Amyotrophic lateral sclerosis (ALS) is a fatal neurologic disease characterized by progressive motor neuron degeneration. Clinical disease management is hindered by both a lengthy diagnostic process and the absence of effective treatments. Reliable panels of diagnostic, surrogate, and prognostic biomarkers are needed to accelerate disease diagnosis and expedite drug development. The cysteine protease inhibitor cystatin C has recently gained interest as a candidate diagnostic biomarker for ALS, but further studies are required to fully characterize its biomarker utility. We used quantitative enzyme-linked immunosorbent assay (ELISA) to assess initial and longitudinal cerebrospinal fluid (CSF) and plasma cystatin C levels in 104 ALS patients and controls. Cystatin C levels in ALS patients were significantly elevated in plasma and reduced in CSF compared to healthy controls, but did not differ significantly from neurologic disease controls. In addition, the direction of longitudinal change in CSF cystatin C levels correlated to the rate of ALS disease progression, and initial CSF cystatin C levels were predictive of patient survival, suggesting that cystatin C may function as a surrogate marker of disease progression and survival. These data verify prior results for reduced cystatin C levels in the CSF of ALS patients, identify increased cystatin C levels in the plasma of ALS patients, and reveal correlations between CSF cystatin C levels to both ALS disease progression and patient survival. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that affects approximately 1.5 to 2.5 per 100,000 individuals of all races and ethnicities throughout the world Biomarkers also hold promise to monitor disease progression and to stratify patient populations for use in clinical trials. One reason new drug therapies have not been successfully translated from ALS model systems to humans is ALS disease heterogeneity One protein that has shown potential for ALS diagnostic utility is cystatin C, a widely expressed cysteine protease inhibitor that is approximately five times more abundant in CSF than in plasma Two prior surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) studies reported significant decreases in CSF cystatin C levels in ALS patients relative to healthy controls The objective of this study was to use quantitative ELISA to further evaluate the utility of cystatin C as a biomarker for ALS using a large subject population. Our subject group size was based on power analysis of previously published mass spectrometry reports on cystatin C in ALS We collected longitudinal CSF and plasma samples from 104 ALS and control subjects  and evalIn order to assess the diagnostic utility of cystatin C, we first compared the mean first-draw cystatin C levels among ALS patients, neurologic disease controls, and healthy controls. A generalized linear model was used to estimate the mean total and percent cystatin C for each diagnostic category, with both gender and age included as co-factors in the model. This statistical design controls for between-group differences in each co-factor when generating estimated means. Therefore, the differences in age and gender among our diagnostic groups should not have affected our results, even in the case that cystatin C varies with these factors. We found that the estimated means for both measures of cystatin C were lower in ALS patients than in disease controls and healthy controls, similar to prior studies . Howeverp-values were reduced for the ALS vs. mimic disease control subgroup comparison . However, the p-values when compared to the analysis including all ALS patients. However, the pair-wise comparisons between these ALS subgroups and mimic disease controls still fell short of statistical significance.We next compared two ALS subgroups to the disease mimic group. Limb-onset ALS (ALS-L) and ALS patients greater than one year from symptom onset both exhibited reduced levels of cystatin C in the CSF when compared to disease mimics , with imFinally, we calculated the diagnostic sensitivity and specificity for several cutoff concentration values of CSF cystatin C. Total cystatin C concentration measurements were found to have better diagnostic parameters than percent cystatin C values. A cutoff value of 2.20 µg/ml identified a small subset of ALS patients (sensitivity: 23%) with relatively high specificity . A cutoff value of 2.70 µg/ml identified a modest subset of ALS patients (sensitivity: 32%) while maintaining high specificity versus controls . A less conservative cutoff value of 3.50 µg/ml identified a majority of ALS patients (sensitivity: 52%), but demonstrated lower specificity .As noted above, cystatin C was previously reported to be significantly reduced in the CSF of ALS patients using mass spectrometry-based proteomics, but the between-group differences based on our ELISA data were less robust. To explore the relationship between CSF cystatin C levels measured by these two techniques, we compared our ELISA results with SELDI-TOF-MS data for the same CSF samples. We found significant, positive correlations between the 13.3 kDa SELDI-TOF-MS mass peak intensity for cystatin C and both total cystatin C and percent cystatin C protein levels as measured by ELISA  or percent cystatin C levels  in CSF and plasma samples drawn from individual patients on the same day . The absWe next examined whether cystatin C levels change over time in ALS patients, and if these changes are associated with clinical disease progression. We compiled the first CSF draws for each of the ALS patients in our study and carried out linear regressions comparing both total and percent cystatin C with the time from symptom onset. Similar to a prior study Next, we collected longitudinal CSF samples from ALS patients and assessed the effect of time on cystatin C levels using a statistical model for repeated measures. This experimental design controls for individual differences in baseline cystatin C levels, but not for individual differences in rate of disease progression. We collected at least three longitudinal CSF draws from 15 ALS patients over a 1–2 year time period for each patient. When all 15 values were averaged for each time point, we did not observe a significant change in cystatin C levels over time , but both measures of CSF cystatin C levels showed a direct correlation, with the results for total cystatin C almost reaching statistical significance . These findings suggest that cystatin C levels in CSF but not plasma may be useful as prognostic indicators of patient survival time.Finally, we assessed the relationship between first-draw cystatin C levels (in CSF and plasma) and patient survival time. Neither measure of plasma cystatin C showed a correlation with subsequent survival time  and a larger high cystatin C group (n = 21). This analysis revealed significantly longer patient survival in the high cystatin C group than in the low cystatin C group . Next, bThe present study represents a comprehensive evaluation of cystatin C as a candidate biomarker in ALS, and is unique in its assessment of two distinct biofluids (CSF and blood plasma), two different measurements of the protein of interest , and longitudinally collected CSF samples. Prior studies using SELDI-TOF-MS found significantly lower cystatin C abundance in the CSF of ALS patients relative to healthy controls To be clinically useful as a diagnostic biomarker, cystatin C must also be able to differentiate between ALS patients and individuals with neurologic diseases that closely resemble ALS, or ALS “mimic diseases.” A recent study reported a significant reduction in CSF cystatin C levels in ALS patients relative to polyneuropathy patients We also assessed the diagnostic utility of plasma cystatin C levels. Plasma cystatin C has been extensively characterized as a peripheral biomarker for kidney function and as a prognostic indicator of the risk of morbidity and mortality relating to cardiovascular disease A recent study examined a single CSF draw per ALS patient, taken at varying times from symptom onset, to indirectly infer the average longitudinal change in cystatin C concentration in the group as a whole, and they reported that cystatin C levels do not change over time We also examined longitudinal CSF data from multiple patients to more accurately assess the changes in cystatin C over time. We found that longitudinal cystatin C concentrations were relatively constant in ALS patients as a combined group. In contrast, the subgroup of patients with slow or absent clinical disease progression exhibited longitudinal increases in cystatin C concentration, and the subgroup with more typical, continuous clinical deterioration exhibited longitudinal decreases in total cystatin C. Interestingly, slow progressors often exhibited lower initial levels of CSF cystatin C than fast progressors . SimilarWe also conducted an analysis to determine the relationship between longitudinal changes in CSF cystatin C levels and time-matched changes in three functional clinical measures of disease progression . However, no significant correlations were found (data not shown). This indicates that cystatin C levels may change independently of the clinical parameters used for monitoring disease progression. However, this finding does not eliminate the possibility that changes in CSF cystatin C levels correlate with more subtle biochemical changes associated with disease progression, as these may not be accurately reflected by overt functional measures of clinical disease status We recently demonstrated a correlation between CSF cystatin C levels and patient survival by SELDI-TOF-MS in vitro, in order to clarify its potential mechanistic role in ALS pathogenesis.The results of this comprehensive biomarker assessment also have implications for the potential mechanistic involvement of cystatin C in the pathogenesis of ALS. The function of cystatin C within the CNS has not been extensively studied, but it appears to have both neurotoxic and neuroprotective properties In summary, we have completed a comprehensive evaluation of cystatin C as a candidate ALS biomarker, including assessments of two complementary measures of cystatin C in two distinct biofluids as well as examinations of both longitudinal CSF samples and patient survival data. Our findings indicate that cystatin C levels, as determined by ELISA, are increased in the plasma and decreased in the CSF of ALS patients relative to healthy controls. CSF cystatin C measurements may possess a more limited diagnostic capacity for ALS than previously proposed, but may still have the potential to improve the diagnostic parameters of a biomarker panel. Additionally, longitudinal changes in CSF cystatin C levels may be useful as a biomarker of fast versus slow rates of disease progression. Our data also demonstrate that CSF cystatin C concentration has prognostic utility in estimating patient survival time. Further validation studies are necessary to confirm these findings and ultimately determine if cystatin C measurements can be used to enhance clinical disease management and clinical trial design. Finally, the association of high or increasing cystatin C levels with slower disease progression and increased survival time suggests a potential neuroprotective role for this protein in the pathobiology of ALS.This study was approved by the institutional review board (IRB) at the University of Pittsburgh, and written informed consent was obtained from all participating subjects. ALS subjects were diagnosed by experienced neurologists specialized in motor neuron disease, using revised El Escorial criteria The disease control group included six patients with multiple sclerosis, one with bilateral facial palsies, one with neurosarcoidosis, one with viral encephalitis, one with CNS lymphoma, one with brain metastases, one with pseudotumor cerebri, one with a seizure disorder, one with complicated migraine, one with paresthesis and possible myelopathy, one with a probable conversion disorder, and nine with neurologic diseases that can clinically resemble ALS at presentation. This ALS-mimic disease subgroup included two patients with primary lateral sclerosis (PLS), two with chronic inflammatory demyelinating polyneuropathy (CIDP), two with progressive muscular atrophy, one with spinocerebellar ataxia, one with small fiber neuropathy, and one with idiopathic sensorimotor polyneuropathy. CSF samples were obtained by lumbar puncture, immediately centrifuged at 450 g for five minutes at 4°C to remove cells and debris, aliquoted, and then frozen at −80°C. Intravenous blood samples were collected in EDTA containing tubes, inverted to mix, and centrifuged at 1,733 g for 10 min at 4°C. The plasma was decanted, aliquoted, and frozen at −80°C. CSF and plasma were aliquoted into small volumes for single use in experiments in order to eliminate any freeze/thaw effects. Samples of either biofluid were thawed on ice immediately prior to use.CSF and plasma samples from individual patients were assigned to random 96-well plate positions, and evaluated in duplicate wells for each ELISA. All samples were independently assayed at least twice. For all experiments, we used a human cystatin C sandwich ELISA kit , according to the manufacturer's instructions. Briefly, diluted CSF and plasma samples  were applied to antibody pre-coated ELISA plates for 30 min with gentle agitation. The wells were washed thoroughly and then the secondary antibody conjugate solution was applied for 30 min with gentle agitation. After a second wash, the 3,3′,5,5′ Tetramethylbenzidine substrate solution  was applied for 10 min, color development was stopped with an acidic stop solution, and the optical density was measured at 450 nm using a plate reader. Total protein concentrations for each sample were calculated using a BCA protein assay (Pierce), according to the manufacturer's instructions.For each ELISA plate, a standard curve was generated by plotting the logarithm of the cystatin C concentration against the logit log of the adjusted optical density . This procedure produced a linear standard curve, which was then used to calculate sample cystatin C concentration from sample optical density. The data points were averaged to determine the absolute cystatin C concentration, or “total cystatin C,” for each sample. Sample cystatin C concentrations were normalized to the sample total protein concentration to determine the percent of total biofluid protein accounted for by cystatin C, or “percent cystatin C.”For the analyses of diagnostic utility, we included only the initial sample collected from each patient, representing the time point closest to symptom onset. Differences between group and subgroup means were identified using the SPSS generalized linear model, with diagnosis and sex as factors in the model and age at draw as a covariate. This model was subsequently used to calculate and compare the estimated marginal group means, in order to determine which pairwise differences among the levels of each factor were responsible for the significant main effects.The relationship between first-draw cystatin C levels and the length of time from symptom onset was assessed by linear regression using GraphPad Prism 5.0 software . For this analysis, the “time from symptom onset” data were transformed by the natural logarithm (Ln) in order to achieve normality as required by the selected statistical test.The effect of time on longitudinal cystatin C levels in ALS patients with multiple biofluid draws was assessed with SPSS software, using the general linear model for repeated measures. The model was applied for all patients combined and for patient subgroups sorted by progression speed. Fast progressors were defined as patients exhibiting above average rates of ALSFRS decline . The Spearman's rank correlation coefficient (r) was calculated and the permutation test was applied to determine if r was significantly different from zero.The relationship between first-draw cystatin C levels and post-draw survival time (for deceased patients only) was assessed by Spearman correlation analysis (GraphPad Prism 5.0). The prognostic utility of CSF cystatin C was further explored by generating Kaplan-Meier survival curves for patients falling above or below several cut-off values of cystatin C. SPSS software was used to calculate the p-values for differential survival time by three different methods: the Log Rank (Mantel-Cox) test, the Breslow  test, and the Tarone-Ware test. For all statistical analyses in this study, the significance level was set at p<0.05.Figure S1Correlation of ELISA-based cystatin C levels and SELDI-TOF-MS 13.3 kDa mass peak intensity levels by Spearman correlation analysis. Both total (A) and percent (B) cystatin C ELISA measurements correlated to the 13.3 kDa cystatin C mass peak.(TIF)Click here for additional data file.Figure S2Correlation analysis for cystatin C levels in CSF and plasma. There was no correlation between total cystatin C concentrations (A)  or percent cystatin C levels (B)  between CSF and plasma.(TIF)Click here for additional data file.


Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations. The mission of regenerative medicine is to repair or replace tissues and organs that have been damaged by trauma, disease, or aging with living bioengineered tissue that restores function , 2. AmonSimilarities and differences among amniotic cells have been classified to a large extent on cell shape. Clones of amniotic cells were first isolated almost 4 decades ago with cloning rings and classified on the basis of colony morphology, see Hoehn and Salk (1982) for a contemporaneous review . F-type http://www.clinicaltrials.gov/). BMMSCs are derived from bone marrow aspirates and adhere to plastic culture wares, in contrast to hematopoietic derivatives of bone marrow that proliferate in suspension  or adipogenic  differentiation [Healthy cells were cultured to ~90% confluence in Chang's media on cultureware pretreated with a 1 : 300 dilution of growth-factor-reduced Matrigel. Media were then switched to ntiation –33. Mediβ (GUSB), Hs00939627, E-Cadherin (CDH1) Hs01023894, and N-cadherin (CDH2), Hs00983056. Ct values of replicate assays were averaged and averages were normalized to expression of GUSB, an internal control that was included in all experiments.Total RNA was extracted with RNAeasy kits (Qiagen) with DNAse treatment to eliminate genomic DNA according to manufacture directions. RNA was converted to cDNA with Superscript III First Strand Synthesis kits (Invitrogen). TaqMan gene expression assays were used to detect transcripts of glucuronidase-Centrifugation is commonly used to concentrate cells in amniotic fluid and minimize dilution of media, but it was unclear whether this manipulation is necessary to recover viable amniotic cell populations. Newly isolated amniotic fluid samples of less than 2.0 ml were diluted 1 : 1 or 1 : 2 in serum-containing media and directly plated in individual wells of a 6-well tissue-culture-treated plate. As early as the first 48 to 72 hrs of plating, a few adherent cells were detected in each sample. These populations, designated as ChM mixed cell populations, were expanded into larger culture wares within a few days. Virtually all primary cultures expanded as monolayers. Subsequent passaging with trypsin generated colonies with centrally located aggregates, reminiscent of descriptions of AF-type colonies in seminal works . With ran = 4) precludes meaningful tests of statistical significance. Nonetheless, 44 independent viable cell lines were generated within 2 to 4 days of plating less than 8 mls of uncultured amniocentesis sample using this simple and highly efficient method.We next tested whether multiple independent populations could be isolated simply by diluting amniocentesis samples into larger volumes of media. The four subsequent samples that we received were diluted in 25 to 125 mL of serum-containing media and plated into one or more 24-well plates treated for tissue culture. Media and nonadherent cells were transferred as replicates to new plates after 48 hr to 72 hr. The 4 amniocentesis samples RC, LB, GW, and PB generated 12, 9, 14, and 9 independent viable cell populations, respectively . Each poCell populations developed as expected of single-cell clones; daily monitoring by phase microscopy showed continual expansion of a small spherical cluster of cells, most often at the edge of the well Figures , but occn = 22) were stromal and half were epithelial. Although AF-type clones fitting the description of Hoehn and Salk [Expanded clonal populations could be classified by phase microscopy on the basis of cell morphology, designated here as stromal or epithelial to use terminology that is widely applied in cell biology and consistent with descriptions of cells in mixed cell populations that were established by others , 34, 35.and Salk  were notβ1 that is detected with CD29 antibodies, the CD44 glycoprotein receptor for hyaluronic acid, and the stage-specific embryonic antigen 4 (SSEA4). Profiles were first established for BMMSCs as a positive control for immunodetection procedures. The results showed that nearly all BMMSCs expressed CD73 as well as CD29, CD44, and SSEA4  protein that is highly expressed in stromal cells, although it is not exclusive to these cells . N-cadhen > 200) as well as smaller cells with more irregular cell shapes . Immunostaining with antibodies against keratin and vimentin or keratin and fibronectin revealed diversity in the size and shape of cells within and between ChM populations. The ChM1 population contained many large spherical cells with prominent networks of keratin and vimentin, representing 85% of cells  is of critical importance to tissue engineering and manufacture of bioengineered organs. Decellularization is perceived to leave behind detergent insoluble ECM that provides form and organization for revascularization and function . While t


Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV’s life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV’s miRNAs. They have been implicated as regulators of most cellular processes with abnormal expression of miRNAs reported in various diseases including cancer Epstein-Barr virus (EBV) is a gamma herpes virus that infects more than 90% of the world’s adult population and was the first virus identified to encode many miRNAs. Its miRNAs are encoded in two transcripts, the BamHI H Rightward Fragment 1 (BHRF1) transcript and the BamHI A Rightward transcript (BART) in vitro in NPC and Burkitt’s lymphomas Currently few mRNAs potentially regulated by EBV’s miRNAs have been identified, and the biological significance of this miRNA-mediated regulation is poorly understood. Among viral targets of the miRNAs, miR-BART2 was reported to decrease the expression of the viral DNA polymerase BALF5, suggesting that miR-BART2 may inhibit the transition from latent to productive viral replication One major impediment to elucidating functions of viral miRNAs is their subtle effects on gene expression and the complex nature of interactions among miRNAs and their target transcripts While the human genome is approximately 10,000 times larger than that of EBV, it encodes only 50 times as many miRNAs. The enrichment for miRNAs by EBV makes the viral miRNAs fascinating to study. Our understanding of the functions and the advantages provided by the viral miRNAs however remains limited due to the lack of functional studies and identified bona fide targets of EBV miRNA-mediated regulation. Identifying cellular gens inhibited by the viral miRNAs will help elucidate pathways regulated by these miRNAs. We have therefore used two approaches, 2D gel separation coupled with mass spectrometry, and RISC immunoprecipitation (RISC IP) coupled with deep sequencing to compare approaches, to understand their advantages and limitations and to identify the method of choice to aid our understanding of the functional importance of viral miRNAs in the life cycle of EBV. The approach using 2D gel separation has the advantage of detecting changes in levels of a protein directly and has been used successfully to identify mRNAs regulated by miRNAs 2 at 37°C. The viability of cell lines was assessed by Trypan Blue staining and by evaluating overall cell morphology. Only cultures with a viability of more than 95% were used in this study. BJAB BARTs cells express EBV’s BART miRNAs constitutively via a retroviral vector, where BJAB Empty cells were transduced with an empty retroviral vector as described previously . Total RNA was isolated using TRIzol reagent (Invitrogen), and precipitated with linear acrylamide (Ambion) as a carrier as previously described EBV-negative Burkitt’s lymphoma cell line, BJAB . Briefly, protein extracts of BJAB BARTs and BJAB Empty cells were covalently linked to Cy3 and Cy5 CyDye respectively, mixed and run on first dimension isoelectric focusing, and second dimension SDS-PAGE in two technical replicates. Image scans were carried out following the SDS-PAGE using Typhoon TRIO (Amersham BioSciences), the scanned images analyzed by Image QuantTL software , and subjected to in-gel analysis and cross-gel analysis using DeCyder software version 6.5 . The ratio of the change of differentially expressed proteins was obtained from in-gel DeCyder analysis. To determine the ID of these differentially expressed proteins, selected spots were picked up by Ettan Spot Picker  following the DeCyder software analysis and spot picking design. The selected protein spots were subjected to in-gel trypsin digestion, peptide extraction, desalting and followed by MALDI-TOF/TOF (Applied Biosystems) analysis to determine the protein identity.mRNA microarrays were carried out as previously described . Briefly, total RNA from BJAB BARTs and BJAB Empty cells was hybridized to whole genome human microarrays  per manufacturer’s instructions. 150 ng of RNA was reverse-transcribed using a d(T)-T7 promoter primer into cDNA, and then transcribed into cRNA containing CTP labeled with either Cy3 or Cy5 by T7 RNA polymerase, using the Agilent Quick Amp Kit, Two color following the manufacturer’s protocol. Equal masses of Cy3- and Cy5- labeled cRNAs were co-hybridized to microarrays for the detection of mRNAs and scanned with an Agilent DNA Microarray Scanner. Microarrays were analyzed with EDGE3 software 8 cells were washed with PBS and lysed with 500 µl of PLB buffer , 1 mM DTT, proteinase inhibitor cocktail) at 4°C for 30 min. Following centrifugation, the lysate was incubated with either anti-GST or anti-human Ago2 (11A9) antibodies (Provided by Dr. Gunter Meister 2)) at 4°C for overnight. The protein-dynabead complexes were washed 6 times with NT-2 buffer, once with NTmS buffer  and resuspended with 1× Turbo DNase buffer and 2 U of Turbo DNase (Ambion) at 37°C for 30 min. The activity of DNase was eliminated with 15 mM of EDTA followed by addition of same volume of proteinase K buffer (2.4 mg/ml of proteinase K and 2% SDS in NT-2 buffer) at 55°C for 30 min. The RNA was extracted with acid-phenol/chloroform (pH 4.5) once, chloroform once and precipitated and resuspended in RNAse-free water.1×10A cDNA library was prepared with Illumina mRNA-Seq sample preparation kit, according to the manufacturer’s protocol (Illumina), and examined by Illumina HiSeq2000 by the UWBC DNA Sequencing Facility. Sequence analysis was carried out using CLC Genomics Workbench 4.9 software as previously described  to identify unique exon reads for each gene. The unique exon reads were used to determine expression values for each gene using the following formula: Expression value = (unique exon reads)×(109)/(exon length)×.EBV BART miRNAs were measured as previously described 1 ug of total RNA was reverse transcribed using Multiscribe  according to manufacturer’s conditions using oligo d(T) at a final concentration of 5 uM. Reverse transcribed cDNAs were amplified and detected by qPCR under the following conditions: 1× Amplitaq Gold PCR Master Mix (Applied Biosystems), 0.5 µM each primer, 0.2 µM probe, 1× ROX reference dye (Invitrogen) and water to the final volume of 20 µl. PCR cycling conditions were 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds. Probes were labeled with 5′FAMRA and 3′TAMRA. The sequences of primers and probes are listed for each mRNA assayed .of firefly luciferase in the pmirGLO expression plasmid, which also contains an expression cassette for renilla luciferase as an internal control . 3′UTR-pmirGLO-derived constructs (100 ng) were transfected into the BJAB Empty and BJAB BARTs cell lines. 2.5×106 cells were harvested 48 hours after electroporation, and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) following manufacturer’s protocol. Firefly activity relative to renilla was normalized to baseline luciferase activity achieved by transfecting pmirGLO construct alone (3′UTR-pmirGLO) and expressed relative to normalized activity in BJAB Empty cells, which was arbitrarily set to 100%. Each sample was measured twice per experiment, and each condition was repeated in three independent experiments.Candidates found enriched in the RISCs by deep sequencing were tested by cloning their 3′UTRs downstream Cell lysates were separated on SDS-PAGE and transferred electrophoretically to PVDF membranes. The membranes were blocked in BLOTTO  and probed with anti-CEBPA (Acris Antibodies), anti-PUMA (Sigma-Aldrich), anti-CNBP1 (Sigma-Aldrich) or anti-Tubulin antibody (Sigma-Aldrich), followed by Horseradish peroxidase conjugated secondary antibody (KPL). The signals were detected using SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific). Western blots were quantified using Image J software. Signals were determined to be in the linear range for their measurements when exposures for increasing times yielded directly proportional increases in signal intensities.Various proteomic approaches such as stable isotope labeling with amino acids in cell culture (SILAC) and two-dimensional gel electrophoresis (2D DIGE) followed by mass spectrometry for protein identification have been widely employed to characterize the effects of differential miRNA(s) expression on global protein abundance 2D DIGE was selected for the comparison of techniques in this study in order to identify proteins that are differentially expressed in the presence of viral BART miRNAs. This method involves labeling two sets of protein samples using two different cyanine dyes, Cy3 and Cy5. The labeled samples can then be mixed allowing simultaneous separation of proteins by their isoelectric point as well as by their mass as a function of the SDS they bind. The samples can be visualized and quantified separately by using the excitation wavelength specific to each fluor. The analysis is reported to be more quantitative than the traditional 2D gel electrophoresis because of wide dynamic linear range and extreme sensitivity of the Cy3 and Cy5 fluors (reported to be 0.025 ng/spot) The EBV-negative Burkitt’s lymphoma cell line, BJAB, which was either transduced with a retrovirus that expresses BART miRNAs constitutively (BJAB BARTs) or an empty virus vector (BJAB Empty) was used to identify cellular proteins whose expression levels are regulated by EBV’s miRNAs. This approach provided as isogenic cell lines as available currently, where changes in protein expression could be attributed to the effects of regulation by BART miRNAs alone, with no other viral genes being present. Expression of 26 BART miRNAs was extensively characterized in BJAB BARTs cells by stem-loop RT-PCR and compared to two EBV-positive Burkitt’s lymphoma cell lines, OkuI and SavI . These m2D DIGE and subsequent protein identification of differentially expressed spots was carried out by Applied Biomics, Inc.  . This apThe 2D DIGE method resolves approximately a fifth to a quarter of the proteins present in a given cell We used immunoprecipitation of RISCs and deep sequencing of the co-immunoprecipitated RNAs as a second approach to identify mRNAs potentially targeted by the BART miRNAs. A strength of this approach is that all mRNAs associated with EBV’s miRNAs in RISCs could be identified. A possible shortcoming of this approach is that it relies on a stable interaction among the Argonaute (Ago) proteins, the miRNAs, and the target mRNAs, in order to be efficiently immunoprecipitated. This difficulty can be overcome by cross-linking the complex by first exposing the cells to UV, though application of this technique has its own limitations such as low cross-linking efficiency and potential induction of the DNA damage response To identify cellular genes targeted by EBV BART miRNAs, RISCs in BJAB BARTs and BJAB Empty cells were immunoprecipitated using anti-human Ago2 antibody or an irrelevant anti-GST antibody, which served as a negative control. The efficiency of immunoprecipitation was validated by stem-loop RT-PCR , where cThe levels of mRNAs that are candidates for being regulated by BART miRNAs may be affected by these miRNAs. If these levels were affected by other means, then differences in them could confuse the identification of the mRNAs as being bona fide targets. Decreases in the levels of a particular candidate mRNA could lead to a decrease in the level of the protein it encodes or to its decrease in a RISC, for example. We measured the levels of all identified candidate mRNAs with microarrays and found no differences in the level of candidates found with 2D DIGE, but found decreases in the levels of 16 mRNAs enriched at low levels (∼2.75-fold or less) in immunoprecipitates of RISC from BJAB BARTs cells . The decLevels of the candidate transcripts  were measured by RT-PCR in the Ago-immunoprecipitated RNA samples from both BJAB BARTs and BJAB Empty cells, in order to assess the degree of enrichment of these transcripts in the RISCs of cells expressing BART miRNAs . Individfirefly luciferase ORF into pGL3-Control (Promega) or a dual-luciferase vector pmirGLO (Promega) respectively. Candidates identified from RISC immunoprecipitations were selected on the basis of their enrichment and predicted binding sites for BART miRNAs   . While nB BARTs) .We tested the direct effects of BART miRNAs on the protein levels of candidate targets by immunoblotting where appropriate antibodies were available using three biological replicates. Levels of CEBPA, PUMA, CNBP1, and IPO7 were measured in the cell extracts of BJAB Empty versus BJAB BARTs cell lines . ExpressWe looked for any insights in comparing our two approaches we could glean from analyzing the candidates they uncovered using the program PITA. 3′UTRs of 35 candidates identified by 2D DIGE and of 135 candidates identified by RISC IPs, which were enriched at least at levels comparable to TOMM22 were screened for predicted binding sites of BART miRNAs. Approximately 46% of candidates identified by 2D DIGE were predicted to have binding sites for BART miRNAs , compareC. elegans to people Studies of levels of proteins and of mRNAs in RISCs have been used to implicate cellular miRNAs as regulators of the levels of specific proteins. These studies have been facilitated by the conservation of miRNAs and of their targets during evolution. For example the let-7 miRNA is conserved from We used an EBV-negative Burkitt’s lymphoma cell line, BJAB, which was transduced either with a retroviral vector encoding viral BART miRNAs or an empty vector in this comparison. We first measured the expression level of all the BART miRNAs by stem-loop RT-PCR in these BJAB cells to ensure that the expression of the miRNAs was comparable to those observed in EBV-positive BLs. Most EBV miRNAs are expressed at low levels relative to many cellular miRNAs  [9]. ManThese cells were analyzed with 2D DIGE followed by mass spectrometry to identify proteins that differed by 1.3-fold or more in their levels between BJAB cells expressing the BARTs and sister cells that did not express them. Of 35 signals that were decreased in the presence of the BART miRNAs, 28 were identified by mass spectrometry as unique proteins, 13 of which had predicted binding sites for BART miRNAs. However, the levels of their mRNAs were the same in each cell type, none was found to be enriched in RISCs from cells expressing the BARTs, and none was found to be regulated by the BART miRNAs in luciferase assays. These cells were analyzed in parallel by immunoprecipitating RISCs from them and identifying mRNAs in the complexes by deep sequencing. 1405 mRNas were enriched by 1.5-fold or more in the RISCs from BJAB cells expressing the BART miRNAs. Immunoprecipitating RISCs followed by deep sequencing of enriched mRNAs was more informative, identifying multiple targets we and others have confirmed We assessed the efficiency of the RISC IPs we used to ones in which RNAs are cross-linked to protein prior to immunoprecipitation by comparing the number of unique reads obtained for the cDNAs derived from the immunoprecipitations for each approach. The RISC IPs without prior cross-linking achieved a comparable number of unique reads to those obtained by two recent reports which have used either UV or PAR crosslinking Click here for additional data file.Figure S2Image scans of the 2D gels along with the overlay image are shown. Protein extracts from BJAB Empty and BJAB BARTs cells were labeled with Cy5 and Cy3 respectively, and analyzed by 2D DIGE. The purple circles represent spots which were selected for identification by mass spectrometry.(TIF)Click here for additional data file.Table S1Full list of 1405 candidates identified as being regulated by the BART miRNAs by virtue of their being enriched in RISCs following immunoprecipitation by anti-Ago2.(XLSX)Click here for additional data file.Table S2Sequences of stem-loop RT-PCR primers and probes used to measure BART miRNAs. Additional sequences used in this study were previously reported (XLSX)Click here for additional data file.Table S3Sequences of RT-PCR primers and probes used to measure expression levels of candidate genes obtained via 2D DIGE and RISC IPs.(XLSX)Click here for additional data file.Table S4A list of candidate genes which decreased in expression in the presence of BART miRNAs as detected by microarrays, and which have also been shown to be enriched in RISCs of BJAB BARTs cells.(XLSX)Click here for additional data file.


Breath hold (BH), a commonly used task to measure cerebrovascular reactivity (CVR) in fMRI studies varies in outcome among individuals due to subject-physiology and/or BH-inspiration/expiration differences . In prior age-related fMRI studies, smaller task-related BOLD response variability is observed among younger than older individuals. Also, a linear CVR versus task relationship exists in younger individuals which maybe useful to test the accuracy of CVR responses in older groups. Hence we hypothesized that subject-related physiological and/or BH differences, if present, may compromise CVR versus task linearity in older individuals. To test the hypothesis, empirical BH versus task relationships from motor and cognitive areas were obtained in younger (mean age = 26 years) and older (mean age = 58 years) human subjects. BH versus task linearity was observed only in the younger group, confirming our hypothesis. Further analysis indicated BH responses and its variability to be similar in both younger and older groups, suggesting that BH may not accurately represent CVR in a large age range. Using the resting state fluctuation of amplitude (RSFA) as an unconstrained alternative to BH, subject-wise correspondence between BH and RSFA was tested. Correlation between BH versus RSFA was significant within the motor but was not significant in the cognitive areas in the younger and was completely disrupted in both areas in the older subjects indicating that BH responses are constrained by subject-related physiology and/or performance-related differences. Contrasting BH to task, RSFA-task relationships were independent of age accompanied by age-related increases in CVR variability as measured by RSFA, not observed with BH. Together the results obtained indicate that RSFA accurately represents CVR in any age range avoiding multiple and yet unknown physiologic and task-related pitfalls of BH. While CBF is the physiological variable defining CVR, its correlate, the blood oxygen level dependent signal (BOLD) has an advantage of high signal to noise ratio for clinical applications 2 changes are negligible during most CVR challenges leading to a linear dependence between CBF and BOLD 2), supplied through a breathing-mask to subjects have been utilized. During scanning, CO2-air mixtures are introduced into the mask. Breathing air-gas mixture leads to perturbation of vasculature, hence avoiding intravenous drug injections 2 perturbation takes a few minutes to produce significant BOLD response. Special groups such as chronic obstructive pulmonary disease (COPD) patients are often excluded from CO2 breathing studies using gas-masks thus narrowing the subject pool. Thus alternatives such as the breath hold (BH) task have been utilized to produce mild hypercapnia in a matter of seconds 2 gas and air and is generally considered a reliable CVR measurement method for fMRI applications 2 saturation in the alveoli and gas exchange between blood and alveoli during the BH episode remain difficult factors to account for. As lung volume contracts during BH, the physiological variables may vary in an unknown manner within large age-ranges. This is further exacerbated by the adoption of different styles of BH such as end-expiration or end-inspiration. Despite no major advantage of one BH style over another for fMRI studies A broad definition of cerebrovascular reactivity (CVR) is the ability of cerebrovasculature to respond to chemical agents or CO22-hypercapnic challenge or BH indicating strong correlation with the resting state BOLD fluctuations and EtCO2 fluctuations in spontaneously breathing humans It is known from prior studies that subject-level CVR versus task responses have a linear relationship for COAll experimental procedures were approved by the Institutional Review Board of the University of Texas at Dallas. Data from twenty four healthy subjects 12 younger  and 12 older  with no history of head trauma and neurological diseases obtained as part of our earlier study 2, BOLD and CBF responses during end-inspiration BH compared to end-expiration BH Experimental tasks are explained in greater detail in our earlier study MRI measurements were performed on a 3T PHILIPS scanner. Gradient echo-EPI images were subsequently obtained during resting state, motor (fingertap), cognitive (DSST) and BH tasks. 32 slices were obtained in the axial plane covering the entire brain. Imaging parameters were: FOV = 220 mm, matrix size = 64×64, TR/TE = 2000/30 msec, and slice thickness = 4 mm. Number of acquisitions were motor task = 110, cognitive task = 150, BH = 120 and resting state = 110.All analysis of the functional images were performed using AFNI 2, resting state fluctuation of amplitude-RSFA, amplitude of low frequency fluctuations-ALFF, BH) that significantly correlate among themselves do not show age-dependent differences in correlation at the voxel-level A subject-wise analysis was followed in this study as previous age-dependent BH-task relationships have shown no apparent differences at the voxel level Significantly activated voxels in every subject during the motor and cognitive tasks were considered as regions of interest and the average motor and cognitive task-induced %BOLD signal change was determined. Similarly, average BH-induced %BOLD signal change was also determined from the respective motor and cognitive task activated regions from every subject. Using the resting-state scan from each subject, subject-wise mean RSFA was calculated from the voxel average of the motor and cognitive task-activated areas. Possible causes for the disruption of the linear relationship between BH versus motor and BH versus cognitive task responses in the older subjects were investigated based on the hypothesis of subject-specific physiology and/or BH-performance difference. The percent-wise distribution of the BH-induced BOLD responses within the motor task activated areas was estimated in the younger and older subjects  along wi−3). A similar trend in RSFA variability was observed in the cognitive-task activated areas (As RSFA is independent of task performance (unlike BH), we applied it as an ‘unconstrained’ (task-free) measure for a comparative analysis of RSFA variability within the motor task activated region. RSFA distributions within the motor task activated areas was estimated for the younger and older subjects  along wied areas  with sigFurther comparisons of the subject-wise relationships between BH and RSFA were performed. As indicated by the results in Since activation volumes represent statistical test parameters depending on both signal and noise levels, we compared the activation volumes during all tasks in both groups. No significant differences were observed in the mean motor, cognitive or BH-induced activation volumes between the younger and older groups . Note thThis study highlight a number of methodological and physiological issues related to the reliable estimation of CVR by BH in human populations of different ages and suggest an internal standard such as RSFA as an alternative.2 gas exchange in addition to performance differences in the BH task itself 2 change can be more accurate when using CO2 inhalation methods 2 during BH durations are not available and need to be assumed as time-linear 2BH has been frequently used in fMRI to test CVR 2 variations vasomotive component related to respiration in spontaneously breathing human subjects 2 fluctuations with low frequency BOLD fluctuations have established RSFA as a strong CVR correlate, available from a simple resting state fMRI scan of spontaneously breathing humans with their eyes closed Resting state low frequency BOLD fluctuations and their temporal characteristics have been frequently used as correlates of intrinsic neural activity per se within brain tissue. A notable result was an increased dispersion in RSFA in the motor areas compared to cognitive areas  in older subjects  than the spontaneous neuronal components present within RSFA.Hemodynamic scaling or normalization of BOLD responses can account for vascular-related changes within the brain and can correct for vascular differences among inhomogeneous population. Scaling or normalization can be performed in different ways. The first is by dividing the task-induced response by the CVR response on a voxel wise manner Subject-related differences in physiological changes during BH can profoundly affect CVR determination in large age ranges. The unconstrained CVR marker such as RSFA available from the resting state fMRI signal is suggested as a better alternative to BH. The unconstrained assessment of subtle CVR differences in the higher and lower order brain areas were sensitive enough to detect regional vascular differences and well-suited for fMRI designs that include comparisons between healthy individuals, older individuals, patient- and special-populations Figure S1A,C. during motor (fingertap) task and B,D. during cognitive (DSST) tasks. A, B. indicate older subjects and C, D. indicate younger subjects. A significant linear correlation was observed between motor task versus RSFA  and cognitive task versus RSFA  in the pooled population of younger and older subjects (n = 22). However, no significant correlation was observed between the motor task versus BH (r = 0.01) and the cognitive task versus BH (r = 0.13) in the pooled population of younger and older subjects.Relationship between the subject-average fractional task-induced BOLD signal change (%) versus BH. (TIF)Click here for additional data file.Figure S2A. in the motor (fingertap) task and B. in the cognitive (DSST) task activated areas pooled from both younger and older subjects. No linear correlation was observed between BH versus RSFA in the motor areas (r = 0.6) and was greatly reduced in the cognitive task activated areas (r = 0.20).Relationship between the subject-average fractional BH-induced BOLD signal change (%) versus RSFA. (TIF)Click here for additional data file.


NLRP3 (CIAS1) gene encoding NLRP3 . Most patients present at a young age with a variety of clinical symptoms including fevers, urticaria-like skin rash, arthropathy, and CNS inflammation. A subset of patients followed at the National Institute of Health’s autoinflammatory disease clinic has adult-onset fevers and urticarial rash but conventional genetic testing has been unremarkable for any mutations in NLRP3. We analyzed one such "mutation-negative" patient. She is a 63y/o female of Irish ancestry who developed a gradually worsening stress-induced urticarial rash in her 40s. Additional clinical history is remarkable for severe arthralgia, myalgia, chills, and occasional conjunctivitis. Initially started on anakinra 100mg/day in 2003, she had dramatic improvement in symptoms; however, her anakinra dose has required periodic adjustments since that time to control her symptoms.The dominantly inherited cryopyrin-associated periodic syndromes (CAPS) are caused by heterozygous missense gain-of-function mutations in the NLRP3 based on the conventional Sanger sequencing.To identify the cause of disease in a patient who presented with late-onset CAPS and who was mutation-negative in NLRP3 somatic mutation. Targeted deep sequencing on NLRP3 will be done using the DNA from blood and buccal cells.We performed whole exome sequencing in the DNA sample extracted from peripheral blood. A total of 192 subclones were randomly selected and subjected to Sanger sequencing to validate the NLRP3. Exome data showed that there are 12 reads carrying the mutant G allele and 64 reads carrying the wild type A allele (15%). This mutation has been previously reported in a NOMID/CINCA patient from Australia. The putative mosaic p.Tyr570Cys mutation was validated by subcloning a 718bp fragment from the patient’s leukocyte DNA and subsequent Sanger sequencing of 192 clones. We found 20 clones carrying the p.Tyr570Cys mutation, which established the level of somatic mosaicism at 10.4%. We are in the process of evaluating this mosaic mutation in other tissues from this patient.Our established exome data analysis pipeline failed to identify any plausible candidate gene in this patient. In order to increase the sensitivity of variant calling, we used a different combination of parameters including calling SNPs/Indels with extreme strand bias, which resulted in a rapid increase in false positive rate but also raised the possibility of identifying somatic mosaicism mutations. Based on this new analytic approach, we identified a possible mutation p.Tyr570Cys (c.1709A>G) in NLRP3 somatic mosaicism in a patient presenting with a late onset CAPS. To our knowledge this is the first time that a NLRP3 mutation has been identified in patients developing symptoms in adulthood. Other possible causal genes have been ruled out based on exome sequencing analysis. Our result contributes significantly to understanding the pathogenesis of disease in patients with adult-onset autoinflammatory diseases.We report None declared.


Toll-like receptors (TLRs) are a family of conserved pattern-recognition molecules responsible for initiating innate and acquired immune responses. Because they play a key role in host defence, these genes have received increasing interest in the evolutionary and population genetics literature, as their variation represents a potential target of adaptive evolution. However, the role of pathogen-mediated selection (i.e. episodic positive selection) in the evolution of these genes remains poorly known and has not been examined outside of mammals. A recent increase in the number of bird species for which TLR sequences are available has enabled us to examine the selective processes that have influenced evolution of the 10 known avian TLR genes. Specifically, we tested for episodic positive selection to identify codons that experience purifying selection for the majority of their evolution, interspersed with bursts of positive selection that may occur only in restricted lineages. We included up to 23 species per gene (mean = 16.0) and observed that, although purifying selection was evident, an average of 4.5% of codons experienced episodic positive selection across all loci. For four genes in which sequence coverage traversed both the extracellular leucine-rich repeat region (LRR) and transmembrane/intracellular domains of the proteins, increased positive selection was observed at the extracellular domain, consistent with theoretical predictions. Our results provide evidence that episodic positive selection has played an important role in the evolution of most avian TLRs, consistent with the role of these loci in pathogen recognition and a mechanism of host-pathogen coevolution. Immune genes are ideal for studying how evolutionary mechanisms influence the genetic diversity of natural populations, as such loci experience a variety of selection pressures from a wide diversity of coevolving pathogens with which animals are associated One important family of innate-immunity genes is the toll-like receptors (TLRs): a group of genes that recognise a wide diversity of pathogens and are responsible for initiating both the innate and acquired immune responses through binding of pathogen-associated molecular patterns (PAMPs) TLR4 and TLR7TLR4 , TLR7 appears to be duplicated in members of the order Passeriformes TLR2A and TLR2B arose by duplication of vertebrate TLR2 early in avian evolution, while the TLR1LA (“TLR1-like”) and TLR1LB gene-duplicates belong to the vertebrate TLR1/6/10 superfamily TLR21 is shared with bony fishes and XenopusTLR15 appears to be unique to birds and reptiles TLR4TLR15TLR21The avian TLR repertoire, first characterised following completion of the chicken genome In addition to examining episodic selection in avian TLRs, we test the hypothesis that greater evidence for positive selection should occur in the extracellular domain characterised by leucine-rich repeats (LRRs) than at a signal/transmembrane domain or an intracellular toll/interleukin I resistance (TIR) domain TLR7 appears to be duplicated in passerines TLR7-type 1 where available (e.g. cloned sequences).To test our hypotheses, we retrieved avian TLR sequences from Genbank . Sequenchttp://www.datamonkey.org, accessed October 2013 To facilitate comparison of our results to previous work, we tested for evidence of positive selection using the single likelihood ancestor counting (SLAC) and random-effects likelihood (REL) methods, as well as making the first examination of episodic selection in avian TLRs using the mixed-effects model of evolution (MEME). Phylogenetic relationships among sequences for each TLR locus were estimated using the neighbour-joining method implemented in the HyPhy package Sd and Nd at each codon position; the REL model uses the observed substitution rates, in comparison to a fitted rate distribution across sites (Kosakovsky Pond and Frost 2005). SLAC and REL both have limitations relative to other likelihood methods in that SLAC lacks power in certain datasets, while REL can be susceptible to false positives; therefore the use of appropriate significance thresholds and basing interpretations on consensus outcomes is generally considered to be a reasonable approach The SLAC model reconstructs ancestral sequences and then uses counts of MEME is a generalisation of the fixed-effects likelihood (FEL) approach, where ω at each codon is free to vary across lineages, thereby identifying residues that have undergone episodic selection  t-test to compare the divergences of all inferred substitutions across the alignment, to the mean distances among the amino acids observed at each positively selected site. We used a null hypothesis of equal means (two-sided test).Because MEME allows the mapping of positively selected sites to branches in a phylogeny, we were able to identify the proportion of positively selected sites that occurred on internal versus terminal branches, and compare this value to a null-hypothesis of uniform distribution. To compare the locations of positively selected sites on each phylogeny, calculations of internal and terminal branch lengths were performed using functions available in the R-package “ape”. Physicochemical distances between inferred amino acid substitutions, or among amino acid states at variable sites, were quantified using Grantham's TLR1LA: BAD67422.1, TLR1LB: ABF67957.1, TLR2A: NP_989609.1, TLR2B: BAB16842.1, TLR3: NP_001011691.3, TLR4: AAL49971.1, TLR5: CAF25167.1, TLR7: NP_001011688.1, TLR15: NP_001032924.1, TLR21: NP_001025729.1). For the four genes for which it was possible to compare selection in LRR and other domains of the gene , we examined whether the mean normalised Nd-Sd values (as determined by the SLAC method) differed between domain types (LRRs and “other”), using a linear model for each gene. In each model, the codon domain (LRR/other) was a categorical predictor variable and the Nd-Sd value for each codon was the response variable.We assessed the functional significance of positively selected codons by examining the distribution of these results across functional domains of each gene, as delineated using the software LRRfinder N≥10 or the exact method for datasets with smaller sample sizes. Both confidence interval methods are implemented in the package “binom” We also tested whether statistically significant findings of selection were disproportionately distributed in LRR or in other domains of the each gene. We used a two-sided Fisher's exact test where the null expectation was that significant findings would be uniformly distributed across LRR and “other” codons. Our observed value was the proportion of significant codons that occurred within LRR domains, from the total number of significant codons detected. We visualised these comparisons with binomial 95% confidence intervals for the observed values, obtained using the Agresti-Coull method TLR3 and TLR4). To facilitate localisation of positively selected sites on the crystal structures of TLR3 and TLR4, we first generated alignments of chicken TLR proteins against each of the mammalian products  using Geneious  compared to the earlier analysis of avian TLR evolution Apteryx mantelli in TLR1LA, TLR2B, TLR3, TLR5, TLR7) and Gruiformes (crane-like birds)   .N = 190 possible amino-acid substitution types total) were inversely correlated with Grantham's Across all genes, we detected 4,441 amino acid substitutions (excluding codons with a nucleotide mixture); the frequencies of each type  than expected . For these four genes, sequencing across LRR and non-LRR regions was uneven, with an average 81% of the data occurring in the LRR region  this trend was statistically significant at α = 0.05 , probably as a result of the aforementioned uneven sequencing coverage. For example, comparisons between LRR and non-LRR domains do not appear to be sensitive to false positives, as increasing the stringency of the significance thresholds used to identify the codons under selection did not change the overall pattern between LRR and other regions of the gene (data not shown).Because our alignments do not traverse the entire coding sequence of each gene, comparisons between LRR and non-LRR domains of the genes were only possible for R region . NeverthTLR3  was associated with juvenile survival in a wild population of a threatened bird TLR4 also had the highest number of haplotypes (compared to other TLRs) in this bottlenecked population TLR4 in particular, and suggest that variation at this gene appears to be of evolutionary significance in wild populations. Population-level studies may be particularly useful for revealing the small-scale pressures exerted on variation at this locus.Our results echo findings from different immunogenetic contexts; for example, episodic positive selection was found to play a key role in the evolution of killer cell Ig-like receptor sequences in New World primates ons e.g. , and whiTLR21  in samples of wild populations of house finch and lesser kestrel TLR21 shows a higher degree of conservation than other avian TLRs.The lowest proportion of selected sites was observed within the analysed region of TLR21 , althougTLR2A) showed a greater degree of positive selection on terminal branches, perhaps indicating a higher degree of species-specific  positive selection. This pattern may also occur if the positively selected substitutions observed in these loci are beneficial to certain species, but deleterious in other contexts, and thus have a lower chance of being incorporated into long-term evolutionary change.Several lines of evidence suggest putatively functional consequences of variation in the LRR domains of avian TLRs, specifically the sites identified here as subject to episodic selection. First, for the four loci where our alignments contain codons in both LRR and other domains of the gene, we observed evidence for generally higher ω values, and greater numbers of positively selected sites, in the LRR domains, consistent with predictions based on gene function A second indication of the functional significance of the positively selected sites identified here is the observation that amino acids at positively selected codons were typically more divergent from each other than inferred substitutions. This finding parallels observations in MHC, where profound amino acid substitutions are more likely to occur in the antigen-binding regions TLR3 and TLR4), we see that the vast majority of positively selected sites are concentrated outside of α and β strands of the molecules, in unbound strands that are more free to vary and able to interact with other macromolecules. In addition, several positively selected sites were observed in the putative PAMP binding region of TLR4Third, where crystallographic structures were available Click here for additional data file.Table S2Sites identified as being under positive selection using three alternative approaches , as compared to sites identified in a previous analysis .(DOCX)Click here for additional data file.Table S3Results of t-tests comparing the physicochemical distances of all inferred amino acid substitutions, to distances among amino acid variants observed at positively-selected sites.(DOCX)Click here for additional data file.Table S4Results of linear regression examining the effect of domain (“LRR” or “other”) on mean normalised Nd-Sd values, for the four TLR loci for which such comparison was possible.(DOCX)Click here for additional data file.Figure S1Neighbour-joining trees of 10 TLR loci, used as the basis for evolutionary analyses. Tip labels are genus names (full species names and Genbank accession numbers provided in (TIF)Click here for additional data file.Figure S2Alignment of chicken (Genbank protein AAL49971) and human (3FXI_A) partial TLR4 proteins. The region examined in this study is shown in bold font; positively selected residues  are shaded grey.(TIF)Click here for additional data file.Figure S3Alignment of chicken (Genbank protein AAL49971) and mouse (3CIY_A) partial TLR3 proteins. The region examined in this study is shown in bold font; positively selected residues  are shaded grey.(TIF)Click here for additional data file.Figure S4Inferred amino acid substitutions observed across all 10 TLR alignments; intensity of shading correlates with the number of substitutions (range 0–212).(TIF)Click here for additional data file.Figure S5Location of positively selected codons  on each TLR gene tree. Codons given refer to positions in the current alignments Click here for additional data file.Figure S6Comparison of mean physicochemical distances between all inferred amino-acid substitutions (open circles) and amino-acid variants observed at positively selected sites (filled circles), at each locus. Error bars are ±1.96× standard error; asterisks indicate pairs of means that differ at α = 0.05 (full test statistics and sample sizes provided in (TIF)Click here for additional data file.Figure S7Approximate positions of positively-selected residues  in the three-dimensional structure of the TLR3  and TLR4  homodimer ectodomains (blue and pink) , and the sequenced region is indicated using a thicker line than the non-sequenced region. Also shown is the association between TLR3 and its dsRNA ligand (green and brown) (in A), and myeloid differentiation factor 2 , which complexes with TLR4 (in B). Both of these molecular representations are provided as interactive, Cn3D graphics, as (TIF)Click here for additional data file.File S1Compressed “zip” file containing all ten alignments used in this analysis, in .fas (fasta) format. Sequences are labelled with Genbank accession numbers and species binom; see also (ZIP)Click here for additional data file.File S2cn3 file for TLR3 structure shown in  This graphic can be viewed using the NCBI application Cn3D (CN3)Click here for additional data file.File S3cn3 file for TLR4 structure shown in  This graphic can be viewed using the NCBI application Cn3D (CN3)Click here for additional data file.