Peripheral blood biomarkers are needed to identify and determine the extent of idiopathic pulmonary fibrosis (IPF). Current physiologic and radiographic prognostic indicators diagnose IPF too late in the course of disease. We hypothesize that peripheral blood biomarkers will identify disease in its early stages, and facilitate monitoring for disease progression.LCO and FVC.Gene expression profiles of peripheral blood RNA from 130 IPF patients were collected on Agilent microarrays. Significance analysis of microarrays (SAM) with a false discovery rate (FDR) of 1% was utilized to identify genes that were differentially-expressed in samples categorized based on percent predicted DLCO >65%) compared to controls and 2790 transcripts were differentially- expressed in severe IPF (DLCO >35%) compared to controls. When categorized by percent predicted DLCO, SAM demonstrated 13 differentially-expressed transcripts between mild and severe IPF (< 5% FDR). These include CAMP, CEACAM6, CTSG, DEFA3 and A4, OLFM4, HLTF, PACSIN1, GABBR1, IGHM, and 3 unknown genes. Principal component analysis (PCA) was performed to determine outliers based on severity of disease, and demonstrated 1 mild case to be clinically misclassified as a severe case of IPF. No differentially-expressed transcripts were identified between mild and severe IPF when categorized by percent predicted FVC.At 1% FDR, 1428 genes were differentially-expressed in mild IPF (DLCO, but not FVC.These results demonstrate that the peripheral blood transcriptome has the potential to distinguish normal individuals from patients with IPF, as well as extent of disease when samples were classified by percent predicted D LCO) and forced vital capacity (FVC) have been utilized as predictive indicators LCO of <35% or a decline in DLCO >15% within a year period are associated with an increased mortality. Similarly, a decline of >10% in FVC over a six month period also indicated an earlier mortality LCO among the placebo arms of the trials indicating there is substantial disease heterogeneity within IPF Idiopathic Pulmonary Fibrosis (IPF) is categorized as an interstitial lung disease (ILD) and is the most common subtype of idiopathic interstitial pneumonias (IIP) comprising nearly 71% of the total cases Rosas and coworkers observed a differential expression of MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A proteins in the peripheral blood between familial interstitial pulmonary fibrosis and controls LCO and FVC, were used to stratify the cases into severe and mild disease categories. Mild disease is defined as either percent predicted DLCO ≥65% (N = 16) or FVC ≥75% (N = 27). Severe disease is defined as either DLCO ≤35% (N = 15), FVC ≤50% (N = 13). All of these were also compared to age and gender matched non-diseased, healthy controls (N = 27). Eight patients categorized as severe were used in both the DLCO and FVC analysis, whereas the mild disease classified by either DLCO or FVC are all distinct cases. Healthy control subjects are family members who participated in screening for the presence of pulmonary fibrosis, and after evaluation of their medical history, lung function, and chest CT, they were found to have no evidence of lung disease. Individual institutional review boards approved this research. All participants in this study provided written IRB-approved informed consent.One hundred thirty peripheral blood RNA specimens were collected from individuals enrolled in either the Interstitial Lung Disease (ILD) or the Familial Pulmonary Fibrosis (FPF) Programs conducted at National Jewish Health and Duke University. Only one individual case per family was utilized from the FPF repository. Individual samples had a consensus diagnosis of probable or definite IPF, and this was based on the ATS/ERS/JRS/ALAT criteria LCO measurement were obtained according to American Thoracic Society standards and guidelines FEV1 and DPeripheral blood samples were collected in PAXgene RNA tubes . RNA extraction and purification was performed manually utilizing the PAXgene Blood RNA kit  according to the manufacturer’s protocol.Quantification of total RNA was measured via the Nanodrop ND-1000 spectrophotometer . Quality of the RNA was assessed with a RNA 6000 NanoChip  on the 2100 Bioanalyzer  by ratio comparison of the 18 S and 28 S rRNA bands.Agilent Whole Human Genome Oligonucleotide Microarrays , were used to determine gene expression levels in peripheral blood. Total RNA was used as a template for synthesis of cDNA utilizing the One Color Low Input Agilent Quick Amp Labeling Kit utilizing the Spike-In Kit to provide positive controls. The Agilent one-color microarray based gene expression analysis was followed per manufacturer’s instructions, and passed Agilent’s quality control (QC).LCO and FVC. All IPF samples were compared to normal controls to identify differentially-expressed genes. Principal component analysis (PCA) was carried out on genes identified by SAM analyses to identify outliers, and gene-based hierarchical clustering was performed to identify relationships among differentially-expressed genes related to mild or severe disease categorized by DLCO. All microarray data is minimal information about a microarray experiment (MIAME) compliant, and raw data has been deposited into the GEO database (GSE33566).Analysis was performed utilizing the Multi-Experiment Viewer (MeV) software package www.ingenuity.com). The Canonical Pathways Analysis identified the pathways, and the significance of these was determined by Fischer’s exact test. Biomarker Analysis was employed to identify the most relevant molecular biomarker candidates.Data were analyzed through the use of Ingenuity Pathways Analysis  using forty cycles of amplification. All assays were performed in duplicate and data were analyzed by the ΔΔCt method utilizing glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as an endogenous control LCO 77.1% predicted in the mild disease group, and a mean FVC 42.5% predicted and 27.5% predicted DLCO in the severe disease group. The mean age is similar between groups when classified by either % predicted FVC (P = 0.18), or % predicted DLCO (P = 0.87), but the mean age of either disease group is slightly older compared to controls (P = 0.15). The majority of patients are male in both mild and severe disease categories, and similar between both disease categories when classified by either % predicted FVC (P = 0.48) or % predicted DLCO (P = 0.29). There are no significant differences between mild and severe disease categories with respect to prior tobacco use. The diagnosis of IPF was confirmed by surgical lung biopsy in 50.7% of the study subjects , both probable and definite, cluster along the first principal component , both probable and definite, distribute together along the first principal component  was performed to determine outliers in the gene expression data based on disease categorization . The majority of mild cases  compared to normal controls (N = 31) revealed 1,428 differentially-expressed transcripts  of the mild IPF cohort, when categorized by percent predicted Dnscripts , and whenscripts  with <1%controls  comparedcontrols . TherefoLCO (DLCO ≥65% [N = 16] and DLCO ≤35% [N = 15]), demonstrated 13 differentially-expressed transcripts with less than a 5% false discovery rate. LCO. When using a FDR of ≤1%, only defensin A3 (DEFA3) is differentially-expressed between both mild or severe cases compared to controls. SAM revealed no differentially-expressed transcripts with less than a 5% false discovery rate between peripheral blood samples when IPF patients were categorized by percent predicted FVC .Principal component analysis was performed to determine outliers in the data set based on severity of disease categorization. Results demonstrate that 1 severe IPF case appears to be clinically misclassified as a mild case of IPF, while other cases are correctly classified as mild or severe . SignifiLCO for the purpose of discovery common potential biomarkers. This analysis showed that at FDR <5% only 2 differentially expressed transcripts, A3 (DEFA3) and hypothetical protein FLJ11710 , were differentially-expressed between control vs mild, mild vs severe, and control vs severe disease (DLCO ≤35%). There is up-regulation in DEFA3 expression compared to controls for severe IPF, while hypothetical protein FLJ11710 demonstrates a down regulation compared to controls for severe IPF. Between the mild and severe IPF cohort , and between normal and severe disease, there is up-regulation of several other host defense genes including defensin A4 (DEFA4), cathelicidin antimicrobial peptide (CAMP), cathespin G (CTSG), and down-regulation of immunoglobulin heavy chain constant mu (IGHM). The greatest increase found distinguishing controls from severe disease, and mild from severe disease is olfactinmedin 4 (OLFM4). We randomly selected 4 of the 13 differentially-expressed genes for qPCR validation, and qPCR confirmed the differential expression of these genes identified by microarray experiments  software was utilized to identify the intersection or common differentially-expressed transcripts between normal and mild IPF, normal and severe IPF, and mild versus severe IPF when classified by Deriments .LCO  LCO).Four of the 13 genes up-regulated in the current study have functions pertaining to host defense. This raises the question of an association of advanced disease and sub-clinical infection, or host-microbe interactions. All of these patients were stable when blood was drawn without overt signs of infection. Viral infection has been put forth as a possible trigger of acute exacerbations of IPF Differential-expression analysis demonstrates up-regulation of the cathelicidin antimicrobial peptide (CAMP) between the severe IPF subgroup compared to controls, and between mild and severe disease groups. Cathelicidin is an antimicrobial protein of the innate immune system stored in peroxidase-negative granules of neutrophils LCO), but not when correlated with by FVC % predicted. The reason for this difference is unclear. FVC is traditionally used to stage the severity of pulmonary fibrosis. However, it has been recognized that a single measurement of the FVC is not useful for predicting disease progression or mortality of IPF LCO <50% predicts an increased mortality LCO% predicted but not a single FVC% predicted measurement due to the better performance of a single measurement of DLCO compared to FVC. A lack of correlation between FVC and severity of IPF can be due to concomitant emphysema, however review of the subject’s HRCT in this study does not demonstrate significant amounts of emphysema. It is possible the signature is a consequence of impaired oxygen transport, but this seems unlikely since two of the thirteen genes also distinguish controls from mild disease where oxygen transport is not a factor. Also, none of the 13 genes have functions related to oxygen transport, aerobic or anaerobic metabolism, or other pathways that might be implicated in the presence of hypoxemia or impaired oxygenation.There are limitations to this study. Given the minimal disease burden and lack of clinical symptoms, five cases of mild IPF were not appropriate candidates for surgical lung biopsy, and therefore did not have surgical tissue. Due to the minimal disease burden, they did not meet HRCT criteria of definite IPF according to the 2011 ATS/ERS/JRS/ALAT consensus statement. However, in the five cases the mean age was 70, and 4 cases were male supporting the diagnosis of IPF. Furthermore, these cases cluster with definite mild cases along the principal components . TherefoLCO, and distinguish IPF patients from normal. Our data is consistent with a role for MMP-7 interacting with α-defensins, and increased expression of host defense proteins in the peripheral blood being associated with deterioration of DLCO in IPF. The ability to use a peripheral blood biomarker to monitor disease progression for IPF could have a substantial impact on the diagnosis, treatment, and management of this disease, and perhaps be generally applicable to other subtypes of idiopathic interstitial pneumonias.In summary, our results demonstrate that the peripheral blood transcriptome can potentially distinguish extent of disease in individuals with IPF when samples are correlated with percent predicted DTable S1†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx): definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S2†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx) :definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S3†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx) :definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S4†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx): definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S5Canonical pathway analysis using Ingenuity Pathway Analysis™ tool to identify associated networks. Network functions with their respective P values and the number of differentially-expressed genes identified are presented.(DOCX)Click here for additional data file.Table S6LCO >65%) using SAM at FDR of <1%.Differentially-expressed genes between controls and mild IPF .(XLSX)Click here for additional data file.Table S7LCO <35%) using SAM at FDR of <1%.Differentially-expressed genes between controls and severe IPF .(XLSX)Click here for additional data file.Table S8Validation of selected differentially-expressed genes by Reatime™ PCR. Primers utilized for PCR and differential expression by Realtime PCR using ΔΔ Ct method.(XLSX)Click here for additional data file.


FoxC2) gene are strongly associated with valve failure in saphenous and deep veins of lower extremities. We explored the association of genetic variants of FoxC2 as well as FoxC2 mRNA and protein expression levels with CVD of lower limbs. We systematically sequenced the single coding exon, 5′ and 3′ flanking regions of FoxC2 gene in 754 study subjects which includes 382 patients with CVD and 372 healthy subjects. Four novel and three reported polymorphisms were identified in our cohort. Three variants in 5′ flanking region and one in 3′ flanking region of FoxC2 gene were significantly associated with CVD risk. FoxC2 mRNA in vein tissues from 22 patients was 4±1.42 fold increased compared to saphenous veins from 20 normal subjects (p<0.01). FoxC2 protein was also significantly upregulated in varicose veins compared to control samples. The c.-512C>T (rs34221221: C>T) variant which is located in the FoxC2 putative promoter region was further analyzed. Functional analysis of c.-512C>T revealed increased mRNA and protein expression in patients with homozygous TT genotype compared to heterozygous CT and wild CC genotypes. Luciferase assay indicated higher transcriptional activity of mutant compared to wild genotype of this variant. These findings suggested that c.-512C>T variant of FoxC2 was strongly associated with susceptibility to CVD and also that this variant resulted in FoxC2 overexpression. To obtain a mechanistic insight into the role of upregulated FoxC2 in varicosities, we overexpressed FoxC2 in venous endothelial cells and observed elevated expression of arterial markers Dll4 and Hey2 and downregulation of venous marker COUP-TFII. Our study indicates altered FoxC2-Notch signaling in saphenous vein wall remodeling in patients with varicose veins.Chronic venous disease (CVD) is one of the most prevalent yet underrated disorders worldwide. High heritability estimates of CVD indicate prominent genetic components in its etiology and pathology. Mutations in human forkhead box C2 ( Chronic venous disease (CVD) of the lower extremities is one of the most prevalent diseases worldwide though the prevalence estimates differ extensively due to the different disease evaluation methods. CVD comprises of visible venous disorders which are not associated with an identifiable mechanism of venous dysfunction Several risk factors contribute to the progression of CVD FoxC2 gene  is located 80 kb distant from this marker. It was also reported that homozygous null mice of FoxC2 (foxc2−/−) had abnormal lymphatic vascular patterning and malfunctioned blood vessels A twin cohort study indicated a link between varicose veins to microsatellite marker D16S520 on chromosome 16 FoxC2 gene variants are strongly associated with lymphedema distichiasis (LD) syndrome where majority of patients develop varicose veins FoxC2 gene is also implicated in the pathogenesis of saphenous vein and deep vein reflux FoxC2 genetic variants in patients with varicose veins. We investigated the role of FoxC2 genetic variants in the development of CVD of lower limbs in a case-control study. We quantified mRNA and protein expression level of FoxC2 gene in saphenous vein from patients with varicose veins and healthy subjects. FoxC2 expression was highly upregulated in varicose vein tissues compared to normal control veins. Our results demonstrate significant correlation between c.-512C>T, a promoter variant of FoxC2 and the expression levels of FoxC2 mRNA and protein in CVD of lower limbs. FoxC2 overexpression in vein endothelial cells in vitro led to the upregulation of arterial markers such as Hey2 and Dll4 and the downregulation of venous marker, COUP TFII.The study was approved by the human ethics committees of Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram and all collaborating hospitals. Samples were collected from patients and healthy subjects after obtaining informed written consent.FoxC2 have been reported to result in susceptibility to diabetes mellitus. Blood samples were collected from age and gender matched 352 healthy controls with no known family history for CVD.382 patients with CVD and 372 control subjects were recruited for this study. Whole blood samples (5 ml) were collected from 360 patients (age - 18 to 60 years) with CVD from St.Thomas Hospital, Kerala, India. Diagnosis of CVD was based on physical examination and Doppler ultrasound test. CVD resulting from obstructions such as neoplasm were excluded from the study. Differential diagnosis was performed by an experienced vascular surgeon and presence of distichiasis was ruled out by an ophthalmologist. Patients with type 2 diabetes mellitus were also excluded since genetic variants of For tissue level expression analysis, varicose vein tissue samples were collected from 22 patients admitted for treatment of CVD by operative treatments at Kempegowda Institute of Medical Sciences, Bangalore, India. Saphenous control vein samples from 20 patients who underwent coronary artery bypass graft surgery at Sri Jayadeva Institute for Cardiovascular Sciences & Research, Bangalore, India were also collected for the study. Whole blood samples were also collected from these 22 patients and 20 controls for sequencing assays. Relevant data regarding the clinical characteristics of patients were collected from medical records of the hospitals participating in the study.Demographic data of all study participants and information regarding symptoms such as pain, itching and throbbing sensation in legs and clinical signs such as hemorrhage, lower limb oedema, hyperpigmentation, thrombophlebitis, cellulitis and ulceration were collected for each patient from medical records. Family history, occupational and lifestyle data were collected to examine their influence in aggravating disease manifestation.Disease phenotypes were categorized according to CEAP  classification system Genomic DNA from whole blood samples was extracted using QIAamp DNA blood mini kit  according to the manufacturer's instructions. Genomic DNA and mRNA from vein tissues were extracted by All Prep DNA/RNA/Protein mini kit . Quantification and purity of DNA and mRNA was measured by nanodrop-1000 spectrophotometer  at 260 nm. RNA was further treated with DNase1  for removing any DNA contamination.FoxC2 gene from DNA of patients with CVD and healthy subjects. Nine primer pairs . PCR conditions were as follows: Initial denaturation for 5 min at 96°C, 20 cycles of denaturing at 96°C for 30 sec, annealing at 70°C for 40 sec with a touchdown of 0.5°C per cycle and extension at 72°C for 1.5 min. This was followed by 20 cycles at same conditions except that annealing was at 60°C for 40 sec. PCR products were purified using gel band elution kit . DNA sequencing was carried out on an ABI 3100 DNA analyzer with Bigdye terminator chemistry .A touch-down PCR was performed to amplify the single coding exon, 3 kb of 5′ flanking and 200 bp of 3′flanking region which includes the 5′ and 3′ untranslated regions (UTR) of er pairs  to ampliFoxC2 and GAPDH genes were designed for real time PCR analysis ].Total RNA (500 ng) from each tissue sample was subjected to reverse transcription with oligodT, dNTPs, and M-MLV reverse transcriptase . Primers for analysis . Quantitg for 20 min at 4°C to collect the supernatant. Proteins were estimated by using Bradford reagent . Protein extracts (30 µg) were subjected to 12% SDS–PAGE and electro transferred to a Hybond C Extra membrane  as per the wet transfer procedure (Bio-Rad Mini Protean II). Membranes were blocked for 1 hour at room temperature in PBS containing 0.5% Tween-20 and 5% BSA, and incubated overnight with anti- FoxC2 antibody (1 µg/ml) at 4°C . Membrane was washed with TBS–Tween-20 and incubated with the 1∶10000 dilution of secondary antibody to rabbit IgG - H&L (HRP)  for 1 h at room temperature. Protein band was developed by enhanced chemiluminescence . The membrane was re-probed with anti GAPDH antibody  for normalization of expression. The densities of immunoreactive bands were quantitated by the Quantity One 1-D image analysis software program .Frozen vein tissues were homogenized and incubated in ice-cold RIPA buffer with protease inhibitor cocktail for 90 minutes followed by centrifugation at 15,000 2O2 in methanol by incubating for 30 minutes. Sections were rehydrated through graded alcohols and antigen retrieval was performed by incubating in 10 mM sodium citrate at 90°C for 15 mins. Sections were washed with TBST and then blocked with 3% BSA for 20 mins. Slides were incubated with anti- FoxC2 antibody (Abcam cat # ab65141) diluted with TBS in 1∶100 ratio. Slides were washed thrice for 5 minutes in TBST and incubated for 1 hour with Horse raddish peroxidase conjugated anti rabbit antibody (Abcam cat # ab97051) diluted with TBS in 1∶200 ratio. After washing, slides were incubated with 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Sigma) and immediately washed under tap water after the color development and were counter stained with haematoxylin. Slides were DPX mounted and observed under light microscope .5 µm paraffin embedded tissue sections were de-paraffinized in xylene and endogenous peroxidase activity was quenched with 3% HFoxC2 gene in the promoter activity of gene, a luciferase reporter gene assay was performed. A 664 bp DNA sequence from the −614 bp to +50 bp from the transcription start site of FoxC2 gene encompassing the putative promoter area was amplified from DNA with CC and TT genotype. Primers including KpnI and HindIII restriction sites  and mutant pGL4-FoxC2 promoter (TT genotype) were transfected into cultured cell lines using Lipofectamine . Renilla luciferase construct (phRL-TK) was used as a control for transfection efficiency. After 48 h, each group of cells was lysed and luciferase assay was carried out using Dual Luciferase assay kit  according to manufacturer's instructions and readings were recorded. Technical replicates were performed in triplicate and biological experiments were performed twice.EA.hy926 FoxC2 – pCAGIG construct was made by inserting FoxC2 coding sequence into EcoRI and XhoI restriction sites of pCAGIG mammalian expression vector (Addgene plasmid 11159) FoxC2 -pCAGIG and control empty vector was performed using lipofectamine-2000  according to the manufacturer's recommendation. The concentrations of constructs used were 1 µg per well. After 6 hours of transfection, 20% FBS supplemented DMEM medium was added. The assays were carried out 8 days post-transfection. Transfection was performed in triplicates and repeated twice. GFP positive cells were sorted on FACS Aria (Becton Dickinson) and used for further analysis.FoxC2, GAPDH, Hey2, Dll4, COUP TFII and Ephrin B4 gene expression. Primers sequences are given in table S2. The conditions for amplifying FoxC2 and GAPDH are as described earlier. For assessing Hey2, Dll4, COUP TFII and Ephrin B4 gene expression, the reactions were performed in triplicate in 96-well plates at 48°C, 30 min; 95°C, 10 min; followed by 40 cycles of 95°C, 15 s; and 61°C, 1 min. The real-time PCR products were re-confirmed by electrophoresis on 2% agarose gels. The amount of the target relative to GAPDH mRNA was expressed as 2 −(ΔCt).Total RNA from untransfected and transfected EA.hy926 cells was isolated by Allprep RNA/protein kit . After the reverse transcription reaction as described earlier, cDNA was used for quantitative real time PCR for Hardy-Weinberg equilibrium was tested for a goodness-of-fit using a Chi square test. Chi-square test was used to investigate the possible association between polymorphisms and CVD in case-control studies. Student's t test was used to analyze the difference in luciferase, mRNA transcripts and protein expression levels. Information collected from answered questionnaires and medical records were entered into MS Excel and analyzed using SPSS 16 . Differences between groups were considered significant for p values less than 0.05.Patients with CVD (n = 382) and healthy subjects (n = 372) had a mean age of 48.17 (SD = 13.37) and 37.86 (SD = 13.63) respectively (p<0.001). 53.7% of patients were females and a positive association was seen between female gender and CVD (p = 0.035). A detailed analysis of patients and controls with respect to different age groups and gender is given in FoxC2 gene in patients with CVD and healthy controls are presented in rs78816814, NG_012025.1∶g.4736G>T), c.-512C>T  and c.-1538A>G ] were observed. After adjusting for other confounding factors (age and gender), a significantly increased risk for CVD was found in patients carrying c.-512C>T, c.-1538A>G, c.-2647A>T and c.*126G>A variants. Allelic frequencies of these four polymorphisms also differed significantly between patients with CVD and controls  risk for CVD compared to first group (table S3).To understand the collective effect of these four significant polymorphisms in the disease, we further classified study subjects into two groups. Subjects with none or either one DNA was isolated from vein specimens and sequenced to check any genotypes discrepancy between whole blood samples and tissues of same patients. The genotype profiles obtained were similar in both the DNA samples from same patients.FoxC2 transcript expression was 4±1.4 folds increased in venous tissues from patients compared to normal subjects (p<0.05) (FoxC2 mRNA compared to patients carrying heterozygous CT genotype (p = 0.02) and wild CC genotype (p<0.001) (FoxC2 in tissue specimens (either at mRNA or protein level) was not significantly altered in patients who had all the four polymorphisms compared to four patients who carried TT genotype of c.-512C>T variant alone (p = 0.65) ((p<0.05) . Patientp<0.001) . The upr = 0.65) .FoxC2 protein expression with FoxC2 genotypes revealed significantly higher protein levels in patients carrying TT genotype compared to patients having heterozygous CT or wild CC genotypes (p<0.01)  .To confirm the overexpression of FoxC2 in varicose veins we performed an immunostaining on tissue sections of varicose veins from 3 patients and control vein from 3 healthy subjects. There was a marked overexpression of FoxC2 protein in the tissue sections of varicose veins compared to control veins .From our disease association results and transcript and protein expression analysis it was evident that c.-512C>T variant was related with altered mRNA and protein expression in vein tissues of CVD. We further examined if gene transcription was affected by this promoter polymorphism. As demonstrated in Dll4 and arterial marker Hey2 in the FoxC2 construct transfected Ea.hy926 cells. Expression of venous specific markers such as COUP TFII and Ephrin B4 were also assessed. Ea.hy926 cells were transiently transfected with pCAGIG vector containing FoxC2 construct (FoxC2-pCAGIG) or empty pCAGIG vector and the expression of Hey2 and Dlll4 genes were determined at mRNA levels. FoxC2 activation upregulated (p<0.05) the expression of both these arterial fate specific markers in Ea.hy926 when compared to empty vector transfected cells (FoxC2 overexpression resulted in a significant downregulation (p = 0.003) of COUP TFII, while Ephrin B4 downregulation was not statistically significant.Based on the reports of a potential FoxC2- Notch signaling ed cells . FoxC2 oFoxC2 is a forkhead/winged helix transcription factor coding gene located on chromosome 16q24.1 (NCBI Reference Sequence No: NG_012025.1). FoxC2 is implicated in vascular development especially in arterial and lymphatic differentiation FoxC2 coding sequences were reported in patients with lymphoedema-distichiasis (LD) which is an autosomal dominant form of primary lymphoedema with majority of patients developing varicose veins in lower limbs FoxC2 gene with LD risk and suggested a role of FoxC2 in the pathogenesis of varicose veins et al linked FoxC2 mutations to venous valve failure and reflux using conventional colour Doppler duplex ultrasound in patients with lymphodema distichiasis FoxC2 gene however has not yet been well-defined in patients with varicose veins or CVD. We report for the first time a positive association between genetic variants of FoxC2 and chronic venous diseases and a mechanistic insight on the role of FoxC2 in pathogenesis of CVD.Human FoxC2 polymorphisms and abnormal protein expression have been implicated with insulin sensitivity in patients with obesity and diabetes mellitus FoxC2 polymorphism pattern in patients with CVD alone. Patients with lymphoedema distichiasis were also not included in our subjects.FoxC2 gene from DNA isolated from whole blood samples of 382 patients with CVD and 372 control subjects. DNA sequencing revealed the presence of only two rare synonymous variants, c.354C>T (novel variation) and c.426G>A (rs41312258) with a frequency of 0.02 of 0.04 respectively. Hence these variations were not followed up for further studies. The analysis was extended to approximately 3 Kb 5′ untranslated flanking region of FoxC2 gene as well as 200 bp of 3′ flanking region which also includes the 3′ UTR region of gene. Seven FoxC2 polymorphisms were observed in patients with CVD and normal subjects. Two reported variants (c.-512C>T: rs34221221 and c.-1538A>G: rs4843162) and two novel variants c.-2647A>T and c.*126G>A were found to be significantly associated with risk of disease.We initially sequenced the 1.5 kb single coding exon of http://www.cbrc.jp/research/db/TFSEARCH. html) C.*126G>A variant is located 126 bp downstream to translation termination codon and 35 bp downstream to 3′UTR sequence of FoxC2 gene. C.*126G>A was consistently present in either heterozygous GA or wild GG genotypes but never in homozygous mutant AA genotype in our cohort. Prediction of microRNA/target duplexes for C.*126G>A variant was analyzed by miRNA prediction tools (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid) in silico analysis by RNAhybrid tool gave a very weak binding probability. C.-512C>T variant is present in the highly conserved proximal promoter of the FoxC2 gene Variants such as c.-2647A>T and c.-1538A>G were not further experimentally validated as they lacked the putative binding sites for transcription factors. Transcription factor binding affinity was evaluated by TF SEARCH version 1.3 computer program Click here for additional data file.Figure S2FoxC2 mRNA expression in vein tissues from patients with CVD. Relative FoxC2 mRNA levels in vein tissues from patients (n = 5) carrying all the four variants such as c.-512C>T, c.-1538A>G, c.-2647A>T and c.*126G>A and FoxC2 mRNA levels in vein tissues from patients (n = 4) carrying only TT genotype of c.-512C>T (rs34221221) variant. The difference in mRNA expression in both groups was statistically insignificant (p = 0.65). Data shown are the mean ± SD in each group.(TIF)Click here for additional data file.Table S1Primers used for PCR and sequencing of 5′ flanking region, 3′flanking regions and coding sequence of FoxC2 gene.(DOC)Click here for additional data file.Table S2Primers used for quantitative real time PCR and luciferase reporter assay.(DOC)Click here for additional data file.Table S3Patients and controls categorized as first group with neither or any single polymorphism and second group with 2, 3 or more polymorphisms. Percentages were taken from the column totals. Chi-square test for measure of association was used to derive p value. a Odds ratio and 95% confidence intervals of individual polymorphisms. b Adjusted odds ratio and 95% confidence intervals is obtained adjusting for age group and sex in multiple logistic regression model.(DOC)Click here for additional data file.


Protein-coding genes in eukaryotes are interrupted by introns, but intron densities widely differ between eukaryotic lineages. Vertebrates, some invertebrates and green plants have intron-rich genes, with 6–7 introns per kilobase of coding sequence, whereas most of the other eukaryotes have intron-poor genes. We reconstructed the history of intron gain and loss using a probabilistic Markov model  on 245 orthologous genes from 99 genomes representing the three of the five supergroups of eukaryotes for which multiple genome sequences are available. Intron-rich ancestors are confidently reconstructed for each major group, with 53 to 74% of the human intron density inferred with 95% confidence for the Last Eukaryotic Common Ancestor (LECA). The results of the MCMC reconstruction are compared with the reconstructions obtained using Maximum Likelihood (ML) and Dollo parsimony methods. An excellent agreement between the MCMC and ML inferences is demonstrated whereas Dollo parsimony introduces a noticeable bias in the estimations, typically yielding lower ancestral intron densities than MCMC and ML. Evolution of eukaryotic genes was dominated by intron loss, with substantial gain only at the bases of several major branches including plants and animals. The highest intron density, 120 to 130% of the human value, is inferred for the last common ancestor of animals. The reconstruction shows that the entire line of descent from LECA to mammals was intron-rich, a state conducive to the evolution of alternative splicing. In eukaryotes, protein-coding genes are interrupted by non-coding introns. The intron densities widely differ, from 6–7 introns per kilobase of coding sequence in vertebrates, some invertebrates and plants, to only a few introns across the entire genome in many unicellular forms. We applied a robust statistical methodology, Markov Chain Monte Carlo, to reconstruct the history of intron gain and loss throughout the evolution of eukaryotes using a set of 245 homologous genes from 99 genomes that represent the diversity of eukaryotes. Intron-rich ancestors were confidently inferred for each major eukaryotic group including 53% to 74% of the human intron density for the last eukaryotic common ancestor, and 120% to 130% of the human value for the last common ancestor of animals. Evolution of eukaryotic genes involved primarily intron loss, with substantial gain only at the bases of several major branches including plants and animals. Thus, the common ancestor of all extant eukaryotes was a complex organism with a gene architecture resembling those in multicellular organisms. The line of descent from the last common ancestor to mammals was an uninterrupted intron-rich state that, given the error-prone splicing in intron-rich organisms, was conducive to the elaboration of functional alternative splicing. Spliceosomal introns that interrupt most of the protein-coding genes and the concurrent splicing machinery that mediates intron excision and exon splicing are defining features of gene architecture and expression in eukaryotes Despite the ubiquity of introns in eukaryotic genomes, their biological status is poorly understood. To what extent introns are “junk DNA” as opposed to being functional parts of the genome, remains an open question and the answers are bound to be complicated and multifaceted. There are many reports on the contribution of introns to the regulation of gene expression Given that most unicellular eukaryotes are intron-poor whereas complex, multicellular organisms are intron-rich, it would seem intuitively plausible that introns accumulated in the course of evolution of eukaryotes. However, comparative analysis of the exon-intron structures of orthologous genes of plants and animals revealed a high level of intron position conservation, with the implication that the common ancestor of these organisms was relatively intron-rich Here we employ a probabilistic Monte Carlo model combined with a Markov Chain Monte Carlo (MCMC) method for the inference of ancestral states including robust estimation of confidence intervals to analyze a representative data set of 99 eukaryotic genomes which extensively covered the three supergroups of eukaryotes, Unikonta, Archaeaplastida (Plantae), and Chromalveolata, for which multiple genome sequences are available. The results clearly show that ancestral eukaryote forms were intron-rich, with LECA having a high intron density, on the order of two-thirds of the introns density in human genes. The subsequent evolution was heavily dominated by intron loss, with several episodes of massive intron gain associated with the emergence of some of the major eukaryote groups, in particular, animals.Naegleria gruberi, with a moderate intron density The present analysis of gene structure evolution included an extensive data set of sequenced and annotated genomes from the Unikonta , the Archaeplastida , and Chromalveolata (Heterokonta and Alveolata). Of the five supergroups of eukaryotes Intron loss and gain were modeled using a probabilistic Markov model encompassing lineage-specific loss and gain rates, as well as rate variation across sites. The Markov Chain Monte Carlo (MCMC) method This reconstruction provides a thorough view of the evolution of gene structure across three eukaryotic supergroups and reveal several general trends . Most liSeveral previous studies, performed on much smaller data sets and with less robust reconstruction methods, have suggested that at least some eukaryotic ancestral forms could have possessed intron-rich genes In addition to the Bayesian MCMC estimates, we inferred ancestral densities by using Dollo parsimony The Dollo and ML estimates show a picture of intron-rich eukaryotic ancestors that is qualitatively similar to the MCMC results. Quantitatively, similarly to the case of ancestral molecular sequence reconstruction Dictyostelium and Entamoeba , pelagophytes (A. anophagefferens), diatoms, and other eukaryotes within or outside chromalveolates , Dikarya (“higher” fungi: 3.7–4.7 introns/kilobase), opisthokonts (4.7–5.5 introns/kilobase) and, most importantly, LECA (see below). The uncertainty is larger in ancestors with subsequent turbulent history in the descendants. A case in point is the amoebozoan ancestor. There was extensive intron loss along the branch leading from the intron-rich unikont ancestor to the extant Amoebozoa. It is unclear, however, whether the losses occurred in parallel in multiple descendant lineages, or prior to the split between Naegleria gruberiNaegleria.The gene architecture of LECA is of special interest. Previous estimates of intron density for LECA were very uncertain due to methodological problems with maximum likelihood inference Strikingly, the greatest intron density among all ancestral and extant eukaryotes was inferred for the last common ancestor of the Metazoa, at 120–130% of the human density, with a 95% confidence  and 2.We validated the inference procedures by simulating the evolution of intron sites  offset by a high gain rate.The choice between these routes of evolution in a particular lineage could depend primarily on the intensity of purifying selection that is linked to the effective population size The results of this work, thanks to the extensive data set of analyzed genomes and the robust reconstruction method that yields inferences of ancestral states with minimal uncertainty, seem to close the debate on the gene architecture of ancestors of extant eukaryotes including LECA. It is now clear that the genes of ancestral eukaryotes possessed high intron density, close to the densities in the most intron-rich modern genomes, those of mammals.This finding has substantial implications for understanding the evolution of eukaryotes. It has been noticed that intron-poor genomes typically possess strong, highly efficient splice signals, whereas intron-rich genomes contain mostly weak, error-prone splice signals Remarkably, the present results indicate that the entire line of descent from LECA to mammals was a continuous intron-rich state  that proNaegleria gruberi were selected using the same procedure, but the large evolutionary distance precluded identification of a sufficient number of orthologs and unambiguous alignment of splice sites. Therefore, sequences from N. gruberi were not included them from the ancestral inference.Orthologous genes were identified using a modification of the previously described procedure The intron positions were mapped onto gene sequences using a previously developed computational pipeline . For a formal treatment, define T as the known phylogeny for the terminal taxon set S, i.e., a rooted tree with n leaves that are bijectively labeled by taxa from S. Internal tree nodes correspond to common ancestors. The history of a potential splice site is modeled as a binary labeling of all tree nodes: ξ = . In a Markov model, the labeling is randomly drawn from a distribution for which the parent-child relationships in the phylogeny define conditional independencies. The distribution of ξ at a site is fully determined by the presence probability at the root π = Pr{ξ[root] = 1}, and edge-specific rates uv, labels change with probabilitiesGene structure evolution was modeled mathematically by assuming that the table columns uv as length and rate ratioS] is a random leaf set labeling. The model defines the likelihood Conversely, −1/875. The conversion formula uses human as a reference: 6.946 is the mean number of human introns per 1000 base pairs (kbp) in the coding sequences of the analyzed genes, and 875 is the number of human introns in the data set. The posterior distribution for ancestral intron counts for a given model parametrization is computable without much difficulty Ancestral intron counts were inferred using three methods. Intron count estimates were converted into densities by the formula density = intron count·6.946 kbpIn order to estimate ancestral intron densities and lineage-specific changes in a Bayesian setting, we adapted mutation mapping techniques commonly employed with molecular sequence evolution models  S1. draw random initial parameters θ by their prior distribution P(θ)repeat S1.  S3. propose new random model parameters θ′ by distribution Q(θ→θ′) S4. with probability  S5. generate random ancestral labeling by posterior probabilitiesj by using the so-called conditional likelihoods for labeling node u with x = 0, 1, given the (possibly unresolved) labelings at the terminal taxa uS within the subtree rooted at u:In Line S4, the acceptance probability includes the likelihoods L(θ) at different model parameters, the prior distribution P(θ) of parameters, and a proposed model distribution Q(θ→θ′). In Line S5, random ancestral labelings The conditional likelihoods are calculated by dynamic programming in a postorder traversal by adapting the pruning algorithm of Felsenstein L1. draw random site-specific rate multipliers γ, νuvL2. set L[u∶x] for all nodes u and labels x = 0,1L3. compute conditional likelihoods L4. set for all non-root nodes v in a preorder traversal doL5. L6. set L7. with probability In Line L1, the rate multipliers are drawn from the posterior distribution for the different discretized rate categories using the shape parameters of the respective Gamma distributions. The SAMPLING algorithm generates a Markov chain for pairs of model parameters and ancestral reconstructions. The equilibrium distribution for the chain is the posterior distributionj, and adding the analogous sum for the sampled histories of all-absent profiles. Intron gains and losses on branches were estimated with a similar calculation.In addition to sampling histories of profiles from the input data, we also generated “all-absent” profiles with introns missing at every terminal taxon The prior distribution P(θ) was uniform for every parameter (and thus absent from the formula in Line S4): over the range  for shape parameters and edge lengths, and over the range  for π and the rate ratios. In a typical MCMC proposal, a subset of model parameters was chosen, and then multiplied by a random value; see q of parameters and ancestral intron densities.The convergence and the mixing efficiency were assessed by running 100 chains in parallel see . EstimatIndividual intron site histories were reconstructed using the Malin software Simulations were performed by generating 100 random data sets of a comparable size to the input data set using the MCMC median model parameters, coupled with an erasure procedure simulating missing orthologs, or randomly generated multipliers for simulating heterotachy : see Figure 13 in Supporting Figure S1Posterior distributions of the ancestral intron densities inferred from the sampling chains for all ancestral forms.(PDF)Click here for additional data file.Text S1Detailed methods and results with the illustrating figures and tables.(PDF)Click here for additional data file.Video S1Dynamic representations of the histories of intron loss and gain for the 245 analyzed clusters of orthologous genes.(MOV)Click here for additional data file.


The debate on human rationality goes to the heart of fundamental questions about human existence. When can we say that a decision is correct? What is the basis for the achievements of the human intellect? What is the most important cognitive distinction between humans and non-human organisms? Proposals of rationality have an interesting status as psychological theories. They are not quite theories of decision making in practice—such theories are referred to as descriptive theories, to imply that they describe what goes on. Rather, proposals of rationality are normative theories, to imply theories of how people ought to reason, if they seek decision outcomes, which are deemed to be correct, on the basis of some absolute standard  theory. This approach was established after a major shift in conceptual thinking about rationality. Before (effectively since antiquity), it was believed that the standard for correctness in decision making was classical logic. But this position came under intense scrutiny, with experimental results showing that naïve participants, even in simple tasks, would not reason in a way consistent with classical logic Wason, . One reacorrect association of probabilities to uncertain events. The Dutch Book Theorem  theory in cognitive modeling. By QP theory, we imply the mathematics for assigning probabilities to events from quantum mechanics, without the physics. QP theory is a formal theory of probability, like CP theory. QP and CP theories are based on different axioms and so their predictions can diverge. QP theory is a plausible contender in decision making . Recent work has shown that QP principles can provide the basis for simple, constrained models for empirical findings, which have been persistently problematic from a classical perspective, such as order effects on choice Moore, , the conThe above points attest to the descriptive status of QP theory in cognitive theory, not its normative status. Nevertheless, they motivate a consideration of explanatory concepts from QP theory in psychological debates. Of relevance presently is the idea of incompatibility, in relation to two (or more) questions (or possibilities etc.). According to classical theory, all questions are compatible, which means that the answer to any set of questions can be known concurrently. Following from Tversky and Kahneman's  experimeN dimensions. A classical space for just 10 binary questions requires over 1000 dimensions. The CP theory requirements for representational capacity appear too stringent. Another way to look at this issue is that, regardless of the number of questions considered, classically it is always possible to construct a complete joint probability distribution. But, where would the information come from to construct such a joint probability distribution, especially when considering unfamiliar combinations of questions (such as being a bank teller or a feminist)? Note, the principle of indifference cannot provide a general solution to this issue . For two questions with binary outcomes, we need a four dimensional space. The consideration of each additional binary question increases the dimensionality of the space by a factor of two, so that, for N binary questions, we require 2Thus, we suggest that cognitively it is more plausible to consider some questions, especially ones not typically considered together, as incompatible. Indeed, there have been suggestions that, with practice, some of the decision making fallacies attenuate . Is it possible to prove a version of the DBT for QP theory as well? Superficially, this may appear not to be the case. First, the axioms of CP theory (on the basis of which the DBT is proved) are very different from those of QP theory. Second, verifiably . However, if some of the questions are incompatible, then QP theory will provide more accurate predictions for probabilistic inference. For example, if some questions are incompatible, then order effects may arise in conjunctions (Trueblood and Busemeyer,


In this work; resistive moisture sensors were obtained by dip coating sulfonated polycarbonate (SPC) onto silver interdigitated electrodes. Commercial polycarbonate was sulfonated with acetyl sulphate at two different sulfonation degrees corresponding to 9.0 and 18.0 mole %. Impedance spectroscopy was used to investigate the humidity sensing properties at controlled relative humidity (RH%) environments generated from standard saline solutions in the range of 11–90 RH%. For the highest sulfonated sample; in the RH% range investigated (11 to 90%); the sensor impedance changed from 4.7 MΩ to 18 kΩ. Humidity sensors made from sulfonated polycarbonate showed exponential decay behavior of the impedance at constant frequency with the environmental relative humidity. Sample 9SPC presented dielectric relaxation response for environmental humidity between 58 and 90 RH% while sample 18SPC presented dielectric relaxation response for the entire measured range between 11 and 90 RH%. Sulfonated polycarbonate could be a promising material for the fabrication of simple and cheap humidity-sensing sensors for the assessment of relative humidity of the surrounding environment, as suggested by experimental results. The pi.e., a resistor that is parallel to the real proton conducting channel. Such a resistor will reduce the impedance range change which means lower sensitivity. By inserting a conductive material on the humidity sensor matrix this will allow protons with very short diffusion lengths to be collected before being bound to an ionic group inserted in the polymeric matrix. Such sensors will behave with improved response for low humidity but at a price to drastically reduce the response at mean and high humidity.There are many kinds of humidity sensors. Capacitive humidity sensors involve a film deposited between electrodes on a substrate. The dielectric constant is proportional to the relative humidity and changes due to water steams –9. The pi.e., exponential change of impedance by many magnitude orders) on the academic point of view the transport of protons on a membrane is not really a simple process and deserves to be investigated without the influence of other processes. Sulfonated polycarbonate with sulfonic acid groups to the degrees of 9.0 and 18 mole % are also insoluble in water and this turns it into interesting to be investigated as humidity sensor. In a previous work, sulfonated polystyrene (SPS) was investigated as a humidity sensor [In the present work, we opted to use a simple proton conducting membrane with improved humidity range sensitivity compared to similar materials. Besides the fact that this guaranties a high sensitivity  has already been evaluated for some applications such as membranes for fuel cell [Due to better mechanical and chemical properties, uel cell , for sepuel cell , and foruel cell . Composiuel cell , polyaniuel cell , SPC-poluel cell  were alsuel cell ,20.3H was investigated as a relative humidity sensor. The preparation of humidity sensors was considered for SPC at two different sulfonation degrees: sensors 9SPC and 18SPC corresponding to 9.0 and 18.0 mole %. The investigations of their humidity sensitivity, vibrational and morphological characteristics are here reported. In order to obtain the sensor impedance dependence with RH% at the frequency range dominated by the resistive behavior, we selected from impedance spectroscopy measurements (carried out from 5 Hz to 13 MHz) the ac frequencies of 25 Hz and 200 Hz for sensor 9SPC and 18SPC respectively. Considering relative humidity generated from standard saline solutions in the range of 11–90 RH%, our previous work in which a 22 mole % SPS sensor presented resistive behavior in the range of 33–90 RH% [i.e., in the range of 11–90 RH%.In the present work, sulfonated polycarbonate, SPC, with the sulfonation group SO3–90 RH%  and, in 2.2.1.4g.mole−1 and The starting polymer was a polycarbonate (PC)  with 2Cl2 at 40 °C with acetyl sulfate, following the method described for the polystyrene [et al. [SPC was obtained by sulfonation of commercial PC in CHystyrene . The sul [et al. . The sul2Cl2 (0.05 g/mL), 9SPC/CH3OH and 18SPC/CH3OH (0.05 g/mL) films were followed by stirring to give homogenous solutions. Each solution was deposited by dip coating on insulating disk-shaped ceramic substrate of 25.0 mm diameter, inert with respect to humidity changes, where an interdigital array of silver electrodes has been defined  and for samples prepared in KBr pellet (2 mg of polymer into 300 mg of KBr) following standard procedures. The FTIR spectra were used to qualitatively characterize the ∼SO3H groups. The surface morphology of the SPC deposited by dip coating was studied by scanning electron microscope (SEM) in JEOL Model JSM T300 equipment, operating at 20 kV. The samples were gold-sputtered prior to the measurements. The impedance measurements were done with an HP4192A impedance analyzer. The experimental parameters were: frequency range of 5 Hz to 13 MHz, sinusoidal voltage amplitude = 1.0 Vrms. All the measurements were carried out at a room temperature .Infrared spectra were recorded using a Perkin-Elmer FTIR 1600 spectrophotometer. Spectra were obtained in the mid-infrared region .Furthermore, two processes are responsible for impedance decreasing: the first is associated with the water electrolysis and the second is related to the transport of H+ ions . This trmobility . Therefomobility –27 the aFor the polyelectrolyte humidity sensors, the conduction mechanism is mainly ionic, and sensing behavior depends on both the kind and concentration of ions and the water absorption ability of the sensing film .The electrical transport phenomenon in polymeric humidity sensors is yet to be fully described. There is much work to carry on in understanding the fundamental physical phenomena presented in such sensors. For instance, protons have high mass and very low mobility and d.c. measurements are impossible to be carried out due to double layer capacitance effects. Under this scenario, Hall effect measurements cannot be easily carried out and due to limitations of water melting and boiling points, temperature dependence of resistivity can be measured in a quite short range, which turns the determination of activation energies associated with carrier statistics a hard task.0 is the projected impedance at 0 RH% and B is the slope obtained from fitting. From the fittings of data for sample 9SPC were obtained Z0 = (10 ± 5) TΩ and B = (0.080 ± 0.006) RH%−1 and for sample 18SPC Z0 = (13 ± 1) MΩ and B = (0.029 ± 0.002) RH%−1.Despite the limitations, an empirical model of impedance exponential decay with relative humidity can lead to reasonable results and was applied to the sensor impedance as a function of RH%. MAX.τ =1 , reduces with increasing RH%  and desorption (changing the environment from 90 RH% to 11 RH%) times for both SPC sensors were carried out. One consideration is that about 30 s was used to change from one environment to the other and to start the measuring program. For sample 9SPC both absorption and desorption times are low (at least lower than the 30 s lost during the environment change). For sample 18SPC a low absorption time and desorption time of 8 min after the changing time was measured.4.Resistive moisture sensors were obtained by dip coating sulfonated polycarbonate onto silver interdigitated electrodes. The SPC was sulfonated to a degree of 9 mole % (9SPC) and 18 mole % (18SPC). Humidity sensors made from SPC show exponential dependence of the impedance with the environmental relative humidity. An empirical model of exponential decay of the impedance measured at fixed low frequencies with the relative humidity was used to fit experimental results. This model allows practical conversion between impedance and relative humidity. Sample 9SPC presented dielectric relaxation response for environmental humidity between 58 and 90 RH% while sample 18SPC presented dielectric relaxation response for the entire measured range between 11 and 90 RH%. The uncertainties of the relative-humidity are 8% and 4% for samples 9SPC and 18SPC respectively. The sensor of 9SPC is suitable for applications that consider high humidity environments and the need of quick responses. The sensor of 18SPC is suitable for applications that consider broad range of humidity environments. These results turn the sulfonated polycarbonate into a very promising material for the fabrication of inexpensive and efficient humidity sensors.


Summary: ADAPT-nuclear magnetic resonance (ADAPT-NMR) offers an automatedapproach to the concurrent acquisition and processing of protein NMR data with the goal ofcomplete backbone and side chain assignments. What the approach lacks is a usefulgraphical interface for reviewing results and for searching for missing peaks that mayhave prevented assignments or led to incorrect assignments. Because most of the dataADAPT-NMR collects are 2D tilted planes used to find peaks in 3D spectra, it would behelpful to have a tool that reconstructs the 3D spectra. The software package reportedhere, ADAPT-NMR Enhancer, supports the visualization of both 2D tilted planes andreconstructed 3D peaks on each tilted plane. ADAPT-NMR Enhancer can be used interactivelywith ADAPT-NMR to automatically assign selected peaks, or it can be used to producePINE-SPARKY-like graphical dialogs that support atom-by-atom and peak-by-peak assignmentstrategies. Results can be exported in various formats, including XEASY proton file(.prot), PINE pre-assignment file (.str), PINE probabilistic output file, SPARKY peak listfile (.list) and TALOS+ input file (.tab). As an example, we show how ADAPT-NMREnhancer was used to extend the automated data collection and assignment results for theprotein Aedes aegypti sterol carrier protein 2.Availability: The program, in the form of binary code along with tutorialsand reference manuals, is available at http://pine.nmrfam.wisc.edu/adapt-nmr-enhancer.Contact:whlee@nmrfam.wisc.edu or markley@nmrfam.wisc.edu One of the goals of protein nuclear magnetic resonance (NMR) spectroscopy is to increaseits throughput by automating the steps of data collection, spectral assignment and structuredetermination. The latest approach towards this goal from our laboratory is ADAPT-NMR (http://qt.nokia.com) for graphical user interface. The software supportsmultiple operating systems . ADAPT-NMR Enhancer offers threeactive dialog boxes: Main Window Dialog, PINE AssignmentDialog and Probable Assignment Dialog. The Main WindowDialog  application written inC++ with QT4 libraries  is the Funding: National Center for ResearchResources (5P41RR002301-27) andNational Institute of General Medical Sciences(8 P41 GM103399-27) from the National Institutes ofHealth.Conflict of Interest: none declared.


We have identified a hydrophobic sequence in the C-terminus that is critical for the TDP-43 aggregation and inclusion formation. The synthetic peptide with this sequence forms an α-helical structure in solution but easily transforms into β-sheet structure. The inclusions formed by the C-terminal 35-kDa fragment (TDP-35) can recruit full-length TDP-43 to cytoplasmic deposition from functionally nuclear localization. TDP-35, rather than TDP-43 and the C-terminal 25-kDa fragment, is prone to aggregation in vitro, and it can further serve as a seed to facilitate aggregation of full-length TDP-43. This suggests that fragmentation of TDP-43 leads to cellular redistribution, inclusion body formation, and altered RNA processing, which are implicated in the molecular pathogenesis of ALS and FTLD.TAR DNA binding protein of 43 kDa (TDP-43) is a nuclear factor functioning in RNA processing. It was also reported that TDP-43 aggregates are deposited in motor neurons implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin (FTLD-U). To understand the mechanism underlying the inclusion body formation and possible functional alteration of TDP-43, we studied TDP-43 and its fragments and their effects on RNA processing


We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila,  The L1 family of cell adhesion molecules is well-known for its divergent roles during early nervous system development, specifically for axonal outgrowth, myelination, fasciculation, as well as long term potentiation, synapse formation and maintenance neuroglian (nrg) gene generates two alternately spliced isoforms 180 is expressed exclusively in the nervous system and Nrg167 mostly in non neuronal tissue. Similar to vertebrates, Nrg plays distinct roles during axonal guidance and synapse formation 180 and Nrg167 isoforms are encoded by distinct exons allowing the characterization of the role of the FIGQY motif for either isoform separately 180 is required for synapse formation and synapse stability but not for axon growth and guidance in the GF circuit as well as at the larval neuromuscular junction 14nrg;P[nrg167ΔFIGQY] mutants in either model neuron.In the central nervous system, the sole L1-type Drosophila 849nrg mutants is analogous to the pathological human L1-H210Q mutation 849nrg mutants exhibit axonal guidance as well as synaptic defects that can be rescued by the transgenic expression of human L1CAM, but not by the expression of the vertebrate paralogs Neurofascin and NrCAM The position of the S213L missense mutation in the second Ig domain of 849nrg along with 14nrg;P[nrg180ΔFIGQY] mutants provide a unique opportunity to study pathological L1CAM missense mutations with respect to their effects on guidance and synapse formation in vivo. Here, we investigated the human L120V, E309K, H210Q, Y1070C, C264Y extracellular missense mutations in addition to a L1CAM protein mutated in the intracellular ezrin–moesin–radixin (ERM) binding site (L1-4A). The human L1 mutant proteins were expressed in both, 849nrg and 14nrg;P[nrg180ΔFIGQY] mutant backgrounds to gauge the abilities of these proteins to rescue axon guidance and synapse development defects, respectively In summary, the GF circuit of Drosophila is a suitable model system for studying L1CAM function and EcoRI-XhoI restriction sites 849nrg were obtained from the Bloomington Stock Center. 14nrg;P(nrg180ΔFIGQY) was generated by the Jan Pielage lab  as previously described 849nrg;OK307/CyO stock and a 849nrg;OK307, UAS-CD8-GFP/CyO stock as well as OK307,UAS-L1/CyO stocks for all L1-constructs were established. Male flies of the UAS-L1 or UAS-L1-variants were crossed to female 849nrg flies. In addition, male flies of the OK307,UAS-L1/CyO (or L1-variants) stocks were crossed to female 14nrg;P(nrg180ΔFIGQY) flies. All genetic crosses were performed at 25°C and the male progeny of the crosses were tested when three to four days old.The following fly stocks generated have been previously described For the developmental Western blot, one pupa or one adult OK307/+;UAS-L1/+ fly homogenized in Laemmli buffer with Halt™ Protease and Phosphatase Inhibitor Cocktail  was loaded per lane. Ten fly heads of offspring from UAS-L1 and UAS-L1-variants crossed to OK307 were used to extract protein for comparing expression between wild type L1CAM and L1CAM with pathological missense mutations. SDS-PAGE was performed according to standard protocol. The Western blots were incubated with the anti-L1 C20 antibody  at a dilution of 1∶200. Anti-Actin  was used as a loading control at a dilution of 1∶200. Secondary donkey anti-goat antibody  was used at a concentration of 1∶1000.849nrg and the mutant L1 rescues was obtained by Chi-square analysis using Microsoft Excel (Microsoft Office 10).Flies (3–5 days old) were dissected to expose the cervical connective and the ventral nerve cord as previously described Electrophysiological recordings from the Dorsal-longitudinal Muscle (DLM) and the TTM Tergo-trochanteral Muscle (TTM) of the GF circuit were obtained as described previously When expressed in different cell lines, several human L1CAM proteins with missense mutations in their extracellular domain are retained in the endoplasmic reticulum 849nrg mutants  in 849nrg animals. In contrast, the expression of L1-C264Y, L1-H210Q, L1-R184Q and L1-4A did not complement the loss of homophilic binding in 849nrg animals. Although 849nrg animals expressing L1-Y1070C were statistically insignificant from negative control animals (849nrg), they were also statistically insignificant from positive control animals (L1CAM in 849nrg background). Therefore, the observed reduction in the guidance defects suggests that expression of L1-Y1070C is able to partially rescue guidance defects in vivo.In wild-type flies each GF cell body in the brain projected an axon through the cervical connective into the second neuromere of the ventral nerve cord (VNC) and made synaptic connections with the TTMn motor neurons  A, B. Intely 30% , while tpecimens . We alsoKGGKYSV,  were sub14nrg;P[nrg180ΔFIGQY] animals, in which the lethality of a nrg null mutant (14nrg) was rescued by a genomic construct in which the neuronal Nrg180 isoform lacks the Ankyrin-binding motif , no improvement of the synaptic phenotypes was observed (14nrg;P[nrg180ΔFIGQY] mutants enabled us to test whether human pathological L1CAM mutations can rescue the synaptic phenotypes or whether the extracellular missense mutations cannot compensate for the lack of outside-in signaling via the FIGQY motif.Mutations in the highly conserved intracellular FIGQY-motif of the sole L1CAM homolog Neuroglian do not affect neurite outgrowth or guidance in the GF circuit or at the larval neuromuscular junction ng motif , only syne ratio  was assone ratio  and withne ratio . We have mutants , Table 1an L1CAM . In contobserved , Table 1849nrg mutants, was able to significantly rescue the synaptic phenotypes of 14nrg;P[nrg180ΔFIGQY] mutants (Expression of L1-Y1070C, L1-H210Q and L1-R184Q failed to restore the GF synapse function , Table 114nrg;P[nrg180ΔFIGQY] animals (14nrg;P[nrg180ΔFIGQY] mutant terminals.Although the average Following Frequency and average Response Latency were significantly improved with the expression of L1-C264Y , Table 1 animals . In orde animals . We foun animals , green d animals , magenta animals  but not  animals . However animals . While i animals , left GF849nrg or 14nrg;P[nrg180ΔFIGQY] mutants is due to the loss of a biological function and not due to a dominant negative or poisonous effect.Finally, in order to test if the expression of L1CAM with pathogenic mutations in Drosophila has any dominant negative or poisonous effects in the GF circuit, we determined putative functional defects in a wild type background . All aniMany different pathological L1CAM mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as on their cell surface expression levels in vitro. However, there are only a few studies on their biological consequences in vivo In vertebrates, L1CAM is proteolytically cleaved by various enzymes, such as neuropsin, plasmin, ADAM10/17 and a yet unidentified serine protease 14nrg;P[nrg180ΔFIGQY] animals suggesting that L1-C264Y protein was expressed at the cell surface. Correlating with the electrophysiological data, we did find that transgenically expressed L1-C264Y protein was present in numerous though not all synaptic terminals at the cell surface as well as in vesicular clusters inside the terminal. Therefore, our results suggest that in the Drosophila GF the pathogenic C264Y mutation not only affects proteolytic processing indirectly by reducing cell surface expression but may also affect it directly in L1-C264Y protein that is expressed at the cell surface.The overall L1-C264Y expression was reduced comparatively but in Western blots no cleavage was observed for L1-C264Y protein even with ten heads . This is849nrg allele is at a site analogous to the position of the H210Q mutation in humans and both mutations affect homophilic L1 binding 180 on either side of 849nrg mutant synapses can partially rescue the synaptic phenotypes. This suggests that Nrg-S213L similar to L1-H210Q is able to bind to a wild-type extracellular domain Homophilic L1-L1 interactions mediated by the extracellular Ig-domains are involved in various L1-dependent signaling processes required for axonal growth, guidance and synapse stability 849nrg mutants. Expression of wild-type human L1CAM, as well as L1-L120V and L1-E309K, both of which mediate normal homophilic binding, was able to efficiently rescue the guidance defects of 849nrg mutants (849 protein induces signaling that is sufficient for GF guidance.Here, we tested whether the expression of different pathological mutations can compensate for the loss of homophilic interaction in  mutants  [15],  mutant animals (ΔFIGQY protein. We identified several L1CAM mutations that had expression levels (14nrg;P[nrg180ΔFIGQY] mutant flies. This suggests that L1-H210Q and L1-R184Q may not interact transcellularly with NrgΔFIGQY and homophilic interactions are also essential for synapse formation. Alternatively, L1-H210Q and L1-R184Q may be able to interact transcellularly with NrgΔFIGQY, which has a wild-type extracellular domain. However, in this scenario the H210Q and R184Q mutations disrupt outside-in signaling processes via the FIGQY motif. The latter hypothesis is supported by our finding that the 849nrg mutants with the analogous L1-H210Q mutation have reduced tyrosine phosphorylation of the FIGQY motif and synaptic phenotypes similar to 14nrg;P[nrg180ΔFIGQY] mutants in addition to guidance defects 14nrg;P[nrg180ΔFIGQY] mutant flies although its homophilic interactions are not affected by the mutation The finding that human L1CAM mutations like L1-L120V, L1-E309K, L1-4A and partially L1-C264Y were able to rescue the synaptic defects of Drosophila  animals , 4 suggen levels  comparabIn summary, we find that extracellular human pathogenic L1CAM missense mutations not only affect adhesive properties but also intracellular signaling pathways distinctly, which are required for axon guidance, synapse formation or both. Mutations that affect homophilic binding are most detrimental because they affect adhesive properties but often also heterophilic interactions and intracellular signaling


Tetrahymena intron more than 30 years ago has been essential to our understanding of group I intron catalysis, higher-order RNA structure, and RNA folding, but other intron models have provided information about the biological role. Nuclear group I introns appear widespread among eukaryotic microorganisms, and the plasmodial slime molds (myxomycetes) contain an abundance of self-splicing introns. Here, we summarize the main conclusions from previous work on the Tetrahymena intron on RNA self-splicing catalysis as well as more recent work on myxomycete intron biology. Group I introns in myxomycetes that represent different evolutionary stages, biological roles, and functional settings are discussed.Group I introns are a distinct class of RNA self-splicing introns with an ancient origin. All known group I introns present in eukaryote nuclei interrupt functional ribosomal RNA genes located in ribosomal DNA loci. The discovery of the  Introns are genetic elements that interrupt functional RNA- or protein-coding genes, and are removed post-transcriptionally in a process termed splicing. Their ability to be spliced out at RNA level makes them almost invisible for the host and limits the phenotypic cost, and introns have often been labeled selfish elements or molecular parasites . A majorGroup I introns in nuclear genomes are exclusively found within functional ribosomal RNA (rRNA) genes of a wide spectrum of eukaryotic microorganisms. Here they are frequently noted among red algae, chlorophyte algae, fungi, and myxomycetes, but only occasionally in ciliates -4. NucleTetrahymena LSU rRNA intron at insertion site L1925 has become the undisputedly most important group I intron model system for the study of RNA catalysis, RNA structure, and RNA folding [Tetrahymena thermophila [Tetrahymena intron has been thoroughly investigated and self-splicing was shown to occur by RNA catalysis based on a two-step transesterification reaction requiring a guanosine cofactor [Despite the fact that thousands of nuclear group I introns have been annotated in sequence databases, only a very few have been submitted to molecular analyses and functional characterizations. The  folding . Tth.L19 folding  for nuclcofactor .Tetrahymena intron, has a more complex structured scaffold domain than the Didymium group IE ribozyme  insertions. The HEGs are located within the peripheral parts of the paired segments such as P1, P2, P6, P8 or P9, and expression of these protein-coding genes embedded in nucleolar rDNA utilizes unconventional strategies . Interestrans-splice group I ribozymes, and most studies have been performed with the Tetrahymena ribozyme. One of the best characterized examples of RNA-based gene repair is the reprogramming of mutant p53 transcripts in human cancer cells [Several group I intron-based biotechnological applications have been suggested and realized. These involve the intron homing endonuclease (HE) as a highly specific and rare-cutting endonuclease, intron splicing as a therapeutic tool in fighting pathogens and engineered group I ribozymes as molecular tools in RNA reprogramming and RNA repair ,17. Riboer cells ,20. The er cells . HoweverTetrahymena intron has proven to be the undisputed prototype in the study of RNA catalysis and RNA structure, other nuclear group I introns have contributed to our understanding of intron functions beyond splicing. Recent studies have shown that the extrachromosomal nuclear rDNA of myxomycetes, eukaryotic microorganisms belonging to the Amoebozoa clade, contain an abundance of group I introns , which is cognate to that of the Tetrahymena intron; it harbors a HEG and is mobile in genetic crosses between intron-lacking and intron-containing strains [Didymium iridis. Dir.S956-1 is mobile in genetic crosses and contains two distinct ribozymes with different functions in splicing and RNA processing, as well as a HEG [Didymium intron (Dir.S956-2) harbors a HEG at the antisense orientation [Diderma niveum has an extremely dense intron content with 20 or more group I introns present within the same rRNA primary transcript  at the 5′ splice site (SS)  Figure B. ExoG i circles ,32. HoweDidymium group I intron Dir.S956-1 [The full-length intron circularization (FLC) pathway has been studied in detail for the r.S956-1 . This par.S956-1 . The 3′OPpoI from P. polycephalum[DirI and I-DirII from D. iridis [Naegleria species [The spreading of group I introns may occur at either the DNA or RNA level Figure . The mosycephalum, I-DirI . iridis , and som species . Nuclear species ,39 and rP. polycephalum, which encodes I-PpoI [D. iridis for the Dir.S956-1 intron [PpoI and the introns Ppo.L1925 and Tth.L1925 (Tetrahymena intron), which were integrated into all the approximately 150 genomic rDNA copies at chromosome XII in an elegant experiment [Experimental evidence for homing in a biological setting has been collected from only two nuclear group I introns, both in the myxomycetes. The first system to be characterized was Ppo.L1925 in s I-PpoI . Here mo1 intron . In addiperiment -43.in vitro, in yeast and in Escherichia coli for both the Tetrahymena intron [Didymium intron [in vitro integration of full-length circular intron RNA has also been noted, suggesting a biological role for the circularization pathway in propagation and intron spread [It has been suggested that intron homing also occurs directly at the RNA level by reverse splicing. Here, an excised intron attacks the ligated exons at the intron-lacking cognate insertion site and integrates into the precursor RNA. Reverse splicing has been reported a intron -46 and tm intron . Interesn spread . Less frn spread ,22,48. HP. polycephalum, D. iridis and D. niveum rDNAs.Recent studies of the rDNA mini-chromosome in myxomycetes have revealed more than 500 group I introns highly divergent in sequence, size and insertion site. Myxomycetes are eukaryotic microorganisms with a complex life cycle, which has several stages from haploid amoebae cells to a multi-nucleated plasmodium with synchronously dividing diploid nuclei . The rDND. niveum is highly unusual since at least 20 group I introns are present . The high abundance of introns is a significant challenge to the host cell since the majority of group I introns have the ability to perform FLC and generate fragmented exons [The rDNA from ed exons . The myxDidymium, L1925 in Physarum, and S529 and S1199 in Diderma). HEGs have been noted in both sense and antisense orientations compared to the group I ribozyme and have been inserted at various locations . The HEG-containing introns belong to both the IC1 and IE subclass, are highly mobile in biological settings [The first category consists of the mobile HEG-containing introns Figure A. Of thesettings ,40, and settings . IntronsDiderma belong to this category (as well as the Tetrahymena intron), and it has been suggested that the all-ribozyme group IC1 and IE introns are remnants of mobile introns after loss of HEGs  and thus the U:G pair at the 5′ SS is replaced by a G:C pair [in vitro[Most introns in s Figure B. This sG:C pair . Further[in vitro.Diderma rDNA is S1389   and in h segment ,60,61. A segment  , which opens the possibility that intron RNA may have gained additional functions beyond splicing.Obligatory introns could have gained new mutual functions that benefit the host, and one possibility is that peripheral insertions are further processed into long non-coding RNAs (lncRNAs). lncRNAs are known to regulate gene expression, translation, splicing and trafficking by acting as guides, scaffolds, decoys or enhancers ,64, and Didymium, the amoebo-flagellates Naegleria and the amoeba Allovahlkampfia [D. iridis, is presented in Figure Didymium intron carries a HEG inserted at P2 of the splicing ribozyme, and is mobile at the DNA level in genetic crosses between intron-containing and intron-lacking strains [One notable example of group I introns that have evolved new biological roles are the twin-ribozyme introns, which are interrupting SSU rRNAs in the myxomycete lkampfia ,60,65,66 strains ,40.Interestingly, a second ribozyme domain is located immediately upstream of the HEG. Detailed structural analysis has shown that the ribozyme is derived from a standard group I intron; it is named GIR1 (group I-like ribozyme 1) . GIR1 laNaegleria and Allovahlkampfia GIR1s [The catalytic part of GIR1 has about 180 to 200 nucleotides, and when activated it catalyzes self-cleaving by branching . This reia GIR1s ,72.DP2 and P10, which depend on base pairing of sequences 5′ and 3′ of the GIR1 core  is a unique example of a group I intron that has gained new catalytic properties and new biological roles in nuclear gene regulation.Self-splicing, RNA structure and folding, and HE-dependent homing are fully described features of the group I introns in BP: branch point; dsDNA: double stranded DNA; eG: exogenous guanosine factor covalently linked at the 5’ end of the free intron RNA; exoG: exogenous guanosine cofactor; FLC: full-length circularization; GIR1: group I-like ribozyme; GIR2: group I splicing ribozyme; HE: homing endonuclease; HEG: homing endonuclease gene; I+: intron-containing allele; I–: intron-lacking allele; IPS: internal processing site; LE: ligated exon; LI: linear intron; LncRNA: long non-coding RNA; LSU rRNA: large subunit ribosomal RNA; Nt: nucleotide; RDNA: ribosomal DNA; RT: reverse transcriptase; SOLiD: sequencing by oligo ligation and detection; SS: splice site; SSU rRNA: small subunit ribosomal RNA.The authors declare that they have no competing interests.AH and SDJ contributed equally to the writing for this article. Both authors read and approved the final manuscript.


H-indol-2-yl)isoxazole-4-carboxamide (AIC) derivatives, and different anions  have been theoretically investigated using DFT approaches. It turned out that the unique selectivity of AIC derivatives for F− is ascribed to their ability of deprotonating the host sensors. Frontier molecular orbital (FMO) analyses have shown that the vertical electronic transitions of absorption and emission for the sensing signals are characterized as intramolecular charge transfer (ICT). The study of substituent effects suggests that all the substituted derivatives are expected to be promising candidates for fluoride chemosensors both in UV-vis and fluorescence spectra except for derivative with benzo[d]thienothiophene fragment that can serve as ratiometric fluorescent fluoride chemosensor only.The interactions between chemosensors, 3-amino-5-(4,5,6,7-tetrahydro-1 Furthermore, it is necessary to understand the unique host-guest interaction of the sensor with F− and other anions. Different signaling mechanisms have been suggested for F−, such as photoinduced electron transfer (PET) thienothiophene fragment that can serve as ratiometric fluorescent fluoride chemosensor only.Our calculated results for both the host-guest interaction and the nature of UV-vis and fluorescent signaling for