Fluorescent Protein-Based Methods for On-Plate Screening of Gene Insertion

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening

Rapid Transient Production in Plants by Replicating and Non-Replicating Vectors Yields High Quality Functional Anti-HIV Antibody

Background: The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product. Methodology/Principal Findings: To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV) RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue) of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER). Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO) cell-produced 2G12. Conclusions: Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for biopharmaceutical development and production

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

[EN] Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplastreplicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.This work was supported by the European Research Council (erc.europa.eu; ERC-2011-StG-281191-VIRMUT to RS), the Spanish Ministerio de Economia y Competitividad (www.mineco.gob.es; BFU2013-41329 grant to RS, BFU2014-56812-P grant to RF, and a predoctoral fellowship to ALC), and the Spanish Junta de Comunidades de Castilla-La Mancha (www.castillalamancha.es;postdoctoral fellowship to CB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.López-Carrasco, MA.; Ballesteros Martínez, C.; Sentandreu, V.; Delgado Villar, SG.; Gago Zachert, SP.; Flores Pedauye, R.; Sanjuan Verdeguer, R. (2017). Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing. PLoS Pathogens. 13(9):1-17. https://doi.org/10.1371/journal.ppat.1006547S117139Ganai, R. A., & Johansson, E. (2016). DNA Replication—A Matter of Fidelity. Molecular Cell, 62(5), 745-755. doi:10.1016/j.molcel.2016.05.003Lynch, M. (2010). Evolution of the mutation rate. Trends in Genetics, 26(8), 345-352. doi:10.1016/j.tig.2010.05.003Sanjuán, R., & Domingo-Calap, P. (2016). Mechanisms of viral mutation. Cellular and Molecular Life Sciences, 73(23), 4433-4448. doi:10.1007/s00018-016-2299-6Gago, S., Elena, S. F., Flores, R., & Sanjuan, R. (2009). Extremely High Mutation Rate of a Hammerhead Viroid. Science, 323(5919), 1308-1308. doi:10.1126/science.1169202Flores, R., Gago-Zachert, S., Serra, P., Sanjuán, R., & Elena, S. F. (2014). Viroids: Survivors from the RNA World? Annual Review of Microbiology, 68(1), 395-414. doi:10.1146/annurev-micro-091313-103416Flores, R., Minoia, S., Carbonell, A., Gisel, A., Delgado, S., López-Carrasco, A., … Di Serio, F. (2015). Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection. Virus Research, 209, 136-145. doi:10.1016/j.virusres.2015.02.027Steger, G., & Perreault, J.-P. (2016). Structure and Associated Biological Functions of Viroids. Advances in Virus Research, 141-172. doi:10.1016/bs.aivir.2015.11.002Diener, T. O. (1989). Circular RNAs: relics of precellular evolution? Proceedings of the National Academy of Sciences, 86(23), 9370-9374. doi:10.1073/pnas.86.23.9370Ambrós, S., Hernández, C., & Flores, R. (1999). Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host The data reported in this paper are in the EMBL nucleotide sequence database and assigned the accession nos AJ241818–AJ241850. Journal of General Virology, 80(8), 2239-2252. doi:10.1099/0022-1317-80-8-2239Navarro, J.-A., Vera, A., & Flores, R. (2000). A Chloroplastic RNA Polymerase Resistant to Tagetitoxin Is Involved in Replication of Avocado Sunblotch Viroid. Virology, 268(1), 218-225. doi:10.1006/viro.1999.0161Rodio, M.-E., Delgado, S., De Stradis, A., Gómez, M.-D., Flores, R., & Di Serio, F. (2007). A Viroid RNA with a Specific Structural Motif Inhibits Chloroplast Development. The Plant Cell, 19(11), 3610-3626. doi:10.1105/tpc.106.049775Carbonell, A., De la Peña, M., Flores, R., & Gago, S. (2006). Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads. Nucleic Acids Research, 34(19), 5613-5622. doi:10.1093/nar/gkl717Hutchins, C. J., Rathjen, P. D., Forster, A. C., & Symons, R. H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Research, 14(9), 3627-3640. doi:10.1093/nar/14.9.3627PRODY, G. A., BAKOS, J. T., BUZAYAN, J. M., SCHNEIDER, I. R., & BRUENING, G. (1986). Autolytic Processing of Dimeric Plant Virus Satellite RNA. Science, 231(4745), 1577-1580. doi:10.1126/science.231.4745.1577Nohales, M.-A., Molina-Serrano, D., Flores, R., & Daros, J.-A. (2012). Involvement of the Chloroplastic Isoform of tRNA Ligase in the Replication of Viroids Belonging to the Family Avsunviroidae. Journal of Virology, 86(15), 8269-8276. doi:10.1128/jvi.00629-12Branch, A. D., Benenfeld, B. J., & Robertson, H. D. (1988). Evidence for a single rolling circle in the replication of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 85(23), 9128-9132. doi:10.1073/pnas.85.23.9128Daros, J.-A., & Flores, R. (2004). Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae. Proceedings of the National Academy of Sciences, 101(17), 6792-6797. doi:10.1073/pnas.0401090101Feldstein, P. A., Hu, Y., & Owens, R. A. (1998). Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 95(11), 6560-6565. doi:10.1073/pnas.95.11.6560Gas, M.-E., Hernández, C., Flores, R., & Daròs, J.-A. (2007). Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations. PLoS Pathogens, 3(11), e182. doi:10.1371/journal.ppat.0030182Nohales, M.-A., Flores, R., & Daros, J.-A. (2012). Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase. Proceedings of the National Academy of Sciences, 109(34), 13805-13810. doi:10.1073/pnas.1206187109Brass, J. R. J., Owens, R. A., Matoušek, J., & Steger, G. (2017). Viroid quasispecies revealed by deep sequencing. RNA Biology, 14(3), 317-325. doi:10.1080/15476286.2016.1272745Bull, J. J., Sanjuán, R., & Wilke, C. O. (2007). Theory of Lethal Mutagenesis for Viruses. Journal of Virology, 81(6), 2930-2939. doi:10.1128/jvi.01624-06Cuevas, J. M., González-Candelas, F., Moya, A., & Sanjuán, R. (2009). Effect of Ribavirin on the Mutation Rate and Spectrum of Hepatitis C Virus In Vivo. Journal of Virology, 83(11), 5760-5764. doi:10.1128/jvi.00201-09Ribeiro, R. M., Li, H., Wang, S., Stoddard, M. B., Learn, G. H., Korber, B. T., … Perelson, A. S. (2012). Quantifying the Diversification of Hepatitis C Virus (HCV) during Primary Infection: Estimates of the In Vivo Mutation Rate. PLoS Pathogens, 8(8), e1002881. doi:10.1371/journal.ppat.1002881Acevedo, A., Brodsky, L., & Andino, R. (2013). Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature, 505(7485), 686-690. doi:10.1038/nature12861Cuevas, J. M., Geller, R., Garijo, R., López-Aldeguer, J., & Sanjuán, R. (2015). Extremely High Mutation Rate of HIV-1 In Vivo. PLOS Biology, 13(9), e1002251. doi:10.1371/journal.pbio.1002251Acevedo, A., & Andino, R. (2014). Library preparation for highly accurate population sequencing of RNA viruses. Nature Protocols, 9(7), 1760-1769. doi:10.1038/nprot.2014.118Kennedy, S. R., Schmitt, M. W., Fox, E. J., Kohrn, B. F., Salk, J. J., Ahn, E. H., … Loeb, L. A. (2014). Detecting ultralow-frequency mutations by Duplex Sequencing. Nature Protocols, 9(11), 2586-2606. doi:10.1038/nprot.2014.170Franklin, R. M. (1966). Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proceedings of the National Academy of Sciences, 55(6), 1504-1511. doi:10.1073/pnas.55.6.1504López-Carrasco, A., Gago-Zachert, S., Mileti, G., Minoia, S., Flores, R., & Delgado, S. (2015). The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in theirin vivoRNA conformations. RNA Biology, 13(1), 83-97. doi:10.1080/15476286.2015.1119365Keese, P., & Symons, R. H. (1985). Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proceedings of the National Academy of Sciences, 82(14), 4582-4586. doi:10.1073/pnas.82.14.4582López-Carrasco, A., & Flores, R. (2016). Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A «naked» rod-like conformation similar but not identical to that observed in vitro. RNA Biology, 14(8), 1046-1054. doi:10.1080/15476286.2016.1223005Flores, R., Hernandez, C., de la Peña, M., Vera, A., & Daros, J.-A. (2001). Hammerhead Ribozyme Structure and Function in Plant RNA Replication. Ribonucleases - Part A, 540-552. doi:10.1016/s0076-6879(01)41175-xMartick, M., & Scott, W. G. (2006). Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis. Cell, 126(2), 309-320. doi:10.1016/j.cell.2006.06.036Ruffner, D. E., Stormo, G. D., & Uhlenbeck, O. C. (1990). Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry, 29(47), 10695-10702. doi:10.1021/bi00499a018Flores, R., Serra, P., Minoia, S., Di Serio, F., & Navarro, B. (2012). Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs. Frontiers in Microbiology, 3. doi:10.3389/fmicb.2012.00217Owens, R. A., Chen, W., Hu, Y., & Hsu, Y.-H. (1995). Suppression of Potato Spindle Tuber Viroid Replication and Symptom Expression by Mutations Which Stabilize the Pathogenicity Domain. Virology, 208(2), 554-564. doi:10.1006/viro.1995.1186Takeda, R., Petrov, A. I., Leontis, N. B., & Ding, B. (2011). A Three-Dimensional RNA Motif in Potato spindle tuber viroid Mediates Trafficking from Palisade Mesophyll to Spongy Mesophyll in Nicotiana benthamiana. The Plant Cell, 23(1), 258-272. doi:10.1105/tpc.110.081414Zhong, X., Leontis, N., Qian, S., Itaya, A., Qi, Y., Boris-Lawrie, K., & Ding, B. (2006). Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication. Journal of Virology, 80(17), 8566-8581. doi:10.1128/jvi.00837-06Zhong, X., Tao, X., Stombaugh, J., Leontis, N., & Ding, B. (2007). Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafficking. The EMBO Journal, 26(16), 3836-3846. doi:10.1038/sj.emboj.7601812Zhong, X., Archual, A. J., Amin, A. A., & Ding, B. (2008). A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking. The Plant Cell, 20(1), 35-47. doi:10.1105/tpc.107.056606Thomas, M. J., Platas, A. A., & Hawley, D. K. (1998). Transcriptional Fidelity and Proofreading by RNA Polymerase II. Cell, 93(4), 627-637. doi:10.1016/s0092-8674(00)81191-5Gout, J.-F., Thomas, W. K., Smith, Z., Okamoto, K., & Lynch, M. (2013). Large-scale detection of in vivo transcription errors. Proceedings of the National Academy of Sciences, 110(46), 18584-18589. doi:10.1073/pnas.1309843110Hedtke, B. (1997). Mitochondrial and Chloroplast Phage-Type RNA Polymerases in Arabidopsis. Science, 277(5327), 809-811. doi:10.1126/science.277.5327.809Lerbs-Mache, S. (1993). The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes? Proceedings of the National Academy of Sciences, 90(12), 5509-5513. doi:10.1073/pnas.90.12.5509Oldenkott, B., Yamaguchi, K., Tsuji-Tsukinoki, S., Knie, N., & Knoop, V. (2014). Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyteSelaginella uncinata. RNA, 20(10), 1499-1506. doi:10.1261/rna.045575.114Codoñer, F. M., Darós, J.-A., Solé, R. V., & Elena, S. F. (2006). The Fittest versus the Flattest: Experimental Confirmation of the Quasispecies Effect with Subviral Pathogens. PLoS Pathogens, 2(12), e136. doi:10.1371/journal.ppat.0020136Eigen, M. (1971). Selforganization of matter and the evolution of biological macromolecules. Die Naturwissenschaften, 58(10), 465-523. doi:10.1007/bf00623322Lynch, M. (2011). The Lower Bound to the Evolution of Mutation Rates. Genome Biology and Evolution, 3, 1107-1118. doi:10.1093/gbe/evr066Bradwell, K., Combe, M., Domingo-Calap, P., & Sanjuán, R. (2013). Correlation Between Mutation Rate and Genome Size in Riboviruses: Mutation Rate of Bacteriophage Qβ. Genetics, 195(1), 243-251. doi:10.1534/genetics.113.154963Drake, J. W. (1991). A constant rate of spontaneous mutation in DNA-based microbes. Proceedings of the National Academy of Sciences, 88(16), 7160-7164. doi:10.1073/pnas.88.16.7160Schmitt, M. W., Kennedy, S. R., Salk, J. J., Fox, E. J., Hiatt, J. B., & Loeb, L. A. (2012). Detection of ultra-rare mutations by next-generation sequencing. Proceedings of the National Academy of Sciences, 109(36), 14508-14513. doi:10.1073/pnas.120871510

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

Background: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Methods: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Results: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. Conclusion: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cytomorphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death

Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

Human African trypanosomiasis, or sleeping sickness, occurs when single-cell trypanosome protozoan parasites spread from the blood to brain over the blood-brain barrier (BBB). This barrier is composed of brain microvascular endothelial cells (BMECs) especially designed to keep pathogens out. Safe drugs for treating sleeping sickness are lacking and alternative treatments are urgently required. Using our human BMEC BBB model, we previously found that a parasite protease, brucipain, induced calcium activation signals that allowed this barrier to open up to parasite crossing. Because human BMECs express protease-activated receptors (PARs) that trigger calcium signals in BMECs, we hypothesized a functional link between parasite brucipain and BMEC PARs. Utilizing RNA interference to block the production of one type of PAR called PAR-2, we hindered the ability of trypanosomes to both open up and cross human BMECs. Using gene-profiling methods to interrogate candidate BMEC pathways specifically triggered by brucipain, several pathways that potentially link brain inflammatory processes were identified, a finding congruent with the known role of PAR-2 as a mediator of inflammation. Overall, our data support a role for brucipain and BMEC PARs in trypanosome BBB transmigration, and as potential triggers for brain inflammation associated with the disease

The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual’s high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients