquid chromatography  linked to a Q-Trap 4000 mass spectrometer . Chromatography was performed on an Aqcuity 100×2.1 mm I.D. column  at 50°C and a flow rate of 0.4 mL/min using reversed-phase elution with water and acetonitrile (both containing 0.1% formic acid v/v) over 10 min. Pooled samples were prepared as quality controls (QCs) and these were analysed between the samples. Mass scan data data (100–850 amu) were collected in +ve and −ve ESI mode. The raw data were extracted with MarkerView. Multivariate data analysis was performed with Simca P  and Orthogonal Partial Least Squares Analysis (OPLS) were applied to project the data).


Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico–basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico–basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the  stardust mRNA regulation. First, the mechanism by which Dynein localizes Stardust may depend, in part, on the apical targeting of the stardust mRNA. Second, some stardust mRNA is apically localized during early stages of epithelial development, but the selective removal of the apical localization signal leads to the sole production of uniformly localized transcripts in mature epithelial cells. Together, these results introduce roles for Dynein in apico–basal polarity regulation and raise important questions about the role of mRNA localization in the targeting of polarity determinant proteins and epithelial maturation.Cells within epithelial sheets are highly polarized with distinct apical and basolateral membrane domains. This cellular organization is critical to both epithelial form and function, and a failure to maintain epithelial polarity is often linked to tumor progression. The protein network that establishes and maintains the two membrane domains relies on the precise subcellular localization of its molecular components, but little is known about how these proteins are targeted to their sites of action. We have shown that the localization of the apical determinant protein Stardust depends on the microtubule motor Dynein. While investigating the relationship between Dynein and Stardust, we also made two unexpected observations about  A polarized architecture increases the level of sophistication at which cells can interact with their neighbors and the environment. Thus, it is not surprising that highly polarized epithelia have become the predominant tissue type among metazoans. Epithelial cells have distinct apical and basolateral membrane domains, each of which contains a unique complement of lipids and proteins to regionalize cellular functions. Intracellular junctions separate the apical from basolateral domains and mediate cell–cell adhesion and selective permeability within the monolayer. Although the mechanisms that specify apico–basal polarity have been a topic of great interest in recent years, there are still many open questions about how this cell architecture is established and maintained.Studies in model organisms and mammalian tissue culture have identified an elaborate protein network regulating epithelial polarity across animal species –3. At thDrosophila ,3534,35]Dhc FC clones and focused our analysis on lateral clones, in which mutant cells retain cuboidal morphology. The results show that, even in Dhc cells with relatively normal shape, Crb is missing from the apical surface , overall protein levels are severely reduced when compared to wild-type cells. These experiments show that Dynein is indeed required for the apical targeting of Crb and suggest that Dynein transport is also necessary for the maintenance of wild-type Crb levels in the FCs.To test whether Dynein is required for the apical targeting of Crb, we performed immunohistochemistry on  surface B. AlthouDhc clones is consistent with our initial hypothesis that Dynein could be transporting Crb-containing exocytic vesicles, but we were struck by the strong general reduction in Crb protein levels. It has been previously shown that Crb levels are reduced in embryonic epithelia when its cytoplasmic binding partner stardust (sdt) is mutated [Dhc cells and is instead found throughout the cytoplasm , as is the localization of Discs-large (Dlg) to the lateral domain . Embryos collected from the cross in which the P10sdt and 4Dhc19− alleles were both segregating showed reduced cuticle production compared to P10sdt mutants alone. In 48% of these embryos, the cuticle was reduced to either a single patch  is overc stages C. These ogenesis F, but beogenesis G. That ssdt transcripts during early oogenic stages. When compared to the ring of apical sdt puncta observed in wild-type FCs . In Dhc FCs, however, only 44% of cytoplasmic puncta were apical (n = 7 ECs). The t value for this data set is 13.49 indicating statistical significance , which contains all 10 coding exons in the final transcript. As expected, transcripts from the sdt A transgene showed dramatic apical localization in the FCs  or the hs-FLPout Act-Gal4 driver and the following UAS constructs: sdt cMAGUK [nod::lacz [DaG32 driver [The Df(3L)Exel6102 and Df(3L)ED4502 lines are from the Bloomington stock center. All wild-type ovaries and embryos were obtained from OreR flies.FC clones were generated as previously described  using th9 FRT80B , FRT82B crb11A22 , sdtXP961 FRT19A , and sdtod::lacz  and the Most ovaries were dissected in PBS and fixed for 15 min at room temperature in 4% EM grade formaldehyde (Polysciences) in PBS. Stainings and washes were performed in PBS with 0.3% triton using the following antibodies: rabbit-α-Baz , rabbi4Dhc19− is larval lethal [sdt alleles are embryonic lethal, mutant embryos collected were presumed to be sdt (−/−). Within the cross segregating both the P10sdt and 4Dhc19− alleles, the sdt (−/−) embryos contain between 0–2 copies of 4Dhc19−.Embryos were collected from the three crosses detailed in l lethal  and the crb (LD13435) and sdt (RE05272) EST clones were obtained from the Drosophila Genome Resource Center (DGRC). The crb clone was linearized with NotI and transcribed with the T7 polymerase and the sdt clone was linearized with MluI and transcribed with the T3 polymerase. To improve probe penetration, both the crb and sdt antisense RNAs were fragmented with carbonate buffer. Because the RE05272 EST contains the entire sdt B mRNA sequence, sdt probe fragments will anneal along the full length of sdt transcripts with the exception of exon 3. The antisense eGFP probe was synthesized from the pBS-eGFPA vector, by linearizing with KpnI and transcribing with the T7 polymerase. The ex3 and ex4–6 antisense probes were synthesized by direct transcription from PCR products where the reverse primer included a T7 promoter. Primer sequences can be found in Antisense RNA Probes were synthesized using a DIG RNA labeling kit (Roche). In situ hybridizations were performed as described in Wilkie (1999) with minor modifications (protocol available upon request). Probes were detected using a horseradish peroxidase conjugated sheep anti-dig antibody  followed by staining with AlexaFluor 488-Tyramide . To detect clones in mosaic egg chambers, in situ hybridization was followed by staining with a rabbit anti-GFP antibody and AlexaFluor 555-conjugated secondary antibody . The nuclear envelopes and basal lamina were illuminated with AlexaFluor647-conjugated wheat germ agglutinin . Images were obtained using a Leica TCS SL confocal microscope and processed with Adobe Illustrator and Photoshop.sdt localization signal were cloned into the pUASBP vector , which contains the BamHI cassette from pUAST [attB element for site-specific recombination using phiC31 integrase [PfuUltra High-Fidelity DNA Polymerase (Stratagene) was used to amplify eGFP, the sdt 3′UTR, and sdt exon3. All other amplifications used the Phusion High-Fidelity DNA Polymerase (Finnzymes). To create the constructs with the eGFP coding sequence, eGFP was amplified from the pBS-eGFPA vector. Directional cloning of this amplicon into pUASBP using 5′ NotI and 3′ XhoI yielded the eGFP-SV40 construct. eGFP-sdt3′UTR was synthesized in two steps. First, the sdt 3′UTR was amplified from sdt cMAGUK [eGFP was extricated from the eGFP-SV40 construct using NotI and XhoI and subsequently ligated into pUASB containing the sdt 3′UTR. For the eGFP-sdtexon3-SV40 construct, exon 3 was amplified from sdt cMAGUK and cloned 5′ XhoI and 3′ KpnI into the eGFP-SV40 plasmid. To create the sdtA-SV40 transgene, the sdt 5′UTR and coding region were amplified from sdt cMAGUK and cloned 5′ NotI 3′ XhoI into pUASBP. The sdt A construct was then created by cutting the 5′UTR and coding sequence from sdtA-SV40 using NotI/XhoI and ligating it into pUASBP containing the sdt 3′UTR. The sdtB transgene was constructed by amplifying the sdt 5′UTR and coding sequence from the RE05252 EST (DGRC) and cloning 5′ NotI and 3′ XhoI into pUASBP containing the sdt 3′UTR. Transgenic animals were produced by Genetic Services, who injected DNA for each construct into the attP2 line [The six transgenes used to map the om pUAST  as well ntegrase . A diagrt cMAGUK . This amtP2 line  and isolTotal RNA was prepped from embryos using TRIzol Reagent (Invitrogen) and first strand cDNA synthesis was performed using the Omniscript reverse transcription kit (Qiagen). Primer sequences can be found in EH681sdt embryonic cuticle phenotype was performed using the maternal P{GAL4-nos.NGT}40 driver [UAS-sdt A and UAS-sdt B transgenes. The UAS-eGFP-sdt 3′UTR transgene was used as a negative control. Embryos were collected as described for the genetic interaction experiment above.Rescue of the 0 driver  and the crumbs mRNA localization to apico–basal polarity regulation can be found in the following reference:A complementary study examining the contributions of Dynein and crumbs transcripts is required for epithelial polarity. J Cell Biol. In press.Li Z, Wang L, Hays TS, Cai Y (2007) Dynein-mediated apical localization of Figure S1crb, Sdt still localizes to the apical surface and junctional region during early oogenic stages, indicating that Sdt is only partially dependent on Crb for its localization in the FCs. This apical localization is gradually lost, however, until Sdt is entirely cytoplasmic by stage 8 (not shown). The bracket denotes Sdt staining in the somatic inner-cyst region.(A) When the entire FC epithelium is mutant for sdt, Crb is never seen at the apical membrane, indicating that Crb absolutely requires Sdt for localization.(B) By contrast, when the entire FC epithelium is mutant for sdt A (C) or sdt B (D) increases the amount of Crb in the apical FC membrane at stage 10, a period when endogenous Crb levels in the FC are low. Cells over-expressing the sdt transgenes are marked with GFP (green). Clonal over-expression of either (5.8 MB JPG)Click here for additional data file.Figure S2D12Dhc−5 clones. This apical reduction could be due to the absence of Crb in D12Dhc−5 clones, as similar reductions are observed in 11A22crb clones (D and F). In occasional D12Dhc−5 clones Baz and aPKC relocalize to the lateral FC surface (A and B), a phenotype never observed in 11A22crb clones. Wild-type cells are marked with GFP (green) and epithelial gaps are indicated with arrowheads.Levels of Baz (A), aPKC (B), and Par6 (C) are mildly reduced at the apical surface of (5.8 MB JPG)Click here for additional data file.Figure S3sdt mRNA (green) forms a crescent posterior to the oocyte nucleus, consistent with localization to MT minus ends.(A) During early oogenic stages, sdt mRNA forms a ring at the anterior of the oocyte. Unlike sdt transcripts, which are apically enriched in the FCs during stages 2–4 (C), crb mRNA is weakly expressed and uniformly localized during these stages (D). Conversely, at stage 10, when sdt transcripts are uniform in the FCs (E), crb transcripts are apical (F).(B) Following the reorganization of the oocyte MTs at stage 7, crb mRNA is mislocalized in Dhc mutant clones (yellow dotted lines) at stage 10.  The nuclear envelope is marked with WGA (blue).(C–F) The apical surface is denoted with a dashed line and the basal surface with a solid line.(4.4 KB JPG)Click here for additional data file.Figure S4sdt mRNA puncta in the FCs.(A–C) A single wild-type egg chamber demonstrates the method used to quantify the subcellular location of sdt mRNA puncta in the egg chamber.(A) Fluorescence in situ hybridization (green) reveals the position of (B) WGA (blue) labels nuclear envelopes and the basal lamina, while an α-Crb antibody  marks the apical surface.D12Dhc5−, the position of the apical surface was inferred from GFP expression in the germline (unpublished data). The green and blue channels were merged, and each green dot was scored as being within the nucleus (pink), the apical cytoplasm (orange), or the basal cytoplasm (white). For egg chambers in which the entire epithelium was mutant for UAS-sdt A (green). Over-expression clones are marked with GFP (red).(D) In situ hybridization on FLPout clones over expressing (2.5 MB JPG)Click here for additional data file.Figure S5(A) Diagram of the pUASBP vector used for transgenic production.sdt 3′UTR is insufficient to target GFP transcripts to the apical surface (B), while sdt exon 3 is sufficient (C). White lines indicate the approximate position of the salivary gland basal surface. Daughterless-Gal4 over-expression followed by in situ hybridization (green) in the embryonic salivary gland reveals that the  Control for the specificity of the exon-specific probes. The exon 3 probe (D) and the exon 4–6 probe (E) recognize localized transcripts in the oocyte with equal efficiency.(B–E) WGA (blue) labels nuclear envelopes.(3.2 MB JPG)Click here for additional data file.Table S1(20 KB DOC)Click here for additional data file.Table S2(35 KB DOC)Click here for additional data file.sdt alleles and sequences for sdt mRNA isoforms can be found on Flybase (http://flybase.org/). In this database, sdt-A is called sdt-RB, and sdt-B is called sdt-RF.Information on


The presence of measles virus (MV) RNA in bowel tissue from children with autism spectrum disorders (ASD) and gastrointestinal (GI) disturbances was reported in 1998. Subsequent investigations found no associations between MV exposure and ASD but did not test for the presence of MV RNA in bowel or focus on children with ASD and GI disturbances. Failure to replicate the original study design may contribute to continued public concern with respect to the safety of the measles, mumps, and rubella (MMR) vaccine.The objective of this case-control study was to determine whether children with GI disturbances and autism are more likely than children with GI disturbances alone to have MV RNA and/or inflammation in bowel tissues and if autism and/or GI episode onset relate temporally to receipt of MMR. The sample was an age-matched group of US children undergoing clinically-indicated ileocolonoscopy. Ileal and cecal tissues from 25 children with autism and GI disturbances and 13 children with GI disturbances alone (controls) were evaluated by real-time reverse transcription (RT)-PCR for presence of MV RNA in three laboratories blinded to diagnosis, including one wherein the original findings suggesting a link between MV and ASD were reported. The temporal order of onset of GI episodes and autism relative to timing of MMR administration was examined. We found no differences between case and control groups in the presence of MV RNA in ileum and cecum. Results were consistent across the three laboratory sites. GI symptom and autism onset were unrelated to MMR timing. Eighty-eight percent of ASD cases had behavioral regression.This study provides strong evidence against association of autism with persistent MV RNA in the GI tract or MMR exposure. Autism with GI disturbances is associated with elevated rates of regression in language or other skills and may represent an endophenotype distinct from other ASD. Beginning in 1998, Wakefield and colleagues reported intestinal abnormalities, including reactive lymphoid hyperplasia in ileum, in children with autism and other developmental disturbances Over 20 epidemiologic studies reported no temporal relationship between MMR and ASD Forty-seven children were recruited. Six recruits did not complete the study: 3 potential cases dropped out prior to colonoscopy; 1 potential case and 2 potential controls completed colonoscopy but had incomplete clinical assessments. No differences were found in age, sex, or case-control status between study completers and non-completers. An additional 2 potential cases were excluded for failure to meet diagnostic inclusion criteria (below cutoffs for autistic disorder [AUT] on ADI-R); and 1 case was excluded because no bowel biopsy material was available. The final study population consisted of 25 cases (AUT/GI group) and 13 controls (GI control group) presenting consecutively for ileocolonoscopy who received at least one dose of MMR and completed all study procedures.P = 0.01). There were no significant differences between cases and controls in their distribution by sex within the three age groups , cases, 5.5 (3.0) years; controls, 5.1 (3.0) years], as was the distribution of cases and controls across the three age strata. Girls were older at biopsy than boys (e groups Table 1.P = 0.27).The clinical indications for endoscopic/colonoscopic procedures commonly noted in both AUT/GI and GI groups included recurrent abdominal pain (RAP), gastroesophageal reflux, vomiting, and food allergies. Although the more subjective factor of RAP was frequently present in both cases (36%) and controls (38%), it was rarely the sole rationale for GI examination in either group . Consistent with the older age of girls in the study, there was a trend toward a higher proportion of girls than boys receiving a second MMR (P = 0.13). None of the children received MV-containing vaccines other than MMR.Median age at receipt of first MMR was similar for cases [15.3 (1.7) months] and controls [16.0 (4.9) months]. The majority of study subjects were in the 3–5 year age stratum and below the age recommended for second MMR (4–6 years p<0.0001).Clearance of MV depends on development of adaptive immunity. As cell-associated MV RNA may be present transiently after receiving MMR P = 0.04, Controls received a greater median number of all types of vaccines than cases .Cases first receiving MMR prior to onset of GI complaints were older at index GI episodes [21.0 (22.0) months] than cases receiving their first MMR after GI episodes already began [1.0 (12.0) months; p<0.0001; bone fide MV RNA produced consistent results across three laboratories, with each laboratory site reporting less than 10 cDNA copies of MV F and H gene in ileal biopsies from one child with autism and one child without neurological disorder.We found no differences between AUT/GI and GI control groups in detection of MV sequences in RNA extracted from ileal or cecal biopsy specimens. Real-time RT-PCR assays with molecular controls engineered to allow differentiation of products arising from synthetic vs. Our results differ with reports noting MV RNA in ileal biopsies of 75% of ASD vs. 6% of control children MV in MMR has been proposed to induce GI inflammation, increasing permeability to neuroactive chemicals that promote developmental neuropathology ASDs comprise a wide range of endophenotypes that may represent different routes to pathogenesis. The work reported here eliminates the remaining support for the hypothesis that ASD with GI complaints is related to MMR exposure. We found no relationship between the timing of MMR and the onset of either GI complaints or autism. We also could not confirm previous work linking the presence of MV RNA in GI tract to ASD with GI complaints. The origin, nature, and frequency of GI disturbances within the larger ASD population remain unclear; focused research strategies are required to define these endophenotypes and determine their significance for causal hypotheses.Families of potential subjects were invited to participate if ileocolonoscopy with biopsy was specifically indicated as part of clinical care. Invited children were scheduled for upper and/or lower endoscopic procedures based on clinical imperative. Routine informed consent for clinical procedures was obtained by the gastroenterologist. Informed consent procedures detailed additional research procedures to be performed, and specific written permission was provided by consenting parents and guardians and children capable of providing assent (7 years or older). Study procedures were approved by the Institutional Review Boards of Partners/MGH, CU Medical Center, and the CDC and by the Ethics Committee of Coombe Women's Hospital prior to study initiation.Children between 3 and 10 years of age were serially and prospectively recruited from the Pediatric Gastroenterology and Nutrition and LADDERS Clinics (MGH) in the years 2003 to 2005 into two groups if they had clinically significant GI disturbances requiring ileocolonoscopic examination and either: 1) presence of Autistic Disorder  (AUT/GI cases), or 2) absence of known or suspected developmental disturbances (GI controls). Potential controls were frequency-matched to potential AUT/GI cases within three age strata: 3–5, 6–7, or 8–10 years. Eligible children received at least one prior immunization containing MV vaccine strain. Children reported by parents to have received MV immunization within 6 months of planned biopsy were excluded.Neuropsychiatric status was established for all subjects by child neurologists, psychiatrists or developmental pediatricians (LADDERS) using Diagnostic and Statistical Manual-Fourth Edition, Text Revision  was established according to well-validated CPEA algorithms A randomized list of linked clinical and laboratory ID codes was prepared by the Biostatistics Core (CU) prior to study initiation and provided to MGH research assistants. The MGH clinical coordinator acquired, recorded and transmitted clinical data on case report forms identified only by clinical ID. To maintain the blind, patient samples were labeled at MGH with only the linked laboratory ID codes and shipped to the Laboratory Core (CU). Samples were maintained under laboratory codes for all assays at all three sites, including any repeat assays required to address inter-laboratory discordance. Linkage of clinical and laboratory ID codes remained with the Biostatistics group until inter-laboratory discordance was resolved and data quality control procedures were complete; a linked dataset was then prepared and final data analysis ensued.Biopsy material was obtained from terminal ileum and cecum under direct supervision of the team gastroenterologist. For analyses of MV RNA, four random samples were taken from superficial mucosae of ileum and cecum. Additional specimens were acquired at sites indicative of inflammatory GI lesions, if present. All samples intended for RNA analysis were frozen immediately in coded tubes in liquid nitrogen and stored at −70°C until shipment to CU on dry ice. Frozen biopsy specimens were stored in a dedicated −70°C freezer until RNA extraction to avoid inadvertent contamination. A portion of each clinical pathology sample also was retained under blind for histopathologic analysis.Total RNA from bowel biopsies was obtained by acid guanidinium thiocyanate-phenol-chloroform extraction  at CU. Aliquots of RNA were prepared to ensure sufficient material for primary analysis at each of the three laboratory sites and for repeated analyses in the event results among sites were discordant. One 16 µg aliquot of RNA was created for each region  and sent to the three analytical laboratories for real-time RT-PCR detection of MV F and H gene sequences using two primer/probe sets each, as well as a control gene. To reduce the possibility that prior results might influence interpretation of repeat laboratory tests, additional aliquots of total RNA were coded with different laboratory IDs and stored at −70°C.Three laboratory sites participated in MV RNA analyses: 1) Coombe Women's Hospital, Trinity College; 2) Center for Infection and Immunity, CU, New York; 3) Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, CDC, Atlanta.et al.Real-time RT-PCR assays employed four primer/probe sets: the sets originally described by Uhlmann bona fide MV sequences. A common cellular gene (β-actin) was used as a control for integrity of RNA template. Negative controls included reactions where water or normal human placental RNA was substituted for clinical sample RNA.Synthetic MV transcripts were used as positive controls. These transcripts contained engineered mutations to allow distinction from Additional details on primer pair design, positive and negative controls, and real-time RT-PCR assay calibration and procedures are provided in A positive finding in a single laboratory for each individual sample was defined as any result above the detection threshold  in two of three triplicate reaction wells in a 96-well plate, for any single set of F or H primers, within that laboratory. The Biostatistics Core assessed coded data from each laboratory for positive results by GI region  and by primer pair, as well as for concordance of positive and negative findings across laboratories. All positive findings for a specific sample  were first evaluated within each laboratory to determine whether both F gene and H gene MV sequences were represented . Concordance with respect to positive findings for a specific primer pair and GI region for an individual subject was next examined across laboratories; findings consistent in two or more laboratories for any primer pair/GI region were defined as concordant results. Samples for which at least two laboratories reported a positive finding with at least one F gene primer set and at least one H gene primer set were defined as positive for the presence of MV sequences , the Biostatistics Core notified the coordinating laboratory that additional aliquots of that sample were to be sent to each of the analytical laboratory sites for repeat assays. This process continued until any such inconsistencies were resolved. Products from positive reaction wells were sequenced to ensure the absence of engineered restriction sites and identify 2X) analyses for nominal data (Fisher's Exact Test to determine significance). One-tailed tests for significance were pursued unless otherwise indicated, given the study objectives of determining whether MV sequences were more likely to be found in biopsy tissues of cases than controls, as previously reported by Wakefield and colleagues and as consistent with the MMR hypothesis Group comparisons were conducted using nonparametric tests  for continuous data deviating from normal distributions, and chi-square Click here for additional data file.Appendix S2Figure S2, study criteria and methods for resolving intra- and inter-laboratory disconcordance(0.11 MB DOC)Click here for additional data file.