Evaluation of homogeneity and genetic stability of REOLYSIN (pelareorep) by complete genome sequencing of reovirus after large scale production.

REOLYSIN (pelareorep) is a proprietary isolate of the reovirus T3D (Type 3 Dearing) strain which is currently being tested in clinical trials as an anticancer therapeutic agent. Reovirus genomes are composed of ten segments of double-stranded ribonucleic acid (RNA) characterized by genome size: large (L1, L2, and L3), medium (M1, M2, and M3), and small (S1, S2, S3, and S4). The objective of this work was to evaluate the homogeneity and genetic stability of REOLYSIN. Sanger sequencing (SS) performed on test articles derived from the Master Virus Bank (MVB) and Working Virus Bank (WVB) identified many modifications when compared to GenBank reference sequences. Massively parallel sequencing (MPS) using Roche-454 sequencing was performed on REOLYSIN (100 L scale) and resulted in 69,821,115 bases and an average of 335 bases per read. Twenty-nine high confidence differences relative to the GenBank reference sequence were identified in REOLYSIN by MPS. Of those, 27 were previously identified by SS in the virus bank-derived test articles. Of the remaining two nucleotide differences, one was predicted to be silent at the amino acid level (L3 genome-T3163C, codon 1054, 86% of the population was "T" and 13% of the population were reported as "C"). The other modification was in the noncoding region (M1 genome-A2284A to A2284G), and A2284G was present in 97% of the population. The results obtained from MPS were comparable to those from SS; both demonstrate a high level of homogeneity at the amino acid level and genetic stability of REOLYSIN. Finally, phylogenetic analysis of the REOLYSIN L1 genome segment showed close evolutionary relationship with its human homologs, serotypes Lang and Dearing.

Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries.

Use of highly potent small interfering RNAs (siRNAs) can substantially reduce dose-dependent cytotoxic and off-target effects. We developed a genetic forward approach by fusing the cytosine deaminase gene with targets for the robust identification of highly potent siRNAs from RNA interference (RNAi) libraries that were directly delivered into cells via bacterial invasion. We demonstrated that two simple drug selection cycles performed conveniently in a single container predominately enriched two siRNAs targets the MVP gene (siMVP) and one siRNA targets the egfp gene (siEGFP) in surviving cells and these proved to be the most effective siRNAs reported. Furthermore, the potent siRNAs isolated from the surviving cells possessed noncellular toxic characteristics. Interestingly, the length of highly potent siMVPs identified could be as short as 16-mer, and increasing the length of their native sequences dramatically reduced RNAi potency. These results suggest that the current approach can robustly discover the most potent and nontoxic siRNAs in the surviving cells, and thus has great potential in facilitating RNAi applications by minimizing the dose-dependent and sequence nonspecific side effects of siRNAs.

Global analysis of microRNA target gene expression reveals that miRNA targets are lower expressed in mature mouse and Drosophila tissues than in the embryos.

MicroRNAs (miRNAs) are non-coding small RNAs of approximately 22 nt that regulate the gene expression by base pairing with target mRNAs, leading to mRNA cleavage or translational repression. It is currently estimated that miRNAs account for approximately 1% of predicted genes in higher eukaryotic genomes and that up to 30% of genes might be regulated by miRNAs. However, only very few miRNAs have been functionally characterized and the general functions of miRNAs are not globally studied. In this study, we systematically analyzed the expression patterns of miRNA targets using several public microarray profiles. We found that the expression levels of miRNA targets are lower in all mouse and Drosophila tissues than in the embryos. We also found miRNAs more preferentially target ubiquitously expressed genes than tissue-specifically expressed genes. These results support the current suggestion that miRNAs are likely to be largely involved in embryo development and maintaining of tissue identity.

Aberrant allele frequencies of the SNPs located in microRNA target sites are potentially associated with human cancers.

MicroRNAs (miRNAs) are a class of noncoding small RNAs that regulate gene expression by base pairing with target mRNAs at the 3'-terminal untranslated regions (3'-UTRs), leading to mRNA cleavage or translational repression. Single-nucleotide polymorphisms (SNPs) located at miRNA-binding sites (miRNA-binding SNPs) are likely to affect the expression of the miRNA target and may contribute to the susceptibility of humans to common diseases. We herein performed a genome-wide analysis of SNPs located in the miRNA-binding sites of the 3'-UTR of various human genes. We found that miRNA-binding SNPs are negatively selected in respect to SNP distribution between the miRNA-binding 'seed' sequence and the entire 3'-UTR sequence. Furthermore, we comprehensively defined the expression of each miRNA-binding SNP in cancers versus normal tissues through mining EST databases. Interestingly, we found that some miRNA-binding SNPs exhibit significant different allele frequencies between the human cancer EST libraries and the dbSNP database. More importantly, using human cancer specimens against the dbSNP database for case-control association studies, we found that twelve miRNA-binding SNPs indeed display an aberrant allele frequency in human cancers. Hence, SNPs located in miRNA-binding sites affect miRNA target expression and function, and are potentially associated with cancers.

Genetic variations of microRNAs in human cancer and their effects on the expression of miRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level to lead to mRNA degradation or repressed protein production. The expression of miRNA is deregulated in many types of cancers. To determine whether genetic alterations in miRNA genes are associated with cancers, we have systematically screened sequence variations in several hundred human miRNAs from >100 human tumor tissues and 20 cancer cell lines. We identified 8 new single-nucleotide polymorphisms (SNPs) and 14 novel mutations (or very rare SNPs) that specifically present in human cancers. These mutations/SNPs are distributed in the regions of pri-, pre- and even mature miRNAs, respectively. Importantly, whereas most of the mutations did not exert detectable effects on miRNA function, a G --> A mutation at 19 nt downstream of miRNA let-7e led to a significant reduction of its expression in vivo, indicating that miRNA mutation could contribute to tumorigenesis. These data suggest that further screening for genetic variations in miRNA genes from a wide variety of human cancers should increase the discovery and identification of molecular diagnostic and therapeutic targets and complement the mutation analysis of consensus coding sequences in human cancers.

Collagen-mediated survival signaling is modulated by CD45 in Jurkat T cells.

T cell activation is a critical step in the development of a proper immune response to infection and inflammation. This dynamic process requires efficient T cell receptor signaling, which in turn is modulated by integrin receptor activation and the actin cytoskeleton. CD45 is a key player in T cell receptor mediated signal transduction. However, its exact role in integrin mediated signaling in T cells remains to be elucidated. The present study addresses the relationship between CD45 and beta1-integrin mediated survival signaling in the human T leukemic cell line Jurkat, in which collagen receptors alpha1 beta1 and alpha2 beta1 integrins are localized. Wild type (WT)-Jurkat T cells treated with collagen demonstrated increased cell proliferation and survival. Monitoring the intracellular signaling pathways activated by collagen in WT-Jurkat cells revealed increased focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) activation. Moreover, examination of the actin cytoskeleton of WT-Jurkat T cells treated with collagen demonstrated the presence of an organized cortical actin structure, reminiscent of the survival phenotype. This is in contrast to CD45-deficient J45.01 T cells, where collagen treatment failed to enhance cell proliferation/survival and was unable to stimulate FAK and ERK activity. In addition, the actin cytoskeleton of collagen treated J45.01 T cells was disorganized with cortical actin aggregates present throughout. The importance of an organized actin cytoskeleton to proper cell signaling and survival was further demonstrated by the inability of collagen treated WT-Jurkat cells to activate the FAK and ERK survival pathway in the presence of cytochalasin D, a cytoskeleton-disrupting drug. Consistently, addition of the CD45 specific inhibitor abolished collagen-stimulated FAK and ERK activation in WT-Jurkat cells, further depicting CD45 as the key mediator. Furthermore, collagen-mediated T cell signaling alone was able to activate IL-2 gene transcription devoid of concomitant T cell receptor activation. Taken together, these results are the first to demonstrate that CD45 is important in promoting cell survival by modulating integrin-mediated FAK/ERK signaling in Jurkat T cells and is involved in a distinct signal transduction pathway, separate from T cell receptor signaling, influencing T cell immune responses. Hence, this study will help further our knowledge about beta1-integrin mediated signaling in T cells, which may prove to be essential for the regulation of various T cell mediated immune responses.

SKAP55 coupled with CD45 positively regulates T-cell receptor-mediated gene transcription.

CD45 plays a critical role in T-cell receptor (TCR)-mediated signaling. In a yeast two-hybrid screen, SKAP55, the Src kinase-associated phosphoprotein of unknown function, was found as a substrate which associated with CD45 in vivo. Mutational analysis demonstrated the pivotal role of Tyr-232 in SKAP55 in the association with CD45. In Jurkat cells, anti-CD3 antibody stimulation promoted SKAP55 tyrosine phosphorylation and translocation from the cytoplasm to the membrane. Overexpression of SKAP55 in these cells induced transcriptional activation of the IL-2 promoter, while mutant SKAP55-Y232F totally suppressed the promoter activity. Furthermore, overexpression of SKAP55-Y232F also caused the tyrosine hyperphosphorylation of Fyn with a decreased kinase activity. Thus, SKAP55 is an essential adapter to couple CD45 with the Src family kinases for dephosphorylation and, thus, positively regulates TCR signaling.

Identification and characterization of hepsin/-TM, a non-transmembrane hepsin isoform.

Type II transmembrane serine proteases (TTSPs), including hepsin, are a new class of cell surface catalytic enzymes. In the present study, a non-transmembrane isoform of hepsin, named hepsin/-TM that originates from alternative splicing, was identified. Unlike the transmembrane hepsin isoform, this non-transmembrane isoform was distributed within the cytoplasm. Real-time PCR experiments revealed that while hepsin was expressed in all tested human tissues, hepsin/-TM was restricted in kidney, brain and lung tissues. Significantly, hepsin/-TM was not expressed in liver where hepsin was originally identified. However, hepsin/-TM was highly expressed in brain where hepsin was expressed at a relatively lower level. Moreover, these two isoforms showed different expression patterns in a number of colon adenocarcinoma cell lines. In addition, in contrast to hepsin, expression of hepsin/-TM in vivo does not exert any apparent inhibitory effect on mammalian cell growth.

Functional dissection of human protease mu-calpain in cell migration using RNAi.

Calpains are a family of calcium-dependent cysteine proteases involved in a variety of cellular functions. Two isoforms, m-calpain and mu-calpain, have been implicated in cell migration. However, since conventional inhibitors used for the studies of the functions of these enzymes lack specificity, the individual physiological function and biochemical mechanism of these two isoforms, especially mu-calpain, are not clear. In contrast, RNA interference has the potential to allow a sequence-specific destruction of target RNA for functional assay of gene of interest. In the present study, we found that small interfering RNAs-mediated knockdown of mu-calpain expression in MCF-7 cells that do not express m-Calpain led to a reduction of cell migration. This isoform-specific function of mu-calpain was further confirmed by the rescue experiment as overexpression of mu-calpain but not m-calpain could restore the cell migration rate. Knockdown of mu-calpain also altered cell morphology with increased filopodial projections and a highly elongated tail that seemed to prevent cell spreading and migration with reduced rear detachment ability. Furthermore, knockdown of mu-calpain decreased the proteolytic products of filamin and talin, which were specifically rescued by overexpression of mu-calpain but not m-calpain, suggesting that their proteolysis could be one of the key mechanisms by which mu-calpain regulates cell migration.

SHP-1 dephosphorylates 3BP2 and potentially downregulates 3BP2-mediated T cell antigen receptor signaling.

Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) is a critical inhibitory regulator in T cell-receptor (TCR) signaling. However, the exact molecular mechanism underlying this is poorly defined, largely because the physiological substrates for SHP-1 in T cells remain elusive. In this study, we showed that adaptor protein 3BP2 serves as a binding protein and a physiological substrate of SHP-1. 3BP2 is phosphorylated on tyrosyl residue 448 in response to TCR activation, and the phosphorylation is required for T cell signalling, as indicated by transcriptional activation of nuclear factor activated in T cells (NFAT). Concurrently, phosphorylation of Tyr566 at the C-terminus of SHP-1 causes specific recruitment of 3BP2 to the phosphatase through the SH2 domain of the adaptor protein. This leads to efficient dephosphorylation of 3BP2 and thereby termination of T cell signaling. The study thus defines a novel function of the C-terminal segment of SHP-1 and reveals a new mechanism by which T cell signaling is regulated.

Disruption of a gene encoding glycerol 3-phosphatase from Candida albicans impairs intracellular glycerol accumulation-mediated salt-tolerance.

Intracellular glycerol accumulation is critical for Candida albicans to maintain osmolarity, and therefore defects in glycerol homeostasis can have severe effects on the morphogenetic plasticity and pathogenicity of this fungus. The final step of glycerol synthesis involves the dephosphorylation of glycerol 3-phosphate by glycerol 3-phosphatase (GPP1). We have identified a single copy of the GPP orthologous gene (GPP1) in the C. albicans haploid genome, as well as the paralogous gene 2-deoxyglucose-6-phosphate phosphatase (DOG1); both belong to a family of low molecular weight phosphatases. A knockout of the GPP1 gene in C. albicans caused increased susceptibility to high salt concentrations, indicating a deficiency in osmoregulation. Reintroduction of the GPP1 gene complemented the impairment of salt-tolerance in the gpp1/gpp1 mutant. Northern blot analysis showed that the GPP1 gene was strongly responsive to osmotic stress, and its transcriptional expression was positively correlated with intracellular glycerol accumulation. These results demonstrate that the GPP1 gene plays an important role in the osmoregulation in C. albicans.

p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2Cdelta.

RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cdelta (protein phosphatase 2Cdelta). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cd is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cdelta (H154D) (His154-->Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1-71 of PP2Cd and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cd associates with RSK1-RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cd in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.

A coumermycin/novobiocin-regulated gene expression system.

Regulated expression of transgenes in mammals is an important technique in both functional genomic studies and clinical applications. Here we describe a regulated gene expression system for mammals, based on coumarin-switched dimerization of the bacterial DNA gyrase B subunit (GyrB). The transactivator was constructed by fusing the GyrB activator to the bacterial lambda repressor-binding domain. The antibiotic coumermycin in nanomolar concentrations activated the transgene through binding of the homodimerized chimeric transactivator to the lambda operator located upstream of a minipromoter. More significantly, addition of novobiocin, an antagonist of coumermycin, promptly switched off expression of the gene by abolishing coumermycin-induced dimerization of the transactivator. Site-directed mutagenesis of the lambda repressor-binding domain resulted in significant reduction of basal expression levels and an induction reaching four orders of magnitude in stably transfected 293A cells in response to coumermycin. The capability of this inducible system for tightly regulated gene expression was demonstrated by the ready generation of stable cell lines inducibly expressing the proapoptotic bax gene in mammalian cells. Hence, this novel coumarin switch-on/switch-off system should broaden the utility of regulated gene expression, particularly when rapid on/off interchange is required.

Development of a Genetically-Engineered, Candidate Polio Vaccine Employing the Self-Assembling Properties of the Tobacco Mosaic Virus Coat Protein.

A synthetic gene coding for the coat protein of tobacco mosaic virus (TMVCP) was expressed in E. coli under the direction of the lacUV5 promoter. Modification of the 3' end of the TMVCP gene by insertion of a region coding for an antigenic epitope from poliovirus type 3 resulted in the production of a hybrid TMVCP (TMVCP-polio 3). Both the E. coli-produced TMVCP and TMVCP-polio 3 were shown to assemble into virus-like rods under acidic conditions in E. coli extracts. Their purification was accomplished in a single step by chromatography on Sepharose 6B. TMVCP-polio 3 induced the formation of poliovirus neutralizing antibodies following injection into rats. The level of immune response was related to the degree of polymerization of the TMVCP-polio 3 preparations.

CD45 recruits adapter protein DOK-1 and negatively regulates JAK-STAT signaling in hematopoietic cells.

It has been extensively documented that CD45 positively regulates T cell receptor-mediated signaling through the activation of Src-family kinases. The mechanism whereby CD45 negatively regulates the JAK/STAT pathway, however, has not been fully elucidated. Here we describe the mechanism by which CD45 negatively regulates the JAK/STAT pathway through the recruitment of the inhibitory molecule Downstream of Kinase 1 (DOK-1) in hematopoietic cells. We present evidences that CD45 recruits DOK-1 to associate with tyrosine-phosphorylated DOK-1, and that the DOK-1-Y296F mutant completely abrogates its interaction with CD45. Moreover, CD45 expression is required for DOK-1 targeting to the plasma membrane in response to anti-CD3 stimulation. Functional studies further showed that stable expression of DOK-1 in K562 cells markedly decreased both JAK-2 and STAT-3/5 phosphorylation following IL-3 and IFN-alpha stimulation. Likewise, stable expression of DOK-1 in Jurkat cells significantly decreased JAK-2 phosphorylation. Similarly, both IL-3 and IFN-alpha-induced JAK-2 phosphorylations were significantly increased in CD45 deficient Jurkat cells. Consistently, silencing of the DOK-1 gene resulted in rescue of MAP kinases and JAKs activities in CD45 positive Jurkat cells. Accordingly, CD45 recruits adaptor DOK-1 to the proximal plasma membrane to serve as a downstream effector, resulting in negative regulation of the JAK/STAT signaling pathway.

A serine/threonine protein phosphatase-like protein, CaPTC8, from Candida albicans defines a new PPM subfamily.

Protein phosphatase M family (PPM; Mg(2+)-dependent protein phosphatases), which specifically dephosphorylates serine/threonine residues, consists of pyruvate dehydrogenase phosphatases, SpoIIE, adenylate cyclase and protein phosphatase type 2Cs (PP2Cs). To identify Candida albicans PP2Cs, the archetype of the PPM Ser/Thr phosphatases, we thoroughly searched the public C. albicans genome database and identified seven PP2C members. One of the PP2Cs in C. albicans, designated as CaPTC8 gene, represents a new member of PP2C genes. Northern blot analysis showed that the expression of CaPTC8 was positively responsive to high osmolarity, temperature or serum-stimulated filamentous growth. Gene disruption further demonstrated that deletion of CaPTC8 gene caused the defect of hyphal formation. Sequence analysis revealed that two conserved amino acids His and Asn in the prototypical members of the PPM family were substituted by Val and Asp in the PTC8p-like proteins. In addition, posterior analysis for site-specific profile showed that seven more sites are under the selection of functional divergence between these two groups of proteins. Three-dimensional homology modeling illustrated the signatures of the two groups in the conserved catalytic region of the protein phosphatases. Hence, CaPTC8 plays a role in stress responses and is required for the yeast-hyphal transition, and the CaPTC8-related genes are evolutionarily conserved. The phylogenetic relationships of all members of the PPM family strongly support the existence of a distinct and new subfamily of the PP2C-related proteins, PP2CR.

Expression of CaPTC7 is developmentally regulated during serum-induced morphogenesis in the human fungal pathogen Candida albicans.

Type 2C protein phosphatases (PP2C) represent a diversified protein phosphatase family and play various roles in cells. We previously identified and characterized a novel PP2C phosphatase encoded by the CaPTC7 gene in the human fungal pathogen Candida albicans. The CaPtc7p has 365 amino acids with a PP2C core domain at the C terminus and an additional 116-residue N-terminal sequence containing a mitochondrion-targeting sequence. Here, we show that CaPtc7p is indeed localized in the mitochondrion, the only eukaryotic PP2C phosphatase that has been directly shown to reside in the mitochondrion, suggesting its potential role in the regulation of mitochondrial physiology. Furthermore, we show that the expression of CaPTC7 at both transcriptional and protein levels is developmentally regulated during the serum-induced morphogenesis of C. albicans cells. However, disruption of the two alleles of CaPTC7 does not affect cell viability or filamentous development in C. albicans.

High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion.

Use of RNA interference (RNAi) as a reverse genetics tool for silencing genes in mammalian cells is achieved by in vitro transfection of small interfering RNAs (siRNAs). For a target gene, several siRNAs must be designed according to the empirical rules. We demonstrated that functional short hairpin RNAs (shRNAs) could be synthesized in Escherichia coli and delivered directly via bacterial invasion to the near entirety of a mammalian cell population to trigger RNAi. Furthermore, using a luciferase-target gene transcript, we identified effective shRNAs and siRNAs from RNAi libraries delivered conveniently through bacterial invasion in 96-well plates without need for preparation, purification and transfection of shRNAs. Notably, several of the most highly effective shRNAs and siRNAs identified do not fit the empirical rules commonly used for siRNA design, suggesting that this approach is a powerful tool for RNAi research, and could be used complementarily to the empirical rules for RNAi applications.

Functional characterization of the Candida albicans homologue of secretion-associated and Ras-related (Sar1) protein.

Secretion-associated and Ras-related protein (Sar1p) plays an essential role during the protein transport from the endoplasmic reticulum to the Golgi apparatus. The cDNA sequence of the Sar1 gene has been identified and characterized from the human yeast pathogen, Candida albicans. This cDNA encodes a protein of 190 amino acids, which shares a 78% sequence identity with Saccharomyces cerevisiae Sar1p and contains the conserved GTP-binding motifs of the small GTPase superfamily. Complementation studies confirmed that this cDNA encodes the functional homologue of ScSar1p. The recombinant C. albicans Sar1p exhibits GTP-binding activity in vitro that was abolished by deletion of one of the three GTP-binding motifs.