Previous studies of perceptual category learning in patients with schizophrenia generally demonstrate impaired perceptual category learning; however, traditional cognitive studies have often failed to address the relationship of different cortical regions to perceptually based category learning and judgments in healthy participants and patients with schizophrenia. In the present study, perceptual category learning was examined in 26 patients with schizophrenia and 25 healthy participants using a dot-pattern category learning task. In the training phase, distortions of a prototypical dot pattern were presented. In the test phase, participants were shown the prototype, low and high distortions of the prototype, and random dot patterns. Participants were required to indicate whether the presented dot pattern was a member of the category of dot-patterns previously presented during the study phase. Patients with schizophrenia displayed an impaired ability to make judgments regarding marginal members of novel, perceptually based categories relative to healthy participants. Category judgment also showed opposite patterns of strong, significant correlations with behavioral measures of prefrontal cortex function in patients relative to healthy participants. These results suggest that impaired judgments regarding novel, perceptually based category membership may be due to abnormal prefrontal cortex function in patients with schizophrenia. Category learning and decisions or judgments regarding ensuing category membership are cognitive processes that are integral to our daily lives. Category formation and decisions regarding category membership have been studied extensively in healthy adults. There are two principal hypotheses regarding the cognitive underpinnings of category learning. The prototype hypothesis suggests that information about category membership yields a prototype (or an average of instances), which is stored separately from the individual items contributing to formation of the prototype –4. ConveSmith et al.  and KoenPrevious studies have demonstrated visual perceptual organization deficits in a portion of patients with schizophrenia, specifically those patients diagnosed with a subtype of schizophrenia referred to as disorganized . These vLearning and judgment of category membership, both semantic and perceptual, is relevant to schizophrenia to the extent that patients with schizophrenia often display disorganized thought processing  and theyStudies using geometric shapes to define membership in perceptually based categories , 25 havePerceptual category learning was examined in patients with schizophrenia using a version of the dot-pattern category learning task described previously , 29. In Twenty-six patients, 23 males and 3 females (81% right handed) with a diagnosis of schizophrenia (21 inpatients and 5 outpatients) participated in this study. Two board-certified psychiatrists concurred on diagnosis by Structured Clinical Interview for the Diagnostic and Statistical Manual-fourth edition without knowledge of cognitive abilities. Patients that received concurrent axis I psychiatric diagnoses other than schizophrenia, or having a history of current substance abuse, head injuries with concomitant loss of consciousness, seizures, central nervous system infection, diabetes, or hypertension were excluded. Patients were classified into undifferentiated 44%, paranoid 24%, disorganized 8%, and residual 8% subtypes, as well as receiving classifications of chronic schizophrenia 4%, and schizoaffective depressed disorder 12%. The majority of patients (88%) were receiving doses of atypical antipsychotic medication such as olanzapine and risperidone at the time of testing and 8% were not receiving antipsychotic medications. In addition to patients with schizophrenia, 25 healthy participants, 12 males and 13 females (100% right handed) recruited through the National Institutes of Health normal volunteer office, participated in this study. Healthy participants with a history of psychiatric disorders, current substance abuse, head injuries with concomitant loss of consciousness, seizures, central nervous system infection, diabetes, or hypertension were excluded. All participants provided informed written consent prior to participation in this study. The Institutional Review Board of the National Institute of Mental Health provided approval for this study.t-tests revealed no significant difference between patients and healthy participants with respect to Reading standard scores, t(38) = 0.74, p = 0.46, visual-spatial abilities (Benton Line Orientation test), t(37) = 0.52, p = 0.61, and a trend toward a significant difference between groups with respect to age, t(48) = 1.91, p = 0.06. As expected, there were significant differences between the groups on the basis of education, t(49) = 2.72, p = 0.01, and current WAIS-R estimated FSIQ, t(39) = 5.20, p < 0.001.A four subtest version of the Wechsler Adult Intelligence Scale-Revised (WAIS-R) , consistThe dot-pattern category learning test used in the current study followed the specifications described previously , 29. TheThe dot-pattern recognition test used in the present study also followed the specifications described previously , 29. ThiTo determine relationships among dot-pattern category judgments and other cognitive abilities known to be dependent on regionally different brain systems, standard neuropsychological tests assessing abilities from different cognitive domains were administered to all patients and a subset of healthy participants over a period of one to three sessions by a psychologist or psychometrician trained in administration and scoring of all tests. Scoring followed standardized procedures. The Wisconsin Card Sorting Test (WCST) , a test Psychotic symptom severity was assessed weekly in patients with schizophrenia using the Positive and Negative Syndrome Scale (PANSS) , by membt-tests since the potential number of correct responses in each condition differed due to experimental design. An identical series of analyses were also applied to the dot-pattern category judgment reaction time data. The mean number of correct responses during dot-pattern recognition was analyzed using independent t-tests since there were only two variables of interest (training pattern and random dot patterns) and the potential number of correct responses in each condition differed due to experimental design. The mean reaction time of correct responding by patients with schizophrenia and healthy participants during dot-pattern recognition for the training pattern and random dot patterns were also analyzed using a series of independent t-tests. Results of all independent t-tests referred to above were determined to be significant only after application of a Bonferroni correction for multiple comparisons with α set at 0.05.The mean number of correct responses made by patients with schizophrenia and healthy participants during dot-pattern category judgments of the prototype, each level of distortion, and random dot patterns were analyzed using a series of independent Correlations of dot-pattern category judgment correct responses with other cognitive variables and PANSS scores were performed to examine the relationship of dot-pattern category judgment scores to other cognitive abilities (primarily but not exclusively representative of different brain regions) and symptom severity. Using a standard test for correlations, correlations among variables representing different non-exclusive regions of cortical function and perceptual category judgment scores were tested to determine differences in correlation strength between groups. A factor analysis  was used to highlight the largest correlations and denote which factors contribute significant additional variance (loadings >0.700 were considered significant). To further analyze the relationship between prefrontal cortex function and perceptual category judgments in patients and controls, the equality of correlation matrices consisting of variables representing prefrontal cortex and perceptual category judgment measures were tested with correlational pattern hypothesis testing using structural equation modeling. Correlation matrices for each group consisting of the following five variables: prototype classification correct, low distortion classification correct, high distortion classification correct, random classification correct, and WCST categories attained, were chosen to minimize the variable-to-subject ratio. In performing the structural equation modeling analysis a Generalized Least Squares (GLSs) followed by a Maximum Likelihood (ML) discrepancy function was used to perform correlational pattern hypothesis tests between samples.t-tests on the mean number of correct responses during category judgments revealed significant differences between patients and healthy participants at all levels of dot-pattern distortion , while WCST categories, dot-pattern category prototype, low distortion, and high distortion number correct produced significant loadings on factor 2 . In the healthy participants, WMS-R verbal paired-associates I total correct, 2 back number correct, and dot-pattern category random number correct produced significant loadings on factor 1 , while F-A-S fluency and CVLT semantic cluster ratio produced significant loadings on factor 2 . Results of the correlational pattern hypothesis testing using structural equation modeling of the correlation matrices suggests abnormal prefrontal cortex function during perceptual category judgment in patients with schizophrenia. There was a lack of equality among the correlation matrices of patients and controls, GLS to ML χThere was no clear differential pattern of correlations among category judgment scores and attention and visual-spatial measures between patients and healthy participants. Judgments of dot-pattern category learning also displayed moderately strong, significant correlations with positive symptoms in the patients.Patients with schizophrenia displayed an impaired ability to make judgments regarding membership in novel, perceptually based categories relative to healthy participants. These results support previous work demonstrating that patients with schizophrenia display impaired judgment regarding novel, perceptually based category membership , 25, 27.Healthy participants appear to rely on different cognitive processes putatively representing different lateralized regions of the prefrontal cortex during spatial versus verbal working memory –52 in whThere are limitations to this study. Although these findings are suggestive of abnormal prefrontal cortical function contributing to impaired novel category judgments in patients with schizophrenia, it can not be conclusively determined which regions of the neural network associated with dot-pattern category learning and judgment are impaired in patients with schizophrenia due, in part, to the multifactorial nature of the cognitive tests used for correlational analyses. Patients with lesions in regions other than the prefrontal cortex have shown impairment on the WCST  and so-cIn relation to the specificity of the deficit to visual category judgment versus an impairment that is related to increasing task difficulty, in the present study, the patients with schizophrenia displayed performances that were not significantly different from healthy participants on the vigilance and distractibility versions of the CPT and the Benton Line Orientation test, each of which represents increasing difficulty level in relation to attention and visual-spatial abilities, respectively. Thus, in this sample of patients with schizophrenia, task difficulty alone may not have been responsible for impaired perceptual category judgment.Additionally, the heterogeneity of cognitive deficits displayed by patients with schizophrenia, often exhibiting wide-spread abnormalities across all cognitive domains tested , can makAnother limitation of the present study pertains to the effects of antipsychotic medication upon the ability to make judgments about category membership. The majority of patients were receiving antipsychotic medication prior to and during dot-pattern category learning and judgment, which may have influenced the patient’s ability to make judgments regarding category membership. However, previous studies typically show generalized improvement in cognitive abilities following the administration of antipsychotic medication –58. NeveRegarding the “normal” recognition observed in patients with schizophrenia in the present study, the purpose of the recognition control task was not to suggest that patients with schizophrenia display normal recognition memory in general, but rather to show that patients with schizophrenia can display normal recognition memory for “over-learned” perceptual patterns. Results of previous recognition studies in patients with schizophrenia have produced varied results: some studies demonstrate normal recognition –63, otheIn summary, patients with schizophrenia displayed an impaired ability to make judgments regarding marginal members of novel, perceptually based categories relative to healthy participants. These findings, in conjunction with correlations among category judgment performance and cognitive measures of prefrontal cortex function in patients with schizophrenia, suggest that impaired judgments regarding novel, perceptually based category membership may be due to abnormal prefrontal cortical function in patients with schizophrenia.Thomas W. Weickert conceived of the project, assessed all participants on the experimental task, ran the statistical analyses, and wrote the manuscript. Alejandro Terrazas supervised computer programing of the experimental and control tasks and contributed to editing the manuscript. Llewellyn B. Bigelow participated in SCID interviews and contributed to editing the manuscript. Jose A. Apud participated in SCID interviews, managed the inpatient unit, and contributed to editing the manuscript. Michael F. Egan participated in SCID interviews, managed the inpatient unit, and contributed to editing the manuscript. Daniel R. Weinberger supervised inpatient unit, the project, and contributed to editing the manuscript.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.


Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase – a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as  Cyanidium caldariumCyanidioschyzon merolaePorphyra purpureaP. umbilicalisGracilaria tenuistipitata var. liuiGrateloupia lanceolaPorphyridium cruentumPyropia yezoensis . Overall, better sampling of red algal plastid genomes and a more detailed comparison between them are needed to fully appreciate the implications of their remarkable characteristics and how these characteristics relate to other photosynthetic eukaryotes, particularly to those with secondary red algal plastids Red algae are an ancient lineage of ecological importance that play a prominent role in our thinking about the evolutionary history of photosynthesis in eukaryotes. Despite their importance, however, genomic data from red algae remain scarce. To date, only one complete nuclear genome and less than a dozen organellar genomes have been characterized and described. Red algal plastid genomes are particularly interesting, because they contain the largest gene complement among all known plastids, are compactly organized, and comparatively slowly evolving. This suggests that among modern plastid DNAs they might best represent the ancestral state of primary plastids also present in land plants, green algae and glaucophytes. However, only five complete plastid genome sequences from red algae have been described: two from the early-branching extremophiles (cox1) and plastid rubisco (rbcL) have become the most commonly used gene for red algal barcoding rbcL already providing lower resolution in at least some cases Red algal plastid genes have also shown promise as markers for reconstructing evolutionary relationships trnMe gene, and a rare case of horizontally acquired leuC/D operon. We demonstrate that whole-plastid genomes are a promising resource to resolve red algal relationships, and that they contain a range of markers potentially useful for species barcoding.Here, we expand the available plastid pool from red algal plastid data by characterizing three new complete and one partial plastid genome. We show that they all have compacted and conserved architectures that are slowly-evolving. They also contain several unique features, such as a group II intron in the plastid Calliarthron tuberculosum, Chondrus crispus, and Grateloupia lanceola, and the partial genome of an unspecified Cruoria species  were assembled from 454-pyrosequencing and Solexa reads, and manually annotated. All previously available plastid genomes from red algae were re-annotated taking advantage of these new genomes for comparison . Altogether, this resulted in 118 corrections to existing annotations affecting the presence of genes and introns, start codon positions, open reading frame (ORF) prediction, and frameshifts  than to other florideophytes. This in turn suggests that the extraordinary compaction of the plastid genome in Cyanidioschyzon has likely resulted from species-specific factors after the divergence of Cyanidioschyzon from other acidothermophilic cyanidiales, and does not relate to ancestral adaptation of cyanidiales to their acidothermophilic environments Grateloupia ; the only outstanding case was a unique 2.1 kb spacer between trnS and trnD in Chondrus of an unknown origin.Red-algal plastid genomes are highly compact, but with the updated annotations we observed that the average and median intergenic lengths varied within lineages see . The medupia see , and theGrateloupia and Calliarthron plastid genomes were co-linear. Chondrus had the smallest re-arrangement distance to the other plastid genomes and differed by only three inversions from the bangialian Porphyra, suggesting that it may most closely reflect the ancestral gene organization in florideophytes. However, even the most divergent genome pair (Gracilaria and Porphyra) could be reconstructed by mere 5 rearrangements. Considering the substantial evolutionary distance among these red algae compared to other photosynthetic groups Porphyra and Pyropia, are co-linear, except for the orientation of five individual genes. Interestingly, all five of these gene inversions occur in otherwise highly conserved gene clusters suggesting they altered ancient polycistronic operons. Three of these inversions  are located in the ribosomal supercluster, an array of 29 genes that is conserved across the whole red plastid lineage  and Gracilaria and Calliarthron retaining the least (6) (ycfs) and might therefore also represent outright loss. Altogether this indicates that plastid-to-nucleus gene transfer has likely been rare in both florideophytes and Bangiales. Cyanidiales contain the next largest plastid gene complements altogether comprising 227 cyanobacterial genes  . Howeveral genes , suggestal genes , 43 of wGracilaria tenustipitata var. liui lacks an essential gene for elongator tRNA-Met (trnMe). Specific searching for this gene revealed it is indeed present, but is difficult to identify because it contains a group II intron. Group II introns in plastid tRNA genes are uncommon and were previously reported only from streptophytes (land plants and charophytes), where they appear evolutionary stable upon acquisition Gracilaria tRNA-Met folded into a canonical structure with the intron inserted after the third anticodon position (mat). The RT/mat contains several putative reverse transcriptase domains and a readily identifiable domain X, which is ubiquitous in RT/mats and required for intron splicing . Interestingly, it has remained present in all five florideophyte species investigated so far, paralleling the stable retention of tRNA gene introns in streptophytes.Atypical group II introns have been reported in several plastid protein-coding genes of unicellular red algae position . The intsplicing  [30]. Thequences . Given tGracilaria tenustipitata var. liui plastid genome contains two genes, leuC and leuD, that are absent in other plastid genomes. The two genes are intact and encode the large and small subunits of 3-isopropylmalate dehydratase (EC 4.2.1.33) catalyzing an isomerization step in leucine biosynthesis and analogous reactions leuC/D subunits are always expressed in the cytoplasm and targeted to the plastid post-translationally leuC and leuD in the G. tenuistipitata plastid genome led us examine their phylogeny using a broad sampling of their closest homologs from bacteria, archaea and plastids. Both single-protein phylogenies and a phylogeny based on both proteins concatenated yielded a very similar picture: G. tenuistipitata leuC and leuD were consistently and strongly related to a clade of six beta- and gamma-proteobacteria, and only distantly related to the clade containing plastid targeted proteins , a single type of leuC and leuD genes was found, and all of these were closely related to the canonical plastid-targeted type found in land plants  compared to bacterial homologs, altogether indicative of plastid-targeting. Intriguingly, both leuC and leuD were found in diverse red algae including leuD in Gracilaria changii, but no significant homologs were detected in the available transcriptomic data from G. tenuistipitata. It would appear then, that ancestrally and up to at least one member of the Gracilariaceae family (G. changii) red algal plastids use the canonical plastid-targeted leuC/D, but that a second proteobacterial-like leuC/D was later acquired in G. tenuistipitata by horizontal gene transfer into the plastid genome. The plastid leuC/D operon is obviously intact and its product is correctly compartmentalized, i.e. potentially functional. If this operon is indeed expressed, then either a canonical plastid-targeted leuC/D is co-localized with it, or has already been lost and replaced by the novel plastid-encoded leuC/D. To our knowledge, either of these scenarios is unprecedented in organellar evolution.Some clue as to this function may be found in a comprehensive search for d plants . ProteinCruoria sp. and the transcriptome of Porphyridium cruentumGracilaria and Grateloupia  using the maximum likelihood approach. Nonsynonymous substitution rates (dN) and their standard deviation  differed substantially among the plastid genes  and lower than the majority (85–91%) of plastid genes. However, dN was also positively correlated with IQR, suggesting that larger dN is associated with a more unequal dN distances among species, which is an undesirable characteristic for a barcoding or a phylogenetic marker (P<2.2e−16). To reflect this variation in both dN and IQR, and highlight the most suitable candidate genes for test barcoding experiments, we sorted all plastid genes into several dN bins and three categories of relative IQR within each dN bin  and other genes that should be sought first when resolving problematic nodes on the red algal tree of life  have already been shown relatively less sensitive in several florideophyte taxa cpeB previously tested for barcoding callithamnioid red algae cox1 fall into the medium IQR category. Similarly, gene length may not be an important factor if a marker is constructed to span a conserved intergenic region  potentially involved in plastid function. At a more general level, our data demonstrates that plastid genomes are both easily-obtainable using high-throughput shotgun sequencing approaches and promising in resolving red algal relationships, and a genome-wide analysis of gene evolutionary rates identified candidate plastid genes for species barcoding and how they compare to well-established markers, such as cox1 and rbcL.The three complete and one partial plastid genome described here fill a substantial gap in plastid data representation among florideophytes, the most diverse group of red algae. The data reveals that most red algal plastid genomes have retained highly compacted and gene-rich architectures that are slow-evolving are most closely resemble the ancestral DNA of all plastids. They are largely devoid of unique uknown ORFs, and display highly conserved gene order, with an exception in Chondrus crispus was collected in Peggy’s Cove, Nova Scotia, Canada (no specific permissions were required for collection of C. crispus at this location). The plastid genome was identified on four scaffolds in Chondrus nuclear genome project sequence that was assembled using Arachne. The four scaffolds were further assembled with Solexa reads into a single contig (87-fold average coverage) and the joins verified by direct sequencing of PCR-amplified fragments. Collection, DNA extraction, sequencing, and sequence assembly of Calliarthron tuberculosum was described previously Chondrus crispus and Calliarthron tuberculosum were deposited publicly in GenBank . The plastid genome of Grateloupia lanceola was identified on two large contigs  in 454 sequence assembly of total genomic DNA trnF(gaa)-clpC and apcB-atpE) and contained at their very ends complementary sequence that was part of stem-loop structures conserved among all florideophytes. This clearly indicated that a complete circularly-mapping sequence of Grateloupia plastid genome was present in the data that was split at loop regions of two intergenic stem-loop structures suggesting only several bases inside these loop regions are likely to be missing. Several frameshifts at homopolymeric regions in Calliarthron and Grateloupia plastid genomes introduced by the 454-pyrosequencing approach were corrected manually by comparison to other florideophytes. A partial plastid genome of an undetermined species from the Cruoriaceae family was recovered on ten contigs 454 sequence contigs of total genomic DNA Cruoria pellita was recovered from the assembly  leading us to refer to this species as Cruoria sp. Altogether, 10 plastid contigs totalling 106493 base pairs of Cruoria sp. were identified .The gametophyte of Porphyra umbilicalis had an identical gene content and order to that of closely related P. purpurea and was excluded from further analysis. Open reading frames longer than 100 amino acids were annotated. Ten protein frameshifts were detected in the published plastid genomes and corrected by using ambiguous nucleotides  and visualized using ClustalX 2.1  and GIMP image editor.All plastid genomes were annotated manually using Artemis 13.2 leotides . Start ctalX 2.1 . GenomestalX 2.1  and MAUVtalX 2.1 . MAUVE wtalX 2.1  was visuLeuC and leuD sequences were extracted from GenBank protein and EST databases using closest homologs from blastp homology searches. Plastid genome dataset was assembled using custom scripts. Protein datasets were aligned using ‘–localpair’ algoritm in MAFFT 6.857b Porphyridium cruentum (21%) and Cruoria sp. (45%). Phylogenetic inferences were done in RAxML 7.28 cox1, whereas the standard genetic code was used for plastid genes. A total of 2956 pairwise dN comparisons were calculated among the 203 genes by using Codeml implemented in PAML www.r-project.org). The Pearson’s product-moment correlation analysis was implemented to examine the association between median dN and interquartile range (IQR) using R.Figure S1Pyropia. Linearized maps of plastid ribosomal super cluster from the Bangiales Porphyra (top) and Pyropia (bottom). Pyropia has three single gene inversions relative to Porphyra, and all other red algal and secondary red alga-derived plastid genomes.Single gene inversions in (TIFF)Click here for additional data file.Figure S2leuC and leuD individually. numbers at nodes correspond to RAxML rapid boostrap (left) and PhyML SH-aLRT supports (right). The topologies are broadly consistent with the concatenated phylogeny shown in Maximum likelihood phylogenies of (TIFF)Click here for additional data file.Figure S3rbcL, mitochondrial cox1 and 5′ coding region of cox1 (5′cox1). Median is indicated by a solid line, the sample minimum and maximum are indicated by dotted lines and bars outside the box, and outliers are indicated by open circles. For comparison, the positions of rbcL and a dN of 0.5 are highlighted.Plastid gene dN rates. Boxplot is showing relative nonsynonymous substitution rates (dN) and their standard deviation  for all selected red algal plastid genes including (TIFF)Click here for additional data file.Table S1Annotation changes in publicly available red algal plastid genomes.(XLS)Click here for additional data file.Table S2Boxplot statistics of nonsynonymous substitution rates estimated using Maximum likelihood.(XLS)Click here for additional data file.


Reward processing abnormalities have been implicated in the pathophysiology of negative symptoms such as anhedonia and avolition in schizophrenia. However, studies examining neural responses to reward anticipation and receipt have largely relied on instrumental tasks, which may confound reward processing abnormalities with deficits in response selection and execution. 25 chronic, medicated outpatients with schizophrenia and 20 healthy controls underwent functional magnetic resonance imaging using a Pavlovian reward prediction paradigm with no response requirements. Subjects passively viewed cues that predicted subsequent receipt of monetary reward or non-reward, and blood-oxygen-level-dependent signal was measured at the time of cue presentation and receipt. At the group level, neural responses to both reward anticipation and receipt were largely similar between groups. At the time of cue presentation, striatal anticipatory responses did not differ between patients and controls. Right anterior insula demonstrated greater activation for nonreward than reward cues in controls, and for reward than nonreward cues in patients. At the time of receipt, robust responses to receipt of reward vs. nonreward were seen in striatum, midbrain, and frontal cortex in both groups. Furthermore, both groups demonstrated responses to unexpected versus expected outcomes in cortical areas including bilateral dorsolateral prefrontal cortex. Individual difference analyses in patients revealed an association between physical anhedonia and activity in ventral striatum and ventromedial prefrontal cortex during anticipation of reward, in which greater anhedonia severity was associated with reduced activation to money versus no-money cues. In ventromedial prefrontal cortex, this relationship held among both controls and patients, suggesting a relationship between anticipatory activity and anhedonia irrespective of diagnosis. These findings suggest that in the absence of response requirements, brain responses to reward receipt are largely intact in medicated individuals with chronic schizophrenia, while reward anticipation responses in left ventral striatum are reduced in those patients with greater anhedonia severity. The role of reward processing in the pathophysiology of schizophrenia has garnered significant attention in recent years. Aberrant reward processing has been implicated in both positive A number of previous neuroimaging studies have examined reward prediction in schizophrenia. Studies using monetary incentive delay paradigms have shown reduced ventral striatal responses to reward-predictive cues in patients who are unmedicated Work examining brain responses to reward receipt has also revealed alterations in schizophrenia. Some studies have shown blunted striatal reward responses or prediction errors (responses to outcomes that do not match expectation) Importantly, the literature examining reward processing in schizophrenia has largely relied on instrumental learning tasks, in which rewards must be earned via correct and/or rapid response execution. In these tasks, the ability to anticipate a reward depends upon the ability to earn the reward by responding appropriately. This requires not only reward prediction, but also action selection and response execution, any of which may be impaired in schizophrenia. Here, we examined reward prediction in schizophrenia in the absence of requirements for response selection and execution. Using a passive Pavlovian paradigm, we examined functional activation in response to rewarding stimuli and to predictive cues that had been associated with rewards based on pre-scan instructions.Previous work using aversive Pavlovian conditioning has revealed abnormal brain responses among individuals with schizophrenia. Using a task in which colored cues were associated with affectively negative or neutral pictures, Romaniuk et al Participants were 29 stable outpatients with DSM-IV schizophrenia or schizoaffective disorder and 22 healthy controls with no personal or family history of psychosis. All patients were taking antipsychotic medications, which were stable for at least two weeks. Participants were group matched on sex, age, parental education, handedness All participants underwent fMRI while performing a Pavlovian reward prediction task. Subjects were presented with one of two visual cues (pink cross or green circle), one predicting receipt of 75¢ for the trial, and one predicting receipt of 0¢ for the trial. The cues were followed by their predicted outcome 75% of the time, and with the opposite outcome 25% of the time. Participants were informed of the cue-outcome associations before the scan, and were told that the cue usually, but not always, predicted its associated outcome. They were also told that they could keep any money they were awarded during the task. In each trial, the visual cue was presented for 10 seconds, followed by a symbol indicating the outcome (+75¢ or +0¢) for 4 seconds. Inter-trial intervals varied pseudorandomly between 4 and 8 seconds. Participants completed four runs of 16 trials each, for a total of 64 trials (32 per cue type). Participants were paid $25/hour for their time, and were awarded an additional $20 in reward money upon session completion.3 isotopic voxels. Stimuli were presented using PsyScope on a G3 Macintosh, with each trial onset triggered directly by a pulse from the scanner. The MR data was normalized across runs by scaling whole-brain signal intensity to a fixed value and removing the linear slope on a voxel-by-voxel basis to counteract effects of drift 3 voxels, registered to Talairach space using 12-parameter affine transformations, and smoothed with a 6 mm FWHM Gaussian filter.Imaging data was acquired on a 3T Siemens TIM TRIO system with a 12-channel head coil. High-resolution T1 images  and T2 images  were acquired to aid in registration to a common atlas space. Functional images were collected in four runs of 182 frames each using an asymmetric spin-echo echo-planar sequence . Functional runs acquired axial images parallel to the anterior-posterior commissure plane with 4 mmAll functional data was analyzed using in-house software. Data analysis was conducted using general linear models (GLMs) These ANOVAs were used in voxelwise whole-brain and ROI analyses. Whole-brain analyses were corrected for multiple comparisons using a p-value/cluster size threshold of p<.003 (two-tailed) and 13 voxels. This correction factor was determined by Monte Carlo simulations to provide a whole-brain false-positive rate of p<.05 We also conducted correlation analyses between BOLD contrasts and anhedonia scores. In these analyses, Cue Money – No-money and Receive Money – No-money contrasts were created for each subject and correlated with Chapman physical and social anhedonia scores. These correlations were conducted voxelwise, and were corrected using the same small-volume and whole-brain correction procedures described above. To explore whether these relationships were unique to the patient group, regions demonstrating significant correlations were also examined within the control group.Participant demographic and clinical characteristics are presented in Results of the voxelwise ROI and whole-brain analyses are reported in Given past findings with similar paradigms, we had expected to see greater activity for money than no-money cues in striatal regions among controls. Thus, to further explore the nature of the striatal responses within each group, we extracted the mean activation across voxels within caudate, putamen, and nucleus accumbens ROIs  for moneResults of voxelwise ANOVA analyses are presented in In the patient group significant negative correlations were found between Chapman physical anhedonia and the Cue Money – No-money contrast in regions in left ventral striatum and VMPFC . As showAt the time of receipt, significant positive correlations were seen between physical anhedonia and the Receive Money – No-money contrast in left superior and middle frontal gyri . These cp values >0.16). Similarly, antipsychotic dosage failed to correlate significantly with physical or social anhedonia scores . To look for more general medication effects, we also conducted voxelwise ROI and whole-brain correlations between antipsychotic dose and activation for the money – no-money cue and receipt contrasts. There were no regions whose cue-related activity correlated significantly with antipsychotic dosage. For receipt, no regions within the ROI mask correlated significantly with medication dose. Whole-brain analysis revealed two regions that correlated positively with dose, such that higher doses were associated with greater responses to receipt of money versus no-money. One of these regions was within left prefrontal cortex  and the other fell within the lateral ventricles adjacent to caudate .To explore the possibility that antipsychotic medications may have influenced the results, we conducted correlations between medication dose in chlorpromazine equivalents In addition, given past results showing effects of typical vs. atypical antipsychotic medications on striatal anticipatory activation, we wished to determine to what extent our results were affected by medication type. To do this, we repeated our analyses with the 5 subjects taking typical antipsychotics removed . All ANOVA and correlation results reported in The goal of this study was to examine cue- and receipt- related brain activation during Pavlovian reward prediction in schizophrenia, and to examine the relationship between this activation and symptoms of anhedonia and amotivation. Our results demonstrate few activation differences at the group level during reward anticipation and receipt. However, we observed that left ventral striatal and VMPFC activation during reward anticipation in schizophrenia was reduced in patients who are higher in anhedonia. These findings are consistent with a number of studies showing similar results in instrumental paradigms, consistent with the interpretation of these studies as reflecting abnormal reward prediction mechanisms in patients experiencing anhedonia and/or motivational deficits.At the group level, we did not see striatal cue-related activity that differed significantly between groups. Upon ROI analysis, there was a main effect of cue in bilateral caudate with greater activity for money than no-money cues, an effect that was driven by the patient group and was not significant in controls alone. We speculate that this relative lack of differential cue-related activity among controls as compared to published studies may reflect a difference between our passive Pavlovian paradigm and the instrumental paradigms typically used in reward prediction studies. Instrumental tasks require that information in the cue be used to plan and execute a correct motor response, which may enhance cue-related activation as compared to Pavlovian paradigms. Our task, modeled after O’Doherty et al 2002 Outside the striatum, we saw group differences in cue-related activity in the right anterior insula, such that patients showed greater activation to money than no-money cues, while controls showed the opposite pattern. The insula has been implicated in the detection of, and allocation of attention to, salient events Patients and controls showed similar activation at the time of reward receipt. A number of regions including bilateral caudate responded more strongly to money than no-money receipt in both groups, suggesting that brain responses to reward receipt were largely intact in this sample of patients. Similarly, we found greater responses to unexpected than expected outcomes in both groups in several regions of the cognitive control network The results reported here are compatible with some, but not all, aspects of the results reported by Morris et al Overall, our results demonstrate a pattern of largely intact responses to reward receipt throughout the brain at the group level among individuals with schizophrenia. The robust striatal responses to reward receipt among individuals with schizophrenia are consistent with recent studies demonstrating intact outcome responses in this region Correlation analyses revealed an inverse relationship between anticipatory activity in left ventral striatum and VMPFC and Chapman physical anhedonia scores in the patient group. This relationship is consistent with the findings of several reward prediction studies in schizophrenia showing that higher negative symptoms, particularly anhedonia and avolition, are associated with reduced striatal responses to reward-predicting cues We also observed relationships between anhedonia scores and activation at the time of receipt. In left frontal cortex, higher physical anhedonia was associated with decreased responses to nonrewarding outcomes. Because this study did not have a punishment condition and nonreward may have been considered a negative outcome in this context, this relationship may perhaps be thought of as a blunted response to negative outcomes among the more severely anhedonic patients. This finding is similar to past results, where anhedonia was found to relate to a reduced experience of both positive and of negative emotion When considering the relationships between anhedonia and brain activity in this study, it is worth noting that our sample had lower mean anhedonia scores than previous studies in the literature There is ample evidence from basic science research that hedonic experience and motivation are dissociable constructs subserved by different neurobiological systems differential responses between Money and No-money cues, we did see robust responses to cue presentation (irrespective of cue type) in regions including visual cortex, anterior insula, DLPFC, and posterior cingulate Click here for additional data file.Figure S2Caudate, Putamen, and Nucleus Accumbens ROIs used in mean-activation ROI analyses.(TIF)Click here for additional data file.Figure S3Receipt-related activation magnitudes for expected and unexpected rewards and nonrewards in left VLPFC . Magnitudes shown represent mean activation across all voxels in the region.(TIF)Click here for additional data file.Figure S4Cue-related activation for both cue types combined (money and no-money) across both groups (patients and controls). Color scale represents Z value in one-sample t-test.(TIF)Click here for additional data file.Text S1Movement and signal-to-noise ratio in fMRI data.(DOC)Click here for additional data file.Table S1Incremental movement and signal-to-noise ratio in patients and controls.(DOC)Click here for additional data file.Table S2Post-hoc paired t-tests for unexpected versus expected rewards and non-rewards within patient and control groups.(XLS)Click here for additional data file.


We present a rare case of epithelioid hemangioendothelioma (EHE) in the nasal cavity of a 40-year-old Indian male who presented with history of intermittent epistaxis. The lesion was tested for a panel of immunohistochemical markers like vimentin, CD31, CD34, Factor VIII, vascular endothelial growth factor (VEGF) and Ki67. Immunohistochemically, the neoplasm showed striking positivity for vimentin, CD31, CD34 and weak positivity for VEGF; positivity was also noted for Factor VIII especially in the miniature intracytoplasmic vascular lumina. Epithelioid hemangioendothelioma (EHE) is a rare vascular tumor, which was described first in 1982 by Weiss and Enzinger. We herebA 40-year-old Indian male presented with 2 months history of a slowly growing firm swelling in the nasal septum associated with intermittent epistaxis. The patient’s medical history was not significant. The routine general examination was also within normal limits. Grossly, the excised lesional mass was globular with cut section showing capsulated grayish white solid mass of 1 × 1 cm size. Histologically, the hematoxylin–eosin (H–E) stained section showed surface pseudostratified ciliated columnar epithelium, thin fibrous encapsulation and presence of seromucinous glands both within and outside the lesional component. The lesion proper comprised epithelioid plump to spindle cells, with large vesicular nuclei and distinct cell border, mild acidophilic to clear cytoplasm, mild to moderate nuclear pleomorphism and scattered mitotic figures. The neoplastic cells were forming miniature capillary buds or vascular channels with little cytoplasmic lumen and many of them were containing red blood cells . Areas oReticulin staining as well as a panel of immunohistochemical markers were performed to confirm the diagnosis. The reticulin staining highlighted a dense reticulin network surrounding individual tumor cells. Immunohistochemically, the tumor was positive for vimentin, CD31, CD 34  and alsoEHE in the head and neck region is extremely rare. The common locations of EHE include soft tissue of extremities, skin, liver, lung and bone. Very few cases of EHE are described in nasal cavity in the current literature.Among the vascular tumors of nasal cavity, lobular capillary hemangioma is quite common. This circumscribed lesion is composed of lobules of capillaries lined by plump endothelial cells. The stroma in between the lobules is fibromyxoid. Though high mitoses may be observed in the cellular areas, atypical ones are not seen yet.Glomangiopericytoma  has predilection for the nasal cavity and paranasal sinuses. This unencapsulated, cellular tumor is usually well-delineated. It comprises elongated to oval neoplastic cells forming short fascicles and occasional ones even exhibiting storiform pattern. Many interspersed vascular channels with “staghorn” or “antler- like” configuration are seen. Characteristically, they exhibit peritheliomatous hyalinization. Though mild nuclear pleomorphism and occasional mitotic figures may be seen, necrosis is distinctly uncommon.In contrast to the above vascular tumors, EHE is composed of a distinctive type of endothelial cells with abundant eosinophilic cytoplasm and many intracellular vascular lumina producing cytoplasmic vacuolations. There are usually minimal mitoses and pleomorphism. HistologEHE is a low-grade malignancy. The poor expression of proliferation marker Ki67 and weak positivity for VEGF, a dimeric polypeptide growth factor known for its specificity for vascular endothelial cells, are indication of the less aggressive nature of the lesion.6 On occaOur patient is symptom free, 9 months after resection of the tumor. This is an additional case of EHE highlighting the immunohistochemical features and to corroborate the borderline malignant nature of the lesion.


In our previous work, which identified Hsp70 as a modulator of sGC, we determined that protein disulfide isomerase (PDI) bound to an sGC-affinity matrix [Nitric oxide (NO) signalling through activation of soluble guanylyl cyclase (sGC) is key for the physiology of the cardiovascular and neuronal systems. Discrepancies in sGC activation and deactivation y matrix . As sevey matrix ,3.in situ PLA (proximity ligation assay) established that endogenous PDI and sGC also interact in the CRL 2018 smooth muscle cells suggesting that the interaction between sGC and PDI is biologically relevant. Notably, both α1 and β1 sGC subunits of sGC showed potential co-localization with PDI [Using protein chemistry and purified sGC and PDI, we first showed that reduced sGC was modified by oxidized PDI in non-reducing Western blot and that sGC thiol titer was altered under these conditions. Moreover a PDI-Flag resin retains specifically sGC and this complex was resolved in the presence of a thiol-reductant. These data suggested that the mechanism of interaction between sGC and PDI is by mixed-disulfide exchange. In addition, PDI modulated NO-stimulated sGC activity while a PDI mutated in its two Cys active sites did not. A relatively novel method, with PDI .These results underlined the significance of redox-thiol modulation of sGC and support past and current research showing that certain sGC Cys are necessary for such functions as enzyme activation, dimerization, and desensitization by S-nitrosation. A novel Mass Spectrometry analysis is currently applied to identify sGC Cys disulfide bonds and combined with mutational analysis and molecular dynamics simulation should unveil their involvement in interaction with PDI and their relevance as a new mechanism of sGC modulation.


High-throughput sequencing is rapidly becoming common practice in clinical diagnosis and cancer research. Many algorithms have been developed for somatic single nucleotide variant (SNV) detection in matched tumor-normal DNA sequencing. Although numerous studies have compared the performance of various algorithms on exome data, there has not yet been a systematic evaluation using PCR-enriched amplicon data with a range of variant allele fractions. The recently developed gold standard variant set for the reference individual NA12878 by the NIST-led “Genome in a Bottle” Consortium (NIST-GIAB) provides a good resource to evaluate admixtures with various SNV fractions.Using the NIST-GIAB gold standard, we compared the performance of five popular somatic SNV calling algorithms  for matched tumor-normal amplicon and exome sequencing data.We demonstrated that the five commonly used somatic SNV calling methods are applicable to both targeted amplicon and exome sequencing data. However, the sensitivities of these methods vary based on the allelic fraction of the mutation in the tumor sample. Our analysis can assist researchers in choosing a somatic SNV calling method suitable for their specific needs. Somatic point mutation calling from matched tumor-normal patient samples is a critical step for cancer genome characterization and clinical genotyping ,2. Over In light of these challenges, there is a need to systematically evaluate the performance of different SNV calling algorithms to help guide best practices in the research community. Recently, several independent studies compared somatic SNV calling tools -17. For Exome capture sequencing is typically limited to ~60x read depth (due to cost), and therefore using exome reads limits the potential to detect low-allelic-fraction SNVs in the sample, especially those at less than 5%. Detecting low-allelic-fraction SNVs in the sample is often important for early diagnosis, prevention of drug resistance, and detection of residual tumors. Targeted panel sequencing has been widely used to increase SNV detection sensitivity by achieving a much higher median read depth (>500x), even on a bench top sequencer ,19. Amonhttp://www.bioplanet.com/gcat), which provides a community platform for comparing variant calling approaches.Another challenge in comparing variant calling methods is to identify a set of true positive and true negative variants in the sample, ideally at various allele fractions. This task is not straightforward, due to the lack of sufficient available validation data. One standard assessment method is to check the overlap between variant sets as a way to assess the concordance and discrepancies among different calling methods. Alternatively, some researchers have randomly chosen a few candidates for Sanger sequencing or used SNP arrays for validation. However, Sanger sequencing involves high cost and labor, making the generation of large-scale validation data difficult. In addition, some of the low-allelic-fraction SNVs are very difficult to validate by Sanger sequencing or SNP array due to the detection sensitivity of these technologies . To overcome these limitations, the NIST-led “Genome in a Bottle” Consortium (NIST-GIAB) [in silico dilution series analysis using publicly available exome data from NA12878. Our results demonstrate the strengths and weaknesses of each method in calling somatic SNVs at diverse levels of allelic fraction, and also the value of the newly established NIST-GIAB gold standard in facilitating such comparisons.In order to evaluate the performance of popular SNV callers on targeted amplicon sequencing reads, especially for low-allelic-fraction SNVs, we mixed reference DNA from NA12878 with DNA from another 1,000 Genomes Project sample (NA19129) at various ratios, thus creating virtual tumor-normal samples with NA12878 “tumor” variant frequencies ranging from 4% to 100%. We then subjected the dilution series samples to multiplex PCR-based enrichment and Illumina MiSeq sequencing. Here we present a comparison of 5 somatic SNV callers using targeted amplicon sequencing from these virtual tumor-normal samples against the NIST-GIAB gold standard variant set. To explore possible performance differences of the SNV callers on amplicon versus exome sequencing data, we also did a similar Our goal was to evaluate the performance of somatic SNV detection methods from matched tumor-normal samples using amplicon sequencing. For this purpose, we generated a dilution series of samples with 0%, 8%, 16%, 36%, and 100% NA12878 mixed in the control NA19129, to mimic production of artificial tumor sample paired with the 100% NA19129 background as the artificial normal sample. Because the majority of variants in the gold standard are heterozygous , the main allele fractions we evaluated are 4%, 8%, 18%, and 50%. Therefore, we labelled the samples as C04 , C08 (8%), C18 (18%), and C50 (50%) in the following descriptions and results. We used QIAGEN’s GeneRead DNAseq Comprehensive Cancer Gene Panel  for enrichment and library construction in triplicate . Then weWe assessed the sensitivity and specificity of each variant calling algorithm on each virtual tumor sample using the NIST-GIAB gold standard variant set Version 2.15). Our analysis region excludes sites where NA19129 is not a homozygous reference (for region of interest (ROI), see Additional file .15. Our Next we further examined the difference between MuTect and Strelka. We chose to focus on these two methods out of the five because they achieved significantly higher sensitivity at the lowest SNV fraction with similar or lower false positive rates than the other methods. Previous studies have compared the performance of MuTect and Strelka in exome sequencing . As showin silico, mixed the resulting subsets of reads to desired “tumor” sample purities. The resulting in silico mixture series is diluted in the same way as the amplicon sequencing experimental dilution series: at NA12878 unique heterozygous  variant positions over the region of interest, the median variant allele fraction is 4% (8%), 8% (16%), 18% (36%) and 50% (100%), respectively  than we used for amplicon sequencing (NA19129), because exome sequencing reads for NA18489 were generated using the same capture procedure used for NA12878. The exome sequencing data of NA12878 and NA18489 are publicly available, as part of the 1,000 Genomes Project . On the other hand, since sequencing read depth is among the factors affecting false positive rate, most algorithms generate more false positives in amplicon sequencing data. However, we also demonstrate through Strelka and MuTect that further tuning the “joint probability” score away from the default setting can reduce the false positives in amplicon sequencing to levels similar to that for exome sequencing, without substantially compromising sensitivity. In summary, the relative performance ranking among five variant callers examined in hybridization capture-based exome sequencing largely holds true for PCR-based amplicon sequencing. Amplicon sequencing can achieve much higher variant detection sensitivity for low-allelic-fraction SNVs. At the same time, default variant calling settings should be further tuned to dampen false positive rates when analyzing amplicon sequencing data.As the first step in analyzing cancer sequencing data, detecting variants with high sensitivity and specificity is of great importance. Our results highlight the differences in performance between five algorithms when used with targeted amplicon versus exome sequencing data. When analyzing different types of data, use of different algorithms may be appropriate. For example, methods using a hypothesis of Poisson/binomial distribution of alternate allele read depth would deviate from actual exome sequencing data, which display a notable overdispersion in the distribution of alternate allele read depth . In termRegarding individual algorithms, MuTect and Strelka show similarly high sensitivity in detecting low-allelic-fraction SNVs in amplicon sequencing reads, with good specificity. For high-allelic-fraction somatic SNVs, VarScan2 and SomaticSniper perform slightly better than MuTect and Strelka in terms of sensitivity in both amplicon sequencing and exome sequencing data.NaiveSubtract is very straightforward in concept. Using NaiveSubtract, variants detected in the tumor but not the normal sample would be considered putative somatic mutations. Early studies applied such a subtraction method in detecting somatic mutations and demonstrated success to some extent . One draThe performance of VarScan2 is closely related to the minimum allele fraction threshold, because VarScan2 will suppress any mutations below this threshold. We could improve VarScan2 sensitivity after tuning the minimum allele fraction cut-off from the default 0.20 to a lower value, but at the cost of significantly compromised specificity (data not shown). In one recent study , VarScanAlthough we set the parameters to maximize sensitivity, Somatic Sniper’s performance still falls behind other algorithms in calling somatic SNVs of low allelic fraction. However, SomaticSniper did achieve the highest sensitivity in the 100% ‘pure tumor’-normal match. The low sensitivity of VarScan2 and SomaticSniper in calling low-allelic-fraction somatic SNVs is consistent with the results in , which sThe merits of using artificial matched tumor-normal sample mixture series data sets created for calling NA12878 “somatic” SNVs are at least two-fold: (1) the NIST-GIAB gold-standard is a well-defined, high-confidence, large set of SNVs for benchmark evaluation, and (2) this setup allowed us to assess variant calling for somatic SNVs of a wide range of allele fractions from real data. The advantages of the NIST-GIAB gold standard lie in the fact that more sites could be assessed as compared to SNP arrays, which are normally limited to common variants. However, one limitation of our analysis is that a high-confidence variant list for the background germline variants is not available. Therefore, using NA12878 for an artificial “somatic” sample is likely more accurate in evaluating sensitivity than in evaluating specificity. Another limitation is that the NA12878 DNA purchased from Coriell is not exactly the same batch used for the NIST-GIAB variant calls. In addition, one constraint of our artificial matched normal samples is the unmatched error distribution between the “normal” and “tumor” samples. So the virtual tumor-normal matches would potentially reduce the performance of methods relative to performance on real tumor samples that have error distributions more similar to the matched normal sample.In principle, characterizing how performance of different methods depends on factors such as allele fraction is necessary for designing experiments. The complexity and systematic noise from sequencing from tumor-normal matched samples affect each algorithm, but does so in different ways. For systematic comparison, here we used the same aligned read file for all the methods, whereas impact of aligners on the methods would further distinguish their performance. Deeper understanding of the sample properties and the ultimate goals of a cancer research project will make a difference in the choice of algorithms. This work can be extended to address questions regarding detecting variants from small amount of target genome mixed in the control samples. Our work provides insights for the community to assist in choosing sensitive and specific methods for somatic SNV calling, given a specific set of data type and experimental conditions.In this work, we demonstrated that the five somatic SNV calling methods are applicable to both matched tumor-normal targeted amplicon and exome sequencing data. Sensitivities vary with allelic fraction of the mutation in the tumor sample. Our results show MuTect and Strelka achieved the highest sensitivities. Our analysis can assist researchers in choosing a somatic SNV calling method for their specific needs.DNA samples of NA12878 and NA19129 were purchased from Coriell Institute. Sample mixtures were created based on the actual amplifiable DNA in each sample, resulting in 0%, 8%, 16%, 36%, and 100% of NA12878 sample mixed in the NA19129 sample, respectively. We treated the mixed samples at 8%, 16%, 36%, and 100% as the virtual tumor samples and the 0% as the virtual normal sample. Therefore, we produced artificially matched tumor-normal samples to call somatic SNVs. QIAGEN’s GeneRead DNAseq Comprehensive Cancer Gene Panel (Version 1) was used to amplify the target region of interest . Targeted enrichment and library construction were done following manufacturer’s user manual (QIAGEN). Briefly, 160 ng of each virtual tumor sample or virtual normal sample were PCR amplified and purified. For each sample, one quarter of the PCR product was used for constructing a barcoded Illumina DNA library. Libraries were quantified using QIAGEN’s GeneRead DNAseq Library Quant System, and were mixed in equal amount. Illumina MiSeq sequencing was performed following manufacturer’s user manual (Illumina) to generate FASTQ files, resulting in median coverage depth of 500–700x for each sample. Experiments were performed in triplicate. The sequencing reads were aligned to the reference genome using BWA [in silico dilution series. Consequently, the primary NA18489 sequencing results represent the virtual normal sample and the NA12878 dilution series were treated as the matched virtual tumor samples.Whole exome sequencing data of NA12878 and NA18489 were obtained from the 1,000 Genomes Project . Raw reaftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/release/20110521/). For exome sequencing data, the region of interest include 33,886,321 bases, resulting from the intersection between the exome capture regions with at least one read coverage in both samples and the NIST-GIAB high-confidence sites, plus masking 12,974 bp of NA18489 variant sites annotated by 1,000 Genomes Project (ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/release/20110521/). For these regions of interest, the NIST-GIAB gold standard contains a 181 and 9,868 ‘somatic’ SNVs for our targeted-sequencing and exome-sequencing evaluation, respectively  includes 661,684 bases, resulting from the intersection between QIAGEN CCP panel regions and NIST-GIAB high-confidence callable regions ), plus masking 213 bp of NA19129 variant sites annotated by 1,000 Genomes Project (Altogether, we included the following methods for comparison (for corresponding command lines for calling the five algorithms, see Additional file 1. NaiveSubtract — SNVs were called separately from virtual tumor and normal samples using GATK UnifiedGenotyper . For exo2. MuTect — MuTect is a method developed for detecting the most likely somatic point mutations in NGS data using a Bayesian classifier approach. The method includes pre-processing aligned reads separately in tumor and normal samples and post-processing resulting variants by applying an additional set of filters. We ran MuTect under the High-Confidence mode with its default parameter settings. We disabled the “Clustered position” filter and the “dbSNP filter” for the amplicon sequencing reads, and we disabled the “dbSNP filter” for the exome sequencing.3. SomaticSniper — SomaticSniper calculates the Bayesian posterior probability of each possible joint genotype across the normal and cancer samples. We tuned the software’s parameters to increase sensitivity and then filtered raw results using a Somatic Score cut-off of 20 to improve specificity.4. Strelka — Strelka reports the most likely genotype for tumor and normal samples based on a Bayesian probability model. Post-calling filters built into the software are based on factors such as read depth, mismatches, and overlap with indels. We skipped depth filtration for exome and amplicon sequencing data as recommended by the Strelka authors. For the amplicon sequencing reads, we set the minimum MAPQ score at 17 for consistency with the defaults in GATK UnifiedGenotyper. We used variants passing Strelka post-calling filters for analysis.5. VarScan2 — VarScan2 performs analyses independently on pileup files from the tumor and normal samples to heuristically call a genotype at positions achieving certain thresholds of coverage and quality. Then, sites of the genotypes not matched in tumor and normal samples are classified into somatic, germline, or ambiguous groups using Fisher’s exact test. We generated the pileup files using SAMtools mpileup command.The compatibility of the output VCF files between different methods as well as the NIST-GIAB gold standard was examined using bcbio.variation tools and manual inspection. The reported SNP call representations between files are comparable to each other.We used sensitivity and false positive rate as the performance metrics:The command line scripts of variant calling algorithms we used in the paper are included in the additional files.All authors are employees of QIAGEN Sciences. We declare that our employment with QIAGEN did not influence our interpretation of data or presentation of information.YW designed the study, managed the project, wrote the paper, and was responsible for the final submission and revisions of the manuscript. HX designed the study, performed various analyses, generated figures and wrote the paper. JD designed the study, managed the project and wrote the paper. RVS performed various analyses. QP performed the experiments and wrote the paper. All authors read and approved the final manuscript.Supplementary Tables and Supplementary Methods.Click here for file


Glutamine may have benefits during immaturity or critical illness in early life but its effects on outcome end hardpoints are controversial. Our aim was to review randomized studies on glutamine supplementation in pups, infants, and children examining whether glutamine affects outcome. Experimental work has proposed various mechanisms of glutamine action but none of the randomized studies in early life showed any effect on mortality and only a few showed some effect on inflammatory response, organ function, and a trend for infection control. Although apparently safe in animal models (pups), premature infants, and critically ill children, glutamine supplementation does not reduce mortality or late onset sepsis, and its routine use cannot be recommended in these sensitive populations. Large prospectively stratified trials are needed to better define the crucial interrelations of “glutamine-heat shock proteins-stress response” in critical illness and to identify the specific subgroups of premature neonates and critically ill infants or children who may have a greater need for glutamine and who may eventually benefit from its supplementation. The methodological problems noted in the reviewed randomized experimental and clinical trials should be seriously considered in any future well-designed large blinded randomized controlled trial involving glutamine supplementation in critical illness. Amino acids have a crucial role in protein synthesis, trigger signaling cascades that regulate various aspects of fuel and energy metabolism, and serve as precursors for important substrates. Glutamine, the most abundant amino acid in the muscle and plasma of humans traditionally considered a nonessential amino acid, now appears to be a conditionally essential nutrient during stress, injury , or illnOne described hypothesis for the release of glutamine following stress is that it provides a vital fuel source for rapidly dividing cells such as those of the immune system  and gastGlutamine may have benefits during experimental neonatal endotoxemia and in premature infants of very low birth weight (VLBW), who are highly stressed and have low energy and protein reserves . UndergoSepsis is the systemic inflammatory response associated with an infectious insult. It is the leading cause of death in critically ill patients, and the predominant cause of multiple organ dysfunctions that is known to develop in response to infection. Sepsis occurs in over 750,000 patients per year in the United States and exhibits a mortality rate of 28% to 48% depending on the age of the patient . NosocomSpecific nutrients known as pharmaconutrients have demonstrated an ability to modulate the immunologic and inflammatory responses in clinical and laboratory studies. Among these substrates, arginine, glutamine, n-3 fatty acids, and nucleotides are the most relevant and exhibit the greatest immune-modulatory action . AlthougκB is dependent on the phosphorylation and degradation of IKB-α, an endogenous inhibitory molecule that binds to NF-κB in the cytoplasm [κB transcriptional activation and translocation to the nucleus and significantly inhibited IKB-α phosphorylation and degradation in lung cytosolic tissue [Activation of NF-ytoplasm . In an ec tissue . Similarc tissue . Thus, gκB binding [The heat shock response is a highly conserved cellular mechanism that protects against injury and environmental stresses. Intracellular heat shock proteins (hsps) function as molecular chaperones governing protein assembly, folding, or transport and as anti-apoptotic regulators of cell signalling pathways leading to cell death . In addi binding . Heat sh binding . Cell cy binding . κB activation and proinflammatory cytokine release [κB, and inflammatory cytokine response whereas the specific absence of hsp70.1/3 gene expression can lead to increased mortality after septic insult [Experimental evidence suggests that hsp70 expression is required for glutamine's protection against tissue injury and for attenuation of NF- release . In the  release , and in  release . It was c insult . The surc insult .β-N-acetylglucosamine (O-GlcNAc) modification and subsequently to increase levels of endonuclear HSF-1 expression and HSF-1 transcription activity [A significant body of preexisting literature has hypothesized a relationship between hsp70 expression and glutamine's protection in both in vitro and in vivo settings –44. Glutactivity . The molactivity . It has activity , causingactivity . κB DNA-binding activity were suppressed, ARDS was attenuated and survival improved [Pioneer studies showed that glutamine supplementation could attenuate lethal heat and oxidant injury and increase hsp72 expression in intestinal epithelial cells (IEC-6 cells) –55. The improved . Similarimproved . Marked attenuation of tissue metabolic dysfunction was observed after glutamine administration as measured by lung tissue adenosine 5′-triphosphate/adenosine 5′-diphosphate ratio and the oxidized form of nicotinamide adenine dinucleotide . Furtherα-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, the heat stress-induction of hsp70 and hsp25 was significantly decreased even in the presence of glutamine [Glutamine is a substrate for polyamine synthesis and stimulates the activity of ornithine decarboxylase (ODC), a key enzyme for polyamine synthesis, in intestinal epithelial cells. In a recent experimental work, polyamines  and their precursor ornithine mediated the induction of hsp expression in IEC-18 rat intestinal epithelial cells . As prevlutamine . Ornithilutamine .Recent work demonstrated that febrile-range temperatures achieved during sepsis and noninfectious SIRS correlated with detectable changes in stress gene expression in vivo (whole blood messenger RNA), thereby suggesting that fever can activate hsp70 gene expression and modify innate immune responses . In addiCritically ill patients display variable physiologic responses when stressed; gene association studies have recently been employed to explain this variability. Genetic variants of hsp70 have also been associated with the development of septic shock in patients , 70. TheDrug interactions were also shown to either suppress hsp protective effects exacerbating therefore drug-induced side effects or to induce hsp beneficial effects by suppressing drug-induced exacerbations. Thus, it was recently shown that bleomycin-induced pulmonary fibrosis is mediated by suppression of pulmonary expression of hsp70 whereas an inducer of hsp70 expression, such as geranylgeranylacetone, may be therapeutically beneficial for the treatment of gefitinib-induced pulmonary fibrosis . Despite the enthusiastic experimental results, suggesting a beneficial effect for the immune-enhancing glutamine, systematic reviews and meta-analyses of randomized studies failed to show any definite benefit of pharmacononutrition in the critically ill adult . Most ofμg/day) or both, for up to seven days [In a multicenter, prospective, double-blind, randomized trial total parenteral nutrition (PN) supplemented with alanine-glutamine in intensive care unit patients was associated with a reduced rate of infectious complications and better glycemic control . In anotven days . The priThe search methods for identification of studies consisted of searches of PubMed database using the search terms: “glutamine” and “critical illness,” or “sepsis,” or “endotoxemia.” The search output was limited with the search filter for ages: pups, infants, and children. References in selected studies were examined also. The title and abstract of all studies identified by the above search strategy were screened and the full text for all potentially relevant studies published in English was obtained. The full text of any potentially relevant studies was assessed by the two authors . The same authors extracted data from the published studies. Study populations, main methodological data and results on outcome are presented in Tables 2, mL/kg/h) of endotoxic suckling rats [Using light and transmission electron microscopy in artificially reared rat pups the greatest blunting of villus height in the ileum and the lowest number of villi per unit length of bowel were in the animals that were treated with inhibition of glutamine and not provided with dietary glutamine . Transmiing rats . Glutamiing rats . Also, ging rats . Importaing rats . Glutamine appears to be safe for use in premature infants and seems to be conditionally essential in premature infants with extremely low birth weights . In ELBWIn a large multicenter, randomized, double-masked, clinical trial in ELBW infants, the safety and efficacy of early PN supplemented with glutamine in decreasing the risk of death or late-onset sepsis were assessed; infants 401 to 1000 g were randomized within 72 hours of birth to receive either TrophAmine (control) or an isonitrogenous study amino acid solution with 20% glutamine whenever they received PN up to 120 days of age, death, or discharge from the hospital . Of the bifidobacteria, lactobacilli, E. coli [Double-blind randomized trials in VLBW infants did not show that enteral glutamine supplementation decreases morbidity or mortality , 103. Bl E. coli  and did  E. coli . Importa E. coli . Studied E. coli . Similar E. coli , 107.Followup of all surviving VLBW infants having received enteral preterm formula or breast milk supplemented with glutamine showed a lower risk of atopic dermatitis but no differences in incidence of bronchial hyperactivity, infections of upper respiratory, lower respiratory, urinary, or gastrointestinal tracts , 109, ofIn a multicenter randomized, double-blinded, comparative effectiveness trial, zinc, selenium, glutamine, and intravenous metoclopramide conferred no advantage in the immune-competent population of children requiring long-term intensive care compared with whey protein supplementation . FurtherIn a blinded, prospective, randomized, controlled clinical trial in critically ill children given an immune-enhancing formula supplemented with glutamine, a favorable effect on nutritional indices and antioxidant catalysts was reported, but none on outcome hard endpoints . Also, aGiven the encouraging experimental results, the absence of glutamine-related adverse effects, and the immune cells need for glutamine to grow and multiply, it has long been hypothesized that high risk patients could benefit from the use of this highly promising pharmaconutrient ; furtherStudies evaluating the effect of specific pharmaconutrients in premature infants and critically ill children are scarce and are insufficient to allow recommendations to be made. Recent evidence from studies in various pediatric diseases and in premature newborns remains inconsistent, partly because of the different effects of enteral and parenteral glutamine supplementation, inefficient treatment doses , co-admiOnce again, laboratory research preliminary beneficial effects failed to be reproduced in clinical randomized (and/or blinded and masked) studies. The lack of beneficial effect of glutamine supplementation in sepsis might be explained by any of the factors influencing glutamine protective role as described above. Various factors during the clinical course might have also influenced results of those studies. In addition, by using isonitrogenous controls (to ensure the specific effect of glutamine), the overall amino acid intake may have been inadequate in the glutamine group, as a consequence of the substitution of 20% of the standard amino acids with glutamine. Although adding more amino acids to the control groups might have prevented the removal of amino acids from the glutamine supplement, the isonitrogenous control groups would have then received more amino acid/nitrogen than the recommended allowances. On the other hand, enteral supplementation of glutamine seems to be locally used by the intestine but may not help entering the systemic circulation to enhance the immune response. All these different research methods along with the use of different feeding guidelines for the introduction or withholding parenteral and enteral feeds among institutions make comparisons between studies difficult . Further


Meningioma is the most common nonglial intracranial primary tumor. It is a slowly growing tumor and presents clinically by causing seizures along with neurological or neuropsychological deficit. However, acute presentation of meningioma is possible. We are reporting a case of cerebral infarction due to a sphenoid wing meningothelial meningioma (with progesterone receptor positivity) leading to an occlusion of the middle cerebral artery (MCA) in a 30-year-old right-handed woman (1 month after childbirth). After surgery, no new neurological event occurred, and she recovered most of her neurological functions. Strokes due to meningioma are a highly rare clinical occurrence but should be given serious consideration, particularly in young patients. Meningioma is the most common nonglial intracranial primary tumor, accounting for 13 to 26% of all primary intracranial tumors  and are We report on a 30-year-old right-handed woman with a personal history of migraine admitted to the Department of Neurology, 1 month after childbirth, for acute onset of headache and complete paresis of her left hemibody, without sensory loss; National Institutes of Health Stroke Scale (NIHSS) was 10. Laboratory examination found no abnormality. T1-weighted magnetic resonance (MR) imaging with gadolinium disclosed a right sphenoid wing meningioma measuring 35 mm in diameter . DiffusiIf encasement and intracranial arterial obstruction are common in cases of meningioma, strokes only rarely occur in association with this slowly growing tumor. In most of these cases, the different manifestations constitute a transient neurological deficit mimicking transient ischemic attacks probably due to hemodynamic stenoses. It has been suggested that meningiomal compression of the carotid artery may produce transient neurological symptoms including loss of consciousness, hemiparesis, paresthesia, and global transient amnesia . HoweverA compressive mechanism is usually suggested as an explanation for this phenomenon. In fact, meningiomas involving the cavernous sinus commonly lead to encasement and narrowing of the internal carotid artery (8 to 100% of cases) . However