We report a case of invasive pulmonary aspergillosis (IPA) following respiratory syncytial virus infection in an allogeneic hematopoietic stem cell transplant (HSCT) recipient with chronic graft-versus-host disease. Delayed diagnosis of IPA resulted in the development of a pneumothorax, a rare consequence of fungal pneumonia. Respiratory virus infections are often harbingers of other infective organisms in HSCT recipients. More aggressive diagnostic investigations such as computed tomography scans of the thorax and bronchoscopy with bronchoalveolar lavage should be considered early in any HSCT patient presenting with respiratory virus pneumonia, particularly if atypical features are present or recovery is delayed. Pulmonary complications develop in 40% to 60% of hematopoietic cell transplant (HSCT) recipients and are recognized as major causes of morbidity and mortality.Aspergillus spp.Community-acquired respiratory viruses (CRVs) such as respiratory syncytial virus (RSV), parainfluenza, and influenza viruses are important causes of acute infectious pulmonary complications, occurring in 15% to 30% of HSCT recipients each year. One-third to one-half of these is due to RSV. The increased mortality seen in RSV infections in this patient population is attributed to both its propensity for causing life-threatening infections in HSCT recipients as well as to a long-term decline in respiratory function in survivors of acute infection.A 40-year-old male of Chinese ethnicity received allogeneic filgastrim-mobilised peripheral blood HSCT from his HLA 6/6 matched sister using reduced-intensity conditioning regimen. He was in his first complete remission from underlying B-cell prolymphocytic leukemia that had been diagnosed 6 months earlier. The course of transplantation was complicated by steroid-refractory grade III acute GVHD of the liver that began 7 months post-transplantation, requiring intensive immunosuppressive therapy with prednisolone followed by subsequent addition of other immunosuppressive agents including tacrolimus, mycopheolate mofetil and sirolimus for steroid-sparing purposes. He continued to receive dapsone and acyclovir prophylaxis, but was not on any antifungal prophylaxis.In December 2007, at 9 months post-transplantation, he presented with acute-onset dry cough and shortness of breath following close contact with a client who had an upper respiratory tract infection (URTI). The initial chest X-ray was unremarkable with no evidence of pneumonia. His symptoms worsened over the ensuing few days, culminating one week later in acute respiratory failure requiring hospitalization and non-invasive mechanical ventilation in the intensive care unit (ICU). The patient was afebrile, with bilateral respiratory rhonchi on chest auscultation. Laboratory studies showed normal blood counts, renal and liver function. A chest x-ray  showed pHigh resolution computed tomography (HRCT) of the thorax  showed bAll microscopy and cultures of clinical samples were negative for aerobic bacteria, acid-fast bacilli or fungi. Reverse-transcriptase polymerase chain reactions (PCR) performed on nasopharyngeal specimens were positive for RSV, although viral culture from the same swab was negative. Serial serum galactomannan screening and quantitative real-time PCR for cytomegalovirus DNA from peripheral blood were all negative. Diagnostic bronchoalveolar lavage (BAL) was not performed at this point owing to the concern of worsening hypoxemia.The patient’s overall condition improved gradually over one week, with reduced oxygen requirement and dyspnea. However, a repeat HRCT thorax  4 weeks Aspergillus fumigatus. He was prescribed voriconazole, which was continued for 6 months. Serial CT thorax showed gradual regression of the left pulmonary cavitary lesion and complete resolution of the ground grass changes. His respiratory symptoms improved despite receiving continued immunosuppressive therapy for his ongoing extensive chronic GVHD.However, he was re-admitted 10 days later with acute onset right pleuritic chest pain, dyspnea and diaphoresis. Chest x-ray  and CT tIPA and respiratory syncytial virus (RSV) pneumonia are important causes of morbidity and mortality in allogeneic HCT recipients. The presence of GVHD and immunosuppression are the main predisposing risk factors for the development of both conditions.3RSV infection in HSCT patients generally presents with the signs and symptoms of a URTI. This may progress to bronchiolitis, pneumonitis, and pneumonia. The frequency of progression of URTI to pneumonia was highest in patients who were <1 month post-transplant or pre-engraftment.2The clinical pulmonary syndromes associated with IPA in allogeneic HSCT recipients are generally similar and usually rapidly progressive, with initial symptoms being that of fever, chest pain, cough, and/or hemoptysis. Respiratory failure can develop with progressive growth of mould and inflammation, which can result in necrotic debris and pulmonary infarction.5The diagnosis of IPA is often made late in the course of disease due to the lack of sensitive and reliable diagnostic techniques. The clinical manifestations are often non-specific. While radiological findings can be suggestive, none are pathognomonic, and cultures of respiratory samples lack sensitivity.7Aspergillus fumigatus was the most common co-pathogen, accounting for 13 of the 29 patients with dual infections, and was associated with higher mortality rate. In another report from the same center, Marr and co-workers showed that patients with respiratory virus infections after day 40 post-HSCT also had a 2.1-fold increased risk of subsequent IPA.9Our case lends weight to previously published reports suggesting that respiratory virus infections may play a role in the pathogenesis of bacterial or fungal pneumonia in HSCT recipients.Mechanisms that can explain the association between respiratory virus and fungal infections have not been elucidated. Potential contributing factors include GVHD and the immunosuppressive agents used to treat this condition such as steroids and calcineurin inhibitors. It is possible that RSV may itself predispose patients to such infections by damaging the respiratory epithelium and allowing other organisms to colonize and invade. Other potential mechanisms include the immune-modulating effects of the virus itself: RSV can impair multiple antimicrobial effector mechanisms, including inhibition of effector T-cell function as well as immunologic memory development in the respiratory tract, and also diminished microbicidal activity of alveolar macrophages.2In conclusion, our case provides further evidence regarding the direct association between RSV infections and aspergillosis in HCT recipients. As respiratory virus infections are often harbingers of other infective organisms, more aggressive diagnostic investigation such bronchoscopy with BAL should be considered early in any HCT patients presenting with respiratory virus pneumonia. Further studies regarding the interaction of RSV with other post-transplant infections are needed.


Pseudomonas viridiflava strain UASWS38, a bacterium species pathogenic to the biological model plant Arabidopsis thaliana but also usable as a biological control agent and thus of great scientific interest for understanding the genetics of plant-microbe interactions.We report here the first whole-genome shotgun sequence of  Pseudomonas viridiflava is a pectinolytic bacterium member of the Pseudomonas syringae group  and incompatible (resistance) responses , which share many gene homologs, have been described for idiflava , 7.P. viridiflava was shown to display a high level of genetic variation worldwide, with all isolated P. viridiflava strains parting into two distinct and deeply diverged clades, with evidence of frequent recombination but little geographic differentiation .This whole-genome shotgun project was deposited at GenBank under the accession no.


Single nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies. In order to provide the best genome coverage for the analysis of performance and production traits, a large number of relatively evenly distributed SNPs are needed. Gene-associated SNPs may fulfill these requirements of large numbers and genome wide distribution. In addition, gene-associated SNPs could themselves be causative SNPs for traits. The objective of this project was to identify large numbers of gene-associated SNPs using high-throughput next generation sequencing.Transcriptome sequencing was conducted for channel catfish and blue catfish using Illumina next generation sequencing technology. Approximately 220 million reads (15.6 Gb) for channel catfish and 280 million reads (19.6 Gb) for blue catfish were obtained by sequencing gene transcripts derived from various tissues of multiple individuals from a diverse genetic background. A total of over 35 billion base pairs of expressed short read sequences were generated. Over two million putative SNPs were identified from channel catfish and almost 2.5 million putative SNPs were identified from blue catfish. Of these putative SNPs, a set of filtered SNPs were identified including 342,104 intra-specific SNPs for channel catfish, 366,269 intra-specific SNPs for blue catfish, and 420,727 inter-specific SNPs between channel catfish and blue catfish. These filtered SNPs are distributed within 16,562 unique genes in channel catfish and 17,423 unique genes in blue catfish.For aquaculture species, transcriptome analysis of pooled RNA samples from multiple individuals using Illumina sequencing technology is both technically efficient and cost-effective for generating expressed sequences. Such an approach is most effective when coupled to existing EST resources generated using traditional sequencing approaches because the reference ESTs facilitate effective assembly of the expressed short reads. When multiple individuals with different genetic backgrounds are used, RNA-Seq is very effective for the identification of SNPs. The SNPs identified in this report will provide a much needed resource for genetic studies in catfish and will contribute to the development of a high-density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for genome association studies and whole genome-based selection in catfish. Single nucleotide polymorphisms (SNPs) are alternative bases at any given position of DNA. They are among the most abundant type of genetic variations and widely distributed within genomes. Theoretically, SNPs can have four alleles in the population, but they most often exist as bi-allelic markers. Because of their potential for high genotyping efficiency, automation, data quality, genome-wide coverage and analytical simplicity , SNPs haIn species where the whole genome has been sequenced, SNPs have been identified from genome sequencing efforts. In most cases, SNPs were identified by sequence variations between the two alleles of a single diploid individual whose genome was sequenced . More reTo identify larger numbers of gene-associated SNPs, higher throughput expressed sequence reads are needed to increase coverage and depth and ensure sequence accuracy. Next generation sequencing technologies such as Roche/454, Illumina/Solexa, and ABI/SOLiD sequencing platforms are particularly adapted to producing high coverage of expressed sequences within contigs . TranscrIctalurus punctatus) and blue catfish (I. furcatus) in order to identify gene-associated SNPs for the development of SNP arrays in catfish.While the 454 sequencing technology has been widely used for transcriptome analysis, Illumina sequencing technology is being gradually accepted for its dramatically improved sequencing throughput and quality ,24. PairIllumina sequencing was conducted to generate short sequence reads of expressed sequences. Two cDNA libraries were made from pooled RNA samples prepared from a total of 11 tissues of 47 channel catfish and 19 blue catfish, respectively, representing major strains used in commercial production. The cDNAs were sequenced with one lane each using Illumina GA-II and Illumina HiSeq 2000 that generated 48.6 million 36-bp paired-end reads and 173.9 million 100-bp paired-end reads for channel catfish, and 66.9 million 36-bp paired-end reads and 216.6 million 100-bp paired-end reads for blue catfish . As shown in Table Assembly of the expressed short reads was conducted in several ways. First, reference assemblies of channel catfish expressed short reads and blue catfish expressed short reads were conducted separately using all existing catfish ESTs as a reference. Such assemblies would allow establishment of contigs for channel catfish expressed short reads and blue catfish expressed short reads separately to allow identification of intra-specific SNPs that are anchored (scaffold) by longer EST reference sequences. Such an assembly is superior to the total de novo assembly of these remaining reads was conducted. As shown in Table de novo, generating 420,165 contigs and 420,953 contigs for channel catfish and blue catfish, respectively. However, the average contig length was much shorter than those in the reference assembly, with 298 bp and 315 bp for channel catfish and blue catfish, respectively. These contigs are also useful resources for the identification of intra-specific SNPs.Despite generating an efficient reference assembly, over 66 million channel catfish reads and 90 million blue catfish reads were not assembled with the reference assembly. These reads could represent additional genes that were not represented by the EST reference sequences, or they could come from gene regions that were not represented by the EST references. In order to make them useful resources for SNP identification, de novo assemblies were conducted using combined channel catfish and blue catfish expressed short reads in order to identify inter-specific SNPs. A total of 493.4 million expressed short reads from both channel catfish and blue catfish  were used. The reference assembly of all catfish expressed short reads placed 336.0 million reads (68%) into 104,870 contigs, with an average contig length of 686 bp. Similarly, the de novo assembly of all catfish expressed short reads generated 421,229 contigs, with an average contig length of 340 bp  had BLAST hits to the Uniprot database, and matched 17,766 unique protein accessions. As expected, a lower percentage of the contigs from de novo assembly had BLAST hits to Uniprot proteins. Of the 421,229 contigs, 24,168 (5.7%) had BLAST hits to the Uniprot database, with matches to 12,331 unique proteins. Altogether, of the 526,099 contigs, 56,518 (10.7%) had significant BLAST hits to the Unitprot database, and matched 24,440 unique protein accessions. Larger numbers of contig hits but fewer matches to unique proteins were observed when compared to the zebrafish Refseq protein database  annotations in catfish was compared with that of zebrafish.The unique genes from the catfish and the zebrafish annotated database were analyzed using generic GO-slim terms with Blast2GO ,27. The As summarized in Table Our previous research suggested that SNPs identified from contigs with at least four sequences at the SNP sites with the minor allele being represented at least twice are more reliable . In thisSince the information on minor allele frequency (MAF) is an important consideration in choosing which SNPs to be included in SNP arrays, the minor allele frequencies of SNPs in the discovery populations were estimated from the sequence data. As shown in Figure While the number of SNPs is important, their distribution in contigs and genes within the genome is also important when used for genetic analysis. A total of 168,458 channel catfish contigs and 190,197 blue catfish contigs were found to contain putative filtered SNPs, of which 13,414 contigs contain SNPs at same positions in both channel catfish and blue catfish. The number of unique Uniprot accessions hit by contigs containing SNPs was 16,562 for channel catfish, and 17,423 for blue catfish, suggesting that putative filtered SNPs were identified from the vast majority of catfish genes.One important aspect of using the inter-specific hybrid system is to identify inter-specific SNPs. From this work, a total of 232,972 contigs were identified to contain 420,727 inter-specific SNPs, i.e., sequence variations between the two species, channel catfish and blue catfish. These SNPs were from at least 18,085 distinct genes as determined by unique hits to the Uniprot protein database , produced significant hits to 6,674 previously uncaptured genes, and covered thousands of additional transcript regions currently without annotation.A second challenge is the over representation of highly expressed gene tags in transcriptome analysis. As shown in Figure Pooling of RNA samples from multiple individuals followed by transcriptome analysis using next generation sequencing is among the most efficient methods for SNP identification. Through many years of efforts, a total of approximately 303,000 putative catfish SNPs were previously identified . HoweverOne major challenge for SNPs is the problem caused by paralogous sequence variants (PSVs) and multisite sequence variants (MSVs) . PutativGiven the large numbers of SNPs generated that meet these minimal requirements, more stringent parameters can be applied in picking SNP sets for different applications. Average depth at putative SNP positions is greater than 100 sequences, providing high confidence in accuracy of identified SNPs within the pooled samples. Re-sequencing or limited validation of these samples by low-throughput SNP genotyping is costly and is unlikely to generate additional information. Ultimately, SNPs need to be validated by genotyping in a variety of reference mapping families and trait-selected populations using a high-density screening array. In catfish, the use of homozygous gynogenetic catfish  as contrGenome-wide association studies of complex traits require a large number of SNPs. However, for research communities focused on non-model organisms, it is cost-prohibitive to genotype all SNPs in an association study with the throughput of current technologies. Selection of uniformly distributed SNPs across the genome for association studies is therefore very important . Gene-asThe approach to sample animals of diverse genetic backgrounds and sequence to sufficient depth for reliable SNP identification allowed the ability to detect many common SNPs across the entire genome. We have demonstrated that transcriptome analysis of pooled RNA samples from multiple individuals using Illumina sequencing technology is both technically efficient and cost-effective for generating expressed sequences. Such an approach is most effective when coupled to existing EST resources generated using traditional sequencing approaches because the reference ESTs facilitate effective assembly of the expressed short reads. The SNPs identified in this report will provide a much needed resource for genetic studies in the catfish scientific community and will contribute to the development of high density, cost-effective genotyping platforms. Validation and testing of SNPs using high-density arrays will subsequently lead to the production of a SNP array with well-spaced SNPs providing a powerful genotyping tool for the study of performance and production traits in catfish.Channel catfish of 47 individuals from five different aquaculture populations/fingerling sources  and blue catfish of 19 individuals from two different strains (7 Rio Grande and 12 D&B) were used for this study. Samples of 11 tissues including brain, gill, head kidney, intestine, liver, muscle, skin, spleen, stomach, heart, and trunk kidney were collected. The fish were euthanized with tricaine methanesulfonate (MS 222) at 300 mg/l before tissue collection. Tissue samples from each species were collected, pooled, immediately placed in 5 ml RNA later™  and kept at 4°C for 2-4 days until RNA extraction. Equal weight of each tissue from individuals of each species were combined, ground to a fine powder with mortar and pestle in the presence of liquid nitrogen and thoroughly mixed. A fraction of the tissue samples was used for RNA isolation. Total RNA was isolated using the RNeasy plus Mini Kit  with DNase I  treatment following the manufacturer's protocol.Sequencing was conducted commercially in HudsonAlpha Genomic Services Lab . Briefly, 100 ng of total RNA was used to prepare amplified cDNA using Ovation RNA-seq, a commercially available kit optimized for RNA sequencing . The produced double-stranded cDNA was subsequently used as the input to the Illumina library preparation protocol starting with the standard end-repair step. The end-repaired DNA with a single 'A'-base overhang is ligated to the adaptors in a standard ligation reaction using T4 DNA ligase and 2 μM-4 μM final adaptor concentration, depending on the DNA yield following purification after the addition of the 'A'-base. Following ligation, the samples were purified and subjected to size selection via gel electrophoresis to isolate 350 bp fragments for ligation-mediated PCR (LM-PCR). Twelve cycles of LM-PCR were used to amplify the ligated material in preparation for cluster generation. For each species of channel catfish and blue catfish, the prepared cDNA library was sequenced with 36-bp paired-end reads on one flow cell lane of the Illumina Genome Analyzer II platform and 100-bp paired-end reads on one flow cell lane of the Hiseq 2000 platform, respectively. The image analysis, base calling and quality score calibration were processed using the Illumina Pipeline Software v1.4.1 according to the manufacturer's instructions. Reads were exported in the FASTQ format and has been deposited at the NCBI Sequence Read Archive (SRA) under accession number SRA025099.de novo assembly. A reference-based assembly was firstly executed using a set of catfish unique sequences generated from ~500,000 Sanger-ESTs of both channel and blue catfish as a reference. For the reference assembly, the default local alignment settings were used to rank all potential matches, with mismatch cost of 2, deletion cost of 3 and insertion cost of 3. The highest scoring matches that shared ≥ 80% similarity with the reference sequence across ≥ 50% of their length were included in the alignment. This permissive alignment ensured that even reads derived from highly mutated orthologs between channel catfish and blue catfish would not be discarded. Reads that were not assembled into contigs in the reference assembly were entered into a subsequent de novo assembly with a higher stringency minimum match similarity (90%). Three separate assemblies were generated: channel catfish assembly, blue catfish assembly, and all catfish assembly . The raw reads were cleaned by trimming of adaptor sequences, ambiguous nucleotides ('N' in the end of reads) and low quality sequences with average quality scores less than 20. Trimmed reads less than 15 bp were also discarded from further analysis, the remaining reads were used in subsequent assembly. The approach of assembly in this study was based on a combination of reference assembly and y Figure .Unique consensus sequences from the all catfish assembly were compared against the Uniprot database and the zebrafish Refseq protein database (NCBI) using BLASTX  to obtain the putative gene identity. To estimate the proportion of annotated contigs that matched to unique genes in the known protein database, all BLASTX hits were filtered for redundancy in protein accessions. Assignment of Gene Ontology terms to annotated unique sequences was conducted using the program Blast2GO . OntologAssembled contigs were scanned for SNPs utilizing SNP detection software included in CLC Genomics Workbench . The central base quality score of ≥25 and average surrounding base quality score of ≥20 were set to assess the quality of reads at positions for SNP detection. Under the criteria of minimum coverage (read depth) of four and the minimum variant frequency of two, the variations compared to the reference sequence were counted as SNPs. Three lists of SNPs were generated from channel catfish, blue catfish and all catfish assembly, respectively. The identification of intra-specific SNPs for both channel and blue catfish, and inter-specific SNP between channel and blue catfish was achieved by comparing these three lists of SNPs. Inter-specific SNPs were defined as those that have sequence variations between channel catfish and blue catfish, but no sequence variations within channel catfish or within blue catfish; similarly, intra-specific SNPs were identified within channel catfish or within blue catfish; and intra-specific SNPs for both channel catfish and blue catfish were identified within both channel catfish and blue catfish at the same SNP position.All the unique sequences were used to search for microsatellite makers using Msatfinder  with a rIn order to identify quality SNPs, putative SNPs identified as mentioned above were further screened following specific criteria based on the read depth, minor allele frequency, the quality of flanking regions and absence of other SNPs within 15-bp flanking regions: only those SNPs with minor allele sequences representing no less than 10% of the reads aligned at the polymorphic loci were declared as quality SNPs; no extra SNPs or indels within 15-bp flanking regions were allowed; SNPs located in repetitive regions were also not considered. Potential repetitive elements were detected by RepeatMasker , SNPs loSL prepared the samples, conducted the bioinformatic analysis and was involved in writing the manuscript. ZZ, JL, FS, SW, HL, YJ, LK and HK were involved in one or more processes of samples collection, RNA extraction or bioinformatic analysis. EP provided assistance for fish collection, data analysis and manuscript preparation. ZL supervised the entire study and provided assistance for data analysis and manuscript preparation. All authors read and approved the final manuscript.Length distribution of contigs from the all catfish assembly with hits to the Uniprot database.Click here for fileCategorization of different types of SNPs identified from the all catfish assembly. (1) Intra-specific SNPs identified from positions where there were SNPs within channel catfish, but not within blue catfish; (2) Intra-specific SNPs identified from positions where there were SNPs within blue catfish, but not within channel catfish; (3) Intra-specific SNPs identified from positions where there were SNPs within both channel catfish and blue catfish; (4) Inter-specific SNPs identified from positions where there were no SNPs in channel catfish or in blue catfish, but the sequence differed between the two species; (5) Intra-specific SNPs identified from positions where there were SNPs within channel catfish and there were fewer than four blue catfish sequences; (6) Intra-specific SNPs identified from positions where there were SNPs within blue catfish and there were fewer than four channel catfish sequences. Intra-specific SNPs in channel catfish = (1) + (3) + (5); Intra-specific SNPs in blue catfish = (2) + (3) + (6); Intra-specific SNPs shared by the two species = (3); Inter-specific SNPs between the two species = (4).Click here for file


Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. Seven custom SNP-assays (384 and 1536-plex), oriented towards broad applications, have been designed. We illustrated here the usefulness of this genotyping technology to address specific questions related to i/ genetic diversity and population structure analysis, ii/ linkage and QTL mapping and iii/ association mapping.Single Nucleotide Polymorphisms (SNPs) are the most abundant form of genetic variation in the genome. In this poster, we reviewed what has been achieved in terms of highly-multiplexed SNP genotyping assay construction in maritime pine  allowed to obtain less blurred, albeit similar, breeding zone boundaries than a set of 12 nuSSRs screened on the same individuals. Levels of diversity were also more accurately estimated, showing clear differences among gene pools. Interestingly, a relatively small subset of SNPs would be enough to develop an application tool for origin certification, which could have a notable impact on current operational practices.Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. Moreover, QTL detection for different traits is underway.In terms of linkage mapping, a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (in vitro SNPs) and from Sanger-derived Expressed Sequenced Tags (in silico SNPs) was established. Offspring from two mapping pedigrees were genotyped. A consensus map comprising 357 SNPs from 292 different loci was constructed and the analysis of sequence homology between mapped markers and their orthologs in a In terms of association mapping, a custom GoldenGate assay with 384 SNPs was built and used to genotype 160 unrelated plus-trees from a half-sib experimental design for which breeding values  were available. Taking into account multiple testing, one single SNP in a gene encoding a putative fasciclin-like arabinogalactan protein was found to be associated with growth traits.We conclude that the VeraCode/GoldenGate assays can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This first generation of SNP-arrays has been recently upgraded to an Infinium-array (containing 10.5k SNPs) thanks to deep sequencing based on new generation sequencing technologies. The Infinium-array also includes SNPs from comparative orthologous sequences with other major conifer species, providing a wider collection of anchor points for comparative genomics among these major groups of forest trees.


A receptor modulators such as zolpidem (ZOL) induce sleep through general inhibition of neural activity. To test the hypothesis that less functional impairment results from HcrtR antagonist-induced sleep, we evaluated the performance of rats in the Morris Water Maze in the presence of ALM vs. ZOL. Performance in spatial reference memory (SRM) and spatial working memory (SWM) tasks were assessed during the dark period after equipotent sleep-promoting doses  following undisturbed and sleep deprivation (SD) conditions. ALM-treated rats were indistinguishable from vehicle (VEH)-treated rats for all SRM performance measures  after both the undisturbed and 6 h SD conditions. In contrast, rats administered ZOL showed impairments in all parameters measured compared to VEH or ALM in the undisturbed conditions. Following SD, ZOL-treated rats also showed impairments in all measures. ALM-treated rats were similar to VEH-treated rats for all SWM measures  after both the undisturbed and SD conditions. In contrast, ZOL-treated rats showed impairments in velocity and in the time to locate the platform. Importantly, ZOL rats only completed the task 23–50% of the time while ALM and VEH rats completed the task 79–100% of the time. Thus, following equipotent sleep-promoting doses, ZOL impaired rats in both memory tasks while ALM rats performed at levels comparable to VEH rats. These results are consistent with the hypothesis that less impairment results from HcrtR antagonism than from GABAA-induced inhibition.The hypocretin receptor (HcrtR) antagonist almorexant (ALM) has potent hypnotic actions but little is known about neurocognitive performance in the presence of ALM. HcrtR antagonists are hypothesized to induce sleep by disfacilitation of wake-promoting systems whereas GABA A receptor such as zolpidem (ZOL) are the most widely prescribed hypnotic medications. Although known to induce sleep, these compounds have been shown to significantly impair psychomotor and memory functions in rodents  receptors has been identified as a target mechanism for the next generation of sleep medications  produces less functional impairment than ZOL. The rationale that underlies this hypothesis is that ZOL causes a general inhibition of neural activity whereas ALM specifically disfacilitates wake-promoting systems. We tested this hypothesis using tests of spatial reference memory (SRM) and spatial working memory (SWM) in the Morris Water Maze. Although the concentrations of ALM and ZOL administered prior to these tests were equipotent in hypnotic efficacy, the performance of rats treated with ALM were superior to that of rats treated with ZOL.ad libitum. All experimental procedures were approved by SRI International's Institutional Animal Care and Use Committee and were in accordance with National Institute of Health (NIH) guidelines.One hundred fifty three male Sprague Dawley rats  were distributed among the 12 groups as described in Table Rats were instrumented with sterile telemetry transmitters  as previously described . Following completion of data collection, the EEG, and EMG recordings were scored in 10 s epochs as waking (W), rapid eye movement sleep (REM), or non-rapid eye movement sleep (NREM) by expert scorers blinded to the treatments using NeuroScore software . Sleep latency was defined as the first 60 s of continuous sleep following drug administration. Recordings were started at Zeitgeber time (ZT) 12 (lights off) and continued until animals performed the water maze tests.Animals were sleep deprived (SD) from ZT12-18 by progressive manual stimulation concurrent with EEG and EMG recording. The rats were continuously observed and, when they appeared to attempt to sleep, progressive interventions were employed to keep them awake: removal of cage tops, tapping on cages, placement of brushes inside the cage, or stroking of vibrissae or fur with an artist's brush.N = 6), ZOL at 30 mg/kg (N = 8) and VEH (N = 7). ZOL is approximately 3X more potent i.p. than p.o., was synthesized at SRI International  according to the patent literature. Zolpidem (ZOL) was a gift from Actelion Pharmaceuticals Ltd. For the SWM task, rats were dosed with ALM , ZOL  or vehicle  at ZT18 and left undisturbed until time to perform memory tasks (see below). For the SRM task, most rats were also administered ALM, ZOL, and VEH p.o. at the concentrations above. However, one cohort of rats was administered drugs i.p. For these rats, ALM was administered at 100 mg/kg (All water maze (WM) tasks occurred in a pool 68″ in diameter and 25″ in depth, containing water at 24 ± 2°C made opaque by the addition of non-toxic, water soluble black paint and milk powder. Since all tests took place during the dark period, distinctive spatial cues were made of small “rice” lights colored blue, yellow, and green. Patterns of lights in distinct shapes  were clearly visible from within the pool. Preliminary studies determined the minimum number of lights that were needed for learning to occur. A 10 cm diameter platform was submerged approximately 1 cm below the surface of the water in one of 6 locations Figure .The acquisition phase occurred in one session consisting of 12–15 consecutive trials with a 60 s inter-trial interval. For each trial, rats were placed in the WM facing the wall in one of three quadrants that did not contain the hidden platform. The location of the hidden platform remained constant across all trials. Rats were given 60 s to locate the platform. If the rats did not locate the platform within this period, they were guided to the platform location. When the rats reached the platform, they were allowed to remain on the platform for approximately 15 s before being placed in a dry holding cage for the next 60 s. This training sequence continued until the rats learned the task, typically 12–15 trials.On the following day, rats were dosed with ALM, ZOL or VEH at ZT18 and a retention probe trial was performed 90 min later in which the rats were returned to the WM but the platform had been removed. A total of 40 rats were subjected to SD for 6 h prior to drug administration, and 42 were left undisturbed during this period . Sleep/wake data  were analyzed in 30 min bins and compared between drug groups using Two-Way mixed-model ANOVA on factors “drug group” (between subjects) and “time” (within subjects). SRM performance parameters  were analyzed using a One-Way ANOVA. SWM performance measures  by delay time were analyzed using Two-Way mixed-model ANOVA on factors “drug group” (between subjects) and “time” (within subjects). Significance levels were set at α = 0.05. When ANOVA indicated significance, Bonferroni Drug concentrations were chosen to be equipotent at sleep promotion based on our previous experience (Morairty et al., For all performance measures analyzed, rats treated with ZOL showed significant impairments while ALM- and VEH-treated rats were indistinguishable Figure . FollowiSwim patterns in the WM were different for ZOL-treated rats compared to VEH- and ALM-treated rats Figure . Both VEZOL-treated rats performed poorly in the SWM task compared to either VEH- or ALM-treated rats Figures , 6. ZOL-The goal for the SWM task was to locate the platform. VEH- and ALM-treated rats found the platform the majority of the time in both SD and undisturbed conditions (83.3–100% for VEH and 79.2–87.5% for ALM; Figure A receptor modulation.Though differing in the latency to induce sleep at the doses tested, ALM, and ZOL were equally effective at promoting sleep during the 90 min period prior to performance testing and both compounds significantly increased sleep compared to VEH. ALM-treated rats were indistinguishable from VEH-treated rats in their performance of both the SRM and SWM tasks. In contrast, ZOL caused significant impairments in both tasks. Specifically, in the SRM task, ZOL increased the latency to, the duration in, and the frequency of entering the target zone. In the SWM task, ZOL increased the time to find the platform, decreased the swim velocity and decreased the success rate in finding the platform. These results support the hypothesis that dual Hcrt receptor antagonism effectively promotes sleep without the functional impairments observed following GABAAn alternative explanation of the results obtained is that ZOL-treated rats were not motivated to perform the tasks rather than having memory/cognitive deficits. ZOL-treated rats had decreased distance traveled during the SRM task and decreased velocity during the SWM task, which could indicate a lack of motivation to escape the WM. Further, the lower success rate in finding the cued platform during the training trials for the SWM task could be interpreted as an absence of motivation to escape. However, ZOL rats did not simply float in the WM; they swam continuously, primarily circling the perimeter of the WM. As mentioned above, this swim pattern is typical of an untrained rat during its first exposure to the WM. Although not measured in this study, it is possible that the decreased distance traveled during the SRM task and decreased velocity during the SWM task are due to motor deficits produced by ZOL. This hypothesis is supported by previous studies that found prominent motor effects following ZOL administration (Depoortere et al., The SD protocol in these studies was included to assess whether moderate increases in sleep drive would exacerbate any cognitive deficits found following ALM or ZOL administration and also produce deficits in VEH-treated rats. While the primary active period of nocturnal rodents such as the rat is during the dark phase, rats still sleep approximately 30% of the time during this period and increasing wake duration during the dark period should create a mild sleep deficit (see Figure ZOL is a widely prescribed hypnotic medication that can be well-tolerated when taken as directed (Greenblatt and Roth, In contrast, the high level of performance following ALM in both of our memory tasks suggests a high degree of safety at concentrations with hypnotic efficacy. Indeed, a recent study found no performance decrements in a variant of the WM SRM task at three-fold the concentration of ALM that we used (Dietrich and Jenck, A modulation.ALM and ZOL are effective hypnotics in multiple mammalian species (Brisbare-Roch 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.


Alternative inclusion of exons increases the functional diversity of proteins. Among alternatively spliced exons, tissue-specific exons play a critical role in maintaining tissue identity. This raises the question of how tissue-specific protein-coding exons influence protein function. Here we investigate the structural, functional, interaction, and evolutionary properties of constitutive, tissue-specific, and other alternative exons in human. We find that tissue-specific protein segments often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. Furthermore, genes containing tissue-specific exons tend to occupy central positions in interaction networks and display distinct interaction partners in the respective tissues, and are enriched in signaling, development, and disease genes. Based on these findings, we propose that tissue-specific inclusion of disordered segments that contain binding motifs rewires interaction networks and signaling pathways. In this way, tissue-specific splicing may contribute to functional versatility of proteins and increases the diversity of interaction networks across tissues. ► Protein segments of tissue-specific (TS) exons frequently contain disordered regions ► TS segments contain modification sites and evolutionarily conserved binding motifs ► Genes with TS exons are hubs in interaction networks and enriched in signaling genes ► TS splicing can rewire protein networks and signaling pathways in different tissues Together with gene duplication and recombination, alternative splicing plays a major role in increasing proteome diversity and organismal complexity . In factAlternative exons for which inclusion levels differ across tissues are referred to as tissue-specific (TS) exons. Studies using microarray data of mouse tissues have shown that, unlike most alternatively spliced exons, the lengths of TS exons are more often in multiples of three nucleotides . Since dWhile individual studies have highlighted the importance of TS exons, genome-scale molecular principles by which such exons influence protein function remain to be elucidated. Toward this goal, we performed a large-scale computational analysis of constitutive, tissue-specific, and other alternative exons and their corresponding protein segments in humans . Our intTo compose a set of TS exons, we used a list of the reported cassette exons whose inclusion levels significantly differed between at least two tissue types or cell lines . By defi−16, chi-square). In other words, Constitutive exons more often mapped to Pfam protein domains . Therefore, the observed high fraction of disorder is not solely a generic property of the proteins encoded by genes with such exons but tends to be specific to TS Cassette exons.Prediction of disordered regions using the IUPred program revealed that alternatively spliced exons were more frequently associated with disordered regions than were constitutive exons: 31%  and 22%  of segments encoded by TS and Other Cassette exons, respectively, had more than half of their residues in intrinsically disordered regions, compared to 16%  of the Constitutive exons D. These In order to assess whether TS Cassette exons are enriched in binding motifs that can be recognized by other proteins, we investigated if they encoded peptide interaction motifs that could be bound by globular domains . Using tWe obtained PTM annotations from the UniProt/Swiss-Prot Database and considered only exons for which representative isoforms had an exact match in the Swiss-Prot Database. The majority (94%) of PTM sites encoded by TS exons correspond to phosphorylation sites, and a majority (74%) were present in disordered regions. We observed that TS Cassette exons encoded PTM sites significantly more often than did other exons E. In a fTo analyze the evolution of TS exons, we first investigated genomic alignments of human and mouse orthologs and found that DNA sequences corresponding to human TS exons were significantly more conserved than the other exon categories A. At theTo analyze the importance of protein functional elements, we investigated evolutionary patterns in the different exon types. The regions encoding disordered segments and predicted binding motifs were both more conserved for the TS exons compared to the other exon types at the DNA level F and S2G−5, Mann-Whitney). Further, a subset of TSE genes that contain predicted disordered binding motifs tend to have more interaction partners than the other TSE genes (We investigated the significance of genes with TS Cassette exons (TSE genes) by mapping them onto the protein interaction network. Using the integrated human protein-protein interaction (PPI) network , we founSE genes A. This iSE genes . The impSE genes , we founSE genes A. This sBy investigating the set of human tissue-specific protein interaction networks in the individual tissues included in the analysis , we obse−8, chi-square) are enriched in genes that cause embryonic lethality in mice when deleted (Mouse Genome Database). Additionally, genes that have been causally implicated in cancer (Cancer Gene Census Database) and genes found to be somatically mutated in different cancer types (COSMIC Database) were both enriched to encode TS exons  experimentally annotated phenotype data for mouse genes with human orthologs and (2) information about cancer-related human genes. This showed that genes whose human orthologs have tissue-specific isoforms . These genes belong to evolutionarily conserved and developmentally important pathways such as the EGF/MAPK, Hedgehog, WNT/wingless, TGF-β, and JAK/STAT pathways , which is crucial for vesicle formation, is mediated by a peptide motif within the disordered segment  Database identified 138 TS segments that had at least one amino acid involved in forming an interaction interface with other proteins, DNA, or RNA in the complex  have more interaction partners on average compared to the other genes and (2) occupy central positions in the protein interaction network. More importantly, genes that contain TS exons tend to make interactions that are distinct in different tissues compared to genes that do not contain TS exons. These observations suggest that tissue-specific splicing may mediate new interactions in a tissue-specific manner through the alternative inclusion of disordered segments that contain binding motifs A. In thiCollectively, these observations raise the question of the molecular mechanisms by which tissue-specific segments influence protein interactions. Based on the observed characteristics of TS segments and the literature examples, we delineate genome-scale molecular principles by which such segments can influence protein function and their interaction networks.Several TS protein segments that encode binding regions have already been implicated in mediating interactions in the literature see A–S3C. FuConsistently, genes with TS segments encoding binding motifs tend to have interaction partners that are distinct in different tissues. Thus, tissue-specific splicing can potentially lead to the recruitment of the same molecular function, often carried out by structured domains encoded by constitutive exons, to different contexts by mediating new molecular interactions through the disordered segment C and S5B2+/calmodulin-dependent protein kinase in We also observe a subset of TS exons to encode disordered segments that do not contain binding motifs. Such segments can still affect interactions with other partner molecules by affecting the linker length between domains or binding motifs , Wilms' tumor gene WT1 , and UltWT1 , respectWT1 C. It is p53 can affect cellular differentiation by titrating full-length p53 and competing for the same DNA sequence . An example is the alternatively spliced acid box (AB) region of several members of the fibroblast growth factor receptor family of kinases see B, right.sequence . The samsequence C are oftFrom the perspective of disease and drug development, de novo mutations in TS exons can result in altered interaction properties and may lead to cell-type-specific diseases such as cancer . While hIn conclusion, one of the prevalent outcomes of splicing of TS exons appears to be modulation of protein-binding properties through the alternative inclusion of disordered segments that contain binding motifs. In this context, our characterized list of TS exons A can guihttp://www.ensembl.org/). The tissues include adipose, brain, breast, cerebellum, colon, heart, liver, lymph node, skeletal muscle, and testes as well as BT474, HME, MB435, MCF7, and T47D cell lines. The set of Other Cassette exons were identified using the following criteria: the exon (1) was alternatively present in at least two transcripts, (2) was not mutually exclusive with an adjacent exon, and (3) did not overlap with TS Cassette exons, and (4) there was another RNA isoform that also contained different exons upstream and downstream of the exon of interest. This was necessary to avoid cases of alternative start and termination sites. Constitutive exons were identified as those that were present in all transcript products with unchanged boundaries. See the TS Cassette exons were composed by mapping cassette exons with differential tissue inclusion levels, as reported by Wang and coworkers , to Ensehttp://pfam.sanger.ac.uk/). Intrinsically disordered regions were predicted with both the IUPred  and VSL2B (http://www.dabi.temple.edu/disprot/readmeVSL2.htm) software. For the structural analysis, TS segments extracted from the Ensembl proteins were first mapped to protein sequences in the UniProt (http://www.uniprot.org/) and SwissProt Knowledgebase (http://www.expasy.org/). Regions that were in PDB (http://www.pdb.org/) were obtained for the UniProt/SwissProt KB canonical sequences.Protein domains were predicted using the Pfam software  software was used to predict binding motifs. PTM sites were obtained from the UniProt/SwissProt KB. Only UniProt entries that were identical to the representative Ensembl proteins were used. Structures of proteins with TS segments in complex with other protein and or DNA and RNA were obtained from the PDB and PISA databases (http://pdbe.org/pisa), and residues at the interface were identified.ANCHOR (Identifiers and sequences of one-to-one human-mouse orthologs were obtained via Ensembl. When more than 90% of the residues in a TS segment were covered in the alignment, the segment was considered as present in the mouse protein. The alignments were obtained using the Needleman-Wunsch algorithm in the EMBOSS package. See the Human tissue-specific PPIs were obtained from the literature . NetworkAll statistical tests employed in this analysis were performed using the R statistical package.


Functional magnetic resonance imaging (MRI) showed that the superior temporal and occipital cortex are involved in multisensory integration. Probabilistic fiber tracking based on diffusion-weighted MRI suggests that multisensory processing is supported by white matter connections between auditory cortex and the temporal and occipital lobe. Here, we present a combined functional MRI and probabilistic fiber tracking study that reveals multisensory processing mechanisms that remained undetected by either technique alone. Ten healthy participants passively observed visually presented lip or body movements, heard speech or body action sounds, or were exposed to a combination of both. Bimodal stimulation engaged a temporal-occipital brain network including the multisensory superior temporal sulcus (msSTS), the lateral superior temporal gyrus (lSTG), and the extrastriate body area (EBA). A region-of-interest (ROI) analysis showed multisensory interactions  in the msSTS, the lSTG, and the EBA region. Moreover, sounds elicited responses in the medial occipital cortex. Probabilistic tracking revealed white matter tracts between the auditory cortex and the medial occipital cortex, the inferior occipital cortex (IOC), and the superior temporal sulcus (STS). However, STS terminations of auditory cortex tracts showed limited overlap with the msSTS region. Instead, msSTS was connected to primary sensory regions via intermediate nodes in the temporal and occipital cortex. Similarly, the lSTG and EBA regions showed limited direct white matter connections but instead were connected via intermediate nodes. Our results suggest that multisensory processing in the STS is mediated by separate brain areas that form a distinct network in the lateral temporal and inferior occipital cortex. Identifying objects or actions in our environment usually relies on multiple sources of sensory cues such as sounds and images. Over the last decade several functional magnetic resonance imaging (fMRI) studies have associated the superior temporal cortex (STC) with multisensory object and action processing. For instance, reliable blood-oxygenation-level-dependent (BOLD) responses to auditory and visual stimuli as well as enhanced BOLD responses to bimodal stimuli were observed in a posterior part of the superior temporal sulcus (STS)  and asynchronous  auditory-visual stimulus pairs , first described in 1985 . All participants reported normal or corrected-to-normal vision and no hearing impairments. Their mean age was 27 years (range from 23 to 40). All participants gave written informed consent prior to the study. The procedure was approved by the ethical board of the University of Regensburg.Unisensory visual and auditory as well as multisensory  brain areas were identified by fMRI while participants passively perceived biological motion stimuli Figure . AttentiEach video clip showed 13 black moving dots on a gray background with a frame rate of 30 Hz. In addition, a blue dot in the center of the display served as a fixation point. The display size was normalized so that the mean dot deviation from the screen center was 2.7 degrees of visual angle (with a dot size of approximately 0.15 degrees). Participants viewed the video clips via a back-mirror (mounted within the head-coil) on a translucent screen positioned about 70 cm distant to the eye. Participants had to view the video clips while fixating the fixation point and press a button with their index finger whenever they detected a red dot. Accordingly, in some videos one dot was colored red for 300 ms during an interval between 300 and 900 ms. Sounds were presented via MR compatible headphones . Sound onset and offset was synchronized with the onset and offset of the video clips. All sounds were matched in root mean square power and presented with a sound pressure level of approximately 65 dB. Participants were asked to listen to the sounds and press a button whenever they detected a beep sound. Accordingly, in some trials a target beep sound  occurred between 300 and 900 ms while the body action or speech sound was presented.Visual and auditory stimuli were presented in nine conditions Figure  that werIn order to monitor attention, participants were asked to respond to a visual (red dot) or auditory (beep) target occurring in 20% of the trials (10% for each target). Targets were balanced across all stimulus conditions. They occurred in a pseudo-random order and never occurred on the same trial. The participants pressed a button on a response box upon target detection.Stimuli were presented with an inter-trial-interval (ITI) of 8000 ms. This relatively large ITI was required, because a sparse-imaging MRI acquisition protocol with a repetition time (TR) of 8000 ms and an acquisition time (TA) of 2070 ms was adopted Figure . Sparse-3, field of view = 256 × 256 mm2). Functional runs were acquired with a T2* weighted echoplanar sparse-imaging sequence  with 36 axial slices . DWI runs were acquired with a single-shot pulsed gradient spin-echo sequence with echoplanar readout . Diffusion was examined along 30 isotropically distributed orientations  were interspersed into the diffusion sequence every six volumes. The 54 axial slices covered the whole brain .All MRI data was acquired by a 3T head-only Allegra scanner  using a one-channel whole-head coil while participants laid supine (head first) in the scanner bore. Head motion during the scans was constrained by cushions. For each participant, one high-resolution structural run, a series of functional runs, and three diffusion-weighted runs were acquired. The structural images were acquired with a magnetization-prepared rapid acquisition gradient echo (MPRAGE) sequence . The parameters were adapted from the Alzheimer's disease Neuroimaging project . The 160 sagittal slices covered the whole brain  as described elsewhere , intensity normalization  were considered.In order to define brain regions relevant for auditory-visual processing, we performed a general linear model (GLM) whole-brain group analysis. The design matrix of the GLM contained separate predictors for all nine conditions  of these five main contrasts. Unisensory contrasts were used to define brain areas that were assumed to be modality-specific. These included the auditory cortex, the visual cortex, and predominantly visual areas within the parietal or frontal cortex. Moreover, a phonological processing region in the lateral superior temporal gyrus (lSTG) . Implications of this relatively liberal definition criterion on our main findings are considered in the discussion (see below). Group labels  were reverse-mapped to individual cortical surfaces based on the spherical registrations. Both functional and structural landmarks (gyri and sulci) were used to segregate neighboring labels (ROIs).Our primary motivation for the group analysis was to identify sensory-specific and putative multisensory regions of interest. Therefore, our analysis focused on five major contrasts: brain areas engaged in combined auditory and visual processing ([ASVS + ABVB + ASVB + ABVS]/4 vs. A0V0), brain areas engaged in auditory processing ([ASV0 + ABV0]/2 vs. A0V0), brain areas associated with phonological processing (ASV0 vs. ABV0), brain areas engaged in visual processing ([A0VS + A0VB]/2 vs. A0V0), and brain areas associated with processing body movements (A0VB vs. A0VS). ROIs were defined based on the group-average cortical significance maps   and posterior (pSTS). As we were interested in their role in multisensory processing, two ROIs (labels) were defined based on the group aggregate track termination maps for tracks seeded in H and PT, respectively. Moreover, additional ROIs (labels) in the inferior occipital cortex (IOC) were defined based on the results of the whole-brain track termination analysis (described in the “Results” section).t-tests were performed on BOLD signals for unimodal auditory and visual stimulus conditions in order to classify ROIs as being primary auditory, visual, or multimodal. In order to detect non-linear multisensory interaction effects (superadditive or subadditive) BOLD signals to combined auditory-visual stimuli were compared to the sum of BOLD signals for unimodal stimulus conditions . In order to detect multisensory congruency effects, BOLD signals to incongruent bimodal stimuli (iAV) were compared to BOLD signals of congruent (cAV) bimodal stimuli . Note that all BOLD signals reflect differences to the baseline (A0V0) condition normalized to percent signal change (see above). For the second analysis, BOLD signals to body movements (B) were subtracted from BOLD signals to speech stimuli (S). The same comparisons were performed as for the feature-unspecific analysis. Note that for incongruent bimodal stimuli  the sign of the S-B difference of visual stimuli (B-S) is opposite of auditory stimuli (S-B). Therefore, responses to bimodal stimuli were compared to the sum of unimodal responses showing the same sign .Pre-processing for the ROI analysis was identical to the whole-brain analysis except that no spatial smoothing was applied. The mean MR signal change for each ROI was extracted by the GLM similar to the whole-brain analysis. In particular, the mean MR signal was estimated for each of the eight stimulus conditions and the baseline condition (A0V0). In addition, target trials (10% beep or 10% red dot) were modeled by separate predictors in order to exclude possible response-related activity. BOLD signals were calculated by subtracting the baseline signal (A0V0) from the MR signal of all eight stimulus conditions . Moreover, all BOLD signals were normalized to percent signal change relative to the ROI-specific global mean (constant predictor of the GLM). Our analysis focused on two aspects: (1) BOLD signals in response to auditory, visual, and bimodal stimuli regardless of stimulus type (speech or body movements) and (2) BOLD signal differences between speech (S) and body (B) stimuli reflecting feature-specific activity. For the first analysis, BOLD signals to speech and body  stimuli were pooled. One-sample White matter connections between ROIs as defined by the whole-brain analysis were estimated by additional trackings. Here, each ROI served as seed for separate trackings. Moreover, each ROI served as target area. In order to derive a measure of pair-wise connectivity strength, we counted the number of tracks emitting from the seed ROI and reaching the target ROI. Note that the number of tracks emitting from the seed is proportionate to the number of seed voxels. Moreover, the number of tracks terminating in the target ROI increases with the number of target voxels. In order to compensate for this dependency on ROI size, a normalized track connectivity index (TCI) was calculated by dividing the number of tracks by the product of the number of voxels in the seed and target ROI, respectively. All ROIs served as both seed and target ROI. The resulting two connectivity indices were averaged. Moreover, connectivity indices from left and right hemispheres were averaged. Finally, TCI values from the whole group were averaged.All participants successfully detected most of the target stimuli . Moreover, no significant differences in response times were observed between visual and auditory targets suggesting that both modalities were attended equally well.In order to identify unisensory and putative multisensory brain regions involved in auditory and visual processing, a whole-brain analysis of the functional data was performed  with responses to the baseline condition (A0V0) revealed a network of brain areas primarily in the occipital and dorsal parietal cortex Figure . Two smaAlthough both unimodal auditory and unimodal visual contrasts showed distinct activity patterns throughout most of the cortex, several brain regions were activated by unimodal auditory, visual, and bimodal contrasts. This putative multisensory brain network included a region in the posterior part of the STS that most likely corresponds to the msSTS area  as determined by functional and structural  criteria (see above). Figure Combined functional and structural criteria were adopted for the PT seed. Only the part of the planum temporale that was functionally active during the auditory or bimodal task was included. Tracks seeded in PT terminated in two distinct regions of the STS: anterior (aSTS) and posterior (pSTS) divisions . Our primary interest was to evaluate the white matter connectivity of this region with auditory and visual cortex. As illustrated in Figure Our functional analysis revealed two regions that were either specific to phonological processing (lSTG) or visual body movements (EBA). Both regions showed signs of multisensory interactions (see below). Therefore, we were interested in the connectivity profile of these two regions. Tracks seeded in the phonological processing area (lSTG) showed wide-spread terminations in the auditory cortex including the H and PT region and the STS Figure . HoweverIn order to compare functional activity maps with track termination maps enlarged views of the cortical surfaces are shown in Figures Several previous studies have shown that activity in the STS is accompanied by activity in the inferior or lateral occipital cortex  or visual stimulation  with the baseline condition (A0V0)  were primarily observed in the posterior part of the STS . Moreover, subadditive responses to congruent bimodal stimuli were observed in the inferior occipito-temporal cortex , parts of the occipital cortex , and the planum temporale. Significant BOLD response differences between congruent and incongruent bimodal stimuli were observed in the planum temporale, EBA, IOC , the OcPo, and anterior insula.As our whole-brain analysis revealed cortical regions that responded preferably to speech sounds (S) or visual body movements (B), an additional ROI analysis was performed on the differences between S and B stimulus types (S-B)  in several brain regions including lSTG, pSTS (but not msSTS), and the EBA. By contrast, incongruent bimodal stimuli (ASVB-ABVS) elicited superadditive responses compared to their unimodal counter-parts in most of these regions including the OcPo. Accordingly, a significant difference in the bimodal response pattern of congruent and incongruent bimodal stimuli was observed in lSTG, aSTS, pSTS, EBA, and vlFC, suggesting that congruent and incongruent bimodal stimuli were processed differently in these areas.Our ROI analysis on functional data and our whole-brain tracking results indicated that several distinct brain areas were involved in the processing of auditory and visual stimuli. Therefore, we performed pair-wise probabilistic tracking in order to quantify the strengths of white matter connections using the ROIs as seed and targets. Track probabilities were normalized by the size of the seed and target regions, respectively. The group average normalized ROI-to-ROI track probabilities (pooled across both hemispheres) are shown in Table The ROI-to-ROI connectivity analysis further revealed the connectivity profile of inferior occipital regions. These regions were primarily connected with the H part rather than the PT part of the auditory cortex. Furthermore, anterior regions of the IOC  were connected with the lSTG and the aSTS, but little connectivity was observed with the msSTS. Moreover, parts of the IOC (lOTG) were connected to the medial occipital cortex (CaS).The ventrolateral frontal cortex as well as the anterior insular region observed in fMRI showed little direct connectivity with the STC. Instead these two regions were connected via inter-mediate nodes such as the dorsolateral frontal cortex or the inferior occipito-temporal cortex .Our whole-brain analysis revealed a large network of brain areas responding to auditory, visual, or multimodal stimuli. Processing of unimodal auditory stimuli Figures  primarilProcessing of unimodal visual stimuli involved a network of brain areas primarily in the occipital and dorsal parietal cortex Figures . This neAlthough auditory and visual processing was associated with distinct brain areas throughout most of the cortex, several cortical sites were activated by both auditory and visual stimuli. In particular, we observed a region in the posterior part of the STS that was activated by auditory, visual, and bimodal stimuli Figures , 5. MoreThe medial occipital cortex showed two clusters in the anterior part of the CaS and the OcPo that were activated by auditory-visual stimuli. These two regions likely reflect peripheral and central representations of the primary visual cortex V1), respectively  implicitly controlled for common response components. Another concern is whether multisensory interactions reflect perceptual or post-perceptual processing . The tracking results of the present study essentially replicated these previous findings by showing that tracks seeded in the Heschl's region and the planum temporale terminated in an anterior (aSTS) and posterior (pSTS) part of the STS. We were interested in the relationship of these structurally-defined regions with the msSTS region that was observed with functional MRI . Our ROI analysis of functional MRI data further suggests that aSTS, pSTS, and msSTS differ by the pattern of multisensory processing. Whereas activity in the aSTS region was sensitive to stimulus type (speech vs. body), no such sensitivity was observed in msSTS. Moreover, some regions  were predominantly involved in auditory processing, but auditory responses were modulated by visual signals. Both our connectivity findings and our functional results suggest that the STC is subdivided into several distinct regions and is best conceived as a multisensory network or complex rather than as a single region. This notion of a multisensory STC network may also help us to understand conventional fMRI findings that are difficult to accommodate with the notion of a single msSTS region. For instance, several brain imaging studies in humans found multiple distinct brain areas within the STS that may be segregated based on their multisensory integration patterns  Figure . These tSeveral studies examining multisensory integration observed multimodal responses in the anterior insula and ventrolateral frontal cortex - in addition to STS activity (Calvert et al., Although combining functional MRI and fiber tracking based on diffusion-weighted MRI provides relevant information that goes beyond the information provided by either method alone, both methods suffer from limitations that should be considered (Ramnani et al., In summary, functional MRI revealed a network of brain regions primarily within the temporal and occipital cortex involved in multisensory object and action processing including the msSTS region, a speech-selective lSTG region, and the EBA. Probabilistic tracking revealed white matter tracks between the auditory and the medial occipital cortex. However, brain areas involved in multisensory processing within the superior temporal and lateral occipital cortex showed little direct connectivity with primary sensory cortices. Instead these brain areas were connected to early sensory cortices via intermediate nodes of the STS and IOC. Our findings suggest that combining structural connectivity and functional imaging reveals mechanisms related to multisensory integration that remain undetected by either technique alone.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.


Morbidity from calcific aortic valve disease (CAVD) is increasing. Recent studies suggest early reversible changes involving inflammation and neoangiogenesis. We hypothesized that microcalcifications, chemokines, and growth factors are present in unaffected regions of calcific aortic valves. We studied aortic valves from 4 patients with CAVD and from 1 control, using immunohistochemistry, scanning electron microscopy, and infrared spectrography. We revealed clusters of capillary neovessels in calcified (ECC), to a lesser extent in noncalcified (ECN) areas. Endothelial cells proved constant expression of SDF-1 in ECC, ECN, and endothelial cells from valvular surface (ECS). Its receptor CXCR4 was expressed in ECC. IL-6 expression correlated with CXCR4 staining and presence of lymphocytes. VEGF was expressed by ECS, its receptor by ECC and ECN. Crystalline ultrastructures were found on the surface of histologically noncalcified areas (HNCAs), spectrography revealed calcium hydroxylapatite. Our results demonstrate that crystalline ultrastructures are present in HNCAs, undergoing neoangiogenesis in an inflammatory context. These alterations could be an early witness of disease and an opening to therapy. Calcific aortic valve disease (CAVD) is the most common valvular lesion in Western countries and the major cause of aortic valve replacement. It is associated with a higher death risk from cardiovascular causes which is increased up to 50 per cent, even in the absence of a hemodynamically significant obstruction , 2. WithChemokines and growth factors play an important role in several pathophysiological processes such as inflammation and immunity. Chemokine receptor CXCR4 is expressed on monocytes, B-lymphocytes, and most T cells –10. It hDue to routine experience with minimal valvular alterations including discrete calcification and associated capillary-like neovessels, we have been interested in possible early stage changes of clinically silent and macroscopically inconspicuous valve areas/valves. We therefore hypothesized that early fibrous remodeling and neoangiogenesis within aortic valves could be initiated by ultrasctructural calcific alterations.n = 4) and aortic insufficiency due to aortic arch aneurysms (n = 1) in the Department of Cardiac Surgery . After resection, specimens were fixed in formalin and dissected. Half of the specimens were processed to paraffin blocks and serially sectioned at 3 μm thickness. Slides were stained with haematoxylin, eosin, and saffron. The other half was discriminated into calcified and noncalcified areas and eventually inserted into X-ray permeable kapton film for submission to X-ray fluorescence and X-ray diffraction experiments. Tissue samples corresponding to aortic valve resections were collected from patients undergoing surgical treatment for CAVD  between 4000 and 400 cmf 4 cm−1 . A ZeissThe study was approved and executed in compliance with the institutional review board of the Pitié-Salpêtrière Hospital and all subjects gave informed consent concerning the use of tissue.The morphologic hallmarks of CAVD were heaped-up calcified masses within the aortic cusps that protruded into the outflow surfaces. Calcific deposits distorted the cuspal architecture, primarily at the bases. The free cuspal edges were usually not involved. On the leaflet profile calcifications were located within the valvular fibrosa, at the point of maximal cusp flexion. Cusps were heavily fibrosed and frequently presented chronic inflammatory infiltrate mainly consisting of lymphocytes, macrophages, and masts cells. Sporadically, foreign body granulomas with giant cells were present near calcifications. Numerous blood microvessels within calcified areas were observed in close association to inflammatory infiltrates and frequently disposed in a nest-like pattern . MicroveEC from all samples and from all compartments expressed vWF and CXCL12. CXCR4 expression was observed in ECC, while ECN and ECS were sporadically stained or completely negative. IL-6 was expressed in ECC and ECS, while mostly negative in ECN. The control sample presented with a different expression profile: as by definition, only ECN and ECS were present; while both EC subgroups were easily detected by vWF expression and stained for CXCL12, ECN were negative for IL-6, while ECS moderately stained.VEGF was constantly expressed in ECS of all valves and, to a lesser extent, in ECC. VEGFR1 was expressed in both ECC and ECN, while ECS was negative. ECN in the control sample revealed expression of VEGFR1.Inflammatory infiltrate in the range of neovessels mainly revealed CD3+ lymphocytes and CD117+ mast cells. In an attempt to differentiate mast-cells from CD117+ bone marrow-derived progenitor cells, slides were secondarily counter stained with Toluidine-Blue; CD117-positive/Toluidine-negative cells were not observed. Immunohistochemical results are presented in Figures μm. These heterogeneously distributed structures where associated with 5–8 μm large sphere- or nodule-like structures, focally covering the leaflet surface. The same ultrasctructural modifications were—to a lesser extent—present in the control sample  analysis of the leaflet surface within noncalcified areas revealed the presence of two types of three-dimensional crystal structures: we differentiated plate-like, facetted microrods which were vertically inserted into the endothelial surface, measuring approximately 10  Figures . In contX-ray fluorescence emission revealed a significant phosphor peak in calcified and a small elevation of phosphor in noncalcified areas. No calcium peak was noticeable for noncalcified areas, whereas present in calcified regions. Light elements, such as carbonate, oxygen, and sodium were present in both regions .Calcium apatite and amorphous carbonated calcium phosphate were the major components in grossly calcified areas. Inconspicuous areas with crystalline ultrastructures only contained calcium apatite. Approximately 80% of the material in grossly calcified areas corresponded to calcium apatite, and 10% of calcium apatite was found in grossly noncalcified areas. Calcium apatite was also present in the control sample . Higher amounts of triglycerides were retrieved within inconspicuous valve areas, as compared to calcific lesions .In order to detect ultrastructural pathological transformations in valve structure, we analyzed noncalcified and calcified areas of human aortic valves which had been surgically removed due to symptomatic calcific stenosis, as well as one totally inconspicuous valve excised in the context of Bentall surgery in a patient with nonatherosclerotic aortic aneurysm. After histological study of the samples, we submitted them to scanning electron microscopic and Fourier transform—infrared spectroscopy in an attempt to eventually define an elementary profile indicating an initial change in structural organization, possibly preceding manifest remodeling.Our scanning electron micrographs suggest that crystalline ultrastructures of different appearance are present on the surface of histologically inconspicuous valve tissue. SEM analysis allowed us to differentiate two types of crystalline ultrastructures, within noncalcified areas: we discriminated smaller, spherical, or nodule-like structures partly covering the surface and accumulations of larger, plate-like facetted microrods radially inserted into the valve leaflet. Our observations match with the ultrastructural aspect of typical biological apatites within calcified lesions of human aortic valves: a physochemical and ultrastructural study by Mikroulis and coworkers of native aortic valves and porcine aortic bioprostheses revealed two types of coexisting valve deposits, that is, large (>20 micrometers) and medium (5–20 micrometers) plate-like crystals as well as microcrystalline (<5 micrometers) calcium phosphate mineral formations . Their rThe mechanism of CAVD remains largely unknown compared to our understanding of the mechanisms underlying atherosclerotic diseases. We hypothesize that the SDF-1/CXCR4 and the VEGF/VEGFR system could have a pivotal role in the initiation and development of CAVD. Our observations demonstrate that expression of SDF-1 and its receptor CXCR4 is present in EC of pathologic microvessels. Moreover expression of CXCR4 appears to correlate with expression of IL-6, a cytokine with potent proinflammatory activity in context of inflammation. Further more, we show that a constant VEGF expression of leaflet surface ECs is associated with VEGFR1 expression of ECs within pathologic neovessels and that those neovessels are not exclusively confined to calcified areas with inflammatory infiltrate but—even if distributed in a sparse pattern—can be observed in noncalcified areas of diseased valves, as well as in nonsymptomatic valves lacking obvious calcification in light-microscopy, but revealing endothelium-adherent crystalline ultrastructures in SEM and IR spectrography.The development of new pharmacologic therapies for this disease requires a clearer understanding of the mechanisms of disease onset. Angiogenic factors and proinflammatory cytokines are present and could represent an early witness of subsequent inflammatory activity, finally leading to fibrous remodeling and symptomatic valvulopathy.


A 68-year-old Caucasian female with a past medical history of a deceased donor kidney transplant four months prior was admitted with a two-day history of anuria and acute kidney injury. A renal ultrasound demonstrated thrombus in the transplanted kidney's renal vein that extended into the left iliac vein as well as into the left femoral venous system. Catheter-guided tissue thrombolytics were infused directly into the clot. Within twelve hours of initiating thrombolytic infusion, there was brisk urine output. Interval venography demonstrated decreasing clot burden. At the time of discharge her creatinine was 0.78 mg/dL, similar to her baseline value prior to presentation. The patient was noted to have May-Thurner syndrome on intravascular ultrasound (IVUS). Angioplasty followed by stent placement was done. Unique to our case report was the timing of the presentation of renal vein thrombosis (four months after transplant) and the predisposing anatomy consistent with May-Thurner syndrome, which was diagnosed with IVUS and successfully treated with local thrombolytics. Renal vein thrombosis secondary to May-Thurner syndrome, postrenal transplantation, is not common. Early diagnosis and intervention are important in preventing renal allograft loss. A 68-year-old nonobese Caucasian female recipient of a left lower quadrant deceased donor kidney transplant due to adult polycystic kidney disease presented four months after transplantation with a two-day history of anuria. Serum creatinine at presentation was 2.20 mg/dL (baseline 0.8 mg/dL). Her immunosuppressant regimen included tacrolimus and prednisone. Renal Doppler ultrasound demonstrated allograft renal vein thrombosis extending into the left iliac and femoral venous systems. Initially, open surgical exploration and thrombectomy of the clotted vein was planned and preoperatively an inferior vena caval filter was placed. However, the patient had minimal urine output since admission and the ultrasound also demonstrated retained transplant arterial flow. Thinking of it as less invasive and having a possibility of success, catheter-based thrombolysis was attempted. Two infusion catheters were placed under fluoroscopic guidance: one to treat the transplant and juxta-anastomotic iliac veins and the other for the iliofemoral venous system distal to the allograft (done within twelve hours after presentation). . Tissue Intraprocedural intravascular ultrasound (IVUS) of left iliac vein demonstrated compression of left iliac vein between the right iliac artery and vertebra . Given tAt the time of discharge creatinine was 0.78 mg/dL, similar to baseline. Followup venography at discharge and 3 months later demonstrated complete stent patency without residual thrombus . After 4Renal vein thrombosis is an important but uncommon cause of allograft failure with an incidence of 1% . Risk faMay-Thurner syndrome, originally described by May and Thurner in 1956, is an anatomic variant where the left common iliac vein is narrowed due to the compression between the overlying right common iliac artery and the underlying lumbar vertebrae . May-ThuOur patient was noted to have renal vein thrombosis in the late posttransplant period secondary to underlying May-Thurner syndrome which was diagnosed with the help of IVUS. Moreover, our case is unique in showing successful catheter-directed treatment of the renal allograft and systemic venous thrombolysis in combination with endovascular stent placement, restoring the patient's allograft function as a treatment modality. Unique to our case was also the sustained success of our intervention at one-year followup.


Oncologic patients who are extreme responders to molecularly targeted therapy provide an important opportunity to better understand the biologic basis of response and, in turn, inform clinical decision making. Malignant neoplasms with an uncertain histologic and immunohistochemical characterization present challenges both on initial diagnostic workups and then later in management, as current treatment algorithms are based on a morphologic diagnosis. Herein, we report a case of a difficult to characterize sarcoma-like lesion for which genomic profiling with clinical next generation sequencing (NGS) identified the molecular underpinnings of arrested progression(stable disease) under combination targeted therapy within a phase I clinical trial.Genomic profiling with clinical next generation sequencing was performed on the FoundationOne™ platform . Histopathology and immunohistochemical studies were performed in the Department of Pathology, MD Anderson Cancer Center . Treatment was administered in the context of a phase I clinical trial ClinicalTrials.gov Identifier: (NCT01187199).KIAA1549-BRAF gene fusion resulting from a tandem duplication event, a homozygous deletion of PTEN, and frameshift insertion/deletions in CDKN2A A68fs*51, SUFU E283fs*3, and MAP3K1 N325fs*3. The patient had a 25% reduction in tumor (RECIST v1.1) following combination therapy consisting of sorafenib, temsirolimus, and bevazicumab within a phase I clinical trial.The histology of the tumor was that of a spindle cell neoplasm, grade 2 by FNCLCC standards. Immunohistochemical staining was positive for S100 and CD34. Genomic profiling identified the following alterations: a KIAA1549-BRAF and PTEN loss within a spindle cell neoplasm, as revealed by genomic profiling based on NGS. This is the first report of a tumor driven by a KIAA1549-BRAF fusion responding to sorafenib-based combination therapy.The patient responded to combination targeted therapy that fortuitously targeted  KIAA1549-BRAF fusion in a PTEN null background as a driving genomic alteration susceptible to targeted therapy.Tumors that defy ready histopathologic characterization are difficult to rationally approach in the setting of treatment algorithms which are based on the anatomic origin of the neoplasm. Spindle cell neoplasms are such tumors that defy characterization even with ancillary methods such as immunohistochemistry. In such cases, genomic profiling based on next generation sequencing (NGS) can identify the genomic alterations (GA) that drive oncogenesis and thus prospectively suggest aberrantly activated pathways for pharmacologic inhibition independent of tumor site origin. Conversely, in the case of exceptional responders to targeted therapy, diagnostic genomic profiling may uncover the linked underlying alterations retrospectively. We report one such case of a patient with a malignant spindle cell neoplasm who exhibited clinical and radiographic improvement with simultaneous RAF kinase inhibition (sorafenib/Nexavar®), mTOR inhibition (temsirolimus/Torisel®) and VEGF (bevacizumab/Avastin®) targeted therapy who had been refractory to standard cyototoxic chemotherapy. A Clinical Laboratory Improvement Amendments (CLIA) laboratory performed NGS-based diagnostic genomic profiling which identified this tumor as the first reported case of the We reviewed the medical records of a patient with spindle cell neoplasm who presented to the Department of Investigational Cancer Therapeutics at The University of Texas MD Anderson Cancer Center after failing standard of care therapy. Treatment and consent on investigational trial, and data collection were performed in accordance with the guidelines of the University of Texas MD Anderson Cancer Center Institutional Review Board (IRB). Tumor response was determined using RECIST (version 1.1) by CT scans obtained about every six to eight weeks. Clinical evaluation and assessments were performed per protocol.3.Next-generation sequencing was performed by using the Clinical Laboratory Improvement Amendments (CLIA)-approved FoundationOne™ platform . FoundationOne™ is a targeted assay utilizing next generation sequencing in routine cancer specimens. The assay simultaneously sequences the entire coding sequence of 236 cancer-related genes  plus 47 introns of 19 genes frequently rearranged in cancer to a minimum coverage depth of 250X. The assay detects all class of genomic alterations  using routine FFPE tissue samples that may be as small as 0.6 mmA 55 year old female presented to the clinical center for targeted therapy to discuss treatment options for a progressive metastatic spindle cell neoplasm. Disease at presentation included a left chest wall mass measuring more than 6 cm in greatest dimension. Extent of disease evaluation also revealed a lytic lesion in the left seventh rib and a second smaller mass centered in the pleura.Pathologic examination of formalin fixed paraffin embedded (FFPE) biopsied tissue from the presumed primary tumor site revealed a spindle cell proliferation, which was diagnosed to be a malignant spindle cell neoplasm, favor sarcoma, which is akin to a diagnosis of exclusion. The diagnosis of a malignant solitary fibrous tumor was also entertained, but the features were not typical for such a diagnosis  75 mg/m2 and ifosfamide (Ifex®) 10 g/m2. She progressed during two cycles of this treatment, and was then started on gemcitabine (Gemzar®) 900 mg/m2 and docetaxel (Taxotere®) 100 mg/m2. The patient then progressed after two cycles of her second regimen.The patient was initially treated with doxorubicin  of bevacizumab and temsirolimus in combination with sorafenib for the treatment of advanced cancer . The patContemporaneously with the entrance of patient into the clinical trial, FFPE tumor cell-predominant tissue from the biopsy was submitted to a commercial CLIA-certified, CAP-accredited laboratory for genomic profiling based on next generation sequencing (NGS) based.At the time of this report, the patient’s overall response was stable disease for eleven cycles. She had the following ongoing toxicities that were felt to be at least possibly related to the study drug: grade 1 altered taste, grade 2 proteinuria, grade 1 hand-foot syndrome, grade 2 hypoalbuminemia, and grade 2 mylagias. The patient did not experience a dose limiting toxicity from the combination therapy. Unfortunately, the patient developed an acute myocardial infarction, hypotension, clinically deteriorated and died because of this co-morbidity.KIAA1549-BRAF fusion gene  due to the positive S100 immunohistochemical stain and histologic phenotype of spindle cells, although that diagnosis was not made here. Interestingly, the known activating V600E ST cases . The ideKIAA1549-BRAF fusion protein, BRAF is a cytosolic serine/threonine kinase that is activated by binding of RAS-GTP. The function of KIAA1549 remains unknown. This fusion joins intron 15 of KIAA1549 and intron 8 of BRAF, although several other configurations of these fused genes have been observed [Of the two components of the KIAA1549-BRAF as the BRAF kinase domain is completely conserved. Interestingly successful targeting by sorafenib of BRAF-fusion bearing cells specifically the AGK-BRAF fusion that contains the first 33 amino acids of AGK and the C-terminal portion of BRAF starting from position 328 of BRAF with loss of the CR1 domain (not KIAA1549-BRAF) has been recently reported [AGK-BRAF expressing melanoma cell line demonstrated an increased sensitivity to sorafenib [RAF fusion proteins reported in prostate cancer [Sorafenib is a small molecule inhibitor of WT BRAF and other kinases including VEGFR 1/2/3, PDGFRB, and RAF family, that has an IC50 for WT BRAF at 25 nm  and bindreported . In the orafenib . RemarkaBRAF fusion is insufficient for tumor response. An analogy can be made to non-melanoma solid tumors expressing the activating V600E mutant BRAF, where vemurafenib treatment is sometimes ineffective, ie as in the colon [KIAA1549-BRAF fusion hyperactivates the mTOR pathway [PTEN [BRAF fusion protein by sorafenib and of the downstream mTOR pathway by temsirolimus synergizes, although the specific mechanism of tumor regression awaits further investigation. Bevacizumab was also administered, but the contribution of this therapy to the patient response is difficult to ascertain. As has been theorized for other tumor types, it is possible that bevacizumab enhanced delivery of the other therapeutics by normalizing the tumoral vasculature [Notably, pediatric astrocytoma patients expressing this fusion progressed on sorafenib monotherapy in a small phase II trial . This obhe colon . In suchhe colon -12. Thishe colon -16 to BRhe colon . Thus, t pathway , as doesay [PTEN . In thisculature .KIAA1549-BRAF fusion such as pediatric pilocytic astrocytomas?What are the implications of this case for treating tumors that express the in vitro and in vivo studies well outside the scope of this report are required. Sorafenib is indeed not a specific inhibitor of BRAF and as such the effects may not be mediated by targeting KIAA1549-BRAF in this tumor.As patients with such tumors on sorafenib monotherapy progressed rapidly in a very small Phase II study , sorafenPTEN loss or other GAs activating the mTOR pathway are present alongside the BRAF fusion in a pilocytic astrocytoma of patient, thus suggesting a potential responsiveness to combined sorafenib/mTOR targeted therapy. As a possible therapeutic alternative, a recent in vitro study suggested that second generation BRAF inhibitors may be capable of effectively inhibiting this fusion protein [Specifically, genomic profiling based on clinical-grade NGS could identify whether  protein .KIAA1549-BRAF fusions typically coincide with alterations in the mTOR pathway, and whether such patients can be effectively treated with targeted therapy on that basis. However, this case already indicates such treatment possibilities exist for patients possessing KIAA1549-BRAF fusions, whether in pediatric astrocytomas or difficult to characterize sarcoma like lesions. In general, genomic profiling based on NGS may be of great relevance in the management of patient with solid tumors that defy pathologic definition, as this case demonstrates that such diagnostics can reveal unexpected but effective avenues of targeted treatment.A prospective clinical trial with prospective diagnostic genomic profiling will best answer whether tumors driven by KIAA1549-BRAF fusion, a recurrent oncogenic driver in sporadic pilocytic astrocytoma, is described here in a spindle cell neoplasm for the first time.• KIAA1549-BRAF fusion with PTEN loss to be successfully treated by targeted therapy.• This case is the first report any tumor expressing • Genomic profiling based on Next-generation sequencing (NGS) may be of great relevance in the management of patient with solid tumors that defy pathologic definition, as this case demonstrates that such diagnostics can reveal unexpected but effective avenues of targeted treatment.KW, SMA, VAM, PJS, GAP, and JSR are all employees of and own stock in Foundation Medicine.All authors contributed to writing the manuscript. VS, VAM and SMA conceived the manuscript. VS and DA provided clinical expertise. SNW is the primary investigator for the clinical trial and VS is the co-primary investigator for the clinical trial. VS, SNW, KW, WW, VAM, JR, PJS, GAP and SMA analyzed the data. WW, JR, SMA provided pathology expertise. VS and SMA wrote the paper. VS and DA provided sarcoma expertise. VS and SMA provided cellular, molecular and targeted therapy expertise. All authors read and approved the final manuscript.