Rhincodon typus) from an aquarium collection was explored using 1H nuclear magnetic resonance (NMR) spectroscopy and direct analysis in real time (DART) mass spectrometry (MS). Principal components analysis (PCA) of spectral data showed that individual animals could be resolved based on the metabolite composition of their serum and that two unhealthy individuals could be discriminated from the remaining healthy animals. The major difference between healthy and unhealthy individuals was the concentration of homarine, here reported for the first time in an elasmobranch, which was present at substantially lower concentrations in unhealthy whale sharks, suggesting that this metabolite may be a useful biomarker of health status in this species. The function(s) of homarine in sharks remain uncertain but it likely plays a significant role as an osmolyte. The presence of trimethylamine oxide (TMAO), another well-known protective osmolyte of elasmobranchs, at 0.1–0.3 mol L−1 was also confirmed using both NMR and MS. Twenty-three additional potential biomarkers were identified based on significant differences in the frequency of their occurrence between samples from healthy and unhealthy animals, as detected by DART MS. Overall, NMR and MS provided complementary data that showed that metabolomics is a useful approach for biomarker prospecting in poorly studied species like elasmobranchs.In a search for biomarkers of health in whale sharks and as exploration of metabolomics as a modern tool for understanding animal physiology, the metabolite composition of serum in six whale sharks ( Rhincodon typus Smith 1828, are circumtropical planktivorous sharks and the largest fish in the world’s oceans Whale sharks, Maintenance of a population of six whale sharks in a large public aquarium in Atlanta, USA has provided opportunities to gather new information about their biology Due to the logistical challenges of working with such large animals rarely kept in captivity, blood samples had not been collected from captive or free-ranging whale sharks prior to this study. This material therefore presented a unique opportunity to research better biomarkers of health in this and other elasmobranch species. Published studies describing biomarkers in elasmobranchs are relatively few in number and have focused on enzymatic indices (see a priori knowledge of the composition of the samples: proton nuclear magnetic resonance (NMR) spectroscopy and direct analysis in real time (DART) mass spectrometry (MS) Prompted in part by the unique nature of the samples, we used discovery-based metabolomic methods to provide the maximum amount of data without Using NMR and MS metabolomic approaches, we sought to characterize variations in the metabolism of healthy and unhealthy whale sharks over a period of several months and thereby identify biomarkers of health in this elasmobranch species. We succeeded in distinguishing healthy and unhealthy animals and identified several promising biomarker compounds.1H NMR spectra of serum extracts revealed the presence of a complex mixture of chemical species in 46 samples collected over a period of months from five whale sharks (N-methyl picolinic acid) is here reported for the first time from any elasmobranch species (see Appendix).NMR and MS analyses of serum samples in this study represent the first examination of the physiology of the world’s largest fish. The e sharks . Consiste sharks . Yet thee sharks . It is aMetabolic profiles of unhealthy whale sharks were significantly different than those of healthy individuals. Pre-processing of these spectra  to removPCA analysis of mass spectra from 53 serum samples from all six whale sharks did not distinguish individuals based upon frequency of occurrence of individual metabolites . However1H NMR spectral analysis, the heteroaromatic metabolite homarine was recognized as the component of whale shark serum contributing the greatest loading to PC1, which best separated healthy from unhealthy animals and was therefore considered a promising biomarker. The identity of this metabolite was first assigned to homarine (N-methyl picolinic acid) by liquid chromatography coupled to tandem MS and 1H NMR spectroscopy, and then confirmed by total synthesis and spectroscopic comparison of synthetic homarine and whale shark serum samples (see Appendix). Comparing peak areas of aromatic proton signals from 46 whale shark serum extracts with peak areas of an internal standard (deuterated trimethylsilylpropionate [TMSP]) of known concentration, we calculated that homarine was present in healthy whale shark serum at a concentration of approximately 1.5 mM, compared with 0.5 mM for unhealthy individuals . In contrast, matching of NMR spectra with publically-available data using an online search engine  1H NMR signals in whale shark serum samples. While the DART MS approach in its present implementation did not provide quantitative data related to concentration of each analyte, the analysis of presence vs. absence of compounds by MS was highly informative because of the better sensitivity and resolving power of MS compared to NMR, and because many metabolites appeared to occur at concentrations near or below the MS detection limit, such that they were apparently “absent” from some samples while detectable in others.Hundreds of metabolites were tentatively identified by their DART mass spectral data using the publically-available metabolite database METLIN  was transferred to a clean 2.0 mL Eppendorf tube on ice and 500 µL of ice-cold acetonitrile was added to precipitate proteins. The tube was immediately sealed to prevent evaporation and the sample vortexed for 15 seconds and then centrifuged for 5 min at 15,000 rpm. The supernatant was then transferred to a clean cryovial tube, and then lyophilized.For NMR studies the samples were re-suspended in 475 µL of deuterium oxide containing 20 mM of 3-trimethylsilyl–2,2,3,3-d4-propionate (TMSP), an internal standard with respect to the resonance frequency and concentration of metabolites. For MS studies the samples were re-suspended in ultrapure water and derivatized following our previously published protocol 1H NMR spectroscopy. NMR spectra were recorded on a Bruker-Biospin AMX400 spectrometer  operating at 400.13 MHz equipped with a 5 mm broadband probe. For each spectrum 128 scans with a repetition time of 5 s, a spectral width of 20.8 ppm and 64K data points were accumulated using 30 degree excitation pulses. The scale of the chemical shift was calibrated with respect to TMSP (0.00 ppm).A total of 46 samples from five whale sharks  were investigated by NMR spectral data were analyzed using the MATLAB Bioinformatics Toolbox . Preporcessing steps required prior to Principal Component Analysis (PCA) were performed using the MATLAB script ProMetab (preliminary distribution of the version ProMetab GUI) Based on the trends observed in the PCA, concentrations of the regions in the NMR spectra corresponding to homarine were compared between whale sharks by ANOVA. The concentrations were extrapolated using the chemical standard and then compared amongst the different whale sharks. Pair-wise comparisons between the concentrations were made using a Tukey-Kramer HSD post-hoc test.−1 heated to 200°C. Accurate mass spectra were acquired within the range of m/z 60–1000 with a spectral recording interval of 1.5 s. Mass drift compensation was performed after analysis of every sample using a 0.20 mM PEG 600 standard in methanol. Prior to PCA, mass spectra were normalized to the base peak intensity in Excel 2003 , imported as csv files, and resampled to 20,000 m/z points between 60 and 990 using the msresample function in the MATLAB Bioinformatics Toolbox.A total of 53 samples from all six whale sharks were investigated by mass spectrometry. MS metabolomics analysis was performed via a DART ion source  coupled to an AccuTOF mass spectrometer  as previously described Metabolomic approaches revealed multivariate data patterns of serum composition that paralleled observed differences in health status of individual whale sharks, indicating that declining health in this species can be recognized by blood chemical (metabolite) profiles –3. Analy1H NMR spectra of whale shark serum extracts showed substantial differences between healthy and unhealthy whale sharks in the overall composition and concentration of metabolites in serum , based on published studies reporting that high concentrations of this protective osmolyte are apparently universal in the blood of elasmobranchs e.g., . ConcentIn contrast to TMAO, homarine was determined to be a potentially useful biomarker because concentrations varied significantly between healthy and unhealthy animals . Homarinsensu Hines et al. In addition to uncertainty regarding the physiological significance of metabolites pinpointed due to their differential concentrations in healthy versus unhealthy whale sharks, the metabolism of whale sharks in general Click here for additional data file.Appendix Figure S1Identification of homarine in whale shark serum using LC-QTOF MS. (a). Total ion chromatogram (TIC) of a partially-purified whale shark serum sample. (b). Mass spectrum of the peak at 3.76 min in (a). Product ion QTOF MS/MS spectrum of the precursor ion at m/z 138 with collision energy of 20 eV from (c): partially-purified whale shark serum samples and (e): synthesized homarine. (d). Extracted ion chromatograms of ions at m/z 138 from whale shark serum samples (black curve) and synthesized homarine (blue curve). (f). Suggested fragmentation pathway for homarine.(DOCX)Click here for additional data file.


The daily clearance of physiologically dying cells is performed safely mainly by cells in the mononuclear phagocyte system. They can recognize and engulf dying cells utilizing several cooperative mechanisms. In our study we show that the expression of a broad range of apopto-phagocytic genes is strongly up-regulated during differentiation of human monocytes to macrophages with different donor variability. The glucocorticoid dexamethasone has a profound effect on this process by selectively up-regulating six genes and down-regulating several others. The key role of the up-regulated mer tyrosine kinase (Mertk) in dexamethasone induced enhancement of phagocytosis could be demonstrated in human monocyte derived macrophages by gene silencing as well as blocking antibodies, and also in a monocyte-macrophage like cell line. However, the additional role of other glucocorticoid induced elements must be also considered since the presence of autologous serum during phagocytosis could almost completely compensate for the blocked function of Mertk. The efficient elimination of apoptotic cells or those dying through necrosis is performed mainly by the cells of the mononuclear phagocyte system Glucocorticoids (GC) have an extensive range of effects in target tissues throughout the organism eliciting both rapid and delayed changes in physiological functions and pathologic tissues environment. Their therapeutic effects are mediated by the classical cytosolic glucocorticoid receptors (cGCRs) which move to the nucleus to regulate gene expression following ligand binding or by membrane-bound GCR and direct interactions with the cell membrane The importance of Mer tyrosine kinase (Mertk), as a member of of the Tyro3/Axl/Mer family of receptor tyrosine kinases in the engulfment and efficient clearance of apoptotic cells has been clearly demonstrated Here, we investigated the effects of differentiation and treatment by DXM on the gene-expression pattern of HMDMs using a custom designed apopto-phagocyte panel. Our data show that during differentiation of monocytes to macrophages most of the apopto-phagocytic genes are highly up-regulated. Dexamethasone led to further up-regulation of 6 genes while some others were significantly down-regulated. Of the up-regulated ones only silencing of Mertk could prevent DXM-mediated increase in phagocytosis of apoptotic cells in a serum-independent manner; this observation was confirmed by applying blocking antibodies against Mertk and showing that in monocytic cell lines low level and lack of Mertk inducibility by DXM is accompanied by their inability to engulf apoptotic cells.Human monocytes were isolated from ‘buffy coats’ of healthy blood donors. Buffy coats were provided anonymously by the Hungarian National Blood Service where blood were taken from healthy volunteers and written informed consent from all participants were obtained. For these studies approval was obtained from the ethics committee of the Medical and Health Science Center, University of Debrecen (DEOEC RKEB/IKEB Prot. No. 2745 -2008). The ethics committee approved this consent procedure.6 cells/ml in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) containing 10% human AB serum in a 5% CO2 atmosphere. Cytokines were added on day 0 and day 3. The glucocorticoid treated cells were differentiated in the presence of 1 µM DXM . Neutrophil granulocytes were isolated by Histopaque (Sigma–Aldrich) fractionation of EDTA-treated venous blood following erythrocyte sedimentation using 3% filtered dextrane solution. Default apoptosis in neutrophils was obtained by culture of freshly isolated cells in IMDM containing 10% human AB serum for 20 h at 37°C in a 5% CO2 atmosphere. Cell death was assessed by the Annexin-V-fluorescein isothio-cyanate Apoptosis Detection Kit  according to manufacturer's recommendations; proportion of stained Annexin-V+ and Annexin-V+ propidium iodide+ (PI+) cells was determined by fluorescence activated cell sorter (FACS) analysis on BD Bioscience flow cytometer. Mono Mac 6 and THP-1 cells were maintained in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS), l-glutamine (300 mg/l), penicillin and streptomycin (Sigma–Aldrich). The cells were washed with phosphate-buffered saline (PBS) three times, followed by incubation in 10% FCS-RPMI, 100 nM PMA for 72 h at 37°C at a cell density of 5×105 cells/ml in a 5% CO2 atmosphere. For vitamin D3 and dexamethasone treated THP-1 cells, 0.1 µM DXM concentration was applied.Human monocytes from ‘buffy coats’ of healthy blood donors were isolated by Ficoll–Paque Plus  gradient and magnetic separation using CD14 human microbeads . Human macrophages were obtained through a 5-day differentiation using 5 ng/ml macrophage colony-stimulating factor (M-CSF)  at 37°C at a cell density of 1×102 atmosphere. The assay was performed in the presence or absence of human 10% AB serum. The whole-cell mixture was collected by trypsin digestion, centrifuging, washing twice in PBS and fixing in 1% PBS-buffered paraformaldehyde (pH 7.4). The net phagocytosis rate was determined by FACS analysis (BD Bioscience) as percent phagocytic cells that have engulfed (positive for both CMTMR and CFDA) as previously described Dying neutrophils were labeled with carboxyfluoresceindiacetate-succinimidyl ester , washed free of conditioned media and resuspended in PBS before their addition to a prewashed Cell Tracker Orange 5-(and-6)-(((4-chloromethyl) benzoyl)amino)tetramethylrhodamine labeled  macrophage monolayer. Before adding dying neutrophils, macrophages were preincubated with anti-Mertk blocking antibodies for 10 min at a concentration of 10 µg/ml. Macrophages and apoptotic neutrophils were mixed at a ratio of 1∶5 and incubated for 30 min at 37°C in 5% COt method, where one sample was designated as calibrator, through which all other samples were analyzed. ΔCt represents the threshold cycle (Ct) of the target minus that of 18S rRNA and ΔΔCt represents the ΔCt of each target minus that of the calibrator. Relative quantities (RQ or fold changes) were determined using the equation where relative quantity equals 2−ΔΔCt (average of three repeated experiments).Total cellular RNA was isolated from untreated and dexamethasone treated human monocyte-derived macrophages using TRIzol Reagent (Invitrogen Life Technologies). Pre-designed, factory-loaded 384- well TaqMan low-density array  was used to determine the level of expression of genes listed in 3′-CGUCUAUGCCUAUAAAAUAtt (sense) and 5′-UAUUUUAUAGGCAUAGACGat (antisense)], AXL receptor tyrosine kinase [3′-GGAACUGCAUGCUGAAUGAtt (sense) and 5′-UCAUUCAGCAUGCAGUUCCtg (antisense)], complement C1q subcomponent subunit A [3′-CCAACCAGGAAGAACCGUAtt (sense) and 5′- UACGGUUCUUCCUGGUUGGtt (antisense)], c-mer proto-oncogene tyrosine kinase  and thrombospondin-1 [3′-GGACUGCGUUGGUGAUGUAtt (sense) and 5′- UACAUCACCAACGCAGUCCtt (antisense)]. Non-targeting siRNA negative control (scrambled) was obtained from Sigma-Genosys (3781976-F/112). Three days after isolation HMDMs were harvested from the 24 well plates and washed once with IMDM and once with PBS . The cells were resuspended in OptiMEM without phenol red (Invitrogen Life Technologies) at a concentration of 4×107/ml. siRNA was transferred to a 4-mm cuvette . A volume of 100 µl of cell suspension was added and incubated for 3 min before being pulsed in a Genepulser Xcell (Bio-Rad). Pulse conditions were square-wave pulse, 500 V, 0.5 ms. Immediately after electroporation, the cells were transferred to human AB serum supplemented IMDM with the previously indicated concentrations of M-CSF and DXM.In order to knock-down each investigated genes, siRNA constructs were obtained from Ambion, targeting adenosine A3 receptor [Total cellular RNA was isolated from electroporated macrophages and THP-1 cells as previously described. Total RNA concentrations were quantified by spectrometry after DNase treatment (Sigma-Aldrich). TaqMan reverse transcription reagent kit (Applied Biosystems) was used for generating cDNA according to manufacturers instructions using 200 ng of total RNA in a 20 µl reaction volume. An ABI Prism 7700 sequence detection system (Applied Biosystems) was used to determine relative gene expression. Gene primers and probes were designed and supplied by Applied Biosystems. Human cyclophyllin was used as endogenous control to normalize the amount of the sample cDNA added to the reaction. The human cyclophyllin primers were labelled with TAMRA and sample primer with FAM. All samples were run in triplicate. Relative mRNA expression was quantified by comparing the cycle threshold (Ct) between control and knock-down cell samples.Mouse monoclonal antibodies against Mertk were purchased from R&D systems  (MAB8911) and ABCAM (ab52591) .Human macrophages were collected and washed with PBS followed by lysing in 50 mMTris–HCl; 0.1% Triton X-100; 1 mM EDTA; 15 mM 2-MEA and proteinase inhibitors. Insoluble cellular material was removed by centrifugation and the lysates were mixed with 5× Laemmli loading buffer (LB), boiled for 10 min and loaded onto a 10% SDS polyacrylamide gel. Proteins were transferred onto PVDF membranes followed by blocking with 5% skimmed milk. Membranes were probed by monoclonal anti-Mertk antibody (ab52591) and β-actin (Sigma-Aldrich) overnight at 4°C, followed by incubation with horseradish-peroxidase (HRP)-conjugated anti-mouse antibody (Sigma-Aldrich) for 1 h at room temperature. Immunoblots were developed with Immobilon Western chemiluminescent substrate (Millipore).Partial Least Squares (PLS) module of Statistica software that is a comprehensive implementation of partial least squares regression analysis.Statistical analysis of phagocytosis data was performed by using the paired Student's t-test (two tailed). Statistical evaluation of the expression changes was performed in “R” using BioConductor implementing a moderate t-test based on Bayesian statistic which allows for a comparably reliable estimation of the SD even in case of few biological replicates. Correlation between gene expression levels and phagocytosis was evaluated using linear regression by Table S1) we examined expression changes during differentiation to explain this phenomenon. During differentiation of monocytes to macrophages, phagocytic ability of mononuclear phagocyte cells dramatically increases The relative expressions of apopto-phagocytic genes in macrophages differentiated from monocytes of different donors were also compared. We observed that there were non-variable genes, whose relative expression level varied only 20–50% amongst donors, and there were highly variable ones, whose expression level varied in 2–3 orders of magnitude . The expGlucocorticoids can highly increase the phagocytic capacity of macrophages toward apoptotic neutrophils To investigate whether DXM induced up-regulation has a direct effect on the enhanced phagocytic capacity of macrophages the up-regulated genes were silenced using siRNA. The silencing efficiency varied in the different genes . AlthougWe also investigated whether blocking of Mertk with an antibody verifies its key role in DXM induced increase of phagocytosis. It was found that while Mertk blocking antibodies did not inhibit phagocytosis by macrophages differentiated in the absence of DXM, the DXM-induced increase of phagocytic capacity of macrophages could be prevented by treatment with this antibody . This efIn order to exclude the possible role of soluble Mer in serum that inhibits macrophage clearance of apoptotic cells To gain further information about the correlation between DXM mediated gene induction in macrophages and their phagocytosis capacity toward apoptotic neutrophils, the effect of DXM treatment on human macrophage like cell lines was also investigated. Mono Mac 6 cells exhibit many characteristics of mature blood monocytes . We found that the expression level of the majority of these genes were altered and most of them up-regulated during differentiation of monocytes to macrophages. Among the highly and moderately up-regulated genes there are several genes whose importance in the phagocytosis of macrophages have already been described either as receptors , as bridging molecules  Differentiation of monocytes to macrophages is associated with an enhancement of phagocytosis of apoptotic cells and glucocorticoids are capable to further augment this process The variability of expression levels of apopto-phagocytic genes among differentiated macrophages of human donors was also investigated and striking differences were found. Although there were several genes with very low variability of SD between 20–50%, the expression levels of another set of genes varied in 2–3 orders of magnitude. The average relative expression level of non-variable genes was typically higher than that of the variable ones. During macrophage differentiation up-regulated genes partly overlapped with non-variable genes in some cases such as calreticulin, PPARγ and CD68 while some with variable ones like FcγRIIB, GAS6, ADORA3. The non-variable gene set might represent permanently switched on tools for the phagocytosis machinery while the variable set may provide alternative ways for phagocytosis under various local conditions to which macrophages may be exposed.Glucocorticoids are still the most potent immunosuppressive agents with complex and cell type specific actions on immune cells kd mice with a truncation of the cytoplasmic part of Mer, where macrophages from these mice were deficient in the clearance of apoptotic thymocytes Mertk is a member of the TAM  receptor protein tyrosine kinase family which have substantial roles in innate immunity. Their known two ligands, growth-arrest-specific 6 (GAS6) and protein S bind to and activate all the three TAM receptors with different affinities and due to their Gla domains they also can bind phosphatidylserine on apoptotic cells simultaneously In a recent paper McColl et al. suggested that glucocorticoids regulate a switch from a serum independent to a serum-dependent apoptotic cell recognition mechanism, which can be recapitulated with protein S, the purified Mertk ligand, and involves macrophage Mertk The discrepancy in the DXM effect in serum free conditions between McColl's and our data can be explained by the application of differently induced apoptotic neutrophils. We exclusively used neutrophils cultured in the presence of serum that mimics physiological processes occurring in the human body. The fact that phagocytosis of neutrophils made apoptotic in the presence of serum could be augmented by glucocorticoids, and this was efficiently inhibited in a Mertk dependent manner, underlines that Mertk has a pivotal role in this phagocytic process which is mainly serum independent. On the other hand, the enhancement effect of glucocorticoid treatment on phagocytosis have additional elements which becomes apparent only when serum is present during engulfment of apoptotic cells; the identification of these molecular pathways warrants further studies. Our data also confirms that phagocytes respond differently even to the same apoptotic cell types if they were induced in different circumstances.in vitro model to investigate this process. Several compounds such as phorbol 12-myristate 13-acetate (PMA), 1,25-dihydroxyvitamin D3, retinoic acid, or eukaryotic cytokines have been used to trigger this differentiation process, which can be monitored by changes in morphology, adherence or expression of surface markers The differentiation of monocytes into macrophages is a crucial step in the early immune response, and the human monocytic leukaemia cell line THP1 has been frequently used as an Efficient phagocytic clearance of apoptotic cells is crucial in many biological processes. If this machinery is impaired the apoptotic cell debris can contribute to the development of sterile inflammation and autoimmunity Table S1http://www.genenames.org).Gene list of the apopto-phagocyte panel. Gene symbol (HUGO), Alias, Gene description, HGNC code, and Location is given for each gene as defined by HUGO Gene Nomenclature Committee Click here for additional data file.Table S2Partial Least Squares (PLS) for correlation between gene expression and phagocytosis data.Correlation data between gene expression levels and phagocytic capacity. It contains HUGO gene ID and R2 calculated with linear regression by (DOC)Click here for additional data file.


We confirmed this finding experimentally using both fragmented and non-fragmented viral RNAs as well as primers differing in random oligomer length (6 or 12 nucleotides) and in the sequence of the amplification tag. The observed oligonucleotide annealing bias can be reduced by extending the random oligomer sequence and by in silico combining sequence data from SISPA experiments using different 5′ defined tag sequences. These findings contribute to the optimization of random nucleic acid amplification protocols that are currently required for downstream applications such as viral metagenomics and microarray analysis.Sequence Independent Single Primer Amplification is one of the most widely used random amplification approaches in virology for sequencing template preparation. This technique relies on oligonucleotides consisting of a 3′ random part used to prime complementary DNA synthesis and a 5′ defined tag sequence for subsequent amplification. Recently, this amplification method was combined with next generation sequencing to obtain viral sequences. However, these studies showed a biased distribution of the resulting sequence reads over the analyzed genomes. The aim of this study was to elucidate the mechanisms that lead to biased sequence depth when using random amplification. Avian paramyxovirus type 8 was used as a model RNA virus to investigate these mechanisms. We showed, based on  The determination of complete viral genome sequences is a growing field in human, animal, and plant virology. Complete genome sequences and their exponential growth in public databases  not only allow for a better understanding of virus evolution, molecular phylogeny (phylogenomics) and epidemiology, but also facilitate functional analysis of virus genes in comparison with other sequences in databases. Traditionally, viral genome sequencing approaches are based on amplification of overlapping genome regions followed by Sanger sequencing Next generation sequencing (NGS) technologies were developed to accommodate the need of higher sequencing capacity and lower cost per nucleotide for large genome sequencing projects e.g. . One mai6 virus particles). This method was also applied to the partial sequencing of a novel paramyxovirus in penguins Truly sequence independent access methods to viral genomes have been developed in the field of viral discovery  was kindly provided by the German reference laboratory for Newcastle disease of the Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany. APMV-8 has a linear single stranded RNA genome of negative orientation with a length of 15 342 bases. Virus propagation was performed in 8–10 day old specific pathogen free embryonated chicken eggs. Allantoic fluid was collected and the virus titer was determined by hemagglutination assays according to the Council Directive 92/66/EC (1992) using 1% chicken erythrocytes.5′-GCCGGAGCTCTGCAGATATC-3′). Purified, size selected (400–1 200 nucleotides [nt]) random PCR fragments were quantified with the Nanodrop-1000 spectrophotometer and used for the preparation of 454 sequencing libraries as described below.APMV-8 virions were purified starting from one milliliter (ml) of allantoic fluid. Centrifugation, filtration with 0.22 µM filters, nuclease treatment with 100 units DNase I, viral RNA extraction and sequence independent single primer amplification (SISPA) were performed as previously described The following modifications of the DNase SISPA protocol (on APMV-8) were performed to test whether annealing effects during cDNA synthesis and/or RNA secondary structures contributed to biased sequence depth.To test whether viral RNA secondary structures assisted in causing the unequal sequencing depth, we fragmented the extracted RNA according to the GS FLX Titanium cDNA Rapid Library Preparation Method Manual protocol  starting from approximately 40 ng of viral RNA. RNA size distribution before and after fragmentation was measured using the RNA 6000 Pico chip  on the Agilent 2100 Bioanalyzer. The fragmented RNA was used in SISPA under identical reaction conditions as described above.5′-GACCATCTAGCGACCTCCAC-3′, modified from To check whether the primer tag sequence (designed for downstream PCR amplification) had an influence on the binding of the random primer along the genome during cDNA synthesis, alternative primer sequences were tested during first and second strand cDNA synthesis , random amplified DNA originating from the different random amplifications of APMV-8 was used to prepare sequencing libraries for the Genome Sequencer FLX . This was performed according to the manufacturer's instructions for Titanium Series reagents, using multiplex identifiers (MID) to identify the different libraries. The resulting libraries were sequenced with a GS FLX with Titanium Series reagents and run protocol (200 cycles).http://www.r-project.org/). To investigate if the 5′ specific amplification tag of SISPA primer influenced the sequence depth distribution, we mapped short sections of the amplification tag of the SISPA primers of increasing length adjacent to the random part of the primer along the genome sequence using R. Consensus sequences from each of the amplification strategies were compared in a clustalW alignment. Variant analysis was performed by mapping of all raw sequencing reads (complete datasets) of a certain condition relative to reference sequence APMV-8/pintail/Wakuya/20/78 (GenBank: FJ215864) using SeqMan NGen® version 3.0 . After mapping, the reference was deleted and single-nucleotide polymorphisms were called. At polymorphic positions, we included a degenerate nucleotide in the consensus sequence if the minor nucleotide alternative was present in at least 30% of the sequence reads.The sequence output file was sorted per sequencing library according their MID sequences. All raw sequence files were submitted to the Sequence Read Archive (SRA) under accession number SRP028373. Sequence reads were trimmed to remove the primer sequence including the random (6N or 12N) part as well as low quality ends. Non-APMV-8 specific reads were filtered out. Of each dataset approximately 7.7 Mb of raw data  were randomly picked to allow direct comparison between all conditions. Reference guided assemblies were performed relative to APMV-8/pintail/Wakuya/20/78 (GenBank: FJ215864) using the GS Reference Mapper software . Data output files were further processed with R (http://www.tbi.univie.ac.at/RNA/) was used to predict partition function and base pairing probability matrix (dot plot). The pair probabilities were extracted and plotted in a mountain plot . This plot represents the secondary structure in a plot of height  versus position. As our SISPA protocol used 95°C to denature the RNA before cDNA synthesis and 50°C at first strand cDNA synthesis, we modeled the minimum free energy secondary structure of the RNA genome sequence at these temperatures.RNAfold from the Vienna RNA Package version 2.0  was used to sort the reads. Subsequently, the percentages of reads identified as viral sequences were calculated as a measure of the virus specificity.Using the 6N SISPA primer FR20RV-6N We could find no evidence of an overall correlation between areas below average sequence depth and global secondary structure of the viral RNA at denaturation and annealing temperatures . HoweverThe unique depth, roughly described as depth where reads with identical starts are omitted, did not show extreme variability over the genome  B.We found no clear correlation between the GC-content  and sequence depth . Mapping of the possible annealing events on the APMV-8 genome during first strand or second cDNA strand synthesis aided by five, six, or even seven consecutive 3′ nucleotides revealed a clear correlation with the variability of the sequence depth  a random annealing sequence extended from 6 to 12 nucleotides, (b) an alternative primer amplification tag sequence, and (c) the combination of the two primers with alternative tag sequences. The amount of raw data used in all assemblies was again kept at about 7.7 Mb to allow direct comparison between all conditions.In order to minimize the impact of the tag sequences on primer annealing we extended the random part of the primer while keeping the tag sequence constant. The 12N oligomer condition was performed on both non-fragmented and fragmented RNA. The use of a 12N SISPA primer resulted in a lower maximum depth, to the advantage of a higher first quartile and median sequence depth . The numUsing a random 6N oligomer with an alternative amplification tag sequence, K-6N, the median and Q3 sequence depth were lower compared to sequences resulting from SISPA primer FR20RV-6N . Again, When comparing the number of enhanced annealing sites on the APMV-8 genome using primer K-6N and FR20RV-6N, we observed fewer enhanced annealing sites at K-6N , the Q1 and median depth was increased, and both the IQR and number of positions with a depth of less than 100 × was decreased compared to the K-6N condition . The extst and 2nd cDNA strand synthesis would most likely solve this problem.Because of the use of an alternative amplification tag sequence resulted in relocation of areas where enhanced annealing induced sequence depth peaks, we tested whether combined libraries from two different SISPA primers might result in a more homogenous sequence depth distribution along the entire genome. We modeled this possibility by performing a combined APMV-8 reference assembly with equal amounts of raw sequence data  resulting from the libraries produced using 6N SISPA primers FR20RV-6N and K-6N. As each of these libraries was repeated independently, we modeled two combined libraries resulting from the two repeats of the respective libraries  C. CombiWhen aligning the consensus sequences of the tested reaction conditions we did not observe any sequence differences. We looked for variation in the largest available dataset (FR20RV-12N condition) making use of all raw data. Only variant positions with depth >100 × and a percentage of minor nucleotide >10% were examined to allow reliable variant analysis . In compA metagenomics analysis of all sequenced raw data showed that all studied conditions were highly specific for APMV-8 RNA . The proThe efficient targeting of the protocol enables sequencing complete viral genomes with very little sequencing effort. To investigate the relation between sequencing effort and sequence depth, we randomly picked increasing amounts of reads from the largest available dataset (FR20RV-12N) and looked at its coverage statistics. This random sampling was repeated 10 times and averages are displayed in Sequence Independent Single Primer Amplification de novo contig assembly and variant analysis. The tendency of the 454 technology to produce duplicate reads as a result of multiple beads present in the same micro-reactor during emulsion PCR When combining random amplification with NGS, its tendency to create a biased distribution of sequence depth becomes evident e.g. . ExtremeVictoria and colleagues Using a different specific amplification tag sequence, we observed a clear shift of the coverage extremes to other genomic regions caused by the shift of possible enhanced annealing sites. Again, the coverage depth was very variable, but the median depth was lower compared to original used primer. This is probably due to the lower number of potential strong annealing places on the APMV-8 genome for the alternative random primer K-6N. This confirms that the distribution of possible enhanced annealing sites depends on the SISPA primer amplification tag sequence and on the target genome sequence, which implies possible differential annealing dynamics when targeting other virus genomes.Using any of these SISPA primers, regions in the genome existed where predicted enhanced annealing sites did not result in extreme sequence depth, for example the region around positions 6 416 and 5 888 at the K-6N primer condition have respectively 6 and 7 nucleotides in common with the tag sequence. Similarly, the region around position 5 843 at the FR20RV-6N primer condition has high similarity with the tag sequence. However, these predicted enhanced annealing sites do not seem to coincide with increased sequence coverage in this region. When looking at the RNA secondary structure mountain plot at first strand cDNA annealing temperature , 50°C weThese findings confirm earlier observations by Wong and colleagues 12 ( = 16 777 216), compared to 46 ( = 4 096) for a hexamer. In our study, increasing the random part of the SISPA primer from 6N to 12N reduced the amplification tag induced sequence depth bias to the advantage of the regions with problematic coverage.Previous studies have used different lengths of the 3′ random annealing part of the SISPA primer. Examples include hexamer We combined two factors affecting the distribution of sequence reads over the genome in a single assembly: (a) alternative SISPA primers are biased to alternative genome regions, and (b) longer oligomer-based SISPA primers tend to have a more equal distribution. The combination of data from libraries resulting from two different 12N SISPA primers (FR20RV-12N and K-12N) resulted in the best distribution of sequence reads over the APMV-8 genome. We suggest that in future efforts , a combination of a longer random annealing part (e.g.12N) and the combined assembly of data resulting from libraries amplified with alternative amplification tags, may result in an improved homogeneity of sequence depth distribution over the genome. Ultimately, a more homogenous sequence depth distribution reduces the sequencing effort (and thus cost) needed for genome completion. In this study, 2 500 GS-FLX titanium reads were enough to sequence 99.9% of the APMV-8 viral genome with a median coverage of 38.3 ×. We have shown that median coverage increased with the increasing sequencing effort. 7 500 reads covered the full genome with a median depth of more than a 100 ×. This confirms our previous experience that only about 5 000 GS-FLX titanium reads from a library of SISPA amplified viral RNA was enough in determining the complete genome of uncharacterized avian paramyxoviruses Next generation sequencing is becoming increasingly accessible to laboratories, both through the evolution of sequencing platforms and chemistries and through the increasing availability of sequencing service providers. Combined with opportunities to multiplex samples during sequencing, this technology is now evolving towards a cost-effective methodology for genome sequencing. Generic, sequence independent access methods such as optimized DNase SISPA may facilitate access to viral genome sequences, without the need for prior sequence knowledge. In addition, our findings may be of value to other technologies requiring random nucleic acid amplification such as DNA microarrays.


Metabolic changes upon antiangiogenic therapy of recurrent glioblastomas (rGBMs) may provide new biomarkers for treatment efficacy. Since in vitro models showed that phospholipid membrane metabolism provides specific information on tumor growth we employed in-vivo MR-spectroscopic imaging (MRSI) of human rGBMs before and under bevacizumab (BVZ) to measure concentrations of phosphocholine (PCho), phosphoethanolamine (PEth), glycerophosphocholine (GPC), and glyceroethanolamine (GPE).1H and 31P MRSI was prospectively performed in 32 patients with rGBMs before and under BVZ therapy at 8 weeks intervals until tumor progression. Patients were dichotomized into subjects with long overall survival (OS) (>median OS) and short OS (<median OS) survival time from BVZ-onset. Metabolite concentrations from tumor tissue and their ratios were compared to contralateral normal-appearing tissue (control).1H-detectable choline signals  in rGBMs were elevated but significance failed after dichotomizing. For metabolite ratios obtained by 31P MRSI, the short-OS group showed higher PCho/GPC (p = 0.004) in rGBMs compared to control tissue before BVZ while PEth/GPE was elevated in rGBMs of both groups . Under BVZ, PCho/GPC and PEth/GPE in the tumor initially decreased (p = 0.04) but only PCho/GPC re-increased upon tumor progression (p = 0.02). Intriguingly, in normal-appearing tissue an initial PEth/GPE decrease (p = 0.047) was followed by an increase at the time of tumor progression (p = 0.031).Before BVZ, An elevated PCho/GPC ratio in the short-OS group suggests that it is a negative predictive marker for BVZ efficacy. These gliomas may represent a malignant phenotype even growing under anti-VEGF treatment. Elevated PEth/GPE may represent an in-vivo biomarker more sensitive to GBM infiltration than MRI. Antiangiogenic treatment, one of the most important new therapies for recurrent glioblastomas (rGBMs), is based on the rationale that cessation of perfusion in the highly vascularized GBMs will induce tumor starvation. Usually, the vascular endothelial growth factor A (VEGF-A), which is mediating the strong neoangiogenesis and increased vascular leakage of GBMs is targeted by the humanized monoclonal IgG antibody bevacizumab (BVZ). Treatment of GBMs is typically monitored with MR imaging, assuming contrast enhancement as the major indicator of recurrent tumor growth 1H) MR spectroscopy (MRS) is an important biomarker for tumor cell proliferation and tumor cell density 1H MRSI just measures the integrated concentration of the membrane lipid catabolite glycerophosphocholine (GPC) and the anabolite phosphocholine (PCho). Extensive studies on tumor models in cell culture showed decreased PCho and increased GPC concentrations upon treatment, a modification not visible by the integrated 1H Cho signal 31P) MRSI offers the discrimination between these two compounds and adds their ethanolamine analogues to the detectable parameters, providing a much more comprehensive characterization of lipid membrane metabolism than 1H MRSI The metabolism of phospholipid cell membrane turnover is one of the major indicators for tumor growth 31P spectroscopic studies of human brain tumors at 1.5 T investigated the phosphomonoester (PME) and the phosphodiester (PDE) region of the spectra without resolving separate resonances 1H decoupled 31P MR spectroscopic studies have been successfully performed at 1.5 T of human brain tumors resolving resonances for PCho, PEth, GPC and glycerophosphoethanolamine (GPE) 1H decoupled 31P MRSI provides a more reliable discrimination and quantification of two PME (PCho and PEth) and two PDE (GPC and GPE) signals The large majority of in vivo In our previous study we found an increased PEth/GPE ratio in recurrent GBMs before BVZ The study was approved by the local ethics committee of the faculty of medicine at the local University , and written informed consent was obtained from each participant prior to inclusion. Spectroscopic examination was part of a prospective registry study performed on a patient cohort receiving BVZ as compassionate treatment 2, while the others received BVZ only. Enrollment was restricted to patients with the histological diagnosis of a GBM with radiologically confirmed recurrence according to the updated response assessment criteria (RANO) for high-grade gliomas All patients received bevacizumab at a dose of 10 mg/kg body weight intravenously every other week. For 9 patients irinotecan was co-administered at a dose of 125 mg/m1H/31P volume head coil . The MRSI protocol was planned on T2-weighted (T2-w) images in three orientations. For 1H MRSI a transversal slice , 30 ms echo-time (TE)) was recorded within a measurement time of 4∶45 min. Visually guided by coronal and sagittal anatomical slices the 2D 1H slab was positioned in the middle of the tumor tissue. The volume of interest (VOI), selected by a combination of point resolved selective spectroscopy (PRESS) and outer volume suppression, covered the center of the recurrent tumor area and the contralateral normal brain tissue. For 31P MRSI, a 3D MRSI slab  aligned to the 1H MRSI slice was recorded. Before spatial Fourier transformation the matrix size was doubled in all dimensions by zero filling. Within this process, the 31P slab was adjusted by grid-shifting to provide an ideal matching of 31P and 1H voxels, i.e. the 1H slice was positioned in the center of a 31P slice and the in-plane 31P grid matched the 1H grid just exhibiting twice the scale.MRSI and MRI of the brain were performed on a 3 T whole body system  using a double tuned A graphical user interface developed at our institute was used for image guided selection of the tumor ROI based on the T2-weighted references images and contrast enhanced T1-weighted images recorded at a previous examination (up to 7 days before starting bevacizumab). An experienced radiologist (E.H.) visually selected the voxels of the tumor area, which was defined as solid contrast enhancing tumor mass avoiding necrotic areas. Whenever possible, control tissue was selected from a region contralateral to the tumor position, provided that there was no obvious tumor or edema. Alternatively, another adjacent healthy region of the contralateral hemisphere was selected. In the follow-up examinations under bevacizumab the tumor area frequently showed no or just faint contrast enhancement. In these cases the tumor area was delineated based on signal changes in T2-weighted images according to following features: intermediate signal intensity between grey matter and vasogenic edema; inhomogeneous signal changes; blurred gray-matter junction with altered cortical ribbon; lack of ‘fingers of edema’ 31P data, a second control area was positioned in the temporo-occipital region of the unaffected hemisphere. For all but two patients this area was sufficiently far apart from the tumor to neglect the signal spreading from adjacent voxels, which was caused by the poor point spread function. Thus, data from regular tissue were provided which were not affected by ROI positioning and therefore were comparable between different patients.For the 31P voxels were selected first, followed by selection of the 1H voxel within the 31P voxel (i.e. 4 1H voxel per 1 31P voxel). The program jMRUI  31P data. The model function was composed of 14 exponentially decaying sinusoids. Six of those had identical damping and were corresponding to peaks assigned to PCr, PEth, PCho, GPC, GPE, and inorganic phosphate (Pi). PCr was adjusted to 0 ppm and constraints for the chemical shifts of the other signals except for Pi were applied as a fixed difference with regard to the position of PCr. ATP was represented by 7 exponentially damped sinusoids, defining each multiplet by the respective number of peaks with identical damping and adequate amplitude ratios. The 1H spectra were analyzed with the software LCModel  1H MRSI spectra from the selected voxels were visually assessed for artifacts according to the criteria described by Kreis et al. 31P spectra, all signals were generally assigned correctly leaving a flat line for the residual with equal distribution of noise.Data were sampled from voxels within the tumor ROI and the control ROIs. For each target area the 1H detectable metabolites were then calculated by referring to an independent measurement in a spherical phantom containing an aqueous solution of 100 mmol/l acetate as calibration standard. A TR of 10 s was applied to avoid T1 saturation. For 31P data, a spherical phantom with 20 mmol/L phosphate served as calibration standard (TR 60 s).Signal intensities were corrected for T1 and T2 relaxations using values reported previously All patients with qualified MRS data were included in a survival analysis (Kaplan-Meier). To answer the question whether there are pre-treatment metabolite changes predicting overall survival under bevacizumab, the entire group was split at the median overall survival time resulting in a group with long overall survival (long-OS) and a group with short overall survival (short-OS). Signal intensities and their ratios for specific metabolites were analyzed with analysis of variance (ANOVA) comparing the groups with long-OS with short-OS for tumor and control tissue of the contralateral hemisphere. Differences between tumor tissue and control tissue were tested using ANOVA with repeated measurements. Significance (p < 0.05) was tested by contrast analysis in ANOVA.For patients with a recurrence-free period of at least 2 follow-up MR examinations, changes in tumor metabolites were determined comparing 3 examinations: the examination before BVZ therapy was commenced, the examination after the first cycle of BVZ and the examination revealing tumor progression under BVZ The median survival time for the entire patient group was 243 days, resulting in 16 patients with OST > 243 days (long-OS) and OST < 243 days (short-OS). For one patient the results regarding energy metabolism deviated considerably from 2 times standard deviation due to an extraordinary high signal of inorganic phosphate. We considered these data as outlier and excluded it from further statistical analysis regarding energy metabolism.From the 32 patients 14 patients were examined at least three times after start of Bevacizumab treatment until the tumor progressed again. Representative spectra from recurrent GBM and from contralateral control tissue are shown in Especially for the tumor area many voxels (about 40%) had to be rejected since they did not meet the quality criteria.1H spectra for the entire group was significantly increased (p = 0.04) in tumor tissue. Likely due to diminished statistical power, the significance is lost after separated into long-OS (p = 0.21) and short-OS (p = 0.09) (a. The concentration of tCho obtained from b. The PCho/GPC ratio was increased for short-OS (p = 0.004), however, patients from the long-OS group showed no difference of PCho/GPC compared to normal-appearing tissue . The dif31P detectable ethanolamine-containing metabolite ratio PEth/GPE for both groups was significantly increased . Changes in 31P metabolites are shown in Neither the For normal-appearing tissue we found a similar pattern for both control areas with changes almost analogue to the tumor tissue. The initial decrease of PEth/GPE after onset of therapy was significant for the contralateral tissue (p = 0.047) while the increase upon tumor progression was significant for the temporo-occipital region (p = 0.031) .Before BVZ, there was a significant difference between tissue of recurrent GBMs and control tissue for PCr/Pi for both groups (p<0.001). It is worth to mention that in the tumor tissue but also in the control tissue the level of PCr/Pi was elevated in patients with long OS compared to the patients with shorter OS . A decreFor tumor tissue the energy metabolite ratios remained decreased during the whole follow-up period .In the normal-appearing control tissue, there was a continuous decrease in the PCr/Pi and ATP/Pi ratio. However, only the ATP/Pi decrease at tumor progression compared to the first post BVZ values was significant  were significantly different in recurrent GBMs before BVZ treatment compared to normal-appearing brain tissue of the respective contralateral hemisphere. While 1H MRSI could detect differences between normal-appearing tissue the 31P data were much more specific: The ratio of PCho/GPC, an indicator for tumor cell proliferation, was increased in patients who survived a shorter period under the following BVZ therapy. In the following we will discuss the pathobiochemical processes underlying the metabolic changes and evaluate the potential of MRSI to monitor antiangiogenic treatment.The 31P MRS reflects changes in tumor metabolism with higher accuracy. In vitro studies showed that PCho is the dominant membrane lipid metabolite in proliferating tumor cells and tumor tissues Although partial volume effects from necrotic and hemorrhagic tissue may affect the overall voxel concentration, an increased tissue concentration of tCho is typical for malignant GBMs The presented study is the first in evaluating lipid metabolism of adult brain tumors in vivo by distinguishing the choline- and ethanolamine-containing metabolites. Referring to the in vitro results, we expected increased PCho concentrations and PCho/GPC ratios in recurrent GBMs compared to normal-appearing brain tissue of the respective contralateral hemisphere before BVZ therapy. For the short-OS patients we observed significantly higher PCho/GPC ratios compared to controls. In contrast no significant PCho/GPC increase was found for the long-OS group . The difPhosphatidylethanolamine is another important phospholipid, also called cephalin because it is abundant in the brain and spinal cord 1H MR spectra, the in vivo signals of the metabolites PEth and GPE are not distinguishable from the background leaving 31P MRS as only resort for measuring ethanolamine membrane metabolites in vivo. Yet, those 31P MRS examinations require high fields (≥ 3T) or at least 1H decoupling 1H decoupled 31P spectra at 1.5 T, Albers et al. observed elevated PEth/GPE ratios in primitive neuroectodermal tumors Knowledge of ethanolamine-containing lipid metabolism from human in vivo data is still limited by methodological problems: In Animal and cell culture studies of experimental in vivo tumors found increased PCho and PEth levels, which were interpreted as elevated rates of membrane synthesis According to in vitro studies In summary, our results corroborate the role of PCho as indicator of malignant tumor behavior, whereas the PtdEth metabolism may be attributed to more ‘inactive’ tumor cells with lower membrane lipid turn-over.31P MRS data clearly indicate a shift in the concentration from the anabolic component to catabolic component (1H detectable choline signal. As discussed above, PCho/GPC and PEth/GPE are indicators for tumor malignancy and growth and are therefore adequate to monitor effects of targeted antiangiogenic therapy. In the time course of BVZ therapy PCho/GPC increased again when the tumor progressed exceeding even the initial values. Venkatesh et al. showed in vitro that PCho acts as metabolic MR biomarker to monitor GBM treatment effects While tCho was increased in the initial measurement, we could not detect any significant changes in the first follow up examination. This is in accordance with a previous study by Kim et al. omponent , 5, whicExperimental and in vivo spectroscopic studies of gliomas and other brain tumors showed a variety of changes in the high-energy phosphate metabolism 31P metabolites upon treatment were not only detected in recurrent tumor but also within the normal-appearing control tissue of the contralateral hemisphere. Under VEGF treatment we found a significant increase of PEth/GPE in normal-appearing brain areas as the tumor progressed preceded by an initial decrease of this ratio. We can exclude that partial volume effects had caused this phenomenon since the PEth/GPE changes were found even in temporo-occipital areas distant from the tumor. The initial metabolite changes may be the result of general treatment effects. However the re-increase of PEth/GPE in the normal-appearing tissue concomitant with tumor progression cannot easily be explained by treatment effects. Its increase together with the decrease in the ATP/Pi ratio may indicate proliferation of glioma cells. This would support the hypothesis that bevacizumab promotes a diffuse infiltrating, gliomatosis-like phenotype at progression An important finding of this study is that changes of Despite the above mentioned evidence, a significant and continuing BVZ effect on energy metabolism in normal brain tissue also is still disputable. The ATP/Pi decrease may be caused by unspecific BVZ effect on normal brain tissue. BVZ is the human antibody acting against the vascular endothelial growth factor VEGF, which is highly expressed in glioblastomas but also in other proliferating tissue. Reparative treatment effects like gliosis could induce some VEGF increase which may be antagonized with BVZ.The visual delineation of tumor areas is challenging in pre-treated rGBMs since no imaging feature may reliably differentiate between vital tumor tissue, therapy related changes or micronecrosis. Especially under antiangiogenic therapy the contrast enhancement may be missed and the tumor area has to be defined on T2-weighted images. Further, tumor borders and tumor edema are often infiltrated by glioma cells. Considering a possible bias of visual tumor delineation, our results strongly suggest that changes of the membrane lipid metabolites may help to detect ‘vital’ tumor tissue under antiangiogenic therapy.31P MRS. Our data show changes in control tissue which were analogue to the tumor metabolite changes. Especially for data from the contralateral side, where vicinity between tumor and control tissue cannot always be avoided, this may indicate bleeding of signal from the tumor voxels. However, the same pattern of longitudinal changes was also observed in the second control area, which was selected far distant from the tumor site, confirming the statement that normal appearing tissue is affected as well. In any case, it is unlikely that the observed metabolite changes demonstrated in this study are just the result of a partial volume effect due to an increase in the proportion of healthy and tumor tissue. Nevertheless, there is an urgent need to improve the spatial resolution. More advanced MR methods which exploit the 31P/1H J-coupling of choline and ethanolamine compounds involved in membrane metabolism by using heteronuclear polarization transfer, present a promising approach to improve the S/N and spatial localization of 31P MRS in the future 31P MRSI.Partial volume effects caused by the rather coarse spatial resolution and signal bleeding between adjacent voxels are a major drawback in


Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the  In a proof-of-principle analysis, we describe a robust protocol for using the barFLEX collection to explore fitness defects caused by gene overexpression in pooled cultures under different genetic or environmental perturbations.To circumvent the aforementioned limitations with existing yeast arrays, we combined the sequence-verified Full-Length EXpression-ready (FLEX) plasmid library of galactose-inducible untagged ORFs . We also tested 55 random strains from the SGA-FLEX array by polymerase chain reaction (PCR) amplification and restriction digestion of the ORFs to ensure that each ORF was represented on the correct location on the array. Almost all amplified ORFs (96%) appeared correct by one or both assays (data available on request).The plasmids from the FLEX collection (MATα haploid strains. The media used throughout SGA were prepared as described previously (Samples (2 μL) of each plasmid were transformed into Y6897 using standard lithium acetate and polyethylene glycol protocol . The traeviously . The trakanMX resistance cassette were transformed into the BY4741 strain background as described previously :To expand the original set of 1141 Barcoders . The transformants were selected by replica-plating onto standard yeast peptone dextrose (YPD) + G418  and patched onto agar plates in a 96-colony format. The Barcoder array was designed to pair perfectly with the FLEX array except for a small number of negative controls that were eliminated through SGA.Figure S3).Genomic DNA from the yeast Barcoder strains was isolated from a pool of the Barcoder collection. UPtag and DNtag PCR products were amplified separately; purified, mixed in equal molar ratios; and sequenced on an Illumina GAIIx, as described previously . In brieMATα were selected by excluding leucine from the medium . At this step, MATa strains also could be selected by changing the media to SD-HIS-URA-ARG-LYS+Canavanine+Thialysine+G418 . The barFLEX collection was maintained in the MATα state to allow for subsequent rounds of SGA with MATa query deletion strains.The FLEX collection was crossed to the new Barcoder collection by robotic replica pinning on a Virtek Colony Arrayer (Bio-Rad Laboratories) in 384 format. The array was mated on YPD omni-trays by pinning once from each collection to the same plate. SGA was performed as described previously  with the2 ratios (treatment/control) were used for analysis. Therefore, strains with decreased abundance in the pool show negative ratios. Pooled experiments in the presence of methyl methanesulfonate  were performed by including either 0.001% or 0.0001% MMS in the growth medium.The pooled yeast cultures were created according to previously described methods (ura3Δ and dun1Δ) were created in the BY4741 background to cross to the barFLEX array. The deletion alleles were marked with natMX providing resistance to nourseothricin (NAT). The ura3Δ::natMX and dun1Δ::natMX deletion strains were crossed to the barFLEX array using robotic pinning and selection through SGA as follows: 2X diploid selection SD-URA+G418 +NAT, 1X sporulation media, 2X haploid selection SD-LEU-URA-ARG-LYS+Canavanine+Thialysine+G418+NAT. Colonies from the final haploid selection plates were pooled and frozen at −80° before testing.Two deletion alleles .To construct a yeast gene overexpression array in a genetic background that is compatible with SGA technology, we made use of the Yeast FLEX array, a unique collection of Gateway-compatible plasmids carrying sequence-verified yeast ORFs  were rearrayed together with the 1141 original strains to create a final array of 5115 Barcoder strains in a format compatible with the SGA-FLEX array .The use of pooled cultures of differentially barcoded mutant strains for genetic screens has a wide range of applications and offers particular advantages for chemical genetics, where small amounts of expensive and scarce chemicals can be screened for effects on the fitness of mutant strains  in a pool. The Barcoder v2 collection can be thus used to “barcode” any SGA-compatible collection of yeast strains. We validated our barFLEX collection by sequencing 54 randomly selected ORFs and 25 uptags. In this test, 96% of barcodes and 96% of ORFs were in the correct strain and in the expected location on the strain array We next used the Barcoder v2 collection to efficiently add barcode identifiers to strains in the SGA-FLEX array . In brieTable S5). The collection can be manipulated to generate either MATa or MATα backgrounds, depending on the applied final haploid selection. The barFLEX strains are compatible with the SGA screening platform to introduce marked alleles of any genes of interest for combinatorial overexpression genetics.In summary, our barFLEX collection contains 5111 strains (MATα barFLEX strains and then pooled them as described previously (Table S5 and Table S6). The undetected barcodes below background (Table S7) reflect those that contain errors in either their common priming sequences or barcodes . We assessed the presence of barcodes in the pool by next generation sequencing. The reads generated by the sequencing of DNA prepared from the pool in a single Illumina flow cell were counted and assigned to the barcodes expected to be in the pool. Global sequence alignments were generated for each of the unidentified barcodes to all unclassified reads. Reads were associated with barcodes for which alignment scores exceeded 80 Due to the ever-decreasing costs of next-generation sequencing, we previously compared barcode microarray data to “bar-seq” data and found them to be generally equivalent  and the supporting website will be updated to include these additional strains. We conclude that the barcodes of the barFLEX collection are suitable for analysis using either next generation sequencing or microarrays. We have generated a list of high scoring alignments of all unique reads in our sequence data .Of the 5106 upstream barcodes, 2518 (49%) were identified as a perfect match and another 1774 (35%) were matched by alignment. For the 5038 downstream barcodes the numbers of perfect matches and aligned reads were 2435 (48%) and 1796 (36%), respectively. Considering both barcodes for the 5106 individual barcoder strains, 1777 (35%) had direct matches for both barcodes and 3176 (62%) for at least one barcode. At least one barcode was identified by alignment in 1506 (22%) strains. For 807 strains (8%), neither barcode was clearly seen in the sequence data generated from this particular sequenced sample. Because these percentages are derived from a single sample, we expect that additional strains will be detectable with further algorithm development . Replicates of this experiment correlated well (r = 0.7897) and the variance further decreased when only the highly toxic genes (defined as those with Log2 ratio < −1) were considered (r = 0.847).Several groups have surveyed the yeast genome for genes that cause an obvious fitness defect when overexpressed . Howeveranalysis . At 20 gTable S11). Despite the obvious technical differences between the two experiments, the competitive growth assay of overexpression toxicity correlated significantly with the measurements of toxicity using growth on solid medium . Also, only 4 of the 95 genes that caused a significant growth defect when overexpressed in liquid medium (Log2 < −3) were not obviously toxic in our plate-based assay. Genes that were highly toxic upon overexpression as assessed in our competitive liquid growth assay  were also identified in the plate assay, with a false-positive rate of 2% (Table S12). As expected, the false-positive rate increased as the toxicity decreased, presumably due to the increased experimental variability associated with assessing a more subtle phenotype.We compared the growth of the barFLEX collection in pooled liquid culture to growth of the array on plates. We examined the collection on solid medium for defects in growth by measuring colony size differences on glucose- or galactose-containing medium. The barFLEX array was pinned in a 1536 strain format onto glucose- and galactose-containing plates and colony size was assessed using an automated colony scoring method (Table S11). Previous assessment of the GST-ORF collection (vs. the high-copy backbone (2 μ) of the GST collection; (2) the quality of each ORF in each collection (sequenced verified in the FLEX collection but not in others); (3) the particular strain background used and; (4) differences in the assays used to measure gene toxicity. For example, the mORF collection was scored for overexpression toxicity by analyzing the size of the colony resulting from spotting a single 3μL culture onto solid medium  are more easily detected using different assays (Table S13).In contrast, the GST-ORF toxic set was identified by serial spot dilution assays and genes were assigned a qualitative score ranging from 1  to 4 (least sick) . This ree.g., mitosis (P = 1.232 × 10−8) and genes involved in cell cycle regulation [P = 2.986 × 10−8 (P = 6.91 × 10−5), which we did not observe with the barFLEX collection, suggesting the GST-ORF enrichment may reflect an effect of the GST tag on protein transport that is less likely to be seen with untagged genes expressed in the barFLEX collection.Previous analysis of genes that cause fitness defects when overexpressed revealed a significant functional enrichment for genes with annotated roles in cell cycle regulation and mitosis . We then used SGA to introduce the kinase deletion allele into the barFLEX collection and selected for haploids carrying the overexpression plasmid, the integrated barcode, and the deletion allele . After the final selection, we pooled the strains and cultured them alongside a negative control strain (ura3Δ::natMX) in glucose- or galactose-containing medium for a total of 20 generations .As noted previously, overexpression of most genes in WT cells is phenotypically benign, but sensitivity to gene dosage can be revealed in strains mutated for an interacting protein or pathway component, a so-called SDL interaction. To develop protocols for using the barFLEX collection for identifying SDL interactions, we performed an SDL screen using a query strain deleted for the k repair . We gene2 ratio < −1), eliminating any genes that were also toxic in the negative control screen (difference of Log2 ratio < −1). Using this statistical cut-off, the dun1Δ screen revealed a list of 52 interactions (Table S14), 25 of which have at least one phosphorylated residue . We used stringent criteria for identifying an SDL interaction in our secondary assays; we demanded an obvious growth difference that was clear over two 10-fold serial spot dilutions or a >10% difference in growth fitness scores, with the fitness defect larger than twice the standard deviation between the control and the kinase deletion strain (dun1Δ using either spot dilutions or automated liquid growth curves (Table S14), corresponding to a confirmation rate of ~38%. This confirmation rate is less than that seen in large-scale SDL screens of the kinome [46%  and the dun1Δ-barFLEX collections throughout 20 generations of growth in three different conditions: (1) galactose (to induce overexpression) for 20 generations; (2) galactose for five generations of growth followed by addition of 0.0001% MMS for 15 generations; (3) galactose for five generations followed by addition of 0.001% MMS for five generations to a total of 20 generations. Glucose (noninduced) controls were performed in parallel. The dose of MMS used in the experiment was selected to induce only a slight fitness defect in the dun1Δ strain. In the presence of 0.001% MMS, the WT (ura3Δ) screen identified 197 additional toxic genes  including known repair genes SAE2, MPH1, and PSO2, which is consistent with published chemogenomic data (Table S16), including MPH1, as well as other DNA repair genes such as TOP1 and IMP2 , including interactions with the known checkpoint gene LCD1 and the repair gene RAD26 (Table S17 and Table S18). The strain overexpressing the known target of Dun1, Sml1, decreased in strain abundance in all three of the conditions compared with WT but was below the stringent statistical cut-off that we chose for our experiments.Screening the kinome using traditional SDL screening approaches revealed that many kinase deletion alleles show few or no SDL interactions  in both standard growth conditions and in the presence of DNA damage through degradation of the ribonucleotide reductase inhibitor Sml1  has enabled functional assessment of genetic perturbations for gain-of-function alleles (Gal4 to the estrogen receptor and the VP16 activator, allowing rapid and specific induction of individual genes using the hormone, β-estradiol (To summarize, induction of gene overexpression using nutrient-inducible promoters (DUN1 with the DNA damaging agent MMS, we uncovered novel genetic interactions that were not apparent in standard conditions. This general approach will be extremely valuable for studying kinases that are known to be conditionally active and other regulators that are required under particular conditions. Conditions that might be useful for SDL and other overexpression screens could be identified by surveying data from systematic phenotypic analysis of the deletion collection (In addition to screening for fitness defects caused by gene overexpression in WT cells, we also validated the barFLEX collection for detection of genes exhibiting conditional SDL using kinases as a test case. By perturbing a barFLEX pool carrying a deletion of the kinase gene


Liver transplantation is the only therapeutic modality for patients with acute-on chronic liver failure (ACLF). These patients are at high risk for bacterial infections while awaiting transplantation. The aim of this study was to elucidate whether an adequately treated bacterial infection influences the outcomes after transplantation in this patient population.P = 0.046) and longer length of pretransplant hospital stay (P = 0.026) in Group 1, similar demographic, laboratory and clinical features were found in both groups. Patients in Group 1  were adequately treated with effective antibiotic(s) before receiving LDLT. All included patients were followed up until one year after transplantation or death. Sixty-one posttransplant infection episodes were found in an overall of 44 ACLF patients . Frequently encountered posttransplant infections were intraabdominal infection, pneumonia, bloodstream infection and urinary tract infection. Two patients died in each group (P = 0.622). No significant difference was found in the length of posttransplant ICU stay, and in one-year survival, graft rejection, and posttransplant infection rate between both groups. The longer overall hospital stay  found in Group 1 resulted from a longer pretransplant hospital stay receiving treatment for pretransplant infection(s) and/or awaiting transplantation.54 recipients  of adult-to-adult living donor liver transplant (LDLT) for ACLF were categorized as those with pretransplant infection  or without pretransplant infection  for retrospective analyses. With the exception of a higher male-female ratio (These data suggested that an adequately treated pretransplant infection do not pose a significant risk for clinical outcomes including posttransplant fatality in recipients in adult-to-adult LDLT for ACLF. Liver transplantation is the only curative modality for patients with relentless liver failure resulting from chronic hepatic decompensation or acute exacerbation of chronic liver disease. Among liver transplant recipients, a substantial number of patients have been of cases of acute-on-chronic liver failure (ACLF) We retrospectively reviewed ACLF patients who underwent adult-to-adult living donor liver transplant (LDLT) at Kaohsiung Chang Gung Memorial Hospital between January 2001 and December 2009. This study was conducted with a waiver of patient consent approved the Institutional Review Board of Chang Gung Memorial Hospital (Document no. 101–0564B).ACLF was defined as an acute hepatic insult manifesting as jaundice (serum bilirubin >5 mg/dl) and coagulopathy  complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease 2 <32 mmHg, and (iv) peripheral white cell count (WBC)> 12,000/ mm3, <4000/ mm3, or consisting of>10% immature (band-form) cell 3 in his or her ascites, regardless bacterial growth from culture A pretransplant infection referred to an infection occurred within 4 weeks prior to liver transplantation, which was diagnosed based on clinical and laboratory  and/or imaging  findings. Pretransplant infections were categorized into bloodstream infection (BSI), pneumonia, spontaneous bacterial peritonitis (SBP), and urinary tract infection (UTI). Sepsis was defined as an infection with the presence of systemic inflammatory response syndrome in the affected patient, which was manifested by two or more of the following conditions: (i) temperature >38°C or <36°C, (ii) heart rate >90 beats/min, (iii) respiratory rate >20 breaths/min or PaCOAll patients with a diagnosed bacterial infection were given antibiotic(s) on empirical basis, which was then adjusted based on recommendations made by an infectious-disease specialist if the infection clinically deteriorated, or was subsequently switched to other antimicrobial(s) as was indicated by the results of culture and susceptibility testing. A pretransplant bacterial infection was considered adequately treated and the affected patient was regarded as an eligible transplant recipient only when he or she fulfilled the criteria as follows: (i) disappearance of symptoms and signs suggestive of sepsis, and (ii) normalization or improvement of laboratory and/or imaging findings indicating bacterial infection.Candida infections, combination of either cefepime or ceftazidime (20 mg/kg/8 hours) and teicoplanin (400 mg/day) was started immediately before operation and lasted for 3–5 days depending upon posttransplant conditions, and fluconazole 100mg/ day was started after transplantation and was used during post-operation stay at ICU. Daily trimethoprim 80mg/sulfamethoxazole 400 mg was started after operation and was used indefinitely as prophylaxis for Pneumocystis jiroveci. Ganciclovir (5 mg/kg/12 hours) was used when cytomegalovirus (CMV) infection was clinically suspected or upon detection of CMV antigenemia.Hepatectomy in donors and recipients and liver engrafting for recipients in LDLT were performed as described previously All recipients were admitted to Liver Intensive Care Unit (LICU) for postoperative care, where laboratory data (mainly hemogram and blood chemistry) were closely monitored. Doppler ultrasound was performed daily to check the vascular flow in the graft for one week after liver transplant and then twice weekly until the patient was discharged. These patients were additionally evaluated by a dedicated infectious-disease specialist (Dr. JW Liu) on daily basis. Once bacterial infection was suspected, empirical antibiotic therapy immediately started.χ2 test or Fisher exact test when appropriate, and comparison of continuous variables was performed using t-test or Mann-Whitney U test, where applicable. To ascertain the impact of pretransplant infection on the postoperative mortality by eliminating potential confounding(s), pretransplant infection and other variables between fatal and survived liver transplant recipients were entered into a multivariate analysis using a logistic regression model. A P value <0.05 was considered statistically significant. All statistical analyses were performed using SPSS for Windows, version 16.0 .Data of the included ACLF patients were retrieved from their medical records. Between different patient groups, comparison of categorical variables was carried out using the P = 0.046) and longer length of pretransplant hospital stay  in Group 1 than in Group 2, similar demographic, laboratory and clinical features were found in both groups, and hepatitis B was the most commonly encountered liver disease among the overall included ACLF patients (P = 0.149) and overall hospital stay  between patients with MELD scores≧25 and those with MELD scores <24 among the overall included patients.Among a total of 408 patients received adult-to-adult LDLT during the study period, 54  were found to be of ACLF cases and were included for analyses. None of these patients was put on an artificial liver support system (ALSS) prior to liver transplant. The included ACLF patients were categorized as those with pretransplant infection  or those without pretransplant infection . With the exception of a higher male-female ratio . All patients with pretransplant infection received effective antibiotic therapy, and extracorporeal shock-wave lithotomy was additionally carried out for one patient with urolithiasis suffering urinary tract infection. The mean time lapse from starting an antimicrobial treatment for pretransplant infection to LDLT was 13.8±12.6 days.A total of 42 pretransplant infection episodes were found in the 34 LDLT recipients. Among these patients, 26 (76.5%) experienced one episode of pretransplant infection, while the rest 8 each experienced 2. Major pretransplant infection entities in decreasing order were UTI (n = 14), SBP (n = 11), pneumonia (n = 7) and BSI (n = 6); pretransplant infection entities and the pathogens are summarized in P = 0.024) in Group 1 than in Group 2, no significant difference was found in the length of posttransplant ICU stay, and in one-year survival, graft rejection, and posttransplant infection rates between both groups (P = 0.622). In Group 1, one patient experienced posttransplant hepatic arterial thrombosis and died of intractable internal bleeding with hypovolumic shock and intraabdominal Candida infection 5 days after retransplantation, while the other died of progressive multiorgan failure one day after LDLT. In Group 2, one patient who experienced biliary leak after LDLT died of biliary tract infection with sepsis 3 months later, while the other who experienced posttransplant graft failure with persistent coagulopathy and internal bleeding died of hepatic failure, pneumonia and bacterial peritonitis with sepsis one month after liver transplantation. Multivariate analysis disclosed that none of the evaluated variables including the length of posttransplant hospital stay, graft rejection, posttransplant infection, and pretransplant infection significantly differed (P>0.050) between fatal and survived liver transplant recipients.With the exception of a longer total hospital stay ; these infection episodes and pathogens were detailed in Acinetobacter species and coagulase-negative staphylococci were the leading pathogens. Intraabdominal infection was the most commonly found posttransplant infection regardless of pretransplant infection. Posttransplant infections developed in patients with pretransplant infection were summarized in Pseudomonas aeruginosa, while another experienced pretransplant primary K. pneumoniae bacteremia and posttransplant peritonitis with a K. pneumoniae being isolated from ascites culture. Molecular differentiation was not performed to clarify whether these P. aeruginosa and K. pneumoniae isolates were of the same clone.A total of 61 posttransplant infection episodes were found in an overall of 44 ACLF patients  of the overall 54 ACLF patients included in this series, and the most common pretransplant infection was UTI, as opposed to SBP that was reported to be the most frequently encountered pretransplant infection in patients with chronic liver failure P = 0.024) and similar lengths of posttransplant ICU and posttransplant hospital stay between patients with pretransplant infection and those without merely suggested that the former had a longer pretransplant hospital stay receiving treatment for their pretransplant infection and/or awaiting transplantation. Our data disclosed that in ACLF patients with an adequately treated pretransplant bacterial infection, irrespective of the anatomic site involved, frequently encountered posttransplant infections were intraabdominal infection, pneumonia, BTI and UTI, which were similar to those reported in other liver transplant series that included patients with chronic liver failure In the circumstances of shortage of organ donated by a deceased donor, liver graft donated by a living adult shortens the waiting time for patients for whom urgent liver transplant is indicated In case a liver graft not being available in a timely fashion, a plasma-exchange-centered ALSS was reported to be helpful in bridging ACLF to liver transplantation per se compromises the immunity of the affected patients ACLF Acinetobacter baumannii, Serratia marcescens, and Stenotrophomonas maltophiliaA. baumannii, a usually multidrug-resistant pathogen Of note, frequently seen pathogens for hospital-acquired infections such as Bacterial infections in general and bacteremia in particular have been reported to be the major causes of posttransplant mortality Limitations of this study include potential biases inherent to the retrospective study and small sample size. Nevertheless, our data suggested that an adequately treated pretransplant infection do not pose a significant risk for clinical outcomes including posttransplant fatality in recipients in adult-to-adult LDLT for ACLF.


An elevation of EC Ca2+ also activates small- and intermediate-conductance (SK and IK) potassium channels. This leads to endothelium-dependent hyperpolarization (“EDH”), which is spread through gap junctions in specialized EC projections to adjacent smooth muscle cells (SMCs) to cause vasodilation of small resistance arteries and arterioles, and this pathway is responsible for the majority of the ACh-induced dilation in resistance arteries [2+ influx events (“sparklets”) through single TRPV4  channels in ECs of intact, small mesenteric arteries. Cooperative opening of as few as 3 TRPV4 channels per EC caused maximum vasodilation primarily through activation of EC IK channels [+ activates EC inward rectifier K+ (Kir) channels to cause vasodilation, and that TRPV4-mediated vasodilation is attenuated by block of Kir channels. Our results support the concept that AKAP150 localization to MEPs ensures the proximity of PKC to TRPV4 channels and enhances channel cooperativity. The resultant activation of MEP IK channels by TRPV4 sparklets causes a hyperpolarizing current as well as local accumulation of K+ ions in the “nanospace” between the MEP and SM membranes. This in turn could activate EC Kir channels to augment EDH. Compartmentalization of signaling elements by anchoring proteins at MEPs, along with a high-amplification network of ion channels at MEPs makes it possible for a small number of ion channels to exert a profound influence on vascular function independent of NO.The endothelial cells (ECs) lining blood vessels are pivotal regulators of vascular tone. Endothelial-dependent vasodilation by classic agents such as acetylcholine (ACh) depends on an elevation of EC Caarteries . We have


Light-emitting diode (LED) fluorescence microscopy offers potential benefits in the diagnosis of human African trypanosomiasis and in other aspects of diseases management, such as detection of drug-resistant strains. To advance such approaches, reliable and specific fluorescent markers to stain parasites in human fluids are needed. Here we describe a series of novel green fluorescent diamidines and their suitability as probes with which to stain trypanosomes. Early diagnosis of human African trypanosomiasis (HAT)  is paramThe development of portable and efficient light-emitting diode (LED)-illuminated microscopes –5 allows–Trypanosoma brucei via specific surface transporters, including the aminopurine P2/TbAT1 transporter mT, measured as described in reference By addition of a series of ultraconjugated ring structures in the linker between the two amidine groups (containing the recognition motif for the parasite's P2 transporter), we generated a series of new diamidines with longer emission wavelengths than the parental UV-emitting DB75 . Spectral measurements confirmed that all novel dications emitted in the green wavelength range . As expeTrypanosoma brucei brucei strains: a wild-type line (bloodstream Lister 427), a line not expressing P2 (tbat1−/−) (50] 25-fold higher for the tbat1−/− line than for the wild type), followed by DB1645 (16-fold higher), DB1465 (10-fold), and DB1464 (8-fold)  characterization demonstrates good pharmacokinetic properties and rules out potential toxicity issues previously observed for some molecules belonging to this class.The dependence of the compounds on P2 transport for uptake into trypanosomes was confirmed by an alamarBlue assay  performebat1−/−) , and a dbat1−/−) . Compoun(8-fold) . Data usT. b. brucei in infected rat blood films using a standard fluorescein isothiocyanate (FITC) filter set  and a Zeiss Axioplan fluorescence microscope , all five diamidines provided specific staining of trypanosomes without labeling erythrocytes or white blood cells. Staining of the parasite nucleus was not observed, but all compounds selectively accumulated inside the kinetoplast  and other cytoplasmic organelles, spread throughout the cell body and also visualized at longer wavelengths , indicating that uptake routes independent of P2 play important roles in their internalization when used under these conditions.Despite the compounds' partial dependence on the P2 transporter for uptake, they did not distinguish wild-type and Implementation of fluorescence tests for trypanosome detection in human fluids must rely on affordable, efficient and preferably specific markers. This work shows the feasibility of generating cheap fluorophores, emitting at differential wavelengths, by simple addition of cyclic groups within the chemical structure of diamidines, the precursor of which, DB75, is fluorescent only in the UV region. The exploitation of surface transporters peculiar to trypanosomes is a straightforward way to specifically target fluorophores to these parasites, and improved compounds may be developed following this approach to target both human and animal infective trypanosomes.


IFNγ ELISPOT analysis demonstrated that 14 of the 81 (17.3%) mutations generated immunogenic CD8+ T cell epitopes, and 5 of the 14 induced robust IFNγ responses (>100 spots per 1x105 T cells). Antitumor efficacy was evaluated by immunizing mice 3 days following subcutaneous Panc02 challenge. Only immunization against the 5 robust peptides was capable of preventing tumor outgrowth, and was significantly more effective than treatment with GM-CSF-secreting whole-cell vaccination (GVAX), another approach currently being investigated in the clinic by our group. We are currently testing the efficacy of this approach against larger established tumors and metastatic liver lesions. We are also working to optimize this approach by testing additional adjuvants and vaccine vectors, including recombinant Listeria monocytogenes that express the mutant gene sequences. Exact epitopes are being mapped in order to evaluate the utility of MHC-binding predictions. Ultimately, we intend to translate our optimized approach into the clinic.A major obstacle to the development of immunotherapy for pancreatic ductal adenocarcinoma (PDA) has been identifying immune relevant antigens that are not expressed by normal cells. DNA sequencing technologies that allow for the genome-wide identification of tumor mutations have shown that human PDAs harbor an average of 63 mutations . Since these mutations are not present prior to the onset of tumorigenesis, each mutation generates a novel and essentially foreign antigen (termed “epitope”). Unlike most classical tumor epitopes, mutome-derived epitopes are uniquely specific to each patient’s tumor. Furthermore, T cells recognizing them may avoid immunological tolerance mechanisms that inhibit or deplete T cells that recognize classical tumor epitopes that are also expressed by normal cells. Vaccines designed to activate mutome-specific T cells have the potential to induce robust and specific antitumor responses and provide a new approach for personalized tumor immunotherapy. We are using the Panc02 murine PDA model to develop and explore the potential of this approach. Sequence analysis identified 596 point mutations in Panc02 cells, significantly more than the number found in human PDAs. The NetMHC prediction server was used to screen for epitopes predicted to bind H2-Db and H2-Kb. In total, 15 strong candidates (NetMHC scores <50) and another 85 candidate epitopes within the limits of prediction (scores <500) were predicted. We analyzed the