BDNF expression in the hippocampus (cKO mice) and have assessed proliferation, survival, differentiation and dendritic development of hippocampal progenitors.Environmental enrichment (EE) is known to enhance BDNF levels and neurogenesis in the adult hippocampus. To examine the role of BDNF in modulating EE-mediated adult hippocampal neurogenesis, we conditionally ablated We show that while the extent of cell proliferation and neuronal fate differentiation in the hippocampus of cKO mice is not different from wild-type (WT) littermates maintained in either standard or enriched conditions, reduced BDNF levels significantly impaired the survival of newborn cells in both housing conditions. In addition, while highly active enriched WT mice exhibited a robust increase in progenitor cell proliferation, highly active cKO mice showed a modest increase in cell proliferation compared to standard housed or underactive cKO mice.There results argue that while BDNF plays a role in exercise-induced cell proliferation, other factors must contribute to this phenomenon. We also show that dendritic development was impaired in cKO mice maintained in standard housing conditions, and that EE rescued this phenotype. Neurons are continuously added to the hippocampal dentate gyrus (DG) throughout life. Studies have correlated increased neurogenesis in the DG with improved performance on hippocampal learning tasks , suggestBDNF knockout (BDNF+/-) mice exhibit reduced [BDNF+/- mice exhibit a multiplicity of growth, metabolic, neuronal maturation and behavioral abnormalities [Brain-derived neurotrophic factor (BDNF) is highly expressed in hippocampus , but the reduced  or enhan reduced  in the D reduced ,5, but n reduced . Howevermalities -10 and iIt is now clear that exposure of adult rodents to environmental enrichment (EE) and exercise induces neurogenesis in the DG ,12 and iPSEN1, encoding presenilin 1, by a CamKII-driven Cre  and male BDNFlox2/BDNFlox 2mice (WT) in EE for 1 month (referred as "enriched" mice) and maintained additional cohorts of cKO and WT mice in standard conditions for 4 months (referred as "standard" mice).We previously demonstrated that conditional ablation of floxed e T29-1, ) transge3 months . Thus, wBDNF alleles are not deleted by the neuronal-specific CamKII-Cre allele, or that this represents BDNF that is axonally transported to the hippocampus from cortical regions in the CNS [We now show that standard housed cKO mice exhibit an approximately 50% reduction of hippocampal BDNF levels compared to standard WT mice Fig. . The res the CNS  and furt the CNS , BDNF isIt has been shown that hippocampal BDNF levels are increased upon long-term  but notBDNF+/- mice exhibit growth retardation, obesity, hyperactivity and aggressiveness [In view of the reports documenting that siveness -10, we fIn order to examine the impact of BDNF on NPC proliferation, cohorts of 3-month-old male cKO or WT mice were subject to EE for 1 month, after which time, mice were injected with BrdU and sacrificed after 1 day. Parallel cohorts of mice were sacrificed 4 weeks after BrdU injection in order to determine the survival rate and differentiation of newborn NPCs.+ cells in cohorts of WT or cKO mice housed in standard conditions failed to reveal any statistically significant differences between groups  other than BDNF might contribute to EE-induced cell proliferation.On the other hand, EE significantly increased the number of BrdUrts Fig. . Notablyort Fig. , the fac+cells in each group. We observed no significant differences in BrdU+cells in standard WT and low-activity WT cohorts .In order to determine the phenotypes of the BrdUFAP Fig. . NeuN waups Fig. . BrdU+ cAdult-generated neurons must develop dendrites to form connections with existing neurons to receive and convey information in order to integrate into the existing neural networks or rebuild neural circuitry. Previous studies have shown marked effects of BDNF on dendrite formation, but the results have not been conclusive. For example, while dendritic complexity in the hippocampal DG is increased in BDNF-overexpressing transgenic mice , BDNF ha+ neurons in the DG of standard WT mice exhibited dendrites projecting to the granule cell layer or crossed the granule cell layer to the molecular layer, with the axis of the cell body perpendicular to the SGL  were more complex than those in standard housed mice at 150-200 μm from the soma  is reduced in cKO mice compared to WT counterparts. Second, reduced BDNF level in cKO mice impairs both basal and EE-mediated survival of newborn cells, but does not affect neuronal fate differentiation in either housing condition. Third, dendritic development is impaired in cKO mice maintained in standard housing conditions, a phenotype that is rescued by EE. Taken together, our data suggest that BDNF expression in the adult hippocampus is required for precursor cell survival, exercise-induced cell proliferation, and dendritic development.BDNF mRNA and encoded polypeptides in the hippocampus [2+ increases generated through the activation of NMDA receptors or voltage-sensitive Ca2+ channels [While hippocampal progenitor cell proliferation was not affected by reduced BDNF levels in the entire cohort of mice housed in either standard or EE conditions, we observe that exercise-induced cell proliferation was markedly impaired in cKO mice. While it has been reported that physical exercise up-regulates pocampus ,26-28 anpocampus , the molchannels ,30. In tchannels . Runningchannels . BDNF plchannels ,34. It schannels . Data frchannels  and perichannels . Taken tTrkB mRNA and encoded polypeptides are expressed in neural progenitor cells and immature neurons in the adult hippocampal neurogenic niche, and that conditional ablation of TrkB from hippocampal neural progenitors using the hGFAP-Cre is sufficient to block running-induced increases in proliferation and neurogenesis [While not established, it is likely that BDNF influences exercise-induced cell proliferation through TrkB that is highly expressed in the hippocampus ; the levogenesis . These oOur finding that a 50% reduction of BDNF in our cKO mice impairs both basal and EE-mediated survival of newborn cells may be related to the known effects of BDNF on activation of phosphatidylinositide 3 kinase (PI3)/AkT (protein kinase B) kinase . In this+ neurons in standard cKO mice exhibited decreases in total dendritic length compared to WT counterparts, similar to findings recently reported by Chan et al (2008) [Quantification and comparison of overall dendritic development revealed that DCXl (2008) . Howeverl (2008) . TogetheBDNF using a CamKII-Cre 159 line [BDNF mRNA expression in the hippocampus, substantially lower than the 50% reductions in BDNF protein levels reported herein. We have previously shown that BDNF regulates eating behavior and locomotor activity [BDNF knockout mice has made it possible to assess how these behaviors are influenced by acute reductions in BDNF levels, in different brain areas and at different developmental stages. However, characterization of BDNF conditional knockouts has yielded mixed behavioral results. For example, the mice used by Chan and colleagues exhibit hyperactivity, obesity and aggressiveness [BDNF+/- mice. In contrast, we failed to observe any perturbations in these parameters in male mice of our strain (cKO-T29-1). Our cKO-T50 mice showed gender differences in depression-related behaviors, and male mice display normal anxiety-related behavior [BDNF cKO mice used by Chan and colleagues is a reflection of alterations in these neurodevelopmental and/or behavioral parameters. We would argue that in our BDNF cKO line, the observed differences in the proliferation of newly-generated hippocampal progenitors in highly-active male WT vs. male cKO mice and reduced survival of these cells reflect bona fide effects of reduced postnatal, neuronal expression of BDNF, and not the result of physiological and behavioral processes resulting from near complete loss of postnatal BDNF, as reported by Chan et al (2008).It is important to note that despite the strengths of our conclusions, studies by Chan et al 2008) have revealed that conditional inactivation of 159 line  led to a159 line . In cont have revactivity . The avasiveness , similarBDNF ablation, the activity of the CamKII promoter in the hippocampus is restricted to "differentiated" neurons and therefore we expect that BDNF will still be present, albeit at low levels in the SGL. We have previously demonstrated that SGL cells and neurospheres derived from these cells express BDNF [CamKII-Cre allele is not expressed in astrocytes, another source of BDNF. Therefore, it is conceivable that the microenvironment of the SGL in our cKO mice could retain BDNF activity in spite of the overall reduction in the entire hippocampus, and that the cells in the SGL have adequate levels of BDNF necessary for specific physiological responses . Thus, it might be also possible that while complete loss of BDNF in the hippocampus affects cell proliferation in adult mice, as observed by Chan et al (2008), approximately 50% reduction in the hippocampus in the postnatal brain does not impair NPC proliferation, but rather, affects NPC survival. In this regard, it is instructive to note that Gao et al (2009) recently reported that mice with conditional deletions of BDNF in postnatal-born granular neurons of the DG using a Pro-opiomelanocortin (POMC)-Cre allele, in which BDNF levels accumulate to ~50% of wild-type levels, also exhibit impaired survival of newborn cells in standard conditions [While our Cre lines have the obvious advantage of avoiding developmental consequences of ess BDNF . Moreovenditions . These lNotwithstanding the lacunae in our understanding of the cellular and molecular mechanism(s) by which BDNF regulates neurogenesis in the adult DG, future insights in this arena will be essential for developing strategies to isolate and maintain progenitors from adult stem cell niches that may serve as therapeutic modalities for acute and chronic neurodegenerative processes in the adult CNS.2lox/BDNF2lox) mice were crossed to mice expressing bacterial Cre recombinase driven by the neuron-specific CaM Kinase II-Cre promoter  and immunofluorescent labelling for Ki67 were described previously [Mice that harbor loxP sites flanking the single BDNF coding exon (Exon 5) were previously described . The home T29-1, ). CamKIIeviously ,38.Animal experiments were conducted in accordance with institutional and NIH guidelines. Cohorts of WT and cKO animals were housed in large cages containing running wheels, tunnels, toys, and chewable materials 3 h a day for 1 month at the age of 3 months. Control groups of animals were maintained in standard laboratory housing conditions. Mice received a single i.p. injection of BrdU  on the last day of the enrichment. Half of the mice in each group were sacrificed 1 day after the injection to determine progenitor proliferation. The remaining mice were allowed to continue under standard or enriched conditions for 4 weeks and processed to determine survival and neuronal differentiation of the newborn cells. Physical activity levels, defined as the percent time spent on the running wheels, of mice during exposure to EE were observed and recorded every week. Additional mice of each group were processed for ELISA analysis for BDNF protein levels as described below.BDNF protein levels were measured using a BDNF ELISA kit  according to the manufacture's protocol. Hippocampal or cortical homogenate samples were acidified to pH < 3 with 1N HCl and incubated for 15 min at room temperature, followed by neutralization to pH ~8 with 1N NaOH, as this treatment has been shown to maximize the yield of free protein . AliquotTissue preparation, and immunofluorescent labelling for BrdU, neuronal nuclei (NeuN), and Glial Fibrillary Acidic Protein (GFAP) were performed as described previously . The ant+ neurons in each mouse were traced in their entirety and total dendritic lengths were analyzed by NIH Image Software . For dendritic complexity, cocentric circle analysis of Sholl (1953) was performed using a separate NIH ImageJ plugin.DCX is exclusively expressed in immature neurons and has been widely used as an immature neuronal marker . The braLocomotor activity was assessed by placing animals in a new home cage and measuring locomotor activity for 2 h by photobeams linked to computer data acquisition software (San Diego Instruments). Aggressive behavior was assessed using the resident intruder test, as described previously , and defData are expressed as mean values ± SEM. Error bars in the figures represent SEM.T-test and ANOVA tests were applied to study the relationship between the different variables where appropriate. Values of p < 0.05 were considered significant.The authors declare that they have no competing interests.SHC and SSS designed the experiments, statistical analysis, interpreted the results and drafted the manuscript. SHC carried out the majority of the experimental work and YL carried out proliferation study in cKO-T50 mice. SSS and LFP supervised the collection and analysis of all the data. All authors read and approved the final manuscript.


Meiotic mapping of quantitative trait loci regulating expression (eQTLs) has allowed the construction of gene networks. However, the limited mapping resolution of these studies has meant that genotype data are largely ignored, leading to undirected networks that fail to capture regulatory hierarchies. Here we use high resolution mapping of copy number eQTLs (ceQTLs) in a mouse-hamster radiation hybrid (RH) panel to construct directed genetic networks in the mammalian cell. The RH network covering 20,145 mouse genes had significant overlap with, and similar topological structures to, existing biological networks. Upregulated edges in the RH network had significantly more overlap than downregulated. This suggests repressive relationships between genes are missed by existing approaches, perhaps because the corresponding proteins are not present in the cell at the same time and therefore unlikely to interact. Gene essentiality was positively correlated with connectivity and betweenness centrality in the RH network, strengthening the centrality-lethality principle in mammals. Consistent with their regulatory role, transcription factors had significantly more outgoing edges (regulating) than incoming (regulated) in the RH network, a feature hidden by conventional undirected networks. Directed RH genetic networks thus showed concordance with pre-existing networks while also yielding information inaccessible to current undirected approaches. An important problem in systems biology is to map gene networks, which help identify gene functions and discover critical disease pathways. Current methods for constructing gene networks have identified a number of biologically significant functional modules. However, these networks do not reveal directionality, that is, which gene regulates which, an important aspect of gene regulation. Radiation hybrid panels are a venerable method for high resolution genetic mapping. Recently we have used radiation hybrids to map loci based on their effects on gene expression. Because these regulatory loci are finely mapped, we can identify which gene turns on another gene, that is, directionality. In this paper, we constructed directed networks from radiation hybrid expression data. We found the radiation hybrid networks concordant with available datasets but also demonstrate that they can reveal information inaccessible to existing approaches. Importantly, directionality can help dissect cause and effect in genetic networks, aiding in understanding and ultimately rational intervention. Interrogating genome-scale datasets is a necessary step to a systems biology of the mammalian cell Caenorhabditis elegansNetworks constructed using these various approaches are correlated, with some exceptions. While a single dataset often has a large number of false positives and false negatives and reflects only one facet of gene function, accessing multiple independent datasets increases the reliability of gene functional annotation. Integrating diverse gene networks has been shown predictive of loss-of-function phenotypes in yeast Recently transcriptional networks have been constructed using expression data from genetically polymorphic individuals A disadvantage of most currently available networks is that it is difficult to infer functional relationships between interacting genes. Consequently, the edges between genes are undirected and have no regulatory hierarchy. This is also true of eQTL networks where, because of limited mapping power, genotype information has been generally ignored and coexpression networks have been constructed instead +Tk1) −Tk1 hamster A23 cells. Selection for the +Tk1 gene using HAT medium resulted in a panel of 100 hybrid cell lines, each of which contained a random sampling of the mouse genome. Mouse autosomal genes retained in a hybrid clone have two hamster copies plus one mouse copy, compared to two copies otherwise.Radiation hybrid panels have been used to construct high resolution maps of mammalian genomes We recently used the T31 RH panel for high-resolution mapping of QTLs for gene expression trans ceQTLs regulating 9,538 genes at a false discovery rate (FDR) = 0.4 in the T31 panel. At the same FDR threshold, we also found 18,810 cis ceQTLs. Consistent with the average fragment length, a ceQTL was identified as trans if >10 Mb from a regulated gene and cis otherwise. The Using regression, we found 29,769 In this paper we evaluate gene networks constructed from ceQTL mapping. In contrast to undirected networks from meiotically mapped eQTLs and protein binding approaches, the high resolution mapping and dense genotyping of ceQTLs in the RH panel allowed the use of genotype information to construct directed networks. This directionality permits insights that cannot be obtained from undirected networks.We previously analyzed a mouse-hamster radiation hybrid panel, T31 Transcript abundance and marker dosage were measured by mouse expression arrays and comparative genomic hybridization arrays (aCGH), respectively. A total of 20,145 transcript levels were assayed by the expression arrays and 232,626 markers by the aCGH. We mapped ceQTLs by regressing the expression array data on the aCGH data. Mouse and hamster genes were detected with comparable efficiency and behaved equivalently in terms of regulation To construct the RH network, the copy number of each gene was estimated by linear interpolation using the two neighboring aCGH markers. The linear interpolation based estimation is reasonable, considering the high density of aCGH markers.Measured transcripts were denoted by Previously, we used an F-statistic, which is monotonic in the absolute value We constructed an adjacency matrix cis ceQTL cis ceQTLs as an artifactual source of edges in the RH network, we omitted all markers within 10 Mb of the gene being considered. Thus, only trans ceQTLs were employed in the analysis.Since nearly all genes show a copy number increase in a portion of the RH panel, the bulk of genes (94%) also showed a We examined the similarity of our network to existing datasets including protein-protein interactions from HPRD (Human Protein Reference Database) To compare the directed RH network and the other undirected networks, we ignored the edge directions in the RH network and calculated the resulting overlap. To test overlap significance, we used a one-sided Fisher's exact test based on a two by two contingency table, replaced with a one-sided chi-square test when the expected values in all table cells exceeded 50 The existing networks we used for comparison vary in size from 20,957 edges (HPRD network) to 18,754,380 (SymAtlas coexpression network) see . Neverth  values  and over  values  imply thThe maximum overlap significance occurred at low correlation coefficient thresholds between 0 and 0.2 . To testNext we investigated how the number of RH clones affects the overlap. The sensitivity and resolution of the RH network should improve as the number of RH clones increases. To test this, we randomly selected a subset of the 99 RH clones  and calculated the significance of overlap with the HPRD network using the one-sided Fisher's exact and chi-square tests . SimilarH clones . HoweverH clones . This obH clones .We assume that for each undirected network there is a hidden directed random network, modeled as in The results of the comparison of the directed RH network and the hidden directed random network are shown in We examined whether upregulation in the RH data, represented by positive correlation coefficients, Unweighted RH networks obtained from left-tailed thresholding, which emphasized downregulation, did not show any significant overlap  of the weighted (unthresholded) RH and HPRD networks were significantly correlated  of the RH and HPRD networks. The diameter of a giant connected component, consisting of 5,433 nodes with 20,859 undirected edges excepting self-loops, of the HPRD network was 4.13. For the RH network, we considered those 5,433 genes that were in the HPRD network and used a right-tailed threshold of 0.37544, yielding 20,859 undirected edges, to make its size (node and edge numbers) comparable to the HPRD network. The diameter of the RH network was 4.11, close to that (4.13) of the HPRD network.We also compared the clustering coefficients of the RH and HPRD networks, a measure of local cliqueness Previous studies in other networks showed that essentiality is positively correlated with connectivity and betweenness centrality Essential genes had significantly more edges than non-essential genes for a range of right-tailed correlation coefficient thresholds from −0.12 to 0.16 (FDR = 0.01) using a one-sided Wilcoxon rank-sum test ) .Similarly, essential genes had significantly larger betweenness centralities for a range of right-tailed correlation coefficient thresholds from −0.14 to 0.16 (FDR = 0.01) using a one-sided Wilcoxon rank-sum test . Figure It is natural to suppose that transcription factors would have more outgoing than incoming edges since transcription factors regulate other genes. This proposition cannot be tested in conventional undirected networks, but can be tested in the directed RH network. Using a one-sided paired signed rank test We used high resolution mapping of ceQTLs in an RH panel to create a directed genetic network. There was significant overlap with existing networks such as HPRD, KEGG, GO annotation and a SymAtlas coexpression network. The RH network also showed similar topological properties to the HPRD network in connectivity, betweenness centrality and diameter.The RH network showed maximum significance of overlap with existing networks at relatively low positive correlation coefficient thresholds between 0 and 0.2. The low thresholds were not simply by chance, since randomly permuted RH networks did not show any significant overlap with existing networks. Also, the low values did not seem to be caused by noise in the array measurements or by lack of sufficient numbers of RH clones, since the correlation coefficient thresholds giving maximum overlap significance remained nearly constant for varying clone number, although the sensitivity of overlap increased with the number of clones. This may reflect the orthogonal nature of the RH network compared to existing networks, suggesting the RH approach will yield complementary information on mammalian genetic networks. Novel and replicated edges in the RH network may thus be balanced in the low correlation coefficient threshold range.The overlap between the RH network and existing interaction networks was greater for edges possessing upregulation than downregulation. This observation may be because the corresponding proteins are unlikely to interact if one gene represses another, since the proteins will not be present in the cell at the same time. It also implies that protein-protein interaction networks may fail to uncover valid edges between genes if they have a repressive relationship.Previous studies found significant associations of essentiality with connectivity and/or betweenness centrality in protein-protein interaction networks We also showed that transcription factors were likely to have more outgoing rather than incoming edges. While this finding is not unexpected and helps validate the RH network, a recent study using naturally occurring polymorphisms in yeast suggested that transcription factors are no more likely to reside close to eQTLs than chance We thresholded the adjacency matrix at different correlation coefficients to compare unweighted RH networks with existing unweighted datasets. However, we chose to leave the RH network weighted rather than finalizing an unweighted form at an optimal threshold. Such an operation is irreversible and would lose information on linkage strength and sign. In other studies, the sensitivity of a coexpression network was limited by thresholding We constructed a directed gene network from radiation hybrids and found it concordant with existing networks. We also showed that RH networks have the potential to provide new insights reflecting orthogonal aspects of gene regulation. The RH networks will be refined as more panels, including those available for other species, are analyzed resulting in improved power and sensitivity.Details on the analysis of the T31 RH panel cells and the preprocessing of aCGH and expression array data can be found in The directed RH network was constructed as described in A protein-protein interaction network was constructed from HPRD (Human Protein Reference Database) A network was constructed from the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database A network was constructed from the GO (Gene Ontology) database We constructed an mRNA coexpression network from the publicly available SymAtlas microarray database The significance of overlap between the RH network obtained from thresholding and, for example, the HPRD network was tested as follows.trans. Fourth, an overlap was defined as the number of gene pairs such that both First, for a given threshold ding see . Second,We similarly calculated the significance of overlaps with the KEGG pathway network, the SymAtlas coexpression network and the GO annotations.trans and performed the overlap significance test (above).We randomly permuted the elements of the weighted and directed adjacency matrix We randomly selected 40, 60 or 80 RH clones out of 99 and constructed an adjacency matrix see  using meFor each existing undirected dataset, for example, the HPRD network, we assume there is a hidden directed random network with adjacency matrix The overlap between the unweighted (thresholded) directed RH network, represented by Ignoring the constant scaling factor without loss of generality, we define an overlap as The node degree of the undirected, weighted adjacency matrix http://www.stanford.edu/~dgleich). When we calculated the betweenness centrality of the RH network, we used a subgraph by taking nodes that were in the HPRD network to reduce computational cost. Then the Spearman's correlation coefficients between the betweenness centralities and also between clustering coefficients for RH and HPRD were calculated.The betweenness centralities and clustering coefficients of the RH adjacency matrix We obtained a list of 1,409 essential genes and 1,979 nonessential genes from the Mouse Genome Database We obtained a list of 1,053 transcription factors by finding genes whose GO description includes a word “transcription.” The number of outgoing edges was calculated by http://labs.pharmacology.ucla.edu/smithlab/RHnetwork.htmlThe network data are available at Text S1Relationship between one-sided chi-square test and Bayesian log-likelihood score (LLS) method(0.08 MB PDF)Click here for additional data file.Table S1Size of RH network constructed from right-tailed, left-tailed and both-tailed thresholding approaches.(0.06 MB PDF)Click here for additional data file.Figure S1Size of RH network. (A) Number of nodes with nonzero degree for RH network constructed from right-tailed thresholding. (B) Number of directed edges for RH network constructed from right-tailed thresholding. (C) Number of nodes with nonzero degree for RH network constructed from left-tailed thresholding. (D) Number of directed edges for RH network constructed from left-tailed thresholding. (E) Number of nodes with nonzero degree for RH network constructed from both-tailed thresholding. (F) Number of directed edges for RH network constructed from both-tailed thresholding.(0.21 MB TIF)Click here for additional data file.Figure S2Overlap between RH network constructed from right-tailed thresholding and existing datasets. Same as (0.26 MB TIF)Click here for additional data file.Figure S3Significance of overlap between RH network constructed from left-tailed thresholding and existing datasets. Same as (0.25 MB TIF)Click here for additional data file.Figure S4Significance of overlap between RH network constructed from both-tailed thresholding and existing datasets. Same as (0.28 MB TIF)Click here for additional data file.Dataset S1Significance of overlap between RH network and existing datasets. (0.78 MB XLS)Click here for additional data file.


ArrayIDer) to retrieve the most recent accession mapping files from public databases based on EST clone names or accessions and rapidly generate database accessions for entire microarrays.Systems biology modeling from microarray data requires the most contemporary structural and functional array annotation. However, microarray annotations, especially for non-commercial, non-traditional biomedical model organisms, are often dated. In addition, most microarray analysis tools do not readily accept EST clone names, which are abundantly represented on arrays. Manual re-annotation of microarrays is impracticable and so we developed a computational re-annotation tool (ArrayIDer could markedly improve annotation. We structurally re-annotated 55% of the entire array. Moreover, we decreased non-chicken functional annotations by 2 fold. One beneficial consequence of our re-annotation was to identify 290 pseudogenes, of which 66 were previously incorrectly annotated.We utilized the Fred Hutchinson Cancer Research Centre 13K chicken cDNA array – a widely-used non-commercial chicken microarray – to demonstrate the principle that ArrayIDer allows rapid automated structural re-annotation of entire arrays and provides multiple accession types for use in subsequent functional analysis. This information is especially valuable for systems biology modeling in the non-traditional biomedical model organisms. Microarrays have become a standard tool for functional genomics allowing analysis of thousands of mRNA transcripts simultaneously and they are widely used for a diverse range of species -4. MicroEasyGO [Although 10 software packages have been developed to map between popular database identifiers ,10,12-18ArrayIDer, a user-friendly program that generates a library of public accessions available from the Gene Expression Omnibus (GEO) browser [ArrayIDer currently accepts data from any microarray containing EST identifiers compatible with the NCBI UniGene database [ArrayIDer generates a library of gene and protein accessions from the latest updated NCBI UniGene [ArrayIDer retrieves identifiers from UniGene and IPI that match the EST input list. All annotations of ESTs to genes (and accompanied proteins) are as assigned by NCBI UniGene. ESTs listed in UniGene are grouped in a UniGene cluster based on their nucleotide overlap. The gene represented by each cluster is determined by the top BlastX hit of the nucleotide sequence. Gene information regarding the EST cluster to gene match is retrieved from the NCBI Homologene database, where known orthologs for genes are mapped through multiple species. The structural annotations retrieved by ArrayIDer are only retrieved from the species-specific UniGene database, which contains pre-assigned structural annotations made according to the methods used at the Homologene database. An online version of ArrayIDer allows rapid identifier searching of EST libraries of several species generated by AgBase.Here we describe  browser  for bothdatabase  from nin UniGene  and Inte UniGene  databaseArrayIDer are available at the AgBase website  → Array Annotation → ArrayIDer) and researchers can use the simple interface to search structural annotations for their microarray ESTs or accessions in the species' EST library. Libraries available online are updated when new versions of the underlying databases are released. Any available library can be extended by users by contacting AgBase directly to request structural annotation for their arrays. Conversely, and especially for those conversant with Perl, ArrayIDer is available for download for researchers to generate a library for species currently not listed on AgBase to avoid requesting the work be done by AgBase staff. ArrayIDer runs locally via the command line console or by execution in a designated directory. To run locally ArrayIDer requires: 1) Perl platform (version 5.8.8 built 8.17 or higher); 2) installation of Archive::Extract, DBI and NET::FTP Perl modules; 3) a text formatted input file of cDNA/EST clone names or GenBank nucleotide sequence accessions; and 4) an internet connection. The script downloads and unpacks the required databases directly from the internet.Microarray libraries for multiple species generated with ArrayIDer standalone version reads the input list and searches each entry against the latest version of NCBI UniGene to retrieve initial gene and protein information  for comprehensive identifier mapping. An example of the online output can be found in Figure Fig. The online ArrayIDer allows researchers to rapidly update the structural annotation of their microarray and use this information in downstream gene expression modeling and pathways analysis. To demonstrate the use of ArrayIDer we selected a widely-used non-commercial array, the Fred Hutchinson Cancer Research Centre (FHCRC) 13K chicken cDNA array [NA array . The arrArrayIDer provided a 6.67-fold increase  in chicken-specific annotations. Among the chicken structural annotations assigned, 55% (4177) are assigned to a Swiss-Prot/TrEMBL accession. 45% (3404) are assigned to a predicted "XP_" accession, which are candidates for further annotation curation. Identification and curation of these XP_ accessions improves the species' genome annotation.Microarray structural re-annotation results were compared to the annotation currently provided for the FHCRC array. In total, 13,234 probe identifiers were submitted as input for the script. Originally, 1136 array probes were structurally annotated to a chicken gene, with a further 7820 structural annotations to other (non-chicken) species . ArrayIDer that mapped to gene elements labelled as pseudogenes. Pseudogenes have been defined as defunct relatives of known genes that are considered non-functional; however, some pseudogenes can be transcribed and play a role in gene regulation and expression [AgBase. Four months after submission of the changes, we re-analyzed the FHCRC array with ArrayIDer and identified the remaining 224 gene elements labelled as pseudogenes, indicating the 66 genes are corrected and updated in the public database.Initially, we identified 290 transcripts on the FHCRC array with pression . MoreoveArrayIDer, we were able to decrease non-chicken functional annotations associated with the array by 2 fold (from 7309 to 3671).The Gene Ontology (GO) is the de facto standard method for functional annotation of gene products . While EArrayIDer allows rapid automated re-annotation of entire arrays and provides the user with multiple accession types for use in functional analysis. Together this information is especially valuable for the non-traditional biomedical model organisms to utilize the wide range of existing tools for systems biology modeling downstream. We focus on expanding the number of public databases used to assign accessions, include up-to-date, curated functional annotations for both commercial and custom designed microarrays (including specific requests) and incorporate this information into the AgBase database for user-friendly online access.Continual structural- and functional re-annotation of microarrays ensures the most up-to-date gene product information for modeling functional genomics datasets. ArrayIDerProject name:  → Array annotation → ArrayIDerProject home page: Operating system(s): Platform independentProgramming language: PerlOther requirements: Perl modules Archive::Extract, DBI and NET::FTPLicense: Freely availableBVDB developed the pipeline, compiled the tool's script and drafted the manuscript. JHK contributed in the tool's script optimization and performance evaluation. FMM and SCB participated in the pipeline development and help draft the manuscript. All authors read and approved the final manuscript.


The syndrome of the sinking skin flap (SSSF) with delayed sensorimotor deficits after craniectomy is not well known and often neglected. Among various postulated causes, there is evidence that disturbed brain perfusion may be related to the observed symptoms, and that cranioplasty reliably alleviates these symptoms. We report a case of sinking skin flap syndrome (SSFS) with recovery from neurological sensorimotor deficits after cranioplasty correlated with pre- and postsurgical MR brain perfusion studies.A 42-year-old woman presented with slowly progressive sensorimotor paresis of her left arm after decompressive extensive craniectomy due to subarachnoid hemorrhage four months ago. Her right cranium showed a "sinking skin flap". After cranioplastic repair of her skull defect, the patient fully recovered from her symptoms. Before cranioplasty, reduced brain perfusion in the right central cortical region was observed in MR-perfusion images. After cranioplasty, a marked increase in brain perfusion was observed which correlated with objective clinical recovery.There is increasing evidence that impaired blood flow is responsible for delayed motor deficits in patients with sinking skin flap syndrome in the area of compressed brain regions. Symptoms should be evaluated by brain perfusion imaging complementing surgical decision-making. There are few reports of SSFS associated with delayed motor deficits, designated as "motor trephine syndrome",. The "sinking skin" is a result of lower intracranial pressure in upright position and higher atmospheric pressure so that the pressure gradient across the skin flap directly acts on the brain resulting in tissue compression. Accordingly, neurological improvement has been reported when this pressure disturbance is normalized temporarily with change in posture,2 or permpression,9; b) hyn posture,7,10; c)in tissue,11-13; detabolism,15.Radiological studies and case series correlating brain perfusion with neurological deficits before and after cranioplastic repair are limited-13,15-18A case of motor trephine syndrome assessed by MR perfusion (MRP) study before and after cranioplasty is presented.A 42-year-old woman presented with slowly progressive weakness and dysesthesia of her left arm after decompressive craniectomy. Neurological examination revealed a slight, distal paresis with increased tendon reflexes, dysesthesia of her left arm, and reduced fine motor skills of the left hand. She suffered from a subarachnoid hemorrhage (SAH) due to a spontaneously ruptured aneurysm of the right middle cerebral artery 4 months ago. A right fronto-temporo-parietal craniectomy with evacuation of the hematoma and coiling of the aneurysm had been performed as initial treatment, and the bone flap had not been replaced. Initial postsurgical left hemiparesis completely disappeared, but late sensorimotor disturbances of the left arm continuously worsened over the last three weeks. The skin of the right temporoparietal region was markedly sunken due to the large skull defect.Computed tomography and magnetic resonance imaging of the brain were unremarkable except for minor focal gliosis. There were no signs of ischemia or edema, new hemorrhage, coil extrusion, brain shift, or disturbed CSF circulation. Three consecutive electroencephalograms did not show epileptiform discharges, an extra- and transcranial Doppler ultrasonography was unrevealing.A secondary neurological deterioration was diagnosed due to SSFS.After replacement of the bone flap the sensorimotor deficits of her left arm continuously improved within a period of 10 to 14 days. At the follow-up examination four weeks after cranioplasty, the patient reported a complete recovery. Only slightly increased tendon reflexes of the left arm were still present.Brain perfusion immediately before and four weeks after cranioplastic repair was assessed by T2*-weighted MR-perfusion imaging  with a double dose of Gadovist-1.0 . Perfusion maps were calculated on a Philips ViewForum workstation from regions of interest (ROI) drawn over the entire left and right hemisphere for each slice, respectively. For objective comparison of perfusion parameters between the two consecutive time points, relative cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) of the right hemisphere compared to normal left side were calculated.Before cranioplasty brain perfusion in the right hemisphere was lower compared to the left side. Perfusion maps demonstrated a focal deficit particularly in the compressed central cortical region beneath the skinflap Fig . Right hSinking skin flap syndrome with delayed motor deficits, or "motor trephine syndrome" is not well known in patients with large skull defects, where progressive neurological deterioration is associated with the sinking skin flap,12. In tEvaluation of SSFS with brain perfusion is not commonly performed; only few cases with perfusion imaging before and after cranioplasty have been reported correlating clinical symptoms with hemodynamics in the affected hemisphere. Winkler et al. demonstrated improved blood flow and cerebrovascular reserve capacity after cranioplasty in 12 cases using transcranial Doppler sonography and 18-FDG positron emission tomography. In smalEven though perfusion imaging generally correlates well with symptoms in SSFS, it must be noted that an inherent limitation may be a result of extensive parenchymal damage or gliosis leading to unspecific perfusion asymmetry.The only systematic prospective study of SSFS revealed an incidence of 30% of SSFS whereas Furthermore, it is to be expected that decompressive craniectomies will be carried out more often in the future due to the improvement of functional outcome after malignant middle cerebral artery infarction and SSFSThis case report contributes to emerging evidence that impaired brain perfusion is a significant mechanism in the sinking skin flap syndrome after decompressive craniectomy. Cranioplasty improves brain perfusion alongside with neurological deficit. Our results emphasize that using brain perfusion imaging could complement surgical decision making for cranioplastic repair.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.AK, TD: have made substantial contributions to conception and design, acquisition, analysis and interpretation of data; have been involved in drafting the manuscript or revising it critically for important intellectual content. LL, TN: have made substantial contributions to acquisition, analysis and interpretation of clinical and imaging data. HW, JM: have been involved in drafting the manuscript or revising it critically for important intellectual content. MM: has given final approval of the version to be published; has made substantial contributions to acquisition, analysis and interpretation of clinical data. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/10/80/prepub


We studied the effects of sheep scrapie on lymphoid tissue in tonsils and lymph nodes by light and electron microscopy. FDCs of sheep were grouped according to morphology as immature, mature or regressing. Scrapie was associated with FDC dendrite hypertrophy and electron dense deposit or vesicles. PrPd was located using immunogold labelling at the plasmalemma of FDC dendrites and, infrequently, mature B cells. Abnormal electron dense deposits surrounding FDC dendrites were identified as immunoglobulins suggesting that excess immune complexes are retained and are indicative of an FDC dysfunction. Within scrapie-affected lymph nodes, macrophages outside the follicle and a proportion of germinal centre TBMs accumulated PrPd within endosomes and lysosomes. In addition, TBMs showed PrPd in association with the cell membrane, non-coated pits and vesicles, and also with discrete, large and random endoplasmic reticulum networks, which co-localised with ubiquitin. These observations suggest that PrPd is internalised via the caveolin-mediated pathway, and causes an abnormal disease-related alteration in endoplasmic reticulum structure. In contrast to current dogma, this study shows that sheep scrapie is associated with cytopathology of germinal centres, which we attribute to abnormal antigen complex trapping by FDCs and abnormal endocytic events in TBMs. The nature of the sub-cellular changes in FDCs and TBMs differs from those of scrapie infected neurones and glial cells suggesting that different PrPd/cell membrane interactions occur in different cell types.Transmissible spongiform encephalopathies (TSEs) or prion diseases often result in accumulation of disease-associated PrP (PrP Scrapie, a disease naturally affecting British sheep and goats for many years, belongs to a group of slowly progressive neurodegenerative disorders, the transmissible spongiform encephalopathies (TSEs) or prion diseases, which include infectious, familial and sporadic forms of disease in animals and man. The TSEs include bovine spongiform encephalopathy (BSE), scrapie of sheep and goats, Creutzfeldt-Jakob disease (CJD), kuru and Gertsmann-Straűssler syndrome (GSS) of humans. TSEs result in abnormal isoforms of a host-coded, cell-surface glycoprotein called prion protein (PrP).sen) abnormal PrP detected by immunoblotting methods is abnormally resistant to proteinase treatment and contains truncated, protease resistant forms of PrP often designated PrPresres. Immunohistochemistry may also be used to detect disease-associated accumulations of PrP (PrPd). Unlike immunoblotting methods, immunohistochemistry detects abnormal PrP forms that may be truncated or full length, protease resistant or protease sensitive d.Unlike the normal proteinase sensitive form of prion protein (PrPd accumulates in the nervous system and lymphoreticular system (LRS) scrapie of sheep and variant Creutzfeldt Jakob disease. In contrast with the CNS, where lesions are well known, it is commonly accepted that despite the presence of infectivity and PrPd accumulation, there is no associated pathology in the lymphoreticular system of naturally occurring TSEs. Studies using severe combined immunodeficient mice and chimaeric mice indicate that follicular dendritic cells (FDCs) are necessary for prion propagation within the LRS d as demonstrated by immunohistochemistry in scrapie PrPd accumulation Within affected lymph nodes of scrapie-affected sheep, most secondary follicles show PrPd immunolabelling only on cells morphologically similar to mature FDCs in the light zone of germinal centres of secondary follicles. In contrast, TBM labelling is present in the light, dark, mantle and paracortical zones d labelling has also been detected within mononuclear cells of the periarteriolar lymphoid sheath (PALS) and within the marginal zone of the spleen d accumulations are truncated with the loss of the N-terminus amino acid sequence from approximately 23–90, while all other types of PrPd accumulation remain full length d is mostly associated with the FDC dendrite plasmalemma and FDC dendrites engaged in plasmablast emperipolesis (i.e. FDC dendrite-encircled plasmablasts). PrPd is also present in TBM lysosomes. The absence of the N-terminus of intra-cellular PrPd in TBMs is consistent with the notion that TBMs internalize and digest extra-cellular PrPd derived from FDCs Labelling of CD21, which is expressed on FDC membranes and on B cells d in scrapie-infected sheep LRS tissues.In previous studies we have suggested that there are pathological changes associated with scrapie infection of splenic secondary follicles. To determine whether similar changes are present in natural disease, we have studied the morphological response and sub-cellular location of PrPd labelling was observed within macrophages of the perifollicular region of the mesenteric and submandibular lymph nodes. Within tonsils, PrPd was associated with macrophages present in thinner regions of the epithelial layer, and in the perifollicular area. This labelling was not revealed by BG4 antibody  from unaffected animals. The intensity of PrPd immunolabelling in scrapie affected animals varied between follicles, tissue blocks and animals, although most follicles showed extensive PrPd accumulations, primarily within the follicle.Secondary follicles from scrapie-affected sheep showed PrPd pattern  labelling were observed interspersed in a curvi-linear pattern between lymphocytes of the germinal centre . A simil pattern .Following application of an anti-ubiquitin antibody, a few granules of immunolabelling were identified within cells morphologically similar to TBMs in scrapie-affected tonsils or lymph nodes.Groups of FDC dendrites, morphologically similar to those found in normal rodents d nor ubiquitin labelling was observed in any sections studied.TBMs containing lysosomes were also present in the light zone and dark zone of all follicles. Occasionally, TBMs contained whole apoptotic lymphocytes. Neither PrPSome normal mature FDCs were occasionally seen within scrapie-affected lymph nodes, but most FDCs showed abnormal morphologic changes of two types, which we categorised as disease-associated mature type 1 or type 2 FDCs. When compared with normal mature FDCs, disease-associated mature type 1 FDCs had more abundant florid dendrites with an excess accumulation of uniform electron dense deposit surrounding them . These dd immunolabelling were observed within lymphoid tissues from scrapie-affected sheep. Primarily, this labelling was confined to the germinal centres, but occasionally, PrPd was present at sites distant from this.Three patterns of PrPd immunolabelling was not abundant. Where present it was clearly limited to the plasmalemma of dendrites  from it Sparse vesicles are associated with the extracellular space between FDC dendrites in uninfected animals (regressing FDCs), but numerous vesicles may be present in scrapie-affected tissues (disease-associated mature type 2 FDCs). Vesicles are 50–200 nm in diameter corresponding in size with exosomes; small membrane vesicles secreted by many cells as a result of endosomes or lysosomes fusing with the plasma-membrane. Vesicles at the surface of FDCs contain MHC-II molecules, but they do not synthesise them, suggesting that they are passively acquired from donor cells in the form of plasma membrane fragments, or exosomes, shed by B cells d was present at the cell membrane of a small proportion of B cells. Survival of B cells within the germinal centre depends on whether their surface immunoglobulins are bound to antigens retained by the FDC. Specific antigens will bind to B cells in the form of immune complexes via the cell surface immunoglobulin receptor. During this process, we hypothesise that PrPd, present at the plasmalemma of the FDC may be passively transferred to the B cell plasmalemma. Were this not so, PrPd would be processed and presented in a similar manner to antigenic proteins in association with MHC-II molecules. As such it would not be recognisable by the PrP antibody, nor would it be in an organised linear manner on the plasmalemma of the cell.PrPd also accumulates on the cell surface in vivo (unpublished observations) and in vitro0/0 mice, which express PrP only on astrocytes but not neurons, show PrPd accumulation and associated pathology on the non PrP-expressing neurons d first accumulates at the plasmalemma of FDCs where it is then either transferred to adjacent cells to be retained on the plasma-membrane as it the case for B cells. Although exosomes transfer infectivity between cultured cells d positive. A mechanism for this transfer process is represented in Pulse-chase experiments were the first to suggest that the transformation of normal to abnormal PrP may take place at the cell surface or at a later stage in the cell cycle d in association with the membranes of random fused tubular extensions of the ER , VV136 (n = 2) and VA136 (n = 4), (136) which were naturally infected with scrapie were killed, Mesenteric Lymph nodes (MLN) removed and immersion fixed in 4% paraformaldehyde. One millimetre thick slices of the lymph nodes or tonsils were taken for paraffin wax embedding and for electron microscopy. Tissues selected for electron microscopy were further trimmed into 1 mm3 blocks, post fixed in osmium tetroxide and embedded in araldite.Ten sheep naturally infected and with clinical signs of scrapie, and four uninfected control animals from New Zealand were perfused via the carotid artery under deep surgical anaesthesia with 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer. All sheep were taken from susceptible PrP genotypes AA(n = 4), . Submandd will be used to indicate all disease-specific PrP accumulations.The light microscopical immunohistochemical procedure was used as described previously As described previously Sections were also immunolabelled with Zymed anti-IgG antibody at a 1∶100 dilution, or Dako anti-Ubiquitin antibody at a 1∶800 dilution using the same immunolabelling procedure with the omission of the formic acid stage d labelling and immunolabelled as described previously 65 nm sections were taken from resin blocks previously found to show PrPAt least 5 follicles were studied from all LRS tissues collected.


The availability of sequenced eukaryotic genomes and commercial oligonucleotide tiling microarrays has enabled many genomics applications. Different from expression microarrays, tiling microarrays have probes that cover the entire genome or contigs of the genome in an unbiased fashion. Currently three commercial sources provide tiling microarrays with different probe lengths and spacing, and array design characteristics. Affymetrix tiles 6 million 25-mer probes per array, which offers the lowest price per probe and the highest resolution . Its arrays use one-color assays, so individual samples are hybridized to different arrays. NimbleGen can tile 385,000 50- to 75-mer probes, and Agilent can tile 244,000 60-mer probes per array. The latter two platforms, with longer oligonucleotide probes and two-color assays for which treatment and control samples are differentially labeled and put on the same array for competitive hybridization, have slightly better sensitivity. They are also flexible for custom array design, especially Agilent's multiplex arrays, which allow multiple samples to hybridize on different subareas of the same array. These tiling arrays offer diverse genomic applications, each with its own data analysis challenges.The most popular application for the tiling array platform is ChIP-chip, which maps the genome-wide binding locations of transcription factors and other DNA-binding proteins. In a ChIP-chip experiment, chromatin is crosslinked and fragmented to approximately 500 bp. An antibody to the protein of interest is used to precipitate the protein together with its interacting DNA . The coprecipitated DNA is detected on a DNA microarray (the “chip”) and mapped back to the genome ,2. In coFor ChIP-chip on Affymetrix tiling microarrays, MAT   is a verhttp://main.g2.bx.psu.edu/) offers more customized and interactive features to analyze additional sequenced genomes.After the ChIP-chip peaks are detected, biologists often want to find the sequence-specific binding motifs of their protein of interests. MEME  and GibbDNA methylation status often controls gene transcription status, and genome-wide DNA methylation sites can be mapped using methyl–DNA immunoprecipitation followed by microarray (MeDIP-chip). MeDIP-chip is similar to ChIP-chip in protocol, except that an antibody against 5-methyl-cytosine is used to directly precipitate methylated DNA ,16. PeakDNase-hypersensitive regions in the genome are often open chromatin harboring transcriptionally active or regulatory regions, which can be located using DNase-chip. Relying on the assumption that open chromatin is cleaved more often by DNase over a short distance, this experiment involves digesting chromatin with DNase I, isolating DNA fragments created by two DNase cleavages less than 1,200 bp apart, and hybridizing the DNA to tiling microarrays . The resA nucleosome, which consists of ∼146 bp of DNA wrapped around eight histone proteins, forms the fundamental structural unit of eukaryotic chromatin. Since nucleosomes limit DNA accessibility to regulatory factors, it is important to map positioned nucleosomes or nucleosome-free regions in the genome. Nucleosome mapping experiments involve digesting the chromatin with micrococcal nuclease to remove the linker DNA between neighboring nucleosomes, and isolating the remaining nucleosomal DNA to be labeled and hybridized to a tiling microarray. The controls for such experiments are often naked genomic DNA (without chromatin structure) cleaved with hydroxyl radicals or micrococcal nuclease to the same size distribution. Unlike ChIP-chip, the occupancy difference between positioned nucleosomes and linker regions is often less than 10-fold, and positioned nucleosomes occupy only about 100–200 bp of DNA. This requires the tiling microarray to have both high sensitivity and high resolution. Long oligonucleotide microarrays tiled at 5–20 bp resolution are often custom-made to cover selected genomic regions  for this application.In a nucleosome mapping study conducted in yeast Chromosome III , a hiddeIn an array-based comparative genome hybridization (arrayCGH) experiment, DNA from normal and diseased individuals are differentially hybridized to microarrays to identify copy number variations in the genome that are potential biomarkers or causal genes of disease . Early mDrosophila embryogenesis stages on genome tiling microarrays, a Kruskal-Wallis test  was used to detect a stretch of probes giving differential expression among conditions [Hybridizing RNA samples to tiling microarrays is gaining popularity for detecting novel transcripts in sequenced genomes. Early studies often called positive probes based on a probe signal cutoff , then denditions . In addiAll commercial tiling microarray companies strive to put more probes on the array at reduced cost. This trend seems to follow the Moore's Law observed in the semiconductor industry, which dictates that chips double their density at half the cost every 18 months. A few years from now might see tiling microarrays covering the whole mammalian genome at single-base resolution that cost only a few thousand dollars. Tiling arrays will have much wider applications, and researchers might use them for different experiments and informatically select a subset of the probes for analysis. At the same time, high-throughput sequencing technologies such as 454, Illumina Solexa, and ABI SOLiD are making fast progress as well. If enough coverage can be achieved at a cost similar to tiling microarrays, they might give more sensitive and unbiased results. These technologies each entail different challenges and opportunities for computational biologists to develop efficient analysis algorithms. The competition between the different technology companies will inevitably benefit researchers regardless of the winner. Therefore, we look forward to a very exciting decade of genomics advances ahead.


Consistent with this hypothesis, pretreatment with either quinidine or verapamil (inhibitors of Pgp-mediated transport) sensitized CHRC5 cells to the antiproliferative effects of flavopiridol. Because of concern that colony forming assays might not accurately reflect cytotoxicity, we also examined flavopiridol-treated cells by trypan blue staining and flow cytometry. These assays confirmed that flavopiridol was less toxic to cells expressing higher levels of Pgp. Further experiments revealed that flavopiridol inhibited the binding of [3H]-azidopine to Pgp in isolated membrane vesicles, but only at high concentrations. Collectively, these results identify flavopiridol as a weak substrate for Pgp. © 2001 Cancer Research Campaign www.bjcancer.comFlavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC


Hymenoptera venoms contain thrombogenic substances that might be responsible for cardiovascular events independent of anaphylactic reactions.We report a 55-year-old man who experienced an acute ST-elevation myocardial infarction after wasp sting. The patient presented without signs of anaphylaxis or shock. The coronary angiography showed an acute stent thrombosis of the right coronary artery. Percutanous coronary intervention was performed immediately and this is an example for a cardiovascular complication associated with a hymenoptera sting, since the vasoactive, inflammatory, and thrombogenic substances of hymenoptera venoms potentially cause stent thrombosis and myocardial ischemia. To the best of our knowledge this is the first report of acute stent thrombosis in a sirolimus-eluting stent following hymenoptera sting.Stent thrombosis is a possible complication after wasp sting induced by thrombogenic substances of the hymenoptera venom. Independent of anaphylactic reactions phospholipase AA 55-year-old Caucasian German man with known ischemic heart disease presented in our emergency room, with an acute inferior myocardial ST-elevation infarction. Three years before stenoses of the proximal left anterior descending and the proximal right coronary artery were treated by PCI and implantation of two bare metal stents, followed by PCI and implantation of a sirolimus-eluting stent (Cypher®) of an In-stent restenosis in the right coronary artery one year later. Afterwards no relevant cardiovascular events or hospitalizations occurred until the current admission. Clopidogrel (75 mg/d) was withdrawn 12 months after implantation of the sirolimus-eluting stent, while aspirin (100 mg/d) was continued. Known and treated cardiovascular risk factors were hypertension, hypercholesterolemia and diabetes mellitus.As hobby beekeeper the patient was used to bee stings and never reported of any related symptoms, except local reactions. One hour before admission he was stung by 5 wasps. Just a few minutes after being stung he started complaining of chest pain and called an emergency physician. ECG revealed an acute myocardial infarction with inferior ST-segment-elevation , blood pImmediately after admission to the emergency room, coronary angiogram was performed showing a thrombosis in the two year old sirolimus-eluting stent of the right coronary artery occluding the vessel sub-totally. PCI was successfully performed with good angiographic result . TherapyMyocardial infarction following hymenoptera stings  is rare but there are some cases reported in the literature -5. HowevDespite from local cutaneous manifestations or generalized anaphylaxis, hymenopterans stings can cause a variety of unusual reactions. There have been reports of neurological reactions such as myasthenia gravis, peripheral neuritis and Guillain-Barré syndrome. Diffuse alveolar hemorrhage, acute renal failure, rhabdomyolysis, thrombocytopenic purpura and vasculitis are other pathologies presumed to be related to insect stings .et al. recently described a protein, magnifin, which was purified from the venom of a wasp Vespa magnifica (Smith), containing phospholipase-like activity and inducing platelet aggregation and thrombosis in vivo [et al. reported a case of thrombosis of the descending aorta and cerebral infarction after massive wasp stings [Myocardial infarction after bites of hymenopterans is mostly reported to be preceded by an allergic reaction, sometimes with angiographic evidence of undamaged coronary arteries . Pathoph in vivo . Chen etp stings .et al. reported of an acute ST-elevation myocardial infarction in the context of an anaphylactic reaction caused by a European hornet sting. In presence of ST-elevations at presentation the patient was not treated with adrenaline. In this patient exogen adrenalin did not contribute to the development of the myocardial infarction, but hypotension due the anaphylactic shock may have contributed to the development or the worsening of myocardial infarction [In the cases reported, the causal relationship between hymenoptera sting and myocardial ischemia was sometimes not very clear. Some patients had signs of anaphylactic shock and therefore were treated with adrenaline. On one hand hypotension cased by anaphylaxis can lead to myocardial hypoperfusion, on the other hand adrenaline itself has thrombogenic effects and is able to provocate vasoconstriction , contribfarction .In our patient no signs of anaphylaxis or shock were present. First symptoms of chest pain occurred a few minutes after the wasp stings. Immediately afterwards the patient called the emergency physician. Initial ECG revealed ST-elevations in leads II, III and aVF, while blood pressure was within normal range (140/85 mmHg). Considering the short time interval between the wasp attack and the beginning of chest pain, it is reasonable to assume, that the wasp venom caused acute stent thrombosis followed by myocardial infarction.1 as phospholipase A2 in contrast to bee venom [et al. that phospholipase A1 isolated from wasp venom inducing platelet aggregation and thrombosis in vivo [It is notable that our patient as beekeeper was used to bee stings and has been stung by bees many times without any symptoms except for local reactions. As bee and wasp venoms are highly complicated mixtures of pharmacologically or biochemically active agents, it remains unclear which components of the wasp venom differing from bee venom induced the acute stent thrombosis. Interestingly wasp venom contains more phospholipase Aee venom . This an in vivo , could b1 of the hymenoptera venom.Stent thrombosis is a possible complication after wasp sting induced by thrombogenic substances as for example phospholipase A


The introduction of new imaging techniques has markedly improved the diagnosis of hepatobiliarydisorders. Due to their anatomic situation, a substantial percentage of malignancies located near thehilus is not suitable for surgical management. We discuss an effective palliative intervention to relievejaundice. In many instances drainage is a superior choice when biliodigestive anastomoses are nottechnically feasible and palliative resection carries a high complication rate.        We present an irrigatable exo-endodrainage method employing a modified port-a-cath system as anew alternative. In four patients, all older than 75 years, this system was implanted because of jaundicedue to unresectable malignant stenosis of the extrahepatic bile duct. One patient (80 years old) died ofpre-existing acute necrotizing pancreatitis, although hyperbilirubinemia was found to decrease on the7th postoperative day. The other three patients showed complete normalization of their bilirubin levelsand their port-a-cath systems remained open until their death .


Autogenous Iliac Crest Bone Graft (ICBG) has been the "gold standard" for spinal fusion. However, bone graft harvest may lead to complications, such as chronic pain, numbness, and poor cosmesis. The long-term impact of these complications on patient function and well-being has not been established but is critical in determining the value of expensive bone graft substitutes such as recombinant bone morphogenic protein. We thus aimed to investigate the long-term complications of ICBG. Our second aim was to evaluate the psychometric properties of a new measure of ICBG morbidity that would be useful for appropriately gauging spinal surgery outcomes.Prospective study of patients undergoing spinal fusion surgery with autologous ICBG. The SF-36v2, Oswestry Disability Index, and a new 14-item follow-up questionnaire addressing persistent pain, functional limitation, and cosmesis were administered with an 83% response rate. Multiple regression analyses examined the independent effect of ICBG complications on physical and mental health and disability.The study population included 170 patients with a mean age of 51.1 years (SD = 12.2) and balanced gender . Lumbar fusion patients predominated . At 3.5 years mean follow-up, 5% of patients reported being bothered by harvest site scar appearance, 24% reported harvest site numbness, and 13% reported the numbness as bothersome. Harvest site pain resulted in difficulty with household chores (19%), recreational activity (18%), walking (16%), sexual activity (16%), work activity (10%), and irritation from clothing (9%). Multivariate regression analyses revealed that persistent ICBG complications 3.5 years post-surgery were associated with significantly worse disability and showed a trend association with worse physical health, after adjusting for age, workers' compensation status, surgical site pain, and arm or leg pain. There was no association between ICBG complications and mental health in the multivariate model.Chronic ICBG harvest site pain and discomfort is reported by a significant percentage of patients undergoing this procedure more than three years following surgery, and these complications are associated with worse patient-reported disability. Future studies should consider employing a control group that does not include autologous bone graft harvest, e.g., a group utilizing rhBMP, to determine whether eliminating harvest-site morbidity does indeed lead to observable improvement in clinical outcome sufficient to justify the increased cost of bone graft substitutes. Autologous iliac crest bone graft (ICBG) harvest is a common component of many spinal surgical procedures. Although historically considered the "gold standard" source of bone graft material, autograft bone is associated with numerous disadvantages, primarily related to additional morbidity incurred at the harvest site. Several studies have identified a surprisingly high rate of complications associated with autologous bone graft harvest, ranging from 9–49% -10. The Various strategies have been applied in an effort to reduce the patient's experience of postoperative donor site pain, including administration of local anesthesia with or without narcotics, as separate injections or as an infusion . PostopeComplete avoidance of autologous bone graft harvest is the only way to eliminate the significant risk of chronic donor site pain. Unfortunately, the high cost of effective alternatives such as rhBMPs prohibits widespread use; and other options such as allograft, synthetic substitutes, and non rhBMP so-called factor- or cell-based substitutes have been associated with less favorable clinical results. The purpose of this study is to investigate the long-term impact of the ICBG complications on patient function and well-being in a cohort of patients undergoing autologous ICBG harvest for various spinal procedures. We also aimed to evaluate the psychometric properties of a new measure of ICBG morbidity that would be useful for appropriately gauging spinal surgery outcomes.This prospective single institution study involved spinal fusion patients who had undergone autologous ICBG harvest as a component of their surgery between 2003 and 2005. During the fusion procedure, a separate incision was made over the posterior iliac crest through the subcutaneous tissue down to the fascia, where subperiosteal dissection was used to expose the outer table of the ilium. A retractor was placed into the wound under direct visualization, and cortical and cancellous bone graft strips were harvested. No patients had indwelling local anesthetic infusion catheters and no patients had bone graft harvest of both tables. There were no hernias related to the bone graft harvest site. There was no measurement of bone graft volume made for each surgery. All wounds were closed in layered fashion with absorbable suture material. Local anesthetic was infiltrated into the bone graft harvest site. The patients with increased post-operative graft harvest site pain were not treated with a pre-specified protocol. They were treated on an individual basis at the discretion of the surgeon. The most common treatments included oral narcotics, nonsteroidal anti-inflammatory medication, topical anesthetic patches , and hot/cold therapy. We were unable to discern a clear advantage in terms of one treatment modality over another, but this was not a predefined question of the study design.We implemented the study using the Dillman Tailored Design Method , a detaiOutcomes were measured using the Short-Form-36v2 (SF-36v2) , the OswOne-hundred-seventy questionnaires were obtained from a total of 205 patients contacted, resulting in a response rate of 83%. Of these 170 patients, longitudinal data for both one-year and final follow-up were available on a subgroup of 139 patients. Although most analyses were done with the full sample of 170 patients, one psychometric analysis utilized the smaller sample of 139 with longitudinal data to assess the stability of the measure.A comparison of the baseline and follow-up samples revealed they were comparable in age, gender distribution, diagnosis distribution, smoking status, worker's compensation status, time since surgery, and pre-operative reporting of chronic pain syndrome, severe pain, and pain tolerance Table .The study population included 170 patients with a mean age of 51.1 years (SD = 12.2) and balanced gender  . We should note that the Exploratory Factor Analysis results and they differ when using tetrachoric and polychoric correlations. With polychoric, two factors are identified (scar issues and ensuing difficulties with item 1 loading weakly on F1); the first factor explains 66% of the variance and the first two factors explain 80%, suggesting two factors. With tetrachoric, one factor is identified (item 1 loading weakly) and it explains 58% of the variance. We formed the scale score on the basis of the tetrachoric because this approach is generally considered more appropriate for dichotomous items. While using tetrachoric correlations might be more appropriate, the results using polychoric appear to be what one would expect looking at the item content.We used a criterion validity approach to evaluate the responsiveness of the ICBG, given the cross-sectional nature of the data. An analysis of variance revealed that the ICBG Complications score was significantly different by ODI score , with the largest group difference being between those with very low ODI scores as compared to the remainder of the sample. To evaluate the stability of the measure, we compared ICBG Complications scores on a subgroup of 139 patients for whom both one-year and final follow-up data were available. There was no difference in ICBG Complication score between the one-year and final follow-up data points . This suggests that the measure is stable during a period when little change is expected. There may, however, be a floor effect; approximately half of the sample reported zero ICBG Complications.An analysis of missing data patterns revealed that one item related to numbness was missing data because of a skip pattern in the questionnaire, and one item about job-related difficulty was missing data because of the number of retired patients. These were recoded to "0" or "1" as appropriate allowing retention of all patients in the analyses without changing overall relationships between ICBG morbidity and clinical outcomes. Validity of this approach was confirmed by scatter plots.At a mean of greater than 3 years following surgery, a relatively large percentage of patients continued to report being troubled by harvest site scar appearance (5%), numbness (24%), and bothersome numbness 13%). In particular, chronic harvest site pain resulted in difficulty with household chores (19%), recreational activity (18%), walking (16%), sexual activity (16%), their job (10%), and irritation from clothing (9%) , after adjusting for age, compensation status, pain at the primary surgical site, and residual extremity pain (Table 2 = 0.40), after adjusting for age, compensation status, pain at surgical site, and pain at extremities. There was no association between ICBG harvest-related complaints and mental health , after adjusting for age, compensation status, pain at surgical site, and pain at extremities. Whereas age was a significant covariate of physical health, it was not related to mental health or disability. Workers' compensation status was a significant covariate of disability, but not of physical or mental health. Persistent pain at the primary surgical site and affecting the extremities were significant predictors of worse scores on all outcomes.Regression analysis revealed that chronic ICBG harvest-related complaints were associated with significantly worse disability on the ODI 3.5 years post surgery , recreational activity 11.9%), work activity (9.7%), activities of daily living (8.2%), sexual activity (7.5%), and household chores (6.7%)[.9%, workThe largest previously reported prospective study of autologous bone graft harvest is by Robertson et al. and involved 106 patients undergoing posterior spinal fusion . AlthougThe strengths and limitations of this study should be noted. The strengths include the development of a psychometrically sound tool for evaluating ICBG morbidity. Further, this is the longest prospective study of ICBG morbidity to date with sufficient sample size to allow multivariate analyses of the independent impact of ICBG morbidity on physical and mental health. The limitations of the study are that 35 (17%) patients did not provide complete data on all of the questionnaires leading to a sample size of 170 in the multivariate regression models. This missing data problem may result in biased estimates of the effect of ICBG on clinical outcomes and may reduce our statistical power to detect clinically important effects on physical health. Second, our measures of key outcomes were limited. The pain measure was based on three individual VAS items, which have lower reliability than multi-item questionnaires. Future studies should consider employing a control group that does not include autologous bone graft harvest, e.g., a group utilizing rhBMP, to determine whether eliminating harvest-site morbidity does indeed lead to observable improvement in clinical outcome sufficient to justify the increased cost of bone graft substitutes. Finally, we had only cross-sectional data available for the full sample analysis and were thus unable to evaluate patterns of change over time. Future research should explore whether the new questionnaire used in this study can be improved by utilizing Likert rather than dichotomous response options. It would be worthwhile to investigate whether this modification in respondent options might reduce the floor effect detected in our data.In summary, the long-term impact of ICBG is substantial and clinically important. Our study suggests that about one-fifth of patients experience substantial pain and disability even three years after surgery, and these symptoms affect functioning. This information would support the use of bone graft substitutes that avoid ICBG harvesting. In addition to this clinical implication, our study provides a useful tool for the continued evaluation of ICBG complications for future research that seeks to evaluate the comparative efficacy of ICBG as compare to rhBMP.(ICBG): Iliac Crest Bone Graft; (rhBMPs): recombinant human bone morphogenetic proteins; (SF-36v2): Short-Form-36v2; (ODI): Oswestry Disability Index; (NDI): Neck Disability Index; (VAS): Visual Analogue Scale.The authors declare that they have no competing interests.CES conceptualized and designed the study, supervised the acquisition of data and data analysis, and drafted the manuscript; JFM, PK, DAW participating in the acquisition of data and participating in revising the manuscript critically for important intellectual content; RB and LL participated in data analysis; DHK conceptualized and designed the study, and drafted the manuscript. All authors read and approved the final manuscript.Appendix 1: ICBG Complications Questionnaire. Questionnaire used to assess the presence or absence of persistent pain, functional limitation, and cosmesis at the ICBG site.Click here for file