Dexmedetomidine is neuroprotective in an in vitro model for traumatic brain injury

<p>Abstract</p> <p>Background</p> <p>The α₂-adrenoreceptor agonist dexmedetomidine is known to provide neuroprotection under ischemic conditions. In this study we investigated whether dexmedetomidine has a protective effect in an <it>in vitro </it>model for traumatic brain injury.</p> <p>Methods</p> <p>Organotypic hippocampal slice cultures were subjected to a focal mechanical trauma and then exposed to varying concentrations of dexmedetomidine. After 72 h cell injury was assessed using propidium iodide. In addition, the effects of delayed dexmedetomidine application, of hypothermia and canonical signalling pathway inhibitors were examined.</p> <p>Results</p> <p>Dexmedetomidine showed a protective effect on traumatically injured hippocampal cells with a maximum effect at a dosage of 1 μM. This effect was partially reversed by the simultaneous administration of the ERK inhibitor PD98059.</p> <p>Conclusion</p> <p>In this TBI model dexmedetomidine had a significant neuroprotective effect. Our results indicate that activation of ERK might be involved in mediating this effect.</p

Tomato TILLING Technology: Development of a Reverse Genetics Tool for the Efficient Isolation of Mutants from Micro-Tom Mutant Libraries

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1–SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato

Novel Insights into Pituitary Tumorigenesis: Genetic and Epigenetic Mechanisms.

Substantial advances have been made recently in the pathobiology of pituitary tumors. Similar to many other endocrine tumors, over the last few years we have recognized the role of germline and somatic mutations in a number of syndromic or nonsyndromic conditions with pituitary tumor predisposition. These include the identification of novel germline variants in patients with familial or simplex pituitary tumors and establishment of novel somatic variants identified through next generation sequencing. Advanced techniques have allowed the exploration of epigenetic mechanisms mediated through DNA methylation, histone modifications and noncoding RNAs, such as microRNA, long noncoding RNAs and circular RNAs. These mechanisms can influence tumor formation, growth, and invasion. While genetic and epigenetic mechanisms often disrupt similar pathways, such as cell cycle regulation, in pituitary tumors there is little overlap between genes altered by germline, somatic, and epigenetic mechanisms. The interplay between these complex mechanisms driving tumorigenesis are best studied in the emerging multiomics studies. Here, we summarize insights from the recent developments in the regulation of pituitary tumorigenesis

First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.Financial support was provided by the Spanish Project INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Almentaria) RTA2011-00044C02-01, the ANR MELODY (ANR-11-BSV7-0024), the European Research Council (ERCSEXYPARTH), FEDER, and FSE funds. NVD has been awarded a grant by the Andalusian Institute of Agronomy Research IFAPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Vicente-Dolera, N.; Troadec, C.; Moya, M.; Río-Celestino, MD.; Pomares-Viciana, T.; Bendahmane, A.; Picó Sirvent, MB.... (2014). First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement. PLoS ONE. 9(11):112743-112743. https://doi.org/10.1371/journal.pone.0112743S112743112743911Paris, H. S., Yonash, N., Portnoy, V., Mozes-Daube, N., Tzuri, G., & Katzir, N. (2002). Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers. Theoretical and Applied Genetics, 106(6), 971-978. doi:10.1007/s00122-002-1157-0Parry, M. A. J., Madgwick, P. J., Bayon, C., Tearall, K., Hernandez-Lopez, A., Baudo, M., … Phillips, A. L. (2009). Mutation discovery for crop improvement. Journal of Experimental Botany, 60(10), 2817-2825. doi:10.1093/jxb/erp189Gilchrist, E., & Haughn, G. (2010). Reverse genetics techniques: engineering loss and gain of gene function in plants. Briefings in Functional Genomics, 9(2), 103-110. doi:10.1093/bfgp/elp059McCallum, C. M., Comai, L., Greene, E. A., & Henikoff, S. (2000). Targeting Induced LocalLesions IN Genomes (TILLING) for Plant Functional Genomics. Plant Physiology, 123(2), 439-442. doi:10.1104/pp.123.2.439Colbert, T., Till, B. J., Tompa, R., Reynolds, S., Steine, M. N., Yeung, A. T., … Henikoff, S. (2001). High-Throughput Screening for Induced Point Mutations. Plant Physiology, 126(2), 480-484. doi:10.1104/pp.126.2.480Wang, T. L., Uauy, C., Robson, F., & Till, B. (2012). TILLINGin extremis. Plant Biotechnology Journal, 10(7), 761-772. doi:10.1111/j.1467-7652.2012.00708.xDong, C., Dalton-Morgan, J., Vincent, K., & Sharp, P. (2009). A Modified TILLING Method for Wheat Breeding. The Plant Genome Journal, 2(1), 39. doi:10.3835/plantgenome2008.10.0012Uauy, C., Paraiso, F., Colasuonno, P., Tran, R. K., Tsai, H., Berardi, S., … Dubcovsky, J. (2009). A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biology, 9(1), 115. doi:10.1186/1471-2229-9-115Kumar, A. P., Boualem, A., Bhattacharya, A., Parikh, S., Desai, N., Zambelli, A., … Bendahmane, A. (2013). SMART -- Sunflower Mutant population And Reverse genetic Tool for crop improvement. BMC Plant Biology, 13(1), 38. doi:10.1186/1471-2229-13-38Kurowska, M., Daszkowska-Golec, A., Gruszka, D., Marzec, M., Szurman, M., Szarejko, I., & Maluszynski, M. (2011). TILLING - a shortcut in functional genomics. Journal of Applied Genetics, 52(4), 371-390. doi:10.1007/s13353-011-0061-1Rigola, D., van Oeveren, J., Janssen, A., Bonné, A., Schneiders, H., van der Poel, H. J. A., … van Eijk, M. J. T. (2009). High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology. PLoS ONE, 4(3), e4761. doi:10.1371/journal.pone.0004761González, M., Xu, M., Esteras, C., Roig, C., Monforte, A. J., Troadec, C., … Picó, B. (2011). Towards a TILLING platform for functional genomics in Piel de Sapo melons. BMC Research Notes, 4(1). doi:10.1186/1756-0500-4-289Elias, R., Till, B. J., Mba, C., & Al-Safadi, B. (2009). Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC Research Notes, 2(1), 141. doi:10.1186/1756-0500-2-141Dahmani-Mardas, F., Troadec, C., Boualem, A., Lévêque, S., Alsadon, A. A., Aldoss, A. A., … Bendahmane, A. (2010). Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach. PLoS ONE, 5(12), e15776. doi:10.1371/journal.pone.0015776Boualem, A., Fleurier, S., Troadec, C., Audigier, P., Kumar, A. P. K., Chatterjee, M., … Bendahmane, A. (2014). Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics. PLoS ONE, 9(5), e97963. doi:10.1371/journal.pone.0097963Blanca, J., Cañizares, J., Roig, C., Ziarsolo, P., Nuez, F., & Picó, B. (2011). Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics, 12(1). doi:10.1186/1471-2164-12-104Esteras, C., Gomez, P., Monforte, A. J., Blanca, J., Vicente-Dolera, N., Roig, C., … Pico, B. (2012). High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genomics, 13(1), 80. doi:10.1186/1471-2164-13-80Vicente-Dólera, N., Pinillos, V., Moya, M., Del Río-Celestino, M., Pomares-Viciana, T., Román, B., & Gómez, P. (2014). An improved method to obtain novel mutants in Cucurbita pepo by pollen viability. Scientia Horticulturae, 169, 14-19. doi:10.1016/j.scienta.2014.01.045Martín, B., Ramiro, M., Martínez-Zapater, J. M., & Alonso-Blanco, C. (2009). A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis. BMC Plant Biology, 9(1), 147. doi:10.1186/1471-2229-9-147Wienholds, E. (2003). Efficient Target-Selected Mutagenesis in Zebrafish. Genome Research, 13(12), 2700-2707. doi:10.1101/gr.1725103Dalmais, M., Schmidt, J., Le Signor, C., Moussy, F., Burstin, J., Savois, V., … Bendahmane, A. (2008). UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biology, 9(2), R43. doi:10.1186/gb-2008-9-2-r43Triques, K., Sturbois, B., Gallais, S., Dalmais, M., Chauvin, S., Clepet, C., … Bendahmane, A. (2007). Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. The Plant Journal, 51(6), 1116-1125. doi:10.1111/j.1365-313x.2007.03201.xTaylor, N. E. (2003). PARSESNP: a tool for the analysis of nucleotide polymorphisms. Nucleic Acids Research, 31(13), 3808-3811. doi:10.1093/nar/gkg574Ng, P. C. (2003). SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Research, 31(13), 3812-3814. doi:10.1093/nar/gkg509Obrero, Á., González-Verdejo, C. I., Die, J. V., Gómez, P., Del Río-Celestino, M., & Román, B. (2013). Carotenogenic Gene Expression and Carotenoid Accumulation in Three Varieties of Cucurbita pepo during Fruit Development. Journal of Agricultural and Food Chemistry, 61(26), 6393-6403. doi:10.1021/jf4004576Cosio, C., Vuillemin, L., De Meyer, M., Kevers, C., Penel, C., & Dunand, C. (2009). An anionic class III peroxidase from zucchini may regulate hypocotyl elongation through its auxin oxidase activity. Planta, 229(4), 823-836. doi:10.1007/s00425-008-0876-0Sisko, M. (2003). Genome size analysis in the genus Cucurbita and its use for determination of interspecific hybrids obtained using the embryo-rescue technique. Plant Science, 165(3), 663-669. doi:10.1016/s0168-9452(03)00256-5Campa A (1991) Biological roles of plant peroxidases: known and potential function. In Peroxidases in Chemistry and Biology Vol. II. (Everse, J., Everse, K.E. &amp; Grisham, M.B., eds), pp. 25–50, CRC Press, Boca Raton, FL.Schuller, D. J., Ban, N., van Huystee, R. B., McPherson, A., & Poulos, T. L. (1996). The crystal structure of peanut peroxidase. Structure, 4(3), 311-321. doi:10.1016/s0969-2126(96)00035-4Stephenson, P., Baker, D., Girin, T., Perez, A., Amoah, S., King, G. J., & Østergaard, L. (2010). A rich TILLING resource for studying gene function in Brassica rapa. BMC Plant Biology, 10(1), 62. doi:10.1186/1471-2229-10-62Suzuki, T., Eiguchi, M., Kumamaru, T., Satoh, H., Matsusaka, H., Moriguchi, K., … Kurata, N. (2007). MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Molecular Genetics and Genomics, 279(3), 213-223. doi:10.1007/s00438-007-0293-2Chantreau, M., Grec, S., Gutierrez, L., Dalmais, M., Pineau, C., Demailly, H., … Hawkins, S. (2013). PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics. BMC Plant Biology, 13(1), 159. doi:10.1186/1471-2229-13-159Rawat, N., Sehgal, S. K., Joshi, A., Rothe, N., Wilson, D. L., McGraw, N., … Gill, B. S. (2012). A diploid wheat TILLING resource for wheat functional genomics. BMC Plant Biology, 12(1), 205. doi:10.1186/1471-2229-12-205Minoia, S., Petrozza, A., D’Onofrio, O., Piron, F., Mosca, G., Sozio, G., … Carriero, F. (2010). A new mutant genetic resource for tomato crop improvement by TILLING technology. BMC Research Notes, 3(1). doi:10.1186/1756-0500-3-69Markiewicz, P., Kleina, L. G., Cruz, C., Ehret, S., & Miller, J. H. (1994). Genetic Studies of the lac Repressor. XIV. Analysis of 4000 Altered Escherichia coli lac Repressors Reveals Essential and Non-essential Residues, as well as «Spacers» which do not Require a Specific Sequence. Journal of Molecular Biology, 240(5), 421-433. doi:10.1006/jmbi.1994.1458Carpin, S., Crèvecoeur, M., Greppin, H., & Penel, C. (1999). Molecular Cloning and Tissue-Specific Expression of an Anionic Peroxidase in Zucchini. Plant Physiology, 120(3), 799-810. doi:10.1104/pp.120.3.799Welinder, K. G., Justesen, A. F., Kjaersgård, I. V. H., Jensen, R. B., Rasmussen, S. K., Jespersen, H. M., & Duroux, L. (2002). Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana. European Journal of Biochemistry, 269(24), 6063-6081. doi:10.1046/j.1432-1033.2002.03311.