Understanding the aetiologies of neurodegenerative diseases such as Alzheimer's disease (AD), Pick's disease (PiD), Progressive Supranuclear Palsy (PSP) and Frontotemporal dementia (FTD) is often hampered by the considerable clinical and molecular overlap between these diseases and normal ageing. The development of high throughput genomic technologies such as microarrays provide a new molecular tool to gain insight in the complexity and relationships between diseases, as they provide data on the simultaneous activity of multiple genes, gene networks and cellular pathways.We have constructed genome wide expression profiles from snap frozen post-mortem tissue from the medial temporal lobe of patients with four neurodegenerative disorders  and 5 control subjects. All patients were matched for age, gender, ApoE-ε and MAPT (tau) haplotype. From all groups a total of 790 probes were shown to be differently expressed when compared to control individuals. The results from these experiments were then used to investigate the correlations between clinical, pathological and molecular findings. From the 790 identified probes we extracted a gene set of 166 probes whose expression could discriminate between these disorders and normal ageing.From genome wide expression profiles we extracted a gene set of 166 probes whose expression could discriminate between neurological disorders and normal ageing. This gene set can be further developed into an accurate microarray-based classification test. Furthermore, from this dataset we extracted a disease specific set of genes and identified two aging related transcription factors (FOXO1A and FOXO3A) as possible drug targets related to neurodegenerative disease. Neurodegenerative diseases are characterised by regional cellular changes associated with progressive loss (of function) of neurons that on careful examination of brain pathology is generally distinctive between diseases. On a clinical level however, large differences exist and in addition symptoms can vary widely during the course of the disease. Although classification of patients is possible into broadly defined clinical and pathological groups, defining the borders of such classifications is often hampered by individual variation between patients, even within families with Mendelian forms of disease . All samples matched the ones from the earlier experiment. RNA was isolated, labelled and run on new Affymetrix GeneChip Human Genome U133 Plus 2.0 microarrays by the LGTC according to protocol. All microarray data was normalised using GeneChip Operating Software (GCOS) 3.0 .n) was transformed to fit between 1 and 0 using the minimal (Expmin) and maximal expression (Expmax) according to following formula: (Expn−Expmin/(Expmax−Expmin)), and hierarchical cluster analysis was done using Cluster3.Considering the results of our first experiment we first performed cluster analysis using the set of 790 significantly different expressed probes. To correct for inter-experimental error, all 790 probes were standardised between the two experiments by dividing the 790 genes from the original dataset and the new dataset by the average expression ratio per probe in Microsoft excel . Since the second set of samples did not include controls, standardisation was only done using patient samples from the first dataset. This correction is necessary since the ratio of gene expression per probe from the first 20 experiments and the latter 6 experiments was on average 1.5 times higher (data not shown). Next, expression of independent genes  to circumvent the issues that a stringently ascertained dataset might miss relevant pathways  or that a larger, less stringently selected set of genes would contain too many false positives. We chose to define only those pathways as interesting when detected in both analyses.The low stringency set was defined by setting the significance threshold such that we accept ∼40 median false positives in the SAM analysis. Since our data contained approximately 40.000 probes this approach corresponds to using a nominal p-value of 0,001. The resulting gene set contained approximately 1600 probes with a false discovery rate (FDR) of 0.025. On the other hand, for the high stringency set we accepted only 1 median false positive. The resulting gene set contained 266 probes at an FDR of 0.004.To determine the likelihood for a pathway to be affected we used the statistics of the Ingenuity Pathway Analysis. This analysis compares the number of user-specified genes that participate in a pathway, relative to the total number these genes in all pathway annotations stored in the Ingenuity Pathways Knowledge Base, and gives significance values using the right-tailed Fisher's Exact Test, to indicate which pathways seem affected by the disease process. Using the less stringent dataset, five pathways were detected to be affected, among which the insulin receptor signalling (p = 0.047) see . A furthNext, to confirm these results and to determine relevant target genes in these pathways, we repeated the analysis with a stringency of 1 median false positive per dataset  to increase confidence that pathways were disease related. With this dataset we obtained again five affected pathways. Three of earlier mentioned pathways were detected to be significantly affected  show that these cases not only have considerable overlap in clinical symptoms and affected brain regions but also in gene expression see  confirmiThe observation that the insulin/IGF-1 signalling pathway is affected  in PSP, FTD and PiD makes this pathway an interesting target for further research. The function of FOXO transcription factors is diverse and depends on activity of other transcription factors and cell type. FOXO transcription factors influence a diverse group of cellular mechanisms, including glucose metabolism Their mode of action however largely depends on interactions with upstream pathways, since these regulate FOXO phosphorylation, acetylation or ubiquitinilation C. elegans a link between aggregation-mediated toxicity and decreased insulin/IGF-1-like signalling was shown. Downstream transcription factors (heat shock factor-1 and Daf-16) regulated (dis-)aggregation activities to promote cellular survival in response to constitutive toxic protein aggregation FOXO proteins have been reported to decrease ROS by increasing the radical scavenging proteins Mn superoxide dismutase (MnSOD) and catalase These results imply that instead of focussing on a single gene only, future experiments to study the involvement of (e.g. these FOXO) factors in PSP, FTD, PiD and other neurodegenerative diseases should be focussed on whole pathways and their interacting pathways, instead of focussing on single genes in order to obtain a more complete insight into the true involvement of these factors in neurodegeneration.In agreement with Dutch law, no ethics statement is required. All research involving human brain material, however was conducted according to the ethical declaration of the Netherlands Brain Bank , FTD In our confirmation experiment two additional PSP patients, two Alzheimer's disease patients , and can be accessed through the following direct link: http://www.ebi.ac.uk/microarray-as/ae/browse.html?keywords=E-MEXP-2280. All microarray data was normalised using GeneChip Operating Software (GCOS) 3.0 . Only marginal (0.06<p<0.04) and present (p<0.04) probe sets were used for subsequent analyses. Data analysis was done using the statistical package Significance Analysis of Microarrays (SAM) http://www.r-project.org) since previous results had shown this test to give valid results All microarray data have been deposited in ArrayExpress, the public archive for transcriptomics and related data at the EBI (n) was transformed to fit between 1 and 0 using the minimal (Expmin) and maximal expression (Expmax) according to following formula: (Expn−Expmin/(Expmax−Expmin)), and hierarchical cluster analysis was done using Cluster3 To correct for inter-experimental error between original and control datasets, all 790 probes obtained in the stringent analysis were standardised between the two experiments by dividing expression values of the 790 genes from the original dataset and the new dataset by the average expression ratio of the two datasets per probe in Microsoft excel . Since the second set of samples did not include controls, standardisation was only done using patient samples from the first dataset. Next, expression of independent genes (Expwww.ingenuity.com) using the “Functional Analysis” option. The significance value associated with this Analysis for a dataset is a measure for the likelihood genes from the dataset file under investigation participate in that function. The significance is expressed as a p-value, which is calculated using a right-tailed Fisher's Exact Test. The p-value is calculated by comparing the number of user-specified genes of interest that participate in a given function or pathway, relative to the total number of occurrences of these genes in all functional/pathway annotations stored in the Ingenuity Pathways Knowledge Base .Significance of analysed pathways was calculated in Ingenuity . To confirm microarray data, primers for MOXD1; ZNF589; FOXC1; SASH1; ACAD2; SEPT2; PNPLA2; TNPO1; CBL; GOLPH4; NRD1; PARD3; PTEN; NPIP; BTBD14A; CENTB5; HPRT and β-ACTIN were designed for quantitative PCR using Primer Express 2.0.0 software . All qPCRs were standardized to β-Actin and HPRT signals that were run in parallel Click here for additional data file.Table S2Compiled table of all 790 probes detected to be significantly different from background and overlapping in at least two different pathologically defined groups. When possible probes are given in official gene symbol names. Gene symbol: official genbank gene symbol. Gene Title: official genbank gene name. ---: Unknown(0.83 MB DOC)Click here for additional data file.Table S3Compiled table of all 166 probes detected to be significantly different from background and overlapping in at least two different pathologically defined groups. When possible probes are given in official gene symbol. Gene Title: official genbank gene name. ---: Unknown(0.20 MB DOC)Click here for additional data file.Table S4Overview of detected probes, signal strengths, background and noise levels.(0.17 MB DOC)Click here for additional data file.Table S5Primer sequences of primers used for quantitative reverse transcriptase confirmation of the microarray data.(0.04 MB DOC)Click here for additional data file.Supplementary File S1Ethical Declaration of The Netherlands Brain Bank(0.09 MB PDF)Click here for additional data file.


ESP. Analysis of Heterogeneous Inbred Families validated seven loci and all of the QTL×DPG (days post-germination) interactions tested, but was complicated by the extensive epistasis. A comparison of transcript accumulation data within 211 of these RILs showed an extensive overlap of gene expression QTLs for structural specifiers and their homologs with the identified glucosinolate activation loci. Finally, we were able to show that two of the QTLs are the result of whole-genome duplications of a glucosinolate activation gene cluster. These data reveal complex age-dependent regulation of structural outcomes and suggest that transcriptional regulation is associated with a significant portion of the underlying ontogenic variation and epistatic interactions in glucosinolate activation.Most phenotypic variation present in natural populations is under polygenic control, largely determined by genetic variation at quantitative trait loci (QTLs). These genetic loci frequently interact with the environment, development, and each other, yet the importance of these interactions on the underlying genetic architecture of quantitative traits is not well characterized. To better study how epistasis and development may influence quantitative traits, we studied genetic variation in Arabidopsis glucosinolate activation using the moderately sized Bayreuth×Shahdara recombinant inbred population, in terms of number of lines. We identified QTLs for glucosinolate activation at three different developmental stages. Numerous QTLs showed developmental dependency, as well as a large epistatic network, centered on the previously cloned large-effect glucosinolate activation QTL,  Arabidopsis thaliana during the development of what is considered a static mature rosette. Variation in glucosinolate activation was principally controlled by epistatic and G×D interactions. Epistatic interactions identified both Mendelian epistasis, where regulatory loci controlled enzymatic loci, and quantitative interactions between regulatory loci. G×D appeared to involve master regulatory loci as determined by trans-eQTL hotspot analysis. Finally, two common glucosinolate activation QTLs appear to have evolved via gene loss and sub-functionalization following quadruplication of an ancestral genomic fragment, potentially by two whole-genome duplications. Thus, genomic duplication events may facilitate the formation of quantitative genetic variation. This study provides insights into the molecular basis of the link between genetic and phenotypic variation in a potentially adaptive trait.A principal interest in biology is to understand how natural genetic variation translates into phenotypic variation. A key component of this connection is how the genetic variation interacts with other sources of variation, such as environment (G×E), development (G×D), or other genetic loci (G×G or epistasis). To analyze the molecular underpinnings of these quantitative genetics interaction terms, we investigated the genetic architecture of an adaptive trait, glucosinolate activation, in  Most phenotypic variation present in natural populations is under polygenic control, largely determined by genetic variation at multiple quantitative trait loci (QTLs), which has motivated considerable efforts to elucidate the genetic basis of these polygenic traits QTL mapping, which measure the association of genetic markers with phenotypic variation, is one of the most common approaches for identifying loci and epistatic interactions controlling polygenic inheritance Accurate characterization of a quantitative trait's underlying genetic architecture is often limited by practical considerations that limit the number of progeny included in a mapping analysis. Small populations are especially problematic in the presence of epistasis between QTLs, as the pair wise comparisons required to detect these interactions rapidly exhausts the available genotypic variation, leading to an underestimation of numbers of loci and interactions, resulting in an incomplete picture of the genetic architecture Arabidopsis thaliana using a moderately sized recombinant inbred population. Glucosinolates are the inert storage form of a two part phytochemical defense system found throughout the Brassicaceae, where biologically active structures are catabolically produced by the enzyme myrosinase To investigate how development and epistasis can interact to control the variation in an adaptive trait, we studied the outcome of glucosinolate activation within Epithiospecifier Protein (ESP) and an as yet unidentified simple nitrile structural specifier (AtNSP), promote the formation of simple and epithionitriles at the expense of the default isothiocyanate rearrangement via two biochemically related yet separate rearrangements (Epithiospecifier Modifier (ESM1) epistatically modulates ESP mediated epithionitrile and simple nitrile rearrangements ×Shahdara (Sha) recombinant inbred lines (RILs) GSL-AOP and GSL-Elong loci, such that Bay-0 has 3-hydroxypropyl glucosinolate as its main short chain aliphatic glucosinolate and Sha has but-3-enyl glucosinolate ESP and produces mixtures of simple nitriles and isothiocyanates, depending on the age of the plant ESP and produces mixtures of epithionitriles, simple nitriles and isothiocyanates  were detected in the RILs at all three DPG, although the additive effect of ESM1 switched direction from promoting nitrile formation at 42 DPG relative to promoting isothiocyanate formation at 30 and 35 DPG. In spite of the fact that Bay-0 lacks functional ESP, four QTLs showed a positive impact of the Bay-0 allele on epithionitrile production, which is consistent with the observed transgressive segregation in the RILs  was detected in all three QTL maps. Increased simple nitrile proportions were fairly evenly distributed between the Bay-0 and Sha alleles (GSL.Activ.IV.16) exhibited significant additive effects in opposite directions at 30 and 35 DPG , confirming the presence of background ESP modifiers. The level of validation observed in the HIF analysis is strongly supportive of the QTL mapping results, as each HIF genotype was only analyzed in six-fold replication per DPG whereas each marker genotype in the RIL study was analyzed in roughly 200-fold replication, lending more power to the RIL analysis.To provide additional support for the identified is study . For thecyanates . HIF-241oduction . InteresGSL.Activ.V.18, showed a significant difference in epithionitrile proportions between alleles at 24 but not 38 DPG . Further, the glucosinolate activation genes had more eQTLs, both cis and trans than the average Arabidopsis transcript. This excess was most dramatic with trans-acting eQTLs, with an average of 3.1 trans-eQTLs detected for each glucosinolate activation gene in comparison to the average Arabidopsis transcript, with 1.5 trans-eQTLs in this population We first obtained the estimated heritability for transcripts encoding these potential glucosinolate activation genes cis and trans-eQTL clusters. The two cis-eQTL clusters are associated with the genomic regions around ESP and ESM1, which contain several ESP, ESM1, and MBP homologues but do not overlap with known trans-eQTL hotspots , suggesting that the GSL.Activ.I.69 may also be a regulatory locus. However, it does not co-locate with a previously identified global trans-eQTL hotspot suggesting that it may be more specific to glucosinolate activation In addition to er genes [37]. ThuESP and its epistatic modifier locus, ESM1The structural outcome of glucosinolate activation strongly influences plant defense, and had been thought to be predominantly controlled by genetic variation in ESP and ESM1 QTLs  flats, and grown in controlled environment chambers at 20°C with 8 h light at 100–120 µEi. Each flat contained one Bay-0 and Sha parents. All plants were free of insect pests by visual inspection. The population was grown three independent times to independently phenotype the structural outcome of glucosinolate activation at 30, 35, and 42 days DPG. These DPG were chosen because day 30 represents the attainment of Stage 1.10 or 10 mature leaves per plant for both the parents and all RILs. Further, day 42 is one week away from the earliest RIL flowering (Stage 5.10) in our environmental conditions Seeds were imbibed and cold stratified at 4 degrees for three days to break dormancy. All seeds were sown directly onto Premier ProMix B potting soil  in 36-cell , by comparison with published mass spectra www.arabidopsis.org) We obtained genotypes and genetic map information for the Bay-0×Sha RIL population from the Arabidopsis Biological Resource Center   . There wEach HIF was planted with twelve independent biological replicates per allele per HIF. These were planted and grown under identical conditions as described above for the RIL population. For each HIF, six replicates of each genotype were assayed for structural outcomes at 24 DPG and six were assayed at 38 DPG to validate each QTL and survey for age dependence. These time points were chosen such that there was a sufficient developmental time difference to detect genotype×DPG effects but before epithionitrile formation reached saturation at the later time points. Due to poor germination, HIF191, 244, 338, and 364 were only assayed at 38 DPG.ESP QTL, to assess background dependent effects upon ESP.The data for each class of glucosinolate activation product were analyzed for the effects of genotype, DPG, and DPG×genotype within each HIF using the general linear model procedure in SAS. Given that each HIF is a separate and independent test, we did not correct for multiple testing within these models. We also directly compared HIF241 to HIF425, two independent HIFs differing at the ESP, ESM1, and Myrosinase Binding Protein 1 (MBP1) and MBP2. For all four gene families we had two criteria to define a gene as potentially associated with glucosinolate activation. First, each included gene had to be similar to known genes based on a BLASTP score of at least e−45. Secondly, we further restricted this list to genes that were phylogenetically limited to Arabidopsis when protein sequences from the poplar, grape and rice plant genomes were included We used previously published sequence data to identify the known myrosinases and structural specifier genes http://www.ebi.ac.uk/arrayexpress/) under numbers E-TABM-126 and E-TABM-224.There are no new accession numbers associated with this dataset. The microarray data set used in this study has been deposited at EBI ArrayExpress Click here for additional data file.Table S1HIF Genotypes. The genotype for each HIF line at the listed markers is provided. Heterozygous scores are in bold.(0.03 MB XLS)Click here for additional data file.Table S2Known and Putative Glucosinolate Activation Genes. Shown are the identified candidate genes for the different glucosinolate activation classes that contain a probeset on the Affymetrix ATH1 microarray.(0.03 MB XLS)Click here for additional data file.Table S3QTL for Glucosinolate Activation Traits. Main effect and DPG interaction provide the P value for the given QTL in relation to the listed trait. For the Epistasis analysis, 1 = a significant epistatic interaction at DPG30, 2 is for significance at DPG35, 3 is for significance at DPG42 and F is significant epistasis across all times. Allele substitution provides the estimated main effect of each QTL on the phenotype.(0.04 MB XLS)Click here for additional data file.Table S4Glucosinolate Activation with Allyl and Butenyl glucosinolates in the RILs. Phenotypic values for glucosinolate activation in the given RIL at the given day-post-germination (DPG).(0.75 MB XLS)Click here for additional data file.Table S5Summary of HIF Results. Shown are all statistical analyses on the HIFs with grey horizontal bars separating the different QTLs. P values for each term in the model as well as mean glucosinolate activation values and standard deviation are provided for each HIF.(0.05 MB XLS)Click here for additional data file.


Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ. © 1999 Cancer Research CampaignDespite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO


Letsbmanta (L.) amazonensis wasinvestigated, as well as the possible correlation between theeffects of these inflammatory mediators with nitric oxideproduction. It was found that pretreatment of macrophages with 10−5 Mof the PAF antagonists, BN-52021 or WEB-2086, increasedmacrophage infection by 17 and 59%, respectively. Thecyclooxygenase inhibitor, indomethacin (10 μg/ml), induced asignificant inhibition which was reversed by addition of PGE (10-3M) to the culture medium. These results suggested that the infectionof macrophages by leisbmanla is inhibited by PAF and enhanced byprostaglandins and that these mediators are produced by macrophagesduring this infection. This was confirmed by addition of thesemediators to the culture medium before infection; PAF  reduced significantly the infection whereas PGE2 (10−5 M)induced a marked enhancement. This effect of exogenous PAF onmacrophage infection was reversed by the two PAF antagonists used inthis study as well as by the inhibitor of nitric oxide synthesis,L-arginine methyl ester (100 mM). Taken together the data suggestthat endogenous production of PAF and PGE2 exert opposing effects onLesbmana–macrophage interaction and that nitric oxide may beinvolved in the augmented destruction of parasites induced by PAF.The role of endogenously synthesized PAF and prostaglandins on theinfection of mouse macrophages by


Functional dyspepsia and non-erosive reflux disease (NERD) are prevalent gastrointestinal conditions with accumulating evidence regarding an overlap between the two. Still, patients with NERD represent a very heterogeneous group and limited data on dyspeptic symptoms in various subgroups of NERD are available.To evaluate the prevalence of dyspeptic symptoms in patients with NERD subclassified by using 24 h impedance-pH monitoring (MII-pH).Patients with typical reflux symptoms and normal endoscopy underwent impedance-pH monitoring off proton pump inhibitor treatment. Oesophageal acid exposure time (AET), type of acid and non-acid reflux episodes, and symptom association probability (SAP) were calculated. A validated dyspepsia questionnaire was used to quantify dyspeptic symptoms prior to reflux monitoring.Of 200 patients with NERD , 81 (41%) had an abnormal oesophageal AET (NERD pH-POS), 65 (32%) had normal oesophageal AET and positive SAP for acid and/or non-acid reflux (hypersensitive oesophagus), and 54 (27%) had normal oesophageal AET and negative SAP . Patients with functional heartburn had more frequent (p<0.01) postprandial fullness, bloating, early satiety and nausea compared to patients with NERD pH-POS and hypersensitive oesophagus.The increased prevalence of dyspeptic symptoms in patients with functional heartburn reinforces the concept that functional gastrointestinal disorders extend beyond the boundaries suggested by the anatomical location of symptoms. This should be regarded as a further argument to test patients with symptoms of gastro-oesophageal reflux disease in order to separate patients with functional heartburn from patients with NERD in whom symptoms are associated with gastro-oesophageal reflux. Gastro-oesophageal reflux disease (GORD) is one of the most common chronic gastrointestinal diseases in Western countries.1358Patients with GORD, both with erosive oesophagitis and NERD, frequently report dyspeptic symptoms.12Given previous reports indicating an inverse relationship between dyspeptic symptoms and the objective criteria for GORD we hypothesised that in patients with functional heartburn dyspeptic symptoms should be more prevalent compared to the rest of NERD patients. To test this hypothesis, we evaluated the prevalence of dyspeptic symptoms in patients with NERD subclassified into three distinct groups by using 24 h MII-pH monitoring.Helicobacter pylori. Patients were asked to discontinue any medication that would influence oesophageal motor function at least 1 week before administering the questionnaires and performing tests of oesophageal function.Between June 2004 and September 2008, patients presenting to the outpatient motility centre at the University of Genoa with predominant typical GORD symptoms  lasting for more than 6 months and occurring at least three times weekly, were prospectively enrolled in the study. All subjects who agreed to participate in our investigation underwent careful history taking physical and clinical examination, upper gastrointestinal endoscopy to assess the presence or not of oesophageal mucosal injury, routine biochemistry, and upper abdominal ultrasound. The medical history included information on treatment with acid suppressive medication (in particular proton pump inhibitors (PPIs)) and symptomatic response to PPI therapy. Patients reporting ⩾50% symptom improvement were considered responders to PPI therapy. Patients treated with antisecretory drugs were asked to discontinue acid suppressive therapy at least 30 days before the endoscopic examination. During the washout period, patients were allowed to use an oral antacid or alginate on an as-needed basis for the relief of heartburn. Based on the results of upper endoscopy, patients were then subdivided into three major groups – Barrett’s oesophagus, erosive oesophagitis and NERD – in cases where the typical symptoms of GORD were present, and where visible oesophageal mucosal injury was absent. Patients with Barrett’s oesophagus and erosive oesophagitis were not included in the present study. Within 1–5 days (median 3 days) from the upper endoscopy, patients with NERD underwent ambulatory combined impedance-pH monitoring. Exclusion criteria were: history of thoracic, oesophageal or gastric surgery; primary or secondary severe oesophageal motility disorders ; underlying psychiatric illness; use of non-steroidal anti-inflammatory drugs (NSAIDs) and aspirin; presence of peptic stricture and duodenal or gastric ulcer on upper endoscopy, evidence of erosive oesophagitis at previous (2–5 years) endoscopy, presence of dyspeptic symptoms as major symptoms. In women of childbearing age, pregnancy was excluded by urine analysis. During upper gastrointestinal endoscopy, biopsies were taken from the antrum and the corpus for assessing the presence of All participants gave written informed consent before entering the study.Before the 24 h pH-impedance study, each patient completed a functional dyspepsia questionnaire as reported and validated previously.Oesophageal impedance-pH monitoring was performed using an ambulatory multichannel intraluminal impedance and pH (MII-pH) monitoring system . The system included a portable data logger and a catheter with one antimony pH electrode and eight impedance electrodes at 2, 4, 6, 8, 10, 14, 16 and 18 cm from the tip of the catheter. Each pair of adjacent electrodes represented an impedance-measuring segment (2 cm length) corresponding to one recording channel. The six impedance and one pH signals were recorded at 50 Hz on a 128 MB CompactFlash .The methodology of probe calibration, catheter placement, patient instruction and performance have been previously described.14The data stored on the CompactFlash card were downloaded into a personal computer and analysed using a semiautomated reflux detection algorithm . Tracings were subsequently reviewed manually by an expert reader (ES) in order to ensure accurate detection and classification of reflux episodes. Meal periods were excluded from the analysis.Simultaneously recorded pH data were used to classify reflux episodes as acid, weakly acidic, or weakly alkaline according to the previously reported criteria:15Acid reflux: impedance-detected reflux episodes with a nadir pH less than 4Weakly acidic reflux: impedance-detected reflux episodes with a nadir pH between 4 and 7Weakly alkaline reflux: impedance-detected reflux episodes with a nadir pH above 7. For symptom analysis, weakly acidic and weakly alkaline reflux were grouped together as non-acid reflux episodes (nadir pH>4)Impedance and pH data were used to determine the number and type of reflux episodes and distal oesophageal acid exposure (reflux time (min) and reflux per cent time) in each patient.Total 24 h oesophageal acid exposure (%) was defined as the total time when the pH was below 4 divided by the time of monitoring. A total distal oesophageal acid exposure  of less than 4.2% over 24 h was considered normal.814In each patient we calculated the symptom association probability (SAP) for typical oesophageal symptoms. In the analysis we separated symptoms associated with acid reflux from those associated with non-acid reflux  and symptoms occurring independent of reflux episodes. Separate analysis was performed for each individual symptom if patients recorded different types of symptoms.The SAP was calculated for acid and non-acid reflux using a custom made Excel macro function (RT). The algorithm counted the number of symptoms preceded by a reflux episode within 2 min (S+R+). The number of symptoms not associated with reflux (S+R−) was calculated by subtracting S+R+ from the total number of symptoms. The study was divided into 2-min segments in order to determine the number reflux containing segments (R+) and reflux-free symptoms (R−). The two other cells of the 2×2 contingency table (S−R− and S−R+) were calculated by performing the corresponding subtractions. A two-sided Fisher exact test was used to calculate the probability (p) that the observed distribution could have been occurred by chance. SAP was calculated as (1−p)×100% and was considered to be positive when ⩾95% was positive.82 test was used when comparing categorical variables . Differences were considered statistically significant when p<0.05.Continuous variables were compared using ANOVA (with post hoc comparisons between groups) or the Kruskal–Wallis test depending on the normal or abnormal distribution of data (assessed by the Kolmogorov–Smirnov test). The χTwo hundred and sixty-six patients with typical symptoms of GORD met the enrolment criteria and entered the study. Based on the results of upper endoscopy, eight patients had Barrett’s oesophagus, 44 had erosive oesophagitis, and 214 had NERD. Patients with Barrett’s oesophagus and erosive oesophagitis were not included in the present study. Of the patients with NERD, 200  reported at least one type of typical gastro-oesophageal reflux symptom during the monitoring period and were included in the final analysis. Thirty-four patients (17%) never received PPIs (“PPI naive” NERD patients). Combined impedance-pH monitoring was well tolerated by all subjects and no important technical failure occurred. The median total recording time was 23.4 h (range 22.9–23.6 h).Acid exposure time and symptom association probability is shown in H pylori infection (p = NS). The prevalence of hiatal hernia was higher in NERD pH-POS patients with positive SAP and negative SAP compared to those with hypersensitive oesophagus or FH . Moreover, patients in the NERD pH-POS subgroups had a higher mean body mass index than patients with hypersensitive oesophagus or FH . Finally, patients of the functional heartburn subgroup were more frequently women than those in the other two subgroups .Detailed demographic and clinical characteristics of the various subgroups of patients with NERD are shown in Patients with NERD had a median heartburn score of 2 (range 1–3) and a median regurgitation score of 2 (range 0–3) recorded in the GORD symptoms questionnaires. Details on the number and percentages of patients on PPIs and the numbers and percentages of patients who responded to PPIs are shown in Symptoms reported during the study day were in agreement with the symptom questionnaires. Indeed, 66 (93%) patients with NERD pH-POS/SAP+ (median heartburn score 2), eight (80%) with NERD pH-POS/SAP− (median heartburn score 2), 58 (89%) NERD pH-NEG/SAP+ (median heartburn score 2) and 50 (93%) NERD pH-NEG/SAP− (median heartburn score 2) reported heartburn, while 43 (61%) patients with NERD pH-POS/SAP+ (median regurgitation score 2), five (50%) with NERD pH-POS/SAP− (median regurgitation score 2), 36 (55%) NERD pH-NEG/SAP+ (median regurgitation score 2) and 18 (33%) NERD pH-NEG/SAP− (median regurgitation score 1) reported regurgitation.The total number of symptoms reported by patients with NERD was 2245 . Patients reported 1465 heartburn events  and 780 regurgitation episodes . Data on the number of heartburn and regurgitation events reported by patients in various subgroups of NERD are summarised in As summarised in Upper gastrointestinal symptoms are remarkably common in the general population, with the majority of patients having GORD or dyspepsia.10171819Non-erosive or negative-endoscopy reflux disease (NERD) may account for up to 70% of patients with GORD in the community.898In our study, prospectively collected 24 h ambulatory impedance-pH monitoring data and functional dyspepsia questionnaires in a large group of unselected NERD patients off PPI treatment led us to show that clinically relevant dyspeptic symptoms are present in 44% of the NERD population, in accordance with other investigations.Patients now diagnosed with FH from the NERD collective reported much more frequently dyspeptic symptoms such as postprandial fullness, bloating, early satiety and nausea compared to the NERD pH-POS with positive SAP and negative SAP and hypersensitive oesophagus subgroups. In particular, postprandial fullness and early satiation were the main symptoms more frequently related to the FH subgroup, suggesting a possible association between this disorder and the post prandial distress syndrome, as defined by Rome III criteria.17et al.et al,Moreover, if we look at the prevalence of typical reflux symptoms in our population, we found that NERD pH-POS with positive SAP and negative SAP patients more often experienced regurgitation episodes than did patients with FH, with an increasing frequency during the recording period, suggesting that they have a GORD symptom pattern more similar to that of patients with erosive oesophagitis and Barrett’s oesophagus than with FH.123H pylori infection could be demonstrated in our population. This is in agreement with various studies20Finally, some demographic features were different in our subgroups. We found that NERD pH-POS patients independently of the symptom association were more frequently male, had a higher mean body mass index and an increased prevalence of hiatal hernia, in agreement with those studies considering these demographic and clinical parameters as risk factors for the development of GORD and, particularly, abnormal pH monitoring.102526172829One limitation of our study is the lack of outcome prospective data as no information on the response to a standardised therapeutic approach in the described patient groups are currently available. However, considering the previous response to PPI treatment referred to by the majority of our patients before entering the study we can argue that patients with abnormal distal oesophageal acid exposure and/or positive symptom association and dyspeptic symptoms do respond better to antisecretive treatment compared to patients with negative symptom association and dyspeptic symptoms. Evaluating patients on a Mediterranean diet could also be regarded as a shortcoming of the present study with the argument that it was not refluxogenic enough to induce symptoms. While being cognisant of this problem we chose the Mediterranean diet in order to be able to compare the results in patients with NERD with those collected in healthy Italian volunteers and, on the other hand, trying to minimise diet-induced variation in the amount of gastro-oesophageal reflux. Last, but not least, the 30 day washout period before the upper endoscopy in patients using acid suppressive medication could have been too short for lesions to develop. Still, in our experience, this represents the maximum we can ask of patients in order to obtain good compliance and without them dropping out from the study. Moreover, the incidence of recurring erosive lesions after a shorter period of PPI cessation remains unknown.In conclusion, the present study underscores the important overlap between functional dyspepsia and NERD. This argues for the need to monitor dyspeptic symptoms and both acid and non-acid reflux episodes in patients primarily diagnosed with NERD to help identify the proportion of patients with true FH, who should, preferably, be included in the overall population with functional gastrointestinal disorders and should not be considered as a GORD subgroup any more. We believe this deliberate separation would spare these patients from wasteful and protracted courses of years of acid suppression and, above all, prevent potentially disastrous exposure to surgical options. Prospective outcome data are now needed to show the clinical significance of these findings in respect of future definition of NERD. Of particular interest would be the prospective evaluation of the response to acid suppressive therapy of NERD pH-POS and NERD pH-NEG/SAP+ for acid patients against NERD pH-NEG/SAP+ for non-acid and the response to reflux reducing therapies of NERD pH-POS and NERD pH-NEG/SAP+ for acid and non-acid patients against NERD pH-NEG/SAP− patients. This possible redefinition as suggested in the present manuscript might impact upon the epidemiology, pathophysiology and natural history of the respective disorders.


Betulinic Acid (BetA) and its derivatives have been extensively studied in the past for their anti-tumor effects, but relatively little is known about its precursor Betulin (BE). We found that BE induces apoptosis utilizing a similar mechanism as BetA and is prevented by cyclosporin A (CsA). BE induces cell death more rapidly as compared to BetA, but to achieve similar amounts of cell death a considerably higher concentration of BE is needed. Interestingly, we observed that cholesterol sensitized cells to BE-induced apoptosis, while there was no effect of cholesterol when combined with BetA. Despite the significantly enhanced cytotoxicity, the mode of cell death was not changed as CsA completely abrogated cell death. These results indicate that BE has potent anti-tumor activity especially in combination with cholesterol. Triterpenoids are extensively studied for the potential use as anticancer agents. One of the most promising compounds in this class is Betulinic Acid (BetA), but its effect is limited by the poor solubility of the compound. A lot of effort is therefore put into the development of derivatives of BetA with the goal to develop even more powerful compounds and to achieve better solubility for enhanced in vivo administration BetA can be found in numerous different plants, but it can also be obtained by a simple 2 step reaction from its more abundantly available precursor molecule Betulin (BE) Betulin  and Betulinic Acid  were dissolved in DMSO at 4 mg/ml, cholesterol (Sigma-Aldrich) was dissolved at 5 mM in DMSO. Aliquots were kept frozen. Propidium iodide (PI), zVAD.fmk , etoposide and cyclosporin A were purchased from Sigma-Aldrich, Mitosox was obtained from Invitrogen .Anti-PARP  and anti-cytochrome c  were used.Cell lines: A549 and Hela were obtained from the ATCC, FADD-deficient, Caspase 8- deficient and control Jurkat cells (JA3) were kindly provided by Dr John Blenis , Jurkat cells over-expressing Bcl-2 by Dr Jannie Borst  and Bax/Bak double knockout (DKO) mouse embryonic fibroblasts (MEFs) and wild-type control MEFs were from Dr Stanley Korsmeyer.Overall cell death was assessed as previously described Cells were incubated in Nicoletti buffer containing 50 µg/ml PI for at least 24 hours before analysis via flow cytometry.Cells were lysed using Triton X-100 buffer and for protein quantification a BCA kit from PIERCE was used. SDS-PAGE was performed and proteins were transferred onto a PVDF transfer membrane (Amersham Biosciences). Blocking of unspecific binding sites was achieved by incubation of the membrane in 5% low fat milk powder in PBS/0.2% Tween-20 (blocking buffer) for 1 hour at room temperature. Primary antibody incubation was performed overnight at 4°C and secondary antibody (HRP labeled) incubation for 2 hours at room temperature. For chemiluminescent detection ECL from Amersham Biosciences was used in combination with a LAS-3000 imaging system.For ROS measurements the highly selective dye for mitochondrial superoxide Mitosox was used. Cells were incubated with 5 µM Mitosox in pre-warmed tissue culture medium at 37°C for 10 min before flow cytometry analysis.Cytochrome c release was measured as previously described by Waterhouse et al cells were incubated in the presence of 40 µg/ml MTT reagent for 2 hours at 37°C. During the incubation period appearance of purple formazan structures was followed by phase-contrast light microscopy.Previously we have shown that BetA induces cell death in Jurkat T leukemia cells in a concentration and time-dependent fashion We have found previously that when using the MTT -2,5-diphenyltetrazolium bromide) assay to measure BetA To identify the nature of cell death induced by BE/Cholesterol we investigated the apoptotic pathway. Apoptosis has been previously reported to be the cell death pathway induced by BE in A549 lung cancer cells Cholesterol is an important constituent of cell membranes where it plays a crucial role in maintaining integrity and fluidity BetA induced apoptosis has been clearly linked to the mitochondria BE/cholesterol showed clear cytochrome c release in Jurkat cells. Importantly, there was only a slight difference in cytochrome c release in the Bcl-2 over-expressing cells , but thiTo further determine the efficacy of BE/cholesterol and to find out if Bax and Bak are involved in BE/cholesterol induced cytotoxicity we used Bax/Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs). DKO MEFs are resistant to drugs such as etoposide, staurosporine, UVC or actinomycin D, all targeting the Bcl-2 family regulated mitochondrial pathway BE is a natural compound, which contains derivatives that have been shown to possess strong anti-tumor properties Despite the strong similarities, and the almost identical structure of BE and BetA, there are also important differences in comparison to BetA induced apoptosis. We previously showed that Bcl-2 over-expression delayed BetA-induced apoptosisThis difference between BetA and BE/cholesterol is even more remarkable when considering the time dependency of cytotoxicity of both molecules: For BetA the maximum effect requires around 48–72 hours and a dose of 7.5–10 µg/ml , while BStriking is the fact, that cholesterol strongly enhances the cytotoxic effects of BE but not BetA  whilst bThe effect on MTT conversion to formazan  mainly consisting of Betulin suggest that it is safe; no signs of toxicity were observed in rats or dogs in a subchronic toxicity study To further evaluate the anti-tumor properties of BE/cholesterol in vivo. Because cholesterol is ubiquitously present in the body it is unlikely that additional applied cholesterol is useful for in vivo effects of BE as an anti-tumor agent. Our results indicate that the amount of cholesterol necessary (5 µM) for enhanced in vitro effects of BE are about 1000 times lower than normal plasma cholesterol levels in humans (5 mM). However the fast majority of this cholesterol is contained in LDL or HDL and it is therefore difficult to assess whether there is sufficient free cholesterol available to potentiate BE-induced apoptosis in vivo. Adding more cholesterol may not bear any significance though, but application of cholesterol containing Betulin-liposomes may be an interesting mode of applying this cytotoxic agent. In summary we conclude that Betulin by itself and in combination with cholesterol is a potent anti-cancer agent in vitro and warrants further investigation in vivo.It will be interesting to explore the combined effects of BE and cholesterol Figure S1MTT conversion. Effects of BetA, cholesterol and BE on MTT assay: Jurkat cells were treated as indicated, incubated with MTT reagent and photographed under a phase-contrast microscope.(6.51 MB TIF)Click here for additional data file.


Recently, the cardiovascular safety of rosiglitazone was brought to center stage following meta analyses and the interim analysis of the RECORD trial. Current evidence points to rosiglitazone having a greater risk of myocardial ischemic events than placebo, metformin, or sulfonylureas. This review article discusses the mechanism of action of PPAR agonists and correlates it with clinical and laboratory outcomes in the published literature. In addition, this review article attempts to discuss some of the molecular mechanisms regarding the association between TZDs therapy and the nontraditional cardiovascular risks.Cardiovascular disease is a major cause of morbidity and mortality among people with type 2 diabetes mellitus. The peroxisome proliferator-activated receptor (PPAR) agonists have a significant role on glucose and fat metabolism. Thiazolidinediones (TZDs) are predominantly PPAR Diabetes Mellitus has emerged as a global burden, as World Health Organization (WHO) has estimated a prevalence of more than 300 million people by the year 2025. Diabetes is associated with a 2–4 fold increased risk for developing unstable angina and myocardial infarction (MI) with subsequent increased mortality and morbidity , 2. Endoγ agonists that improve glycemic control and have been shown to exert possible cardiovascular benefits. However, recent studies have shown that rosiglitazone is associated with an increased risk of heart failure, acute MI (AMI) and death as a result of cardiovascular complications . Si. Si19]. γ agonists have reinforced the need to identify additional therapies that improve insulin sensitivity and treat hyperlipidemia in addition to lowering blood pressure. In this regard, several lines of clinical evidence support the use of two Angiotensin receptor blockers (ARBs), telmisartan and irbesartan, in treating hyperlipidemia and insulin resistance [n = 2954) or placebo (n = 2972). The primary endpoint was the composite of cardiovascular death, MI, stroke or hospitalization due to heart failure. The secondary outcome excluded heart failure. Median follow-up was 56 months [The adverse effects of full PPARsistance . In the sistance . This st6 months . There wα by regulating the expression of proteins involved in the transport and β-oxidation of free fatty acids (FFAs) plays a pivotal role in the regulation of lipid and glucose metabolism [α. They lower circulating triglyceride levels by increasing the activity of lipoprotein lipase (LPL) which hydrolyzes triglycerides [α agonists increase the gene expresson of LPL and up regulate Apo A-I and A-II synthesis which are major apoproteins of the HDL fraction in the liver and resulting in increased serum HDL levels [α agonists are possibly beneficial in the clinical scenario [PPARtabolism . Fibrateycerides . PPARα aL levels , 30. DatL levels . In the L levels , 33. Simscenario . α activation [α binding.Several studies have shown that pioglitazone increased serum HDL-cholesterol and decreased triglycerides, and pioglitazone produced more favorable lipid profiles than rosiglitazone in patients with type 2 diabetes mellitus , 35. Nottivation . They haγ and PPARα agonists which ultimately inhibit atherosclerosis by decreasing the expression of several inflammatory mediators involved in macrophage activation and vascular smooth muscle cells (VSMCs) proliferation  and lipoprotein (Lp(a)). In recent years it has been established that inflammation has a pathogenic role in atherosclerosis. Several studies described the antiinflammatory properties of PPARferation . Again, δ agonists are clinically approved at the present moment, but they may be beneficial for the treatment of cardiovascular disorders and improve overall cardiovascular risk assessment. The cardiovascular benefit-risk ratio of individual PPAR agonists is not completely clear. The results of data analysis of the RECORD study showed an increased risk of heart failure in the rosiglitazone group compared with metformin and sulfonylurea. It did not show any increase in AMI , 18. Theδ agonist for the potential treatment of dyslipidemia by GlaxoSmithKline and Ligand are soon to be released. GW-501516 may prove to be a suitable alternative for the treatment of the cardiovascular disease and improve overall cardiovascular risk assessment. Interestingly, GW501516 suppresses IL-6-mediated hepatocyte acute phase reaction via STAT3 inhibition [Ongoing Phase II clinical trials of GW-501516, a PPARhibition .α activity. A larger population study is needed to understand PPAR agonists antiatherogenic properties. However, their adverse effects should be taken into considerations.The findings reviewed in this article suggest that the mechanisms of PPAR agonists have an excellent glucose and lipid control by their effects on vasculature, muscle and adipose tissue. The in vitro studies also show their effects by improving lipid profile, maintaining euglycemia, suppressing various inflammatory mediators, HCT, Lp(a), and preventing the progression of atherosclerosis. However, when clinical trials are reviewed, they do not reflect very well with the beneficial actions in cell and animal models. Rosiglitazone has shown adverse effects on heart failure, mortality, and abnormal lipid profile. Pioglitazone is associated with increased risk of heart failure due to edema. Furthermore, pioglitazone has been shown to reduce the cardiovascular risk in patients with established atherosclerotic vascular disease. Pioglitazone has also a favorable effect on lipid profile by stimulating apoA1 production because it has some additional PPAR


Neuronal activity is mediated through changes in the probability of stochastic transitions between open and closed states of ion channels. While differences in morphology define neuronal cell types and may underlie neurological disorders, very little is known about influences of stochastic ion channel gating in neurons with complex morphology. We introduce and validate new computational tools that enable efficient generation and simulation of models containing stochastic ion channels distributed across dendritic and axonal membranes. Comparison of five morphologically distinct neuronal cell types reveals that when all simulated neurons contain identical densities of stochastic ion channels, the amplitude of stochastic membrane potential fluctuations differs between cell types and depends on sub-cellular location. For typical neurons, the amplitude of membrane potential fluctuations depends on channel kinetics as well as open probability. Using a detailed model of a hippocampal CA1 pyramidal neuron, we show that when intrinsic ion channels gate stochastically, the probability of initiation of dendritic or somatic spikes by dendritic synaptic input varies continuously between zero and one, whereas when ion channels gate deterministically, the probability is either zero or one. At physiological firing rates, stochastic gating of dendritic ion channels almost completely accounts for probabilistic somatic and dendritic spikes generated by the fully stochastic model. These results suggest that the consequences of stochastic ion channel gating differ globally between neuronal cell-types and locally between neuronal compartments. Whereas dendritic neurons are often assumed to behave deterministically, our simulations suggest that a direct consequence of stochastic gating of intrinsic ion channels is that spike output may instead be a probabilistic function of patterns of synaptic input to dendrites. The activity of neurons in the brain is mediated through changes in the probability of random transitions between open and closed states of ion channels. Since differences in morphology define distinct types of neuron and may underlie neurological disorders, it is important to understand how morphology influences the functional consequences of these random transitions. However, the complexities of neuronal morphology, together with the large number of ion channels expressed by a single neuron, have made this issue difficult to explore systematically. We introduce and validate new computational tools that enable efficient generation and simulation of models containing ion channels distributed across complex neuronal morphologies. Using these tools we demonstrate that the impact of random ion channel opening depends on neuronal morphology and ion channel kinetics. We show that in a realistic model of a neuron important for navigation and memory random gating of ion channels substantially modifies responses to synaptic input. Our results suggest a new and general perspective, whereby output from a neuron is a probabilistic rather than a fixed function of synaptic input to its dendrites. These results and new tools will contribute to the understanding of how intrinsic properties of neurons influence computations carried out within the brain. The appropriate level of physical detail required to understand how complex processes such as cognition and behavior emerge from more simple biological structures is unclear Investigation of stochastic ion channel gating using numerical simulations has been limited by trades-offs between simulation accuracy and computation time http://www.psics.org).To address the functional consequences of stochastic ion channel gating in neurons with extensive dendritic or axonal arborizations we developed a parallel stochastic ion channel simulator (PSICS), which enables efficient simulation of the electrical activity of neurons with complex morphologies and arbitrary localization of stochastic ion channels on the extracellular membrane, while also addressing limitations of previous approaches. We have also developed an interactive tool (ICING) for visualization and development of models of neurons containing uniquely located ion channels. Here, we illustrate the use of PSICS and ICING, outline the computational strategies used and provide benchmark data for evaluation. We then identify previously unappreciated differences between the effects of stochastic ion channel gating on somatic and dendritic membrane potential activity in several different morphological classes of neuron. We show that the consequences of stochastic gating depend on dendritic morphology and suggest novel functional roles for the kinetics of ion channel gating. Using a previously well-validated realistic model of a CA1 pyramidal neuron we demonstrate that stochastic ion channel gating influences spike output in response to dendritic synaptic input. We show that stochastic gating of axonal or dendritic ion channels substantially modifies synaptically driven dendritic and axonal spike output, with stochastic gating of voltage-dependent sodium and potassium channels having the greatest impact and hyperpolarization-activated channels the least. By demonstrating that neuronal responses to dendritic synaptic input can be intrinsically probabilistic, these results offer a new and general perspective on synaptic integration by central neurons. Full documentation for PSICS/ICING as well as the software, source code and examples are available from the project website (http://www.psics.org). Here, we briefly outline novel features of model specification and visualization, before describing key benchmark data and simulation experiments that evaluate the functional impact of stochastic ion channel gating in different neuronal cell types. The new software uses a simple XML file structure that enables components of a model either to be constructed manually, to be configured using a graphical interface  and exported from NEURON as a .xml file. Similar methods can be used to import models developed with Neuroconstruct (http://www.neuroconstruct.org/) To investigate the functional consequences of stochastic ion channel gating for neurons with complex dendritic or axonal morphologies, we first developed new software tools that enable accurate, fast simulation (PSICS) and visualization (ICING) of neuronal models that contain stochastically gating ion channels. The organization and development of the new software tools are described in nterface , or in tTo specify the membrane conductance we adopted a new approach in which the location of each individual ion channel is first uniquely determined . This aphttp://neuromorpho.org/). This enables components of a PSICS model to be visualized and edited. For example, to: 1) specify the size of compartments to use for the simulation, 2) select ion channels to be included in the model neuron, 3) select sections of the model for insertion of a particular ion channel class, 4) set rules that dictate the distribution of ion channels in their designated sections. The model neuron and its associated ion channels can be displayed in a variety of formats, for example to emphasize labeled sub-regions of the model  to allow display and manipulation of neuronal models with complex three-dimensional architectures and many discrete membrane ion channels . ICING rhe model , to illuhe model , or to phe model .We represent ion channels using Markov models, in which each ion channel may be in one of a number of discrete states with the probability of transition to any other state determined independently of the channel's previous history + channel model recorded with an ideal voltage-clamp  with a version in which the corresponding rate constants were 7 ms−1 and 3 ms−1 (fast gating). The model containing the slow gating channels produced membrane currents in voltage-clamp, or membrane potentials in current-clamp, that fluctuated at frequencies below approximately 15 Hz te model , whereas there were fewer “extra” somatic and dendritic spikes (p<1e-4 in both cases). Models in which only one of the other ion channel types gated stochastically differed significantly from the fully stochastic model in all measures of “extra” and “dropped” spikes (p<1e-3). Nevertheless, models containing stochastic gating voltage-dependent K+ channels generated considerably more than 50% of the number of “extra” and “dropped” spikes observed in the fully stochastic model. Interestingly, stochastic gating of Ih channels alone had very little impact on axonal spikes or spike jitter, but nevertheless increased the number of “extra” dendritic spikes. The relative lack of effect of Ih can be explained by the small single channel conductance, while the primary effect on additional dendritic spikes may reflect slow gating kinetics and dendritic localization of these channels  unless indicated otherwise. Both the PSICS shell and ICING are written in Java and can run on Windows, OS X and Linux operating systems. To minimize the time required to run simulations, the default version of the PSICS core is written in Fortran and has been compiled separately for each operating system. A slower version of the core that runs in Java is also available. The core of PSICS performs equivalent deterministic or stochastic simulations of all models. In PSICS ion channels are treated as distinct entities rather than as a conductance density, and channel gating can be simulated either stochastically or in the deterministic limit. Most of the methods involved in such calculations are well documented elsewhere All calculations were performed with PSICS . Neurons were identified in the database as follows. Layer V pyramidal cells: p18 and p22 from Dendritica; g0692P1, g0699P1 and gR002P1 from Svoboda lab. Dentate gyrus granule cells: n271, n272 and n518 from Turner lab; 428883, B106885 from Claibourne lab. Purkinje cells: alxP, e4cb3a1 and e1cb4a1 from Martone lab; p19 and p20 from Dendritica. CA1 pyramidal cells: n409 from Turner lab; NM1 from Ascoli lab; ri04 and ri06 from Spruston lab; pc4c from Gulyas lab. Substantia nigra dopaminergic neurons: Nigra2a955, Nigra11h941-1, Nigra24a953, Nigra12h945 from Dendritica. Parvalbumin interneurons: pv08e, pv22b, pv22e, pv22j and pv22m from Gulyas lab. In these simulations the densities of voltage-gated channels were based on previously published studies + and K+ channels with open probabilities of 0.7. The following channels were included: fast Na+ channels (1/µm2); non-inactivating K+ channels (0.05/µm2); high-voltage Ca2+ channels (0.15/µm2); Na+ and K+ leak channels (0.016/µm2). The resting membrane potential was set by modifying the ratio of Na+ to K+ leak channels. In all simulations reported here this was −60 mV. We chose single-channel conductances of 20 pS for all ion channels, as this is similar to values reported for single channel recordings made from neuronal dendrites 2 and axial resistivity to 150 Ω cm For simulations illustrated in Ih conductance and channel distribution taken from a different publication from the same group + and K+ leak channels were automatically adjusted to achieve a resting potential of approximately −70 mV throughout the cell, while maintaining a total leak conductance consistent with the original model. The single channel conductance of the delayed rectifier K+ channels and voltage-dependent Na+ channels were set to 20 pS, which is similar to estimates from single channel recordings + channels and leak channels were also set to 20 pS, which is similar to experimental measurements for D-type channels + and delayed rectifier K+ channels can be considered as fully constrained predictions given currently available data, while our simulations of the fully stochastic model likely estimate a lower limit for the consequences of stochastic ion channel gating. This is because our results from simulations when A/D type K+ channels are deterministic, but voltage-dependent Na+ or delayed rectifier K+ channels are stochastic, nevertheless demonstrate highly probabilistic spike firing, indicating that a smaller single channel conductance for A/D type K+ channels would have little impact on the results, while a possible larger single channel conductance for the leak channels would be expected to increase the impact of stochastic gating. Our simulations of A/D type stochastic gating alone should be considered as setting an upper limit for stochastic effects based on known properties of these channels, whereas the simulations of leak channels alone are less well constrained and serve as an illustrative example. Unlike other ion channels, the single channel conductance of Ih channels is set at 1 pS, which is consistent with noise-analysis of dendritic Ih recorded from cortical neurons Ih obtained with cell-attached recordings from CA1 pyramidal neurons 2 . Each synapse was activated independently according to a Poisson process with a mean frequency of 5.5 Hz. For analysis dendritic spike times were calculated as upward voltage crossings above a −60 mV threshold. Visual inspection of traces confirmed that this threshold successfully isolated all-or-nothing dendritic events.The simulations of a detailed model of CA1 pyramidal neuron  and 9 ushttp://www.r-project.org/). Comparisons of group data use ANOVAs. For analysis of data in www.neuron.yale.edu). For comparisons between NEURON and PSICS, simulations were run on the same hardware with minimal competing activity. Although other factors such as cache size and output options may contribute to performance differences in some cases, the simulation algorithm was the main factor determining simulation time. Parallel simulations were run on multi-processor PCs, or on a cluster of servers (maximum 1456 processors) at the Edinburgh Compute and Data Facility (ECDF).Additional analysis of simulation data was carried out using IGORpro (Wavemetrics). Statistical analysis used R Click here for additional data file.Figure S2Sodium channel model. (A) The sodium channel model used to illustrate ion channel simulation with PSICS has a single open state (O1) connected as shown to two closed states (C1 and C2). (B) The transitions between states of the model are governed by forward α and backward β rate constants that vary as a function of membrane potential (upper graph). The time constants (Taux) and steady-state distribution (Xinf) for each transition are plotted as a function of membrane potential (lower graph). (C) Deterministic currents (bottom) generated by the gating scheme in response to step changes in membrane potential from a holding potential of 80 mV (top). Inset shows the activation phase of the currents on an expanded time base.(0.25 MB JPG)Click here for additional data file.Figure S3All-or-nothing dendritic responses of a fully stochastic CA1 pyramidal neuron model to synaptic stimulation. (A–B) Membrane potentials recordings from the soma (top) and indicated basal dendrite (bottom) from twenty consecutive trials as in (0.24 MB TIF)Click here for additional data file.Figure S4+ channels (A), delayed rectifier K+ channels (B), A/D type K+ channels (C) and leak channels (D).Synaptically driven spike output is modified by stochastic gating of single types of ion channel. Raster plots as in (0.50 MB JPG)Click here for additional data file.Text S1Stochastic simulation framework; software development; estimate of the number of ion channels in a central neuron.(0.05 MB PDF)Click here for additional data file.


Nxf1 (Tap), a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that CASTNxf1 suppresses a specific and frequent class of intracisternal A particle (IAP)-induced mutations, including mh2JAp3d1, a model for Hermansky-Pudlak syndrome, and hesAtcay, an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes ∼two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of CASTNxf1 alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse  Mus musculus castaneus) suppresses most insertions of this class (six of seven tested). To our knowledge, these results make Nxf1 the most broadly interacting modifier gene yet documented in this well-studied species. These results have significant implications for manipulating gene expression in mouse models of disease, the role of Nxf1 in pre-mRNA processing and in the dynamic range for therapeutic intervention in Cayman ataxia.Retroviruses and transposable elements are molecular parasites that integrate into the host genome and require host cell machinery for gene expression, replication and dissemination. Integrating elements can alter the expression of nearby host genes through both transcriptional and post-transcriptional mechanisms. Components of the host cell machinery that can adapt to favor genetic programs of the host cell over those of the parasite may afford one level of innate immunity. In laboratory mice, endogenous retroviruses are virus-derived mobile elements that account for many spontaneous mutations. A frequent class involves retrotransposition into introns of genes in the transcriptional sense orientation, which alters host gene pre-mRNA splicing. Here we show that for the intracisternal A particle (IAP) family of endogenous retroviruses, an allele of the canonical mRNA export factor Nxf1 found in wild Asiatic mice ( Nxf1, which encodes the major mRNA nuclear export factor, can significantly suppress two mutations caused by insertions of endogenous retroviruses into introns of cellular genes by modulating their mature transcript levels ∼2 fold Nxf1 haplotype, but no other recognized gene, was able to confer the modifier phenotype. Whether this interaction could be generalized to a broader class of insertional events, and if so for what range of insertions, was limited by the relatively small number of events examined.Retroviruses and transposable elements both utilize host cell factors for their own expression and influence the expression of adjacent host genes through a variety of mechanisms. Components of host cell gene regulatory machinery that interact with molecular parasites may be regarded as components of innate immunity if they can discriminate between host and parasite expression Nxf1 haplotype shows hallmarks of recent positive selection in wild Mus musculus castaneus accessions Nxf1  was first described as a cellular factor that interacts with the Tip protein of herpesvirus saimiri Endogenous retroviruses (ERVs) are non-infectious molecular parasites that are frequent mutagens in mice. Several families of ERV are highly polymorphic among classical inbred strains and among wild accessions CASTNxf1 is effective, we examined gene expression, visible phenotypes, or both for five additional IAP, one LINE, and seven ETn insertion alleles. The host genes cover a wide range of phenotypes, expression patterns, and biochemical pathways:To test the range of insertion events for which the modifier activity of mahogany (Atrn) and mahoganoid (Mgrn) both mediate intercellular signaling by secreted agouti protein. Atrn encodes a transmembrane accessory receptor Mgrn encodes an E3 ubiquitin-protein ligase that participates in endosomal trafficking mgLAtrn and mdMgrn are de novo IAP insertions into introns in the transcriptional sense orientation The genes mutated in classical coat color mutations mocha alleles of the intracellular trafficking adapter protein gene Ap3d12Jmh) that reduces levels of wild-type RNA and protein. In addition to coat color dilution caused by sorting defects in melanosomes, 2Jmh and more severe alleles show substantial mortality, neurological and behavioral impairments mocha mice have been used to model this disease Spontaneous Jataxia mutation is an IAP insertion into an intron of the ubiquitin specific protease gene Usp14. Although the protein targets have not been systematically identified, loss of Usp14 activity results in synaptic defects that manifest behaviorally as tremor and ataxic gait in axJUsp14 mice The jittery is orthologous to the CRAL-TRIO domain gene ATCAY mutated in human Cayman ataxia hesitant mutation (hesAtcay ) is an IAP insertion into the first intron, resulting in profound locomotor deficits with no obvious neuroanatomical correlates The classical mouse locus Mitf transcription factor gene block melanocyte development, causing white-spotting and other defects in mice and Waardenburg syndrome in humans. The mouse black-eyed white allele is a L1 LINE element inserted into an intron that disrupts splicing of one alternative 5′ exon Mitf show larger and more frequent area of pigmented fur, providing a sensitive phenotypic readout for allele strength and modifier genes Mutations of the Zhx2 is a transcriptional repressor required to down-regulate expression of Afp fetal globin RNA. Loss of Zhx2 expression in BALB/cJ (but not other BALB/c lines) due to an ETn-II insertion results in persistent Afp expression into adulthood Dysf, in A/J mice results in loss of expression and creates an orthologous gene model for human limb-girdle muscular dystrophy 2B The MusD/ETn family are endogenous retroviruses that are more closely related to the IAP superfamily than most other currently active mouse retroelements CASTNxf1 suppresses six of six IAP insertions of the IΔ1 class CASTNxf1 provides a genetic rheostat for gene activity in situ.Here we show that CASTNxf1 can suppress the RNA processing defects in mgLAtrn and mdMgrn, we examined whole brain RNA of progeny from genetic crosses to CASTNxf1, comparing homozygous mutant littermates that differ in Nxf1 genotype. Because each of these crosses also segregated other loci contributing to coat color, we did not assess pigmentation phenotypes for these two mutants.To test whether Atrn . Quantitative RT-PCR across the inserted intron confirms a ∼2-fold increase in correctly-spliced transcript levels by CASTNxf1  dilution and tremor severity of mh2JAp3d1 mutant animals  IAP, while each of CASTNxf1-sensitive elements includes the 1.9 kb deletion of gag-pol sequence typical of type IΔ1 elements www.repeatmasker.org), and contain an RTE-D transport element mgLAtrn element is not otherwise an outlier in overall sequence composition, except for the undeleted region of the gag gene .Our results show that  to date . The oneCASTNxf1 would be for Nxf1 to participate in pre-mRNA processing prior to the completion of splicing. This could occur by recruitment of Nxf1 to the nascent transcript by sequences in the IAP  and subsequent interactions between Nxf1 and other components of the mRNP. Under such a model, amino acid differences (S48P and E610G) between the allelic Nxf1 proteins would alter the balance of alternative splicing either directly through interactions with splicing machinery or indirectly through an effect on transcriptional elongation rate or preference for termination site in the insertion. An alternative explanation might be for the export activity of Nxf1 to drive the nascent RNP into a territory with different relative activities for splicing and degradation, but this seems more difficult to reconcile with simultaneously increased levels of the correctly spliced message and decreased levels of the mutant splice form in five of the six suppression events.The simplest explanation for the molecular data from the six mutations suppressed by MEX67-deficient strains cluster with transcriptional elongation factors Nxf1 protein interacts with several factors that could influence alternative splicing, including U2AF35 CASTNxf1 should be able to modify other mutations that involve similar IAP insertions, for which new examples continue to be reported Pofut1 notes variable reduction of phenotype among F2 progeny in a cross to CAST/Ei, the strain from which the suppressing allele of Nxf1 was derived CASTNxf1 stock we have developed should be a useful tool to allow in situ titration of gene expression from either spontaneous or engineered alleles involving such insertions. Second, the range of titration in each of the six cases we have examined is ∼1.5 to 2-fold and semi-dominant. This holds over a fairly broad range of mutational effects on gene expression, ranging from ∼2% and 4% of wild-type levels  for hesAtcay to 50% and 75% for BOREya1. Finally, our in vivo gene titration results across six different mutations suggests that for a wide range of loci and allele strengths, even modest recovery of function may have dramatic phenotypic benefits. This is strikingly true in the case of hesAtcay, where even a 2% increment of expression has a dramatic impact on behavioral phenotype –Nxf1 and each insertional mutation were determined by custom PCR assays for each locus. Conditions for PCR of full-length insertions were optimized using a commercial kit  and primers in unique flanking sequences. DNA sequence analysis from the resulting PCR products used standard methods, as previously implemented in our laboratory http://www.ebi.ac.uk/).Genotypes for + RNA was purified by oligo(dT) cellulose chromatography. Northern blots were prepared from formaldehyde-agarose gels by capillary transfer to Hybond-N membranes and crosslinked by exposure to 2400 J UV light. Probes were prepared from cDNA fragments by random primer labeling. Hybridizations to each filter were quantified by phosphorimage analysis  and normalized to subsequent hybridization of Gapd to the same membrane as an internal control. Quantitative PCR assays were performed on total RNA. TaqMan assays for Atrn  and Atcay (Mm01172843_m1) were performed by the UCSD Center for AIDS Research Genomics Core Laboratory and normalized to a Gapd TaqMan assay. All other quantitative RT-PCR experiments were performed using intron-spanning primers that flank the inserted intron, detected by SYBR green fluorescence in a Bio-Rad CFX96 instrument, and quantified by the ΔΔCt method. Measurements were performed in triplicate for each sample. Samples to be compared were measured on the same plate during a single run. Custom primer sequences and conditions are provided as Freshly dissected tissues were homogenized in Trizol reagent (Invitrogen) and processed for RNA according to the manufacturers instructions. Poly(A)Freshly dissected tissues were homogenized in CelLytic M Cell Lysis reagent (Sigma #C2978) plus protease inhibitors and quantified using a bichromate assay . Samples were subjected to SDS-PAGE and Western blotting onto Hybond-ECL membranes. Antibodies and dilutions used were goat anti-Ap3d1 , rabbit anti-Caytaxin/BNIP  or aggregates . For variables expected to have non-normal distributions across trials  hypotheses were tested using a nonparametric Wilcoxon signed-ranks test applied to replicates of paired experimental measures. Statistical calculations were carried out in Microsoft Excel or SISA online, http://www.quantitativeskills.com/sisa/http://faculty.vassar.edu/lowry/VassarStats.html (Wilcoxon tests).Summary data are plotted in figures as mean values, with error bars indicating standard deviations. For variables with expected normal distributions, including quantitative PCR experiments and behavioral observations in which several observers rated performance against a calibrated scale, hypotheses were tested using paired or unpaired Table S1Genotype assays.(0.02 MB XLS)Click here for additional data file.Table S2qPCR assays.(0.02 MB XLS)Click here for additional data file.Table S3IAP insertions.(0.01 MB XLS)Click here for additional data file.Table S4Long PCR conditions.(0.01 MB XLS)Click here for additional data file.Video S1Usp14axJ with Nxf1B6.Video of behavioral phenotypes for (0.26 MB MOV)Click here for additional data file.Video S2Usp14axJ with Nxf1CAST.Video of behavioral phenotypes for (0.58 MB MOV)Click here for additional data file.Video S3Atcayhes with Nxf1B6.Video of behavioral phenotypes for (1.20 MB MOV)Click here for additional data file.Video S4Atcayhes with Nxf1CAST.Video of behavioral phenotypes for (0.51 MB MOV)Click here for additional data file.


Saccharomyces cerevisiae respond by activating a massive transcriptional program that reflects the fact that “DNA damaging” agents also damage other cellular macromolecules. To identify the transcriptional response that is specific to DNA damage, we have modulated the first two enzymes in the base excision repair (BER) pathway generating yeast strains with varied levels of the repair intermediates, abasic sites or strand breaks. We show that the number of abasic sites is significantly increased when the 3-methyladenine DNA glycosylase (Mag): AP endonuclease (Apn1) ratio is increased and that spontaneous frame shift mutation is considerably elevated when either Mag, or Mag plus Apn1, expression is elevated. Expression profiling identified 633 ORFs with differential expression associated with BER modulation. Analysis of transcriptional networks associated with the accumulation of DNA repair intermediates identifies an enrichment for numerous biological processes. Moreover, most of the BER-activated transcriptional response was independent of the classical yeast environmental stress response (ESR). This study highlights that DNA damage in the form of abasic sites or strand breaks resulting from BER modulation is a trigger for substantial genome-wide change and that this response is largely ESR-independent. Taken together, these results suggest that a branch point exists in the current model for DNA damage-signaled transcription in S. cerevisiae.Upon exposure to DNA damaging agents,  Previously, we have shown that yeast exposed to alkylating agents initiate a broad gene expression response modulating a staggering 30% of the yeast genome, i.e. up to 2000 genes N-glycosidic bond to initiate BER. In S. cerevisiae there are at least five distinct DNA glycosylases, namely Ung, Nth1, Nth2, Ogg1, and Mag, that collectively recognize damaged bases and process them to generate abasic sites. The abasic site is substrate for AP endonucleases that carry out the next step in BER. S. cerevisiae has two distinct AP endonucleases, the major enzyme Apn1 and a damage-inducible enzyme, Apn2 In its simplest form, the first two steps of the BER pathway are represented by a DNA glycosylase and an AP endonuclease. DNA glycosylases can recognize damaged bases and remove them via cleavage of the E. coli, yeast, mouse and human cells, treatments with xenobiotics can induce the transcription of BER genes S. cerevisiae and E. coli by imbalanced base excision repair Rev1/Rev3/Rev7 lesion bypass polymerases Across phylogeny, the regulation of enzymes involved in the first two steps of BER has been noted at the level of transcription. In S. cerevisiae produces a 2- to 5-fold increase in AP sites, depending on the Mag:Apn1 ratio, an effect that mirrors the changes in spontaneous base substitution mutation rates in the same strains of yeast In these studies, we explore how imbalanced BER affects the persistence of AP sites and frameshift mutations in yeast and how the accumulation of BER intermediates is linked to a DNA damage specific transcriptional response. We show that Mag expression in MAG), (ii) maintaining wild type levels of Mag in an Apn1 deficient strain (Δapn1), or (iii) increased expression of Mag in an Apn1 deficient strain (Δapn1/pMAG). The impact of accumulating gaps and nicks was examined by (i) expressing Apn1 (Δapn1/pAPN1), or (ii) expressing both Mag and Apn1 (Δapn1/pMAG/pAPN1)  .apn1 strain, AP sites increased ∼2-fold, an effect that was essentially reversed by expressing Apn1 from a plasmid  and (−1) frame shift mutations. We find that Apn1 expression does not affect frame shift mutation rates . HoweverMAG) showing the most transcriptional change, specifically 334 modulated ORFs , at two doses 0.05% and 0.1% To identify whether significant molecular interactions exist among the gene products of the transcripts that were modulated by BER intermediates, the 633 ORFs that were transcriptionally modulated in at least one BER condition were overlayed on a functional yeast network comprised of 4,691 nodes (proteins) and 34,000 edges (interactions) To identify transcriptionally enriched modules within the protein network, we further analyzed this large interactome for clusters of proteins that interact and also are modulated by BER intermediate accumulation; five subnetworks were identified . It shouThe current model of DNA damage-induced transcriptional response in yeast suggests that DNA damage signals for the modulation of three groups of genes; (i) the activation or repression of a battery of ∼900 genes classified as the environmental stress response (ESR), (ii) the modulation of ∼50 cell cycle associated genes, and (iii) the modulation of 9 genes termed the “DNA damage signature” It should be mentioned that contained within these BER intermediate modulated transcriptional networks are a few members of the “DNA damage signature” and transcripts that encode members of the cell-cycle related genes. Specifically, the fifth subnetwork that is enriched for DNA replication/metabolism integrates two members of the yeast “DNA damage signature,” namely the ribonucleotide reductases Rnr2 and Rnr4. The third subnetwork that is enriched for chromatin assembly/disassembly-related proteins contains two cell-cycle related transcripts that encode histones, Htb1 and Htb2, found previously to be repressed by MMS and IR Our network findings suggest that the current model of DNA damage-induced transcriptional activation in yeast should be expanded in a few ways . First, apn1 strain, AP sites were further elevated to ∼5-fold that in wild type, whereas coexpression of Apn1 with Mag in the Δapn1 strain resulted in a dramatic decrease in the steady state level of AP sites. These results complement our findings for single base substitution mutation rates in the same strains of yeast where an increase in Mag: Apn1 ratio leads to the generation of a strong mutator phenotype in yeast In this study we set out to identify the transcriptional response in yeast that is specifically caused by damage to DNA and not to other cellular molecules. As an alternative to introducing damage with reactive electrophilic agents , we generated DNA damage by imbalancing the base excision repair (BER) pathway such that abasic sites or gaps and nicks accumulate in the genome during the process of repairing endogenous DNA damage. Five yeast strains with altered expression levels of Apn1 and Mag were analyzed for the persistence of AP sites. Mag expression in the wild type host increased the steady-state level of genomic AP sites by ∼2.5 fold. As expected, when Mag was expressed in the ΔMAG) showing the most transcriptional change. It is noteworthy that even without exposure to any exogenous DNA damaging agents there is considerable gene expression change.To determine the impact of the accumulation of DNA repair intermediates on gene expression, the five yeast strains with altered expression of BER repair enzymes were analyzed for differential gene expression. A total of 633 ORFs were identified with statistically significant changes in at least one condition compared to wild type yeast. The extent of change across the yeast genome in response to modulated BER expression was quite varied across the five experiments, with yeast expressing Mag  To classify the biological processes and pathways that were affected by BER intermediate accumulation, the gene products of the transcripts that were modulated by BER intermediates, were overlayed on a functional yeast network The panel of biological processes that in this study are modulated by BER intermediate accumulation is strikingly absent of a number of cellular processes previously shown to be modulated in yeast exposed to the alkylating agent MMS To date, the proposed model of DNA damage-induced transcriptional response in yeast suggests that DNA damage signals for the modulation of ∼900 genes classified as the environmental stress response (ESR), but also the modulation of ∼50 cell cycle associated genes, and 9 genes termed the “DNA damage signature” S. cerevisiae. This DNA damage-specific expression pattern of ∼600 ORFs varies markedly from the expression pattern produced by exogenous exposure to the alkyating agent MMS. The biological processes modulated by the presence of DNA repair intermediates include protein synthesis, protein targeting, amino acid biosynthesis, and chromatin assembly. Notably lacking from the biological processes modulated by DNA repair intermediates is that of the protein degradation machinery (e.g. 26S proteasome) which is robustly induced upon exposure to exogenous DNA damaging agents; this suggests that the trigger for upregulating the proteasome emanates from damaged molecules other than DNA, most likely from damaged proteins. The DNA damage-triggered transcripts encode molecular networks that are comprised of proteins involved, not surprisingly, in DNA replication/metabolism, but also in numerous other biological processes but that the triggered responses is largely ESR-independent. As the DNA damage-specific response to BER intermediates is largely ESR independent, these data suggest that a branch point in the current model of DNA damage-induced transcriptional signaling exists. This study highlights that DNA damage, specifically, is the trigger for the modulation of transcripts that encode proteins with broad biological functions.Our results show that imbalancing the BER pathway leads to an accumulation of repair intermediates, such as AP sites, and stimulates the induction of frame shift mutations. Furthermore, we show that the induction of DNA repair intermediates, even in the absence of an exogenous damaging agent, causes a profound change in gene expression ura+GAL media at 30°C with constant shaking until cell density of 107 cells/ml. They were then pelleted by centrifugation, quick-frozen using a dry ice/ethanol bath, and stored at −80°C for use in the assays described below. All experiments were performed in biological triplicate.DNA was extracted by a procedure slightly modified from the method reported previously Abasic (AP) sites were measured based on a procedure reported by Nakamura and Swenberg apn1 null E133 and E134 yeast strains InsE inserts inactivate the LYS2 gene with 12 and 14 nucleotide runs of A, respectively, resulting in a Lys− phenotype. E133 reverts to Lys+ solely by a +1 frameshift, whereas E134 reverts by a −1 frameshift mutation. S. cerevisiae 3MeA DNA glycosylase (MAG) was expressed in yeast when galactose, at final concentration of 2%, was added to SC drop-out media lacking uracil . The S. cerevisiae AP endonuclease 1 (APN1) was expressed from the YEpAPN1 plasmid maintained in culture by excluding leucine from the SC drop-out media. Transformant cultures were grown overnight in 2% synthetic glucose media without uracil for pYES2.0 and pYES-MAG or without uracil and leucine when YEpAPN1 was additionally included in combination with the above plasmids. When cultures reached saturation, they were diluted to a density of ∼4,000 cells/ml in 2% galactose SC media and divided into ten parallel 10 ml cultures. After incubation at 30°C for ∼4 days, cells were harvested, washed, and resuspended in 1 ml water. Dilutions were spread on SC–uracil or SC–uracil–leucine plates, respectively, to determine the average number of cells per culture for three randomly chosen samples per each condition. Duplicates of 0.1 ml aliquots of each culture were spread on SC +galactose plates lacking lysine to select for Lys+ revertants. After ∼4–5 day incubation at 30°C, colonies were counted. The median number of revertants per 108 cells was obtained and is expressed as mean±standard deviation from at least 3 independent experiments.The plasmids used in the study were described previously www.ncbi.nlm.nih.gov/geo/).Total RNA was isolated using hot phenol extraction and hybridized to Affymetrix®  Yeast Genome S98 arrays in biological triplicate. RNA clean-up, cRNA labeling and fragmentation, array hybridization and staining were done as described in the Affymetrix® eukaryotic labeling protocol . Processed microarrays were scanned and raw image intensity data files were processed and normalized using robust multi-array average (RMA) software Network analysis was carried out using the Cytoscape software Figure S1S. cerevisiae. A) The transcripts modulated under BER intermediate accumulation were integrated with their gene products and overlayed on all known protein-protein interactions in yeast . Of the BER modulated ORFs, 501 ORFs were identified in the database of known protein-protein interactions in the large interacting network (indicated in red). B) Analysis requiring that all interacting nodes are transcriptionally modulated by BER intermediate accumulation results in the identification of five subnetworks (1–5) significantly enriched for GO biological processes.BER modulated transcriptional networks in (7.69 MB EPS)Click here for additional data file.Dataset S1List of 633 ORFs modulated by BER imbalance. ORFs modulated in at least one condition of base excision repair imbalance in yeast were identified using differential expression testing.(0.21 MB XLS)Click here for additional data file.


Bleeding from pancreatic pseudocyst’s rupture into adjacent organs is a rare, but potentially fatal, complication of chronic pancreatitis requiring quick management. Timing of the rupture is unpredictable; early diagnosis and correct management is essential in preventing the bleeding.We describe the case of a 53 years old male patient successfully treated with emergency surgery for massive hematemesis due to a rupture of a bleeding pseudocyst into the stomach. Patient underwent emergency laparotomy and suture of the bleeding vessel. At 5 years follow-up patient is in healthy condition.This case shows to surgeons that pancreatic pseudocyst cannot be managed strictly with one rule and prompt surgical treatment is mandatory in case of haemodinamic instability. Pancreatic pseudocyst is a common finding in patients with previous acute or chronic pancreatitis, developing respectively in up to 15% and 40% of these groups of patients -4.This report detail a case of sudden haemorragic complication of pancreatic pseudocyst and its emergency surgical management.A 53-years Italian, white Caucasian, old male former alcoholic (stop drinking since 11 years) with known chronic pancreatitis came at our Department for mild abdominal pain, anorexia and bowel obstruction. Physical examination was unremarkable except for a mesogastric tenderness. Laboratory findings revealed an increase value of Amilase (148 mg/dl), with slightly increase levels of bilirubin (2.1 mg/dl) and transaminase . A CT scan revealed a 10 × 10 × 7 cm tail cystic esofitic lesion of the pancreatic tail in a pattern of chronic pancreatitis . PatientEcographic evaluation coupled with CT scan did not diagnose any fluid collection within the abdomen. A CT scan performed 4 weeks from the first diagnosis of acute on chronic pancreatitis, the day before hospital discharge, revealed a reduction of diameters (10 × 10 × 10 cm) and disappearing of infarcted areas within the spleen if compared with the previous one. Few hours after the examination patient presented hypotension, sweating, diaphoresis, massive hematemesis and subsequent haemorrhagic shock.Emergency laparotomy was performed. Surgical exploration of the pseudocyst was performed through an anterior gastrotomy, revealing a bleeding coming directly from the main trunk of the splenic artery; after adequate surgical vessel isolation the defect was sutured by means of not reabsorbable stitches (4/0 prolene).Post-operative course was uneventful. A color Doppler flow of splancic vessels performed 2 weeks after surgery demonstrated normal flow within all arteries with little reduced flow in part of the branches at the splenic hilum. CT scan examination revealed splenic diffuse hypodensity due to reduced blood flow . PatientPancreatic pseudocysts are common conditions following both acute and chronic pancreatitis or pancreatic trauma . DiffereThis case reminds to surgeons that pancreatic pseudocyst cannot be managed strictly with one rule , but str