Animesh Sharma

The genome sequence of Atlantic cod reveals a unique immune system

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Characteristics of 454 pyrosequencing data—enabling realistic simulation with flowsim

Motivation: The commercial launch of 454 pyrosequencing in 2005 was a milestone in genome sequencing in terms of performance and cost. Throughout the three available releases, average read lengths have increased to ∼500 base pairs and are thus approaching read lengths obtained from traditional Sanger sequencing. Study design of sequencing projects would benefit from being able to simulate experiments. Results: We explore 454 raw data to investigate its characteristics and derive empirical distributions for the flow values generated by pyrosequencing. Based on our findings, we implement Flowsim, a simulator that generates realistic pyrosequencing data files of arbitrary size from a given set of input DNA sequences. We finally use our simulator to examine the impact of sequence lengths on the results of concrete whole-genome assemblies, and we suggest its use in planning of sequencing projects, benchmarking of assembly methods and other fields. Availability: Flowsim is freely available under the General Public License from http://blog.malde.org/index.php/flowsim/ Contact: susanne.balzer@imr.no; ketil.malde@imr.no

Discovering biomarkers from gene expression data for predicting cancer subgroups using neural networks and relational fuzzy clustering

BackgroundThe four heterogeneous childhood cancers, neuroblastoma, non-Hodgkin lymphoma, rhabdomyosarcoma, and Ewing sarcoma present a similar histology of small round blue cell tumor (SRBCT) and thus often leads to misdiagnosis. Identification of biomarkers for distinguishing these cancers is a well studied problem. Existing methods typically evaluate each gene separately and do not take into account the nonlinear interaction between genes and the tools that are used to design the diagnostic prediction system. Consequently, more genes are usually identified as necessary for prediction. We propose a general scheme for finding a small set of biomarkers to design a diagnostic system for accurate classification of the cancer subgroups. We use multilayer networks with online gene selection ability and relational fuzzy clustering to identify a small set of biomarkers for accurate classification of the training and blind test cases of a well studied data set.ResultsOur method discerned just seven biomarkers that precisely categorized the four subgroups of cancer both in training and blind samples. For the same problem, others suggested 19–94 genes. These seven biomarkers include three novel genes (NAB2, LSP1 and EHD1 – not identified by others) with distinct class-specific signatures and important role in cancer biology, including cellular proliferation, transendothelial migration and trafficking of MHC class antigens. Interestingly, NAB2 is downregulated in other tumors including Non-Hodgkin lymphoma and Neuroblastoma but we observed moderate to high upregulation in a few cases of Ewing sarcoma and Rabhdomyosarcoma, suggesting that NAB2 might be mutated in these tumors. These genes can discover the subgroups correctly with unsupervised learning, can differentiate non-SRBCT samples and they perform equally well with other machine learning tools including support vector machines. These biomarkers lead to four simple human interpretable rules for the diagnostic task.ConclusionAlthough the proposed method is tested on a SRBCT data set, it is quite general and can be applied to other cancer data sets. Our scheme takes into account the interaction between genes as well as that between genes and the tool and thus is able find a very small set and can discover novel genes. Our findings suggest the possibility of developing specialized microarray chips or use of real-time qPCR assays or antibody based methods such as ELISA and western blot analysis for an easy and low cost diagnosis of the subgroups.

An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an ∼58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of ∼3.6 μg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed α-chitin binding activity with Kd ∼ 0.64 μm and Bmax ∼ 4.68 μmol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an ∼8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

Whole genome sequencing of the fish pathogen Francisella noatunensis subsp. orientalis Toba04 gives novel insights into Francisella evolution and pathogenecity

BackgroundFrancisella is a genus of gram-negative bacterium highly virulent in fishes and human where F. tularensis is causing the serious disease tularaemia in human. Recently Francisella species have been reported to cause mortality in aquaculture species like Atlantic cod and tilapia. We have completed the sequencing and draft assembly of the Francisella noatunensis subsp. orientalisToba04 strain isolated from farmed Tilapia. Compared to other available Francisella genomes, it is most similar to the genome of Francisella philomiragia subsp. philomiragia, a free-living bacterium not virulent to human.ResultsThe genome is rearranged compared to the available Francisella genomes even though we found no IS-elements in the genome. Nearly 16% percent of the predicted ORFs are pseudogenes. Computational pathway analysis indicates that a number of the metabolic pathways are disrupted due to pseudogenes. Comparing the novel genome with other available Francisella genomes, we found around 2.5% of unique genes present in Francisella noatunensis subsp. orientalis Toba04 and a list of genes uniquely present in the human-pathogenic Francisella subspecies. Most of these genes might have transferred from bacterial species through horizontal gene transfer. Comparative analysis between human and fish pathogen also provide insights into genes responsible for pathogenecity. Our analysis of pseudogenes indicates that the evolution of Francisella subspecies’s pseudogenes from Tilapia is old with large number of pseudogenes having more than one inactivating mutation.ConclusionsThe fish pathogen has lost non-essential genes some time ago. Evolutionary analysis of the Francisella genomes, strongly suggests that human and fish pathogenic Francisella species have evolved independently from free-living metabolically competent Francisella species. These findings will contribute to understanding the evolution of Francisella species and pathogenesis.

Analysis of polymeric immunoglobulin receptor- and CD300-like molecules from Atlantic salmon

The polymeric immunoglobulin receptor (pIgR) plays a pivotal role in mucosal immune protection by transporting secretory immunoglobulins to mucosal epithelia, and protecting them from proteolytic degradation. It has been reported that a homolog of the pIgR has a similar role in teleost fish. Considering the role pIgR has in mucosal defenses, this study was initiated to characterize a possible pIgR homolog in Atlantic salmon (Salmo salar) and its relatedness to pIgR of other vertebrates and similar molecules. Two pIgR-like cDNAs and genes of Atlantic salmon (Salsal pIgR and Salsal pIgRL) were cloned and analyzed. In addition, we gathered sequence information of CMRF35-like molecules (CLM) 1, 7, and 8 (designated as CD300 in humans) and made a comparative evaluation to that of the Salsal pIgR and Salsal pIgRL polypeptides. Salsal pIgR and Salsal pIgRL, like pIgR in other teleosts, are composed of two IG V domains, a connecting, a transmembrane, and a cytoplasmic region. The same holds true for Atlantic salmon CLM1 and CLM7, except that they possess putative immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic tails. The abundance of Salsal pIgR transcript is significantly higher than Salsal pIgRL and CLM in the skin, while Salsal pIgRL transcripts were abundant in the gills, depicting their possible tissue-specific role in mucosal immunity. To further highlight the roles of these molecules in cutaneous mucosal defence, we compared their transcriptional changes in salmon skin and spleen infected with the ectoparasite Lepeophtheirus salmonis which targets skin and mucus of salmonid fish (sampled 3, 14 and 28 days post infection (dpi)). Salsal pIgR and Salsal pIgRL transcripts significantly increased after 14dpi in skin and spleen. CLM1 was up-regulated in skin and down-regulated in spleen, possibly indicating that CLM1 expressing cells had migrated to the target site. Homology modeling using human pIgR domain 1 (PDB 1xed) identified structurally equivalent residues on both Salsal pIgR and Salsal pIgRL, and the same domain disulphide bridge topology. Cysteines 42 and 50 (IMGT numbering) are 7 residues apart in all V domains of Salsal pIgR, Salsal pIgRL, and mammalian [D1].

Modulation of Cell Metabolic Pathways and Oxidative Stress Signaling Contribute to Acquired Melphalan Resistance in Multiple Myeloma Cells

Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70–80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance.

A monoclonal antibody distinguishes between two IgM heavy chain isotypes in Atlantic salmon and brown trout: protein characterization, 3D modeling and epitope mapping.

Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) possess two distinct subpopulations of IgM which can be separated by anion exchange chromatography. Accordingly, there are two isotypic μ genes in these species, related to ancestral tetraploidy. In the present work it was verified by mass spectrometry that IgM of peak 1 (subpopulation 1) have heavy chains previously designated as μB type whereas IgM of peak 2 (subpopulation 2) have heavy chains of μA type. Two adjacent cysteine residues are present near the C-terminal part of μB, in contrast to one cysteine residue in μA. Salmon IgM of both peak 1 and peak 2 contain light chains of the two most common isotypes: IgL1 and IgL3. In contrast to salmon and brown trout, IgM of rainbow trout (Oncorhynchus mykiss) is eluted in a single peak when subjected to anion exchange chromatography. Surprisingly, a monoclonal antibody MAb4C10 against rainbow trout IgM, reacted with μA in salmon, whereas in brown trout it reacted with μB. It is plausible to assume that DNA has been exchanged between the paralogous A and B loci during evolution while maintaining the two sub-variants, with and without the extra cysteine. MAb4C10 was conjugated to magnetic beads and used to separate cells, demonstrating that μ transcripts residing from captured cells were primarily of A type in salmon and B type in brown trout. An analysis of amino acid substitutions in μA and μB of salmon and brown trout indicated that the third constant domain is essential for MAb4C10 binding. This was supported by 3D modeling and was finally verified by studies of MAb4C10 reactivity with a series of recombinant μ3 constructs.

Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication.

Abstract Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process.

On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling.