Atherosclerosis is the leading cause of cardiovascular disease (CVD). Traditional risk factors can be used to identify individuals at high risk for developing CVD and are generally associated with the extent of atherosclerosis; however, substantial numbers of individuals at low or intermediate risk still develop atherosclerosis.A case-control study was performed using microarray gene expression profiling of peripheral blood from 119 healthy women in the Multi-Ethnic Study of Atherosclerosis cohort aged 50 or above. All participants had low (<10%) to intermediate (10% to 20%) predicted Framingham risk; cases (N = 48) had coronary artery calcium (CAC) score >100 and carotid intima-media thickness (IMT) >1.0 mm, whereas controls (N = 71) had CAC<10 and IMT <0.65 mm. We identified two major expression profiles significantly associated with significant atherosclerosis ; among those with Framingham risk score <10%, the odds ratio was 5.30 (P<0.001). Ontology analysis of the gene signature reveals activation of a major innate immune pathway, toll-like receptors and IL-1R signaling, in individuals with significant atherosclerosis.Gene expression profiles of peripheral blood may be a useful tool to identify individuals with significant burden of atherosclerosis, even among those with low predicted risk by clinical factors. Furthermore, our data suggest an intimate connection between atherosclerosis and the innate immune system and inflammation via TLR signaling in lower risk individuals. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the United States and the developed world, and it will soon be the leading cause in the developing world. Atherosclerosis accounts for the vast majority of fatal and non-fatal CVD events. Multivariable (MV) risk equations Atherosclerosis is a disease of inflammation characterized by interactions among platelets, leukocytes, and endothelial cells In the present study, we performed microarray gene expression profiling of whole blood from a population of healthy women with low to intermediate FRS to test whether gene expression profiles could distinguish those with and without significant atherosclerosis. We further sought to identify genes and pathways associated with significant burden of atherosclerosis among these women predicted to be at lower risk for CVD events.Among all 119 MESA women, 48 had significant subclinical atherosclerosis (cases) and 71 had no evident subclinical atherosclerosis (controls); among those with FRS<10%, there were 39 cases and 69 controls. The characteristics of all 119 participants at the fourth examination with and without evident subclinical atherosclerosis are shown in The gene expression patterns differ greatly between cases and controls. The sample dendrogram generated by hierarchical clustering shows that the samples separate into two major branches indicated by BL and BR . The larThe distribution of the 2,057 P-values  using t The multiple random validation procedure using top 50–60 genes with smallest p-values identified two major gene expression profiling (or molecular) subtypes with an odds ratio of 4.85  for the association with atherosclerosis see  and S3. A sensitivity analysis was further performed by excluding those on lipid lowering medication, leaving 25 cases and 61 controls for comparison. The multiple random validation procedure on this subset of samples resulted in an association similar to the results from the entire 119 samples (data not shown). Similarly, the association did not change substantially by excluding smokers or those taking medications for hypertension.In order to better characterize pathways associated with the AWA profile, we performed ontology analysis on the top 344 probes representing 325 unique genes that had an FDR <0.1% from the comparison between the two molecular profiles. Information on these 325 genes is provided in −5 and P<10−15). More importantly, a significant concordance of fold changes was observed between both studies (−15).To further validate the observation that TLR pathway is activated in AWA profile, we compared our gene expression data with Calvano’s study studies , with a −8∼1.8•10−15, see Quantitative real-time RT-PCR was performed to confirm the RNA expression levels of eight selected genes involved in various functions including lipid metabolism, immune and inflammatory response, apoptosis, cell proliferation, and hypoxia signaling . All eight genes showed high correlation . The left branch consisted of all eight men with high CAC and two men with low CAC, while the right branch consisted of six men, and all had CAC<1. Of note, the two men with low CAC on the left branch had the highest CAC scores (1.2 and 0.9) among the eight men with low-CACs. Because CAC score is highly correlated with the sums of histological artery plaque areasWe further characterized the association of atherosclerosis with the AIIG signature described above, employing peripheral blood from 16 white men aged 65–80. The clinical characteristics of these 16 men are shown in Atherosclerosis is the main cause of clinical CVD events. Inflammation plays a central role at all stages of this disease Prior studies have demonstrated the increased expression of TLR2 and TLR4 on monocytes from patients with angina and acute coronary syndrome Experimental studies have consistently demonstrated the significant role of innate immunity in atherogenesis. Genetic deletion of TLR4 or TLR2 resulted in significant reduction of arterial plaques in atherosclerosis-prone apoE or LDLR deficient mice T cell activation has been implicated in the pathogenesis of atherosclerosis. T cells are enriched in atherosclerotic lesions Recently, two microarray gene expression studies The findings from the present study have several potential clinical implications. Higher burden of atherosclerosis, as measured by CAC or carotid IMT, is associated with significantly greater risk for CVD events in the short term, independent of traditional risk factors Some limitations of the current study should be acknowledged. First, because of the cross-sectional nature of our study, the causal relationship of TLR signaling with atherosclerosis cannot be established in the present data. Second, our study only included 119 women aged 50∼86 and 16 men aged 65∼80. It is unknown whether our gene signature is applicable to identify atherosclerosis among younger adults. Larger and perspective studies are warranted to examine expression alterations of members of TLR and IL-1R signaling pathways in association with developing subclinical atherosclerosis. Nonetheless, our translational finding is unique in revealing an AIIG signature that was associated with significant atherosclerotic burden, even among a group predicted to be at low risk for atherosclerosis and clinical CVD.We used global gene expression profiles of whole blood to systematically examine the dynamic transcriptional alterations associated with atherosclerosis among women with low to intermediate predicted risk for the disease. Our results suggest the involvement of the innate immune system in atherogenesis and that whole blood provides an accessible and informative source of transcriptomal information regarding the inflammatory status of atherosclerosis in the preclinical phase of the diseases. Because of the easy access of blood samples, expression profiles of whole blood may be a useful tool to assess the burden of atherosclerosis and could potentially be used to enhance the risk prediction of CVD beyond the traditional risk factors. Future studies will be needed to validate our findings.The Institutional Review Boards at all Multi-Ethnic Study of Atherosclerosis (MESA) sites  as well as the Chicago Health Aging Study (CHAS) center (Northwestern University) approved the study, and all participants gave written informed consent.A subset of 119 study participants was selected from the MESA cohort. MESA is designed to study the prevalence, risk factors, and progression of subclinical cardiovascular disease in a multiethnic cohort. A detailed description of the study design and methods has been published previously and common carotid intima-media thickness (CC-IMT) ≥1.0 mm. Controls were selected as having a CAC score ≤10 and CC-IMT ≤0.65 mm. Additionally, both cases and controls were selected from those without diabetes or history of CVD who had an FRS less than 10% at baseline examination (2000∼2002). Among all MESA women, only 48 women agreed to participate and met the selection criteria as cases with significant atherosclerosis burden despite low predicted risk for CHD. We also selected potential control participants from among MESA women based on similar age and race –71 women were identified as controls and agreed to participate, giving us a ratio of 1.5 controls for each case. Out of these 119 participants, the FRS of 11 participants had increased from 11% to 20% when the MESA data for the fourth examination  became available. Carotid IMT was measured for all MESA participants only at the baseline examination. Measurement of CAC was performed for all MESA participants at the baseline examination. Follow-up testing for CAC was performed in two stages: 50% of participants returned from September 2002 to January 2004 (examination 2), and the remainder returned from March 2004 to July 2005 (examination 3). Therefore, we used the CAC score from examination 2 or 3 for these 119 participants. All these 119 participants were called back during April-July 2007 to have 5 ml whole blood drawn into two PAXgene tubes for RNA isolation.In our case-control study, cases were selected as having a coronary artery calcium (CAC) score ≥100 PAXgene blood samples of additional 16 white men without diabetes or history of CVD from the CHAS cohort were selected for gene expression profiling. The blood samples of these men were collected during their clinical visits between April 2009 and July 2009. Because IMT data were not available in CHAS, cases and controls were identified based on their CAC scores. Eight of these 16 men had CAC scores 446∼1,206. The other eight had CAC scores <2. The Institutional Review Boards at each MESA site as well as the CHAS center  approved the study, and all participants gave informed consent.Scanning centers assessed CAC by chest-computed tomography using either a cardiac-gated electron-beam computed tomography scanner at three MESA field centers or a multi-detector computed tomography system at the other three MESA field centers. Each participant was scanned twice, and these scans were read independently at a central reading center. The amount of calcium was quantified with the Agatston scoring method. The average Agatston score was used for data analysis. Carotid IMT was measured using high-resolution B-mode ultrasonography of the right and left near and far walls of the internal carotid and common carotid arteries. Images were acquired bilaterally for both the common and internal carotid arteries. The CHAS used a similar method to assess CAC. The methodology for acquisition and interpretation of the scans has been documented previously Five ml whole blood from each participant was drawn into two PAXgene tubes and incubated at room temperature for three hours before being frozen at −70°C. RNAs were extracted using the PAXgene blood RNA extraction kit according to the manufacture’s protocol. The concentration of the extracted total RNA and their quality were measured by NanoDrop (Thermofisher) and Bioanalzyer 2100 (Agilent), respectively. Total RNA preparations with 260/280 ratio between 1.98–2.22 and RIN number >7.4 with enough quantity were used for the expression analysis. Globin reduction was performed using the Ambion GLOBIN clear kit. The quality of the globin-reduced RNA samples was assessed by Agilent 2100 Bioanalyzer. High quality samples were used to make first and second strand DNA followed by an IVT reaction. The size distribution of the resulting biotin labeled cRNA and the yield was checked by Agilent 2100 Bioanalyzer and NanoDrop, respectively. A normalized amount of labeled cRNA was hybridized) for 18 hours at 55°C to the Sentrix Human Ref-8 v2 Expression BeadChip for the 119 MESA samples and HumanHT-12 V4 Expression BeadChip for the 16 CHAS samples . After washing and staining with Cy3, the chips were scanned on the Illumina iScan. The Illumina Human Ref-8 v2 BeadChip allows genome-wide expression profiling of more than 22,000 transcripts of genes and known alternative splice variants from the RefSeq database while the HumanHT-12 V4 Expression BeadChip targets more than 47,000 transcripts of genes, gene candidates, and splice variants. The microarray analysis was performed at the Northwestern Genomic Core Facility at the Center for Genetic Medicine. All the 119 MESA RNA samples were run on 5 different days within a week. Samples collected from six MESA field centers were randomly assigned to different chips and days. All case and control samples in each MESA field center were also randomly assigned to different chips and days. The 16 CHAS RNA samples were also randomly assigned to two different chips and run on the same day.T) method.RNA levels of eight selected genes were analyzed by quantitative real time RT-PCR using the TaqMan chemistry on the 7300 Real-Time PCR System from Life Technologies. GAPDH was used as the endogenous control. Relative expression levels of these genes were determined by the delta-delta threshold cycle (DDChttp://rana.lbl.gov/EisenSoftware.htm) using Pearson correlation as a distance metric. Significance analysis of microarray (SAM) http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE20129.BeadStudio software was used to translate the scanned images into expression data, which were further log transformed and normalized by the quantile normalization procedure using the Bioconductor package: affy. Gene expression data from microarray experiments are known to have substantial background noise to signal ratios for genes expressed at the lower levels. To reduce the chance of identifying such genes, genes with a coefficient of variation less than 0.03 across all 119 MESA samples or a P value for expression detection call less than 0.01 for 60 samples or fewer were excluded, resulting in 2,057 probes for data analysis. Average linkage hierarchical clustering was performed for these 2,057 probes in the 119 MESA samples using Cluster 3.0 software .(TIF)Click here for additional data file.Figure S2Association of atherosclerosis with gene expression profiles of peripheral blood. The optimal association from the multiple random validation procedure was achieved using a set of 50–60 genes, resulting in an odds ratio of 4.85. When only the 108 women with FRS<10% were considered, the odds ratio was 5.3. SVM: support vector machine. FRS: Framingham risk score.(TIF)Click here for additional data file.Figure S3Multiple random validation using 50–60 genes identified 2 major gene expression molecular subtypes: 29 true positives (cases) + 17 false positives (controls); 54 true negatives (controls) + 19 false negatives (cases). Distribution of 2,057 P-values from t tests for comparisons between (a) true positives  vs false negatives  ; (b) true negatives  vs false positives ; (c) TP vs FP; (d) TN vs FN. A large proportion of genes (45%) were estimated to be differentially expressed between the TPs and the FNs. Similar result was observed between the TNs and the FPs. On the other hand, no differentially expressed genes can be identified between the TPs and the FPs or between the TNs and the FNs.(TIF)Click here for additional data file.Table S1344 atherosclerosis-associated probes.(DOC)Click here for additional data file.Table S2Selected enriched pathways and functional categories.(DOC)Click here for additional data file.Table S3Correlation of gene expression between microarray and RT-PCR experiments.(DOC)Click here for additional data file.Table S4Characteristics of 16 CHAS non-diabetic white men with low or high coronary artery calcium (CAC) score.(DOC)Click here for additional data file.


Orbicella annularis, formerly of the genus MontastraeaAurantimonas coralicida, which has been described as the causative agent of white plague type II. The microarrays revealed a large amount of bacterial heterogeneity within the healthy corals and less diversity in the diseased corals. Gram-positive bacterial groups  comprised a greater proportion of the operational taxonomic units (OTUs) unique to healthy samples. Diseased samples were enriched in OTUs from the families Corynebacteriaceae, Lachnospiraceae, Rhodobacteraceae, and Streptococcaceae. Much previous coral disease work has used clone libraries, which seem to be methodologically biased toward recovery of Gram-negative bacterial sequences and may therefore have missed the importance of Gram-positive groups. The PhyloChip™data presented here provide a broader characterization of the bacterial community changes that occur within Orbicella annularis during the shift from a healthy to diseased state.Coral disease is a global problem. Diseases are typically named or described based on macroscopic changes, but broad signs of coral distress such as tissue loss or discoloration are unlikely to be specific to a particular pathogen. For example, there appear to be multiple diseases that manifest the rapid tissue loss that characterizes ‘white plague.’ PhyloChip™ G3 microarrays were used to compare the bacterial community composition of both healthy and white plague-like diseased corals. Samples of lobed star coral ( Coral disease is now recognized as one of the major causes of reef degradation and coral mortality. The Caribbean has been particularly hard hit, with disease mortality reducing coral cover by an order of magnitude in some areas White plague is a perfect example. This disease is visibly ‘identified’ by a sharp boundary between pigmented tissue and bare skeleton that is bright white due to recent tissue loss Aurantimonas coralicida, for WPII in the Caribbean Thalassomonas loyana for white plague-like disease in the Red Sea Orbicella annularis  and DRTO-2009-SCI-0018 (study DRTO-00074), granted to the first author by the Virgin Islands National Park and Dry Tortugas National Park, respectively. No ethical approval was required for the experimental research described here.Orbicella annularis  samples were collected in two National Parks during the summer of 2009. Five healthy and six diseased colonies were sampled at Tektite Reef in Great Lameshur Bay , St. John, Virgin Islands National Park (VIIS) on July 16–17  5′-GGTTACCTTGTTACGACTT-3′) 2, 300 nM each primer (27F and 1492R), 200 µM each dNTP, 25 µg bovine serum albumin and 0.625 units of Ex Taq polymerase. The reactions were amplified using an iCycler and under the following thermocycling conditions: 95°C for 3 min for initial denaturation, 30 cycles of 95°C for 30 sec, 48–58°C for 30 sec, and 72°C for 2 min, and then final extension for 10 min at 72°C. PCR products from each annealing temperature for a sample were combined and concentrated using Amicon Ultra-0.5, Ultracel-30 Membrane, 30 kDa centrifugal filter units and quantified before application to the PhyloChip™. The concentrated 1 µl PCR product was quantified on a 2% agarose E-gel using the Low Range Quantitative DNA Ladder as reference. In some cases the primers amplified both coral and bacterial DNA Amplification was carried out as previously described th percentile was used as the summary intensity for the feature. Array fluorescence intensities could range from 1 to 65,536. The hybridization score  for an OTU was calculated as the mean intensity of the perfectly matching probes exclusive of the maximum and minimum. HybScore data obtained from the CEL files  were scaled by setting the spike-mix control intensity to 10,000 to correct for slight differences in scan intensities or potential uneven hybridization efficiencies across microarrays.Details about probe selection, probe scoring, data acquisition and preliminary data analysis are according to Hazen et al. http://greengenes.lbl.gov/Download/Microarray_Data/Orbicella_Kellogg/). Intensity values were log2 transformed before hybridization data were further analyzed.An updated taxonomy PRIMER 6 version 6.1.13 software A custom PERL script was created  is the representation of many coral-associated sequences in the additional OTUs. Through keyword searches followed by manual screening of the results, we determined that there are 545 total OTUs on the PhyloChip™ G3 that include at least one coral-associated sequence . The bul samples , only 38 samples . These w samples .Given such high representation of coral-associated sequences in the subset of OTUs unique to diseased samples, we reran the NMDS plot using only the 313 coral-associated OTUs that had been identified within the total dataset of 13,441 OTUs . The conA large number of taxa are shared by healthy and diseased samples at all phylogenetic levels , but somWhen comparing entire bacterial communities, the white plague-like disease samples all clustered together and there was no significant difference between the two sampling locations  to generate the OTUs Using presence/absence of OTUs in our PhyloChip™results, we constructed non-metric multidimensional scaling plots that showed strong clustering of all the white plague-like samples, regardless of collection site . A PhyloMany studies, including the two prior studies of coral disease employing PhyloChips™ Micrococcus, 81 for Bacillus, and 9 for Vibrio. This matches well with Bacillus have been shown to fix nitrogen, while others may stimulate nitrogen-fixing activities of other bacteria Bacillus sp. in healthy corals that is disrupted by the transition to a diseased state. Recent studies of white plague-like disease in other coral species give conflicting results in terms of Vibrios: a lower number of Vibrionales sequences was seen in diseased Diploria strigosa and Siderastrea siderea (both Caribbean species) Pavona duerdeni and Porites lutea (Pacific species) Vibrio species have been implicated in several coral diseases Out of the 13,441 OTUs present across all samples, 106 OTUs were unique to healthy samples and present in more than half of the healthy replicates . Of noteDesulfovibrio, providing some evidence for the ‘atypical black band disease’ that Ainesworth et al. There were eight families that occurred in diseased corals but were not present in any of the healthy samples : BSV26/SOrbicella faveolata by Sunagawa et al. Diploria strigosa and Siderastrea siderea compared to healthy Rhodobacter spp. to be associated with several coral diseases, not just white plague. This seems to corroborate the suggestion by Sunagawa et al. There is also noticeable representation of the Rhodobacteraceae family 8 OTUs; ; this faAcropora when surrounded by an outbreak of white plague-like disease in other coral species In the recent PhyloChip™ G3 comparison of healthy and white plague-like diseased Indo-Pacific corals, several of the bacterial families that showed higher abundance in diseased corals were Clostridiaceae, Corynebacteriaceae, Lachnospiraceae, and Ruminococcaceae Two recent coral disease studies Aurantimonas coralicida has been described as the causative agent of white plague type II (WPII) A. coralicida sequence. Using the second generation (G2) PhyloChip™and clone libraries, Sunagawa et al. A. coralicida in either healthy or white plague-affected Orbicella faveolata. More recent work using the PhyloChip™ G3 and clone libraries to examine a white plague-like disease in Pavona duerdeni and Porites lutea also did not find any indication of A. coralicida. Similarly, previous work on O. annularis using clone libraries failed to detect A. coralicidaA. coralicida using specific PCR primers (on other Caribbean species Diploria strigosa and Siderastrea siderea) also failed A. coralicida and other bacteria with a high percentage of GC bases in their DNA, such that they are not detected in a mixed community even when present O. annularisThe PhyloChip™ G3 microarray provides a broader taxonomic overview than can be achieved by a traditionally sized clone library and also provides data that can be analyzed without a steep learning curve of bioinformatics, such as is required to handle pyrosequencing datasets. Our results show no evidence for geographically distinct disease states of coral-associated microbiota or for a single causative agent of this white plague-like disease. The higher taxonomic diversity observed in the healthy samples is likely indicative of the natural spatial heterogeneity that has been documented in coral-associated bacterial communities on Table S1PhyloChip™ G3 operational taxonomic units (OTUs) that contain one or more coral-associated sequences.(XLS)Click here for additional data file.File S1OTU sorting program, QueryOTU.pl, written in Perl.(PL)Click here for additional data file.File S2Comma-delineated OTU table provided as input for the QueryOTU.pl program.(CSV)Click here for additional data file.File S3Detailed example for using QueryOTU.pl program to sort PhyloChip™ G3 OTUs.(DOC)Click here for additional data file.


Overall, we evaluated 4,926 CNVs in 2,373 ASD subjects from 48 scientific reports, encompassing ∼2.12×109 bp of genomic data. Remarkable variation was seen in CNV size, with duplications being significantly larger than deletions, . Examination of the CNV burden across the genome revealed 11 loci with a significant excess of CNVs among ASD subjects (P<7×10−7). Altogether, these loci covered 15,610 kb of the genome and contained 166 genes. Remarkable variation was seen both in locus size (20 - 4950 kb), and gene content, with seven multigenic (≥3 genes) and four monogenic loci. CNV data from control populations was used to further refine the boundaries of these ASD susceptibility loci. Interestingly, our analysis indicates that 15q11.2-13.3, a genomic region prone to chromosomal rearrangements of various sizes, contains three distinct ASD susceptibility CNV loci that vary in their genomic boundaries, CNV types, inheritance patterns, and overlap with CNVs from control populations. In summary, our analysis of AutDB CNV data provides valuable insights into the genomic characteristics of ASD susceptibility CNV loci and could therefore be utilized in various clinical settings and facilitate future genetic research of this disorder.Copy number variants (CNVs) are thought to play an important role in the predisposition to autism spectrum disorder (ASD). However, their relatively low frequency and widespread genomic distribution complicates their accurate characterization and utilization for clinical genetics purposes. Here we present a comprehensive analysis of multi-study, genome-wide CNV data from AutDB ( Copy number variations (CNVs) are structural chromosomal aberrations, which are giving rise to gains or losses of certain genomic loci across the human genome Autism spectrum disorder (ASD) constitutes a collection of clinically heterogeneous disorders that are characterized by impairments in social interactions, deficits in language and communication, and increased repetitive or stereotypic movements http://www.mindspec.org/autdb.html) which, to the best of our knowledge, is the most comprehensive online resource of curated genetic data of ASD from published scientific reports. Using a range of statistical and bioinformatics analyses we performed a comprehensive and rigorous assessment of CNVs that were observed in ASD cohorts across multiple published reports consisting of both large-scale whole-genome studies and smaller-scale case studies. We subsequently determined the genomic boundaries and genetic characteristics of 11 loci demonstrating significant CNV burden among ASD subjects.Given the important role of CNVs in ASD genetics and the increasing usage of CMA screening for the genetic evaluation of ASD individuals, there is a tremendous need to consolidate the large amounts of CNV data that were generated from ASD subjects and subsequently prioritize the most consistent genomic loci associated with ASD susceptibility. To this end, we explored the CNV data available at the online autism genetic database AutDB (http://www.ncbi.nlm.nih.gov/pubmed/) with the following keywords: “autism/autistic/ASD” and “copy number/CNV/deletion/duplication/chromosome/structural variant”. Furthermore, CNV reports listed in ASD review articles that were not identified in the initial PubMed search were included for consideration. Next, the initial candidate CNV reports were filtered to remove those reports that did not contain at least one ASD individual in which one or more CNVS were identified. This restriction has since been relaxed to include reports describing patients with other neurodevelopmental or neuropsychiatric disorders, such as mental retardation/intellectual disability and developmental delay; in some cases, these patients also display ASD traits, but no formal diagnosis of ASD. Detailed information on ASD and control subjects from the studied cohorts was extracted from each selected CNV report for inclusion in the CNV module database.For this study, we used CNV data available at the CNV module of the AutDB database (data freeze of October 2011)et. al study For the purpose of CNV prioritization we aimed at analyzing a homogeneous subset of the CNV data set from AutDB by using several filtering criteria (number of CNVs removed from the data are in square brackets): only ASD cases were used ; patients with a disease other than ASD, such as schizophrenia, developmental delay, etc. were excluded [74 CNVs]; a genome-wide CNV discovery method  was required for inclusion; CNVs discovered by a targeted discovery method such as FISH or qPCR were excluded [215 CNVs]; we manually screened individuals to ensure minimal overlap of patients; in some cases a given CNV report may include individuals that had previously been described in another report [104 CNVs]. In such cases, we used patient ID information included in the scientific report to identify duplicate patient entries in the CNV module dataset and subsequently use the CNV data for a given individual that contained the largest number of CNV loci [104 CNVs]. Furthermore, we restricted our analysis to CNVs with defined start and end points [218 CNVs]. Finally, to maintain a uniform CNV/individual ratio in our data, we used CNVs identified in a control cohort of unaffected matched siblings as a filter to generate a set of “case-specific” CNVs in the accompanying case cohort of the Sanders To search for genomic loci demonstrating excess of CNVs among ASD subjects, we divided the genome into consecutively distributed regions of 10 kb, and evaluated the CNV burden in each region as follows: i,jC  =  1 if a CNV (i) from a particular study (j) of all CNVs (N) in the data, overlaps with the 10 kb genomic region, and ,jW is the weight associated with the study (j) calculated as the Loci/CNVs ratio in the study.where 4. Hence, we used this number to set a Bonferroni corrected cutoff for genome-wide significance of P<7×10−7 (0.05/7×104). All analyses were performed using a commercial software package .To assess the statistical significance of θ, we first randomly distributed the CNVs in our dataset in the human genome and then calculated their corresponding 10 kb θs. We repeated this procedure 10,000 times to generate a null genome-wide distribution of θs that fitted a Poisson distribution with λ  =  0.8363. We then used this Poisson distribution to calculate the statistical significance of θ associated with each 10 kb region among individual with ASD in our data. Given the median CNV length in our data was ∼ 43 kb, the maximal number of non-overlapping CNV loci in the human genome is ∼ 7×10http://genome.ucsc.edu) to locate genes overlapping with ASD susceptibility CNV loci. Further, we used the December 2012 release of the Human Gene module of AutDB We used the RefSeq genes track in the UCSC Genome Browser on Human Mar. 2006 (NCBI36/hg18) assembly,  using data from three genome-wide CNV analyses varying in their sample sizes, studied cohorts, and CNV detection method/platform The CNV module of AutDB as of October 2011 consisted of CNV data from 72 annotated publications, encompassing 30,989 CNVs from 4,359 individuals . These have been summarized into 2,429 unique CNV reports classified as 'major'  or ‘minor' , and distributed across 1035 CNV loci genome-wide . The med9 bp of genomic data. The maximal number of CNVs per individuals seen in our data was 18. Of the 2,373 ASD subjects in our data, 1,532 were males and 330 females, which is consistent with the 4:1 reported male-to-female ratio of ASD prevalence in the general population de novo, and 1,310 (26.6%) had no indicated inheritance .For the purpose of CNV prioritization, we applied a set of stringent filtering criteria (see methods) to the AutDB data to generate a uniform subset of CNVs. This resulted in a dataset containing 4,926 CNVs in 2,373 ASD subjects  collected from 48 scientific reports  that enceritance . We obseP<7×10−7) distributing along eight chromosomes , one locus contained three genes  and six loci contained ≥ seven genes . The genomic distribution of CNV burden among individuals with ASD is depicted in omosomes ; Table 2P <1×10−20), with 27 duplications and 36 deletions identified in ASD subjects from 10 different studies  have been reported as containing susceptibility genetic variants in ASD individuals de novo in origin  have already been associated with ASD. The locus on 22q13.32-q13.33 was primarily enriched in deletions among individuals with ASD (nine deletions vs. three duplications), and did not include CNVs in controls  prioritize ASD susceptibility loci based on their CNV burden among ASD individuals, and (B) determine the genomic and genetic characteristics of these loci. The CNV dataset used for our analysis was curated from published scientific reports and primarily included CNVs that have been exclusively seen among individual with ASD in each of these studies. Yet, we compared these data with a control CNV dataset derived from three genome-wide CNV reports SEZ6L2, MAPK3, and KCTD13) have been independently identified as ASD genetic risk factors KCTD13, led to decreased proliferation of neural progenitor cells and reduced head size, mirroring the microcephaly phenotype commonly seen in cases with 16p11.2 duplication. Alternatively, suppression of KCTD13 led to increased neural progenitor cell proliferation and increased head size, mirroring the macrocephaly phenotype observed in many cases with 16p11.2 deletion. Likewise, another gene within the 16p11.2 locus, TAOK2, was recently shown to influence the formation of basal dendrites in the developing cortex The highest CNV burden in our study was seen in the 16p11.2 locus. This genomic locus have long been known as, a genetic risk factor for ASD Our analysis implicated three distinct ASD loci within the 15q11-q13 genomic locus. Duplications within the 15q11.2-q13.1 region, located between chromosomal breakpoints (BPs) BP2-BP3, have long been strongly implicated in ASD pathogenesis DOCK8 gene overlaps with linkage regions identified in large autism extended pedigrees DOCK8 has previously been implicated in two cases of intellectual disability and developmental delay DOCK8 in ASD, these previous results and our findings argue for a potentially critical role for this gene in ASD susceptibility. The BCL9 gene resides within a ∼1.5–Mb genomic region of the 1q21.1 locus in which both deletions and duplications can result in syndromes associated with numerous phenotypes, including autism BCL9 gene are associated with schizophrenia, bipolar disorder, and major depressive disorder BCL9 is a component of the canonical Wnt signaling pathway, which has been proposed to be affected in ASD BCL9 gene confers genetic risk to ASD susceptibility, our analysis in combination with previous findings strongly implies a potential pathogenic role for this gene. Duplications of the 3q26.31 locus within the NLGN1 gene were also seen among control individuals, thus questioning the association of this locus with ASD susceptibility NLGN1 to ASD is supported by a recent report identified a duplication of the NLGN1 gene in an autistic patient with mild intellectual disability KPNA3 gene at the 13q14.3 locus, has also been implicated as a potential schizophrenia susceptibility gene KPNA3 to these two related psychiatric disorders.Four monogenic genomic loci were identified in our analysis that merit further investigation. The 9p24.3 locus, which contains the The incorporation of CNV data from both large-scale whole-genome studies and smaller-scale case studies as a framework for ASD-related CNV loci characterization is the major strength of our study. However, this approach has some limitations. First, some of the studies in our dataset included genome-wide CNV data, while other studies focused on the identification of CNVs within specific genomic loci or with a specific mechanism of CNV inheritance. To account for the potential bias arising from this variation, we assigned different weights to studies based on the CNVs/Loci ratio reported in them, and incorporated this weight in the calculation of the CNV burden score (See methods). Another potential source for variation in our data is the differing CNV detection technologies used by the different studies. Accordingly, one might suspect that the higher burden scores observed among larger genomic regions (>500 kb) would be due to the greater likelihood of CNVs within these regions to be detected by all CNV detection methods, whereas smaller CNVs would not be detected by many of the older lower-resolution microarray platforms. While we cannot rule out this possibility, Sanders et. al The results of this analysis could have broad clinical and scientific implications. For example, one could use these loci as a guideline for the evaluation of chromosomal microarray (CMA) screening, a procedure that is being increasingly used in genetic evaluation of ASD subjects. Alternatively, the detailed characteristics provided for each of the CNV loci highlighted in the study may be used for further, in-depth exploration of the biological mechanism underlying their role in ASD susceptibility. An intriguing premise is whether the different genetic mechanisms trigger ASD susceptibility in subjects containing CNVs at distinct genomic loci, and whether these genetically diverse individuals present different ASD related phenotypes. We anticipate to increase the resolution of these analyses with continued updates to AutDB and additional control CNV data, which will subsequently provide both scientists and clinicians with a valuable resource for genetics research and clinical diagnostic efforts of ASD.Figure S1Distribution of CNV loci reports. Distribution of ‘major' and ‘minor' reports across CNV loci in AutDB(TIF)Click here for additional data file.Figure S2Distribution of CNV sizes. (A) Histogram of the log10 (CNV size) indicate that CNV sizes in our data have a lognormal distribution with a mean  =  42.8 kb. (B) CDF plots for the sizes of copy number gains (green), and copy number losses (red).(TIF)Click here for additional data file.Figure S3ASD susceptibility CNV loci on human chromosomes 3 & 4. The number of individuals with duplications (green) and deletions (red) are plotted for both ASD cases (continuous lines) and controls (broken lines) along human chromosomes 3q26.31 & 4q35.2. RefSeq genes overlapping with these regions are depicted in blue rectangles. Genes that have been associated with ASD according to AutDB (TIF)Click here for additional data file.Table S1Scientific reports of CNVs in ASD individuals. Details of the 48 scientific reports and their CNV data used in this study are listed in the table.(XLS)Click here for additional data file.Table S2CNV loci associated with ASD across different studies. A list of CNV loci associated with ASD from four different studies are depicted in the table. CNV loci highlighted in multiple studies are highlighted in bold.(DOCX)Click here for additional data file.


Recent molecular dynamics simulation results have increased conceptual understanding of the grazing and the ploughing friction at elevated temperatures, particularly near the substrate’s melting point. In this commentary we address a major constraint concerning its experimental verification. It was postulated some time ago that a component sliding under lightly loaded conditions should experience very low friction and nearly zero wear . Recent 3N4) [However, diamond is known to exhibit poor thermo-chemo-mechanical stability particularly against low carbon ferrous alloys  and at e3N4) ), wears 3N4) . A hypot3N4)  ascribin3N4) .This raises a key question concerning the stability of diamond tips during nanoscratch tests at elevated temperatures and more categorically against low carbon ferrous alloys. The wear of the diamond tip will change the contact area which will alter the frictional force as the latter depends linearly on the number of atoms that chemically interact across the surface . Then, t3N4, B4C and the recently recognized C3N4.Principally, ultra-hard materials – materials whose hardness is attributable to covalent bonding – can be represented using the “composition cycle” shown in 3-bonded state. Two commercially available materials from this composition cycle, diamond (C) and cubic boron nitride (CBN), possess ultra-high hardness (attributed both to sp3-bonding and relatively short bond lengths) and, for this reason, they are frequently used to manufacture cutting tips. While diamond resides in a cubic lattice structure, CBN possesses a zinc-blende structure having boron atoms at the crystal site  and nitrogen atoms in the respective centers of the boron tetrahedra. Although, a chemical bond between the carbon atoms in diamond is stronger than the corresponding isoelectronic bond between nitrogen and boron atoms, we propose that cubic boron nitride “CBN” could be an alternative appropriate choice for high-temperature nanotribology applications because of its superior thermal and chemical stability compared to that of diamond. Even though diamond and CBN have similar lattice structures, their surface chemistry is different. In a CBN lattice, boron atoms have only three valence electrons on the surface while nitrogen atoms have five. However, two of these five electrons of nitrogen can form a stable pair, leaving three valence electrons to bond with boron. On the contrary, a diamond lattice has four valence electrons; therefore, only a maximum of three electrons on the surface can have stable bonding between them. Consequently, this leads to the possibility that the remaining one or two electrons of each surface atom in diamond react readily with other materials like iron, nickel and even silicon [Under ambient conditions – while nitrogen reside in a gaseous form as individual chemical species – boron, silicon and diamond are known to prefer a solid state. Due to its abundance and its capability to form better oxides, silicon dominated the electronic consumer market  while th silicon  in a tri silicon  and sili silicon  in compaIt is proposed that the results for the ploughing friction and grazing friction in a high temperature environment must be retested using a CBN probe to reconfirm the state-of-the-art knowledge. Moreover, the influence of the rake angle of the tip must necessarily be accounted for when generalizing the results of grazing friction, as it is an integral part in governing atomic scale friction –20.


BRCA1 cDNA. The effect of puromycin, DNase I and two different DNA cleaning protocols were tested in the RNA analysis of lymphocyte cultures. We used two samples from unrelated families with two different BRCA1 exon deletion events, two healthy unrelated controls and six samples from hereditary breast/ovarian cancer syndrome (HBOC) patients without BRCA1/2 mutations. Using RNA treated with DNase I and cleaned in a column system from puromycin-treated fractions, we were able to identify the two BRCA1 deletions. Additional HBOC patients did not show additional splice events. However, we were not able to get reproducible results. Therefore, the cDNA-MLPA technique using kit BRCA1 P002 is in our hands currently not reliable enough for routine RNA analysis and needs further optimization.A rapid and easy method to screen for aberrant cDNA would be a very useful diagnostic tool in genetics since a fraction of the DNA variants found affect RNA splicing. The currently used RT-PCR methods require new primer combinations to study each variant that might affect splicing. Since MLPA is routinely used to detect large genomic deletions and successfully detected exon skipping events in Duchenne muscular dystrophy in cDNA, we performed a pilot study to evaluate its value for BRCA1 and BRCA2 genes is offered to families with high risk of breast and ovarian cancer. Besides clear pathogenic mutations and polymorphisms, unclassified variants (UVs) of unclear clinical relevance are found. Some of these UVs may result in aberrant splicing, by affecting the donor or acceptor splice sites, or exonic splice site enhancer (ESE) sites [in silico. Additionally, deep intronic variants, which are normally ignored, may also affect splicing. One example of a deep intronic pathogenic variant is the variant CDKN2A IVS2-105A>G, which causes retention of intronic sequence [BRCA1 MLPA kit [BRCA1 MLPA is a multiplex assay based on the hybridization of a large set of primers throughout the entire coding part of the BRCA1 gene. Therefore the assay should potentially also be able to detect all exon skipping events in cDNA in the presence of a variant affecting splicing, without the need to design a specific RT-PCR assay for each variant. Although these are likely rare events, using a rapid and relatively cheap assay to assess them would be valuable in a diagnostic setting to rule out their presence.Genetic screening of the sequence . Experimsequence –7. It issequence . After tsequence , who werBRCA1 exon 13 skipping (c.4242-1643del3835) or exon 22 skipping (c.5333-36del510) [BRCA1 or BRCA2 mutation was identified in the standard diagnostic screening. Here we show that the MLPA method was able to detect the skipping events, but it was not reproducible enough for use in clinical testing despite the optimization attempts which are here described.For this pilot study, samples with 6del510)  were sel l-glutamine (Gibco) and 12.5% FCS with additional supplements and antibiotics. Lymphocyte growth was stimulated with 50 μL/mL PHA (Gibco) and 10 U/mL of IL-2 (Roche). At day 7, 4–6 h before harvesting the cells, cultures were treated with 200 μg/mL of puromycin (Sigma), to enrich for transcripts containing premature stop codons by the inhibition of NMD [White blood cells were isolated and cultured in complete medium consisting of: RPMI 1640 supplemented withTotal RNA was isolated using TRIzol (Invitrogen) or TRIpure (Roche) reagent. RNA samples used were either not subjected to DNase I treatment or treated with DNA-free kit (AMBION) or with DNase I treatment followed by purification in the column system RNeasy MinElute Kit (Qiagen). First-strand cDNA was obtained with Reverse Transcriptase M-MUL (Finnzymes) using random hexamers (Invitrogen) following the manufacturers’ instructions. The cDNA was amplified with the SALSA MLPA P002 probe mix (MRC-Holland) according to the manufacturer’s protocol. Fragment analysis was performed by capillary electrophoresis in an ABI PRISM 3730 automatic sequencer (Applied Biosystems).BRCA1 mRNA. The normalization of the data was performed using a spreadsheet according to the Manual spreadsheet-based MLPA analysis instructions (available on the MRC-Holland website: www.MLPA.com). The threshold values for deletions and duplications were set to 0.75–1.25, respectively, which are also used for DNA analysis [The size calling and the peak areas were assessed using the Genemarker software (Softgenetics) and exported to a “.txt” file. The values of the antisense probes were extremely low compared to the sense probes, and they do not have known biological meaning. Therefore, the data was filtered to leave only the data from probes corresponding in sequence to that of sense analysis –16.With the SALSA MLPA P002 kit, strong signals were obtained for 21 out of 25 probes. These probes contained more than 85% nucleotides hybridizing to the exon sequence in the correct orientation. The signals for the probes with less than 85% matching exonic sequence  or in antisense (23) were extremely weak and often not even detectable by the software. This also confirms the absence of contaminating genomic DNA in the RNA samples.Initially, we have compared the results from puromycin-treated and non-treated samples Fig. , withoutBRCA1/2 mutation were also analyzed (data not shown) using the puromycin-treated fractions and RNAs treated with DNase I and cleaned in a column system. None of these samples showed an exon skipping event, in the 20 exons tested. However, in an independent third experiment we observed increased interindividual variability in some exon signals. Many exons had normalized values outside the 0.75–1.25 thresholds (Fig. Six samples from high risk families without a BRCA1 and BRCA2 mutation negative patients with strong breast and/or ovarian cancer history. This group of patients may carry variants outside the screened intronic region flanking the exons which could affect splicing. Since exon skipping is the most common alternative splice event [The MLPA method is widely used in diagnostics, mainly to test genomic events such as deletions and duplications. Although there are a few commercial RT-MLPA kits, these are designed to test the expression of genes associated with certain biological processes, MRC-Holland has not developed RT-MLPA kits to test splice events. Besides the use of the MLPA, or other multiplex approaches, to test the effect of genetic variants predicted to affect splicing at the RNA level, it would be useful to test for ce event , developDMD gene [ BRCA1 exon skipping events. The most optimal results were obtained from puromycin-treated samples and when RNA was treated with DNase I and subsequently purified in a column system. However, despite efforts to optimize the technique further, we were not able to get reliable, reproducible results for unequivocal interpretation using the kit BRCA1 P002. This variation was also observed in healthy control samples, which showed both deletion and duplication events in one out of three experiments performed.One single study has previously shown that MLPA could be used to test exon skipping events in RNA transcripts of the DMD gene . Here weMLPA test is a flexible multiplex assay which allows for up to a total of 50 probes and in principle, it should be possible to use it for detection of alternative splicing events other than exon skipping. To be able to test also for intron retention or insertion of pseudoexons, probes crossing over exon–exon boundaries should also be included in the assay. Although mRNA-seq technology  will als


Spillover of nitric oxide between neurons may have a major impact on central nervous system diseases and particularly on neurodegeneration. There is evidence whereby communication between nitrergic and dopaminergic systems plays an essential role in the control of the nigrostriatal pathway. However, there is sparse information for either the coexistence or overlap of nitric oxide and dopaminergic structures. The dual localization of immunoreactivity for nitric oxide synthase (NOS) and tyrosine hydroxylase, enzymes responsible for the synthesis of nitric oxide and dopamine, respectively, was examined in neurons of the nigrostriatal pathway in the rat brain by means of a double-immunohistochemical method and confocal laser scanning microscopy, acquired at the resolution limit. After perfusional fixation, the brains were cut and double-immunostained. A proximity analysis of tyrosine hydroxylase and NOS structures was done using binary masks generated from the respective maximum projections, using confocal laser microscopy. Unrevealed regions were determined somatodendritic positive for both NOS and tyrosine hydroxylase, within an image limit resolution at 2 μm-wide margin. The described interconnected localization of nNOS(+) and TH(+) containing neuronal fibers and cells bodies in the nigrostriatal pathway propose a close anatomical link between the two neurotransmitters.Nitric oxide is unconstrained by cell membranes and can therefore act along a broad distance as a  The mammalian basal ganglia  are a group of subcortical nuclei implicated in a multiplicity of functions including motor, cognitive, and mnemonic behaviors  after N-Methyl d-Aspartate (NMDA) receptor activation and calcium influx , a rate-limiting enzyme for catecholamine synthesis that catalyzes the conversion of tyrosine to Our aim is to explore the hypothesis that the spatial relationships between either axons or somata of the nitric oxide and dopaminergic cells may represent selective associations between the nitrergic and dopaminergic systems. For this purpose, the dual localization of immunoreactivity for NOS and tyrosine hydroxylase in neurons of the nigrostriatal pathway in the rat brain was examined by means of a double-immunohistochemical method and confocal laser scanning microscopy (CLSM).Wistar rats (200–250 g) were housed in groups of five per cage in a temperature-controlled room (23°C), under 12 h light-dark cycles with free access to food and water. Experiments were conducted according to the principles and procedures described by the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research  and Guidelines from the School of Medicine . The institutions housing conditions and experimental procedures were previously approved by the local Animal Ethics Committee.Adult male Rats were deeply anesthetized with urethane (25 mg/kg) and then rapidly perfused transcardially with 250 ml of cold saline and 400 ml of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Brains were immediately removed and fixed in 4% PFA for 24 h and cryoprotected in 30% sucrose solution. Brains were snap frozen in isopenthane  and stored at −70°C till use. The tissues were cut at 40 μm on a cryostat. Sections through the brain regions were collected in 0.01 M PBS solution containing 0.02% sodium azide and stored at 4°C until use.Herbison et al.  reportedImmunohistochemistry was performed using a standard peroxidase-based method  followed by a 2 h incubation at room temperature with a secondary donkey anti-mouse IgG (H + L) antibody coupled to Alexa Fluor 488 . After TH labeling, the tissue was washed three times with PBS and incubated overnight with a polyclonal antibody specific for rat nNOS . The primary nNOS antibody was then revealed with a Cy3-coupled donkey anti-sheep IgG . The primary antibodies were diluted in PBS  containing 0.02% thimerosal, 5% normal goat serum , 0.2% Triton X-100, while the secondary antibodies were diluted only in PBS .pars compacta and reticulate, ventral tegmental area, and pedunculopontine nuclei were examined, as previously described  equipped with a 60x objective  and connected to an image analysis system . A total of six sections of each selected brain region, encompassing rostral, middle, and caudal levels of the striatum, subthalamic nuclei, substantia nigra 2O stock solution; Invitrogen D1306) was diluted 1:5000 with PBS (DAPI labeling solution). The slides were incubated for 1–5 min at room temperature in DAPI labeling solution. The slides were rinsed three times in PBS+ and the coverslips mounted  to obtain an improved signal-to-noise ratio. The Alexa Fluor 488 fluorophore  was excited with the 488 nm laser line of an Ar-laser, while the Cy3 fluorophore  was excited at 561 nm, with emission being collected in a line-by-line fashion at 500–530 and 570–650 nm, respectively.Sections double-stained for nNOS and TH were inspected using a Leica SP2 and Leica SP5 confocal laser scanning microscopes . Z-stacks of the frontal cortex were acquired with a 40× oil objective , while regions from the striatum and substantia nigra were acquired at the optical resolution limit (43 nm× 43 nm × 130 nm) with a 63× (HCX PL APO) oil objective and subsequently deconvolved with AutoQuantz-axis of the stack. A maximum intensity projection was then generated with Fiji software package  and TH(+) immunoreactivity revealed a considerable overlap of their topographies. The distribution of nNOS(+) immunoreactivity is generally in agreement with the data published by others using either an immunocytochemical  and nNOS(+) immunoreactive cells and fibers within the striatum-nigra pathway. Using CLSM optical sections acquired at the resolution limit, colocalized regions (Pearson correlation coefficient ≥0.7) were identified (yellow). The yellow pseudo-color for these structures visible in CLSM images could represent the superimposition of TH(+) immunoreactivity (green) on nNOS(+) immunoreactivity (red) neurons/fibers and The prefrontal, anterior cingulate, insular, piriform, perirhinal, entorhinal, motor, premotor, parietal, temporal, and posterior cingulate cortices had a dense distribution of TH(+) immunoreactive fibers. A small population of medium-large, intensely nNOS(+) immunoreactive multipolar neurons was found scattered throughout layers 2–4 of all examined cortices. The reaction product for nNOS(+) immunohistochemistry in the cortical neurons formed dense, conspicuous masses with no nuclei labeling.A qualitative analysis of the area 2 μm proximal to the nNOS(+) cell body was carried out Figure  in the aIn general, nNOS(+) immunolabeling was detected in the cytoplasmic domains of the cell body and dendrites. The nucleus was unstained and the reaction product was evenly distributed in the cytoplasm. A dense network of NOS(+) varicose fibers was observed in the striatal complex. TH(+) immunohistochemistry demonstrated TH(+) immunoreactive terminals or varicose fibers in the neuropil of the striatum Figures C, and 8DTH(+) immunolabeling in the striatum appeared as a dense, inhomogeneous plexus of fibers with an absence of cell bodies at any level  displayed TH(+) immunoreactivity and nNOS(+) immunoreactivity similar to that of the dorsal striatum Figure . There wThe TH(+) and nNOS(+) immunoreactivity double-labeling in the pallidus external and internal (entopeduncular nucleus in the rat) is presented in Figures In the external globus pallidus Figures D and 9, pars compacta, where it is spread forming a dense band that lies dorsally from the ventral tegmental area  immunoreactive cells are present, some of them arranged in linear bridges which cross the substantia nigra pars reticulata perpendicularly to the substantia nigra pars compacta  immunoreactive cells and fibers next to a small number of fibers and cell somata with strong NOS(+) immunoreactivity  immunoreactivity is grouped in the substantia nigra  Figures A,B. In t Figures A,B. A doTH(+) immunoreactive and NOS(+) immunoreactive cells could be observed adjacent to each other, with areas of perfect match Figure . Fibers Functional importance of the subthalamic nucleus neurons is reflected by its anatomical connections to the main output nuclei of the basal ganglia, including both segments of the globus pallidus and the substantia nigra. In the subthalamic nucleus we observed numerous NOS(+) immunoreactive cell bodies and an intense pattern in the TH(+) immunoreactive fiber staining Figure , correspThe pedunculopontine tegmental nuclei are a loosely defined aggregate of cholinergic and non-cholinergic neurons in the midbrain which neurons project to substantia nigra compacta dopaminergic neurons. Our observations revealed clusters of intensely labeled NOS(+) immunoreactive neurons Figures A,C and TWe compared the spatial relationship between dopaminergic and nitrergic nigrostriatal fibers/cell body through the dual localization of the immunoreactivity for nNOS(+) and TH(+). The approach was based on double-immunohistochemical staining method using CLSM optical sections acquired at the resolution limit. A proximity analysis of TH(+) and nNOS(+) structures was done using binary masks generated from the respective maximum projections and revealed regions positive for both, nNOS(+) and TH(+) immunoreactivity, within a 2 μm-wide margin. Colocalized regions were identified with a Pearson correlation coefficient ≥0.7. A large proportion of nNOS(+) immunoreactive soma/axon/dendrite themselves were directly apposed by TH(+) immunoreactive ones, within a radius of 1 and 2 μm.In the dopaminergic synapses, dopamine is released from pre-synaptic structures by exocytose, binds post-synaptic receptors and generates pre- and post-synaptic cascades of events  and NOS(+) immunoreactive neurons interact with each other in all analyzed regions. Together, our observations corroborate evidence of dopamine and nitric oxide being intertwined in the anatomy in addition to physiology and pathology of the nigrostriatal pathway  and TH(+) containing fibers and cells bodies in the nigrostriatal pathway propose an anatomical link between the two neurotransmitters. Nitric oxide in combination with dopamine may represent suitable targets for therapeutic intervention in neurodegenerative diseases as for example Parkinson’s (for review see Przedborski et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. The pathogenesis underlying the increased predisposition to the development of basal cell carcinomas (BCCs) in the context of Gorlin-Goltz syndrome is linked to molecular mechanisms that differ from sporadic BCCs. Patients with Gorlin syndrome tend to develop multiple BCCs at an early age and present with tumors of non-sun-exposed skin. The aim of this study was to compare the proteomic profile of cultured fibroblast and fibroblast conditioned culture media of PTCH1+ and nonmutated fibroblasts. Results. Proteomic analysis was performed using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry in PTCH1+ fibroblast conditioned media isolated from not affected sun-protected skin areas of Gorlin patients and from healthy subjects. 12 protein cluster peaks, >5 kDa, had significant differences in their peak intensities between PTCH1+ and PTCH1− subject groups. We detected a strongly MMP1 overexpression in PTCH1+ fibroblasts obtained from NBCCS patients with respect to healthy donors. Conclusion. Protein profiles in the fibroblast conditioned media revealed statistically significant differences between two different types (missense versus nonsense) of PTCH1 mutations. These differences could be useful as signatures to identify PTCH1 gene carriers at high risk for the development of NBCCS-associated malignancies and to develop novel experimental molecular tailored therapies based on these druggable targets. PTCH1 mutations lead to complex syndromes such as the Gorlin syndrome (GS) also named nevoid basal cell carcinoma syndrome . GS is a rare autosomal dominant disorder characterized by striking predisposition to the development of basal cell carcinomas (BCCs) (up to hundreds) [Patients with germ-line mutations in tumor suppressor genes represent an intriguing heredofamilial model of cancer susceptibility and genotype-phenotype correlation . PTCH1 mundreds) , keratocPTCH1 germ line mutation have a variable phenotype [In this hereditary setting, the genotype-phenotype correlation is not always present: gene carriers and family members sharing the same henotype . Howeverhenotype , as wellhenotype .A systemic tailored therapy has been introduced in clinical practice, for GS patients with multiple BCCs that cannot undergo surgery , showingm/z) ratio and intensities of the bound peptide/protein [Among the modern technologies proteomics can be employed in the discovery and identification of protein profiles possibly related to different sporadic and hereditary phenotypes. Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-ToF MS) is a proteomic tool for differential expression profiling which permits to detect a large number of low-molecular weight proteins (<20 kDa) –10. Sele/protein .PTCH1 mutated fibroblasts, we compared the proteomic expression of PTCH1+ fibroblast conditioned culture media to that of healthy control fibroblasts under the same conditions. We also investigated patients with different subtypes of germ-line PTCH1 mutations, through the comparison of both genetic and proteomic profiles.In this study, we postulated that fibroblasts in patients with GS may possess characteristics determining BCC development being responsible for the production of proteins (cytokines and growth factors) promoting the basaloid proliferation. To validate the existence of specific protein patterns in culture media of This study was approved by the Ethical Committee of the University Hospital of Modena, Italy, and was conducted after informed written consent of NBCCS patients.PTCH1− patients included Wilde Type healthy controls and PTCH1 not mutated NBCCS patients, PTCH1+ patients (germline mutated patients). The definition of “aggressive” phenotype is defined by a large number of big diameter BCCs and/or OKCTs and other NBCCS clinical criteria.Patients were characterized for their phenotypic aspects as studied through clinical examination. n = 3; NBCCS, n = 8) . NBCCS patients were separately analyzed according to the different types of PTCH1 mutation (nonsense versus missense).Biological samples were collected from all NBCCS and control persons  and fixed with paraformaldehyde 4% for 10 min.To block nonspecific binding, cells were incubated with 0.5% Bovine Serum Albumin, 5% Goat Serum for 15 min, and then incubated with primary antibodies: Monoclonal Mouse Anti-Vimentin  and Monoclonal Mouse Anti-Matrix Metalloproteinases (MMP) 1  for 1 hour at room temperature. Subsequently, cells were incubated with secondary antibody anti-mouse AlexaFluor 488 (1 : 100 dilution) and AlexaFluor 546 (1 : 100 dilution)  for 45 min at room temperature. Fluorescent specimens were analyzed by a confocal scanning laser microscope Nikon A1 .Samples were obtained from cultured fibroblast previously isolated by healthy skin biopsies of all NBCCS patients and control individuals, from sun-protected healthy areas. Primary dermal fibroblasts were isolated by explant and cultured as previously described . Experimμm) and concentrated by Amicon Ultra 3 K  according to the manufacturer's recommendations.The CM collected was filtrated  plus a cocktail of proteinase inhibitors . The samples were freeze/thawed and sonicated. The cell proteins were obtained through centrifugation at 15000 ×g for 15 min to remove insoluble debris.The fibroblasts were washed with phosphate buffer pH 7.4  and lysated with 300 The total protein in cell lysates and in CM was quantified using the classical Bradford method  according to the manufacturer's instructions . There wμL of binding buffer  was mixed with 20 μL of fibroblasts conditioned medium or 5 μL of cell lysates to obtain a 0.3 μg/μL final protein concentration that was then loaded onto IMAC30-Cu ProteinChip Array spot surfaces, according to the manufacturer protocols. After 45 min incubation at room temperature with shaking, the unbound proteins were removed by three washing steps using 200 μL of the same binding buffer. Finally, 1 μL of saturated sinapinic acid solution in 0.5% trifluoroacetic acid and 50% acetonitrile  was applied to each spot twice, allowing the surface to dry between each application.Lysate and conditioned culture medium samples were analyzed with SELDI-ToF-MS, with the purpose to investigate the protein profile in both the low (2–30 kDa) and the high (30–100 kDa) molecular weights (MW). In a preliminary study, in order to set up the experimental conditions, pooled samples were loaded onto two different types of ProteinChip Arrays : CM10 (weak cation-exchanger) and IMAC30-Cu . The IMAC30-Cu array gave the higher number of peaks detected and the higher total signal intensity compared to CM10, so this array was used in the main study. After preequilibration, 90 The ProteinChip Arrays were analyzed with a SELDI-ToF-MS reader , by protocols optimized for low (1.5–30 kDa) and high (30–150 kDa) molecular weight (MW) ranges. Mass spectra were acquired using low (3500 nJ) and high (7500 nJ) laser energies averaging 901 laser shots onto the spot surface.The “All-in-one protein standard II”  was used to obtain protein standard spectra for mass accuracy calibration.U test. A P value less than 0.05 was accepted as statistically significant.Statistical analysis was performed using the ProteinChip Data Manager 3.0 software  as previously reported . BrieflyFibroblasts were extracted from eleven human skin samples obtained from surgical resection. The skin was dissociated, and various cell types were separated to obtain populations of fibroblasts. The cultures reached confluence after two weeks and were stored in −80°C at cell passage 6.The cultured primary cells showed typical fibroblast-like features, with spindle-like shapes and elongated projections. Performing immunofluorescence analysis of vimentin expression, we confirm the fibroblast nature of isolated cells .PTCH1 mutated fibroblasts obtained from NBCCS patients with respect to healthy donors , obtained with IMAC30 Cu pretreated ProteinChip arrays.We compared the proteomic expression profiles of 11 fibroblasts conditioned cultures media and total cell lysates .The statistical analysis of peak intensities of total cell lysates of fibroblast NBCCS patients and control individuals revealed an elevated number of differentially expressed protein masses , comparing PTCH1+ nonsense and missense mutations, respectively  are overexpressed while two under-expressed  while only one (m/z 35968) in NBCCS not aggressive phenotype for culture media, and lysates, respectively. Also these peaks are not in common with those discovered in the previous comparison, indicating their specific role in determining a different phenotype of the disease.It was also possible to find differences in protein expression comparing NBCCS patients according to their aggressive or not aggressive phenotype. PTCH1 mutations through the comparison of proteomic profiles supports the hypothesis that epithelial oncogenesis could be induced by a dermal-epidermal cross-talk based on proteins that are directly produced by dermal fibroblasts. This mechanism is mainly involved in the development of multiple early onset BCCs arising on non-sun-damaged skin, which is not usually affected in nonsyndromic patients.The investigation of different cohorts of healthy individuals and patients carrying different germ-line PTCH1+/− fibroblasts and/or NBCCS patients with aggressive/mild clinical behavior.SELDI-ToF-MS analysis is a proteomic technology regarded as one of the most powerful tools useful to investigate a differential expression profile in the low (2–30 kDa) and the high (30–100 kDa) molecular weights (MW). Our method represents a novel approach based on the study of conditioned media through the extended proteomic analysis. In fact, the application of SELDI-ToF technique had so far been limited to cell lysates for which the huge quantity of proteins represents a consistent obstacle to the clear identification of specific protein clusters specifically associated to PTCH1 mutations and 2 PTCH1 WT) and 3 healthy controls, obtaining peculiar clusters of protein peaks for IMAC30-Cu and 95 for CM10 ProteinChip Arrays, respectively.In our study we analyzed by the SELDI-ToF technique samples of fibroblasts conditioned media from 8 GS patients  , 15. A pPTCH1 have overall very similar consequences on the transcriptome of dermal fibroblasts, we detected characteristic protein peaks of the conditioned media discriminating between these two PTCH1 mutation patients groups. There is still no evidence concerning PTCH1 associated transcriptome for nonsense PTCH1 mutations, but we can speculate that such mutations codify for decay mRNA linked to soluble cytosolic PTCH1 variants, instead of transmembranous proteins. The absence of this transmembranous protein receptor can be related to the constitutional activation of SHH pathway, which increases protein synthesis and secretion. On the contrary, missense PTCH1 mutations may lead to the production of defective receptors that may explain the reduced synthetic ability of WT fibroblasts.However, in contrast to Valin et al.  who descPTCH1+ fibroblasts may include peptides or small proteins with low abundance, which can scarcely be revealed using lysate cells, in which proteins are present in large quantity. However, the main SELDI-ToF limits are that it does not allow protein detection of higher molecular weight proteins and does not consent peaks identification such as the postulated growth factors or matrix proteins. A specific analysis through the TagIdent tool with pI and mass information was conducted, in order to obtain matching information for each protein peak. However, an excess of meaningful matching proteins was found, indicating that the functions of these putative uncharacterized proteins are undefined. At this regard, we have preliminary data showing that MMP1 is overexpressed in the PTCH1 mutated fibroblasts, confirming that there is a relationship between PTCH1 mutation and MMP1-related ability to induce neoplastic transformation of the epithelial cells. In addition, MMP1 can be a novel target for experimental tailored therapy of malignancies in the setting of NBCCS. A future characterization of these peculiar proteomic signature can be useful for the clinical, prognostic, and therapeutic evaluation of those patients.The protein peaks identified in conditioned media of PTCH1 germline mutations; in the latter, it has been hypothesized that BCCs in advanced stage would have higher chances of developing resistant mutations. This might be due to both higher mutation rates and/or the presence of an increased number of tumor cells [Patients affected from multiple BCCs that cannot or do not want to undergo surgery can benefit from the treatment with Vismodegib, a systemic drug that has provided good response rates in the trials . Howeveror cells . A furthEach target therapy has its own resistance mechanisms, which are due to acquired or initially present secondary mutations that affect genes involved in the target pathways. Their occurrence can be prevented through the introduction of multitarget inhibitors that help avoid resistance mechanisms, finally leading to an increased disease-free survival. For both BCCs and medulloblastomas in GS, determining specific proteomic patterns in individuals with particularly aggressive phenotypes might lead to both experimental approaches and clinical trials on multiple smoothed therapies. Those should be pointed towards the simultaneous inhibition of several molecular targets involved in the pathogenesis and clinical progression of syndromic tumors.PTCH1 and control samples of fibroblast conditioned media. Statistical analysis of differentially expressed protein peaks has given the possibility to define algorithms, which could distinguish between NBCCS patients and control individuals.In this study, we used SELDI-ToF-MS analysis to evaluate the protein profiles of PTCH1+ fibroblasts and keratinocytes cocoltures are required to recognize and corroborate the revealed protein peaks.Our data prove that the SELDI-ToF-MS technology is capable to detect a set of protein peaks as candidate biomarkers, rapidly and with high throughput. protein peaks profiling provided a better understanding of the complex skin cancer microenvironment and could be useful to select patients at risk to develop multiple and aggressive BCCs and/or other NBCCS-associated malignancies. These are the patients which could benefit from a systemic drug approach and/or require specific clinical and instrumental followup. However, further studies, involving Comparison of peak intensities between NBCCS and controls fibroblast lysates for IMAC30-Cu. For each cluster peak comparison the p-value was <0.05.Click here for additional data file.


EIF2AK3 gene were described in patients with WRS and defects in this gene lead to increased susceptibility to apoptotic cell death. Mutations in IER3IP1 have been reported in patients with MEDS and similarly, loss of activity results in apoptosis of neurons and pancreatic beta cells in patients. Here we report on a homozygous mutation of the IER3IP1 gene in four patients from two unrelated consanguineous Egyptian families presenting with MEDS who display burst suppression patterns on EEG. All patients presented with mildly elevated liver enzymes, microalbuminuria, and skeletal changes such as scoliosis and osteopenia, leading to repeated bone fractures. We expand the phenotypic spectrum of MEDS caused by IER3IP1 gene mutations and propose that WRS and MEDS are overlapping clinical syndromes, displaying significant gene-dependent clinical variability. © 2012 Wiley Periodicals, Inc.Wolcott–Rallison syndrome (WRS) and the recently delineated microcephaly with simplified gyration, epilepsy, and permanent neonatal diabetes syndrome (MEDS) are clinically overlapping autosomal recessive disorders characterized by early onset diabetes, skeletal defects, and growth retardation. While liver and renal symptoms are more severe in WRS, neurodevelopmental characteristics are more pronounced in MEDS patients, in which microcephaly and uncontrolled epilepsy are uniformly present. Mutations in the  PTF1A mutations) [Sellick et al., GLIS3 mutations) [Senee et al., KCNJ11 mutations), and epilepsy (DEND syndrome) [Gloyn et al., Microcephaly with simplified gyration is defined as congenital microcephaly associated with a reduced number of gyri, thin or normal cortical thickness, and reduced number and depth of sulci. It may be associated with abnormal myelination or additional brain malformations, and it is classified into five groups according to associated brain imaging findings [Basel-Vanagaite and Dobyns, EIF2AK3 gene [Delepine et al., EIF2AK3 is one of four kinases known to phosphorylate EIF2A, a translation initiation factor crucial for protein synthesis, and mutations in this gene are thought to cause Wolcott–Rallison syndrome by increasing endoplasmic reticulum stress and inducing subsequent apoptotic cell death [Delepine et al., IER3IP1), an ER stress response protein that mediates cell differentiation [Poulton et al., Wolcott–Rallison syndrome  is a rare autosomal recessive condition characterized by permanent neonatal diabetes, epiphyseal dysplasia, hepatic dysfunction, and renal impairment and is caused by mutations in the IER3IP1 mutations who display burst suppression on EEG, elevated liver enzymes, and microalbuminuria. Our work provides independent confirmation that mutations in IER2IP1 underlie MEDS and expands the phenotypic spectrum of this syndrome. We propose MEDS and Wolcott–Rallison syndrome as overlapping clinical syndromes that display significant gene-dependent clinical variability.Here, we report on the clinical, radiological, and molecular findings of four patients with MEDS and This male patient was the first child born to first-cousin healthy Egyptian parents , mother 4), and liver enzymes were mildly elevated on different occasions. Echocardiography and abdominal sonography were normal. There was no evidence of pancreatic or renal anomalies; however, urine analysis consistently showed microalbuminuria. G-banded karyotype at 550–600 band levels revealed normal male. Brain magnetic resonance imaging , and GATK [McKenna et al., EIF2AK3 gene [de Wit et al., MCPH 1-7, known to cause primary microcephaly. We then performed whole exome sequencing [Park et al., IER3IP1 was identified in both families [Poulton et al., IER3IP1 and converts a leucine to a proline at amino acid position 78. This homozygous variant was confirmed to be present in all affected children, while the healthy siblings were both homozygous for the wildtype allele  is a rare condition known to progress quickly to a vegetative state or death. Despite some progress in understanding the molecular basis of epilepsy, the pathogenesis of EIEE remains unclear [Molinari, All of our patients had dysmorphic facial features similar to those found in patients with progressive encephalopathy, edema, hypsarrhythmia, and optic atrophy (PEHO) syndrome Somer,  Table ITable I. Our results suggest that Wolcott–Rallison syndrome patients and patients with MEDS represent a continuum of phenotypically related neurodevelopmental disorders in which the mutant gene and the nature of the mutation can often predict the severity of the phenotype. Thus, it is possible that inadequate knowledge and the rarity of cases may account, in part, for the confusion in the literature regarding these disorders. Further detailed reports of similar cases along with molecular data hopefully will clarify the questions raised on genotype–phenotype correlation in this group of related disorders.