Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells

<p>Abstract</p> <p>Background</p> <p>The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells.</p> <p>Methods</p> <p>Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay.</p> <p>Results</p> <p>DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure.</p> <p>Conclusion</p> <p>DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.</p

SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

[EN] Background The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 euro/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted fromSolanum elaeagnifoliumusing this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.This research has been funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No 677379 (Linking genetic resources, genomes and phenotypes of Solanaceous crops; G2P-SOL). David Alonso is grateful to Universitat Politecnica de Valencia for a predoctoral (PAID-01-16) contract under the Programa de Ayudas de Investigacion y Desarrollo initiative. Mariola Plazas is grateful to Generalitat Valenciana and Fondo Social Europeo for a postdoctoral grant (APOSTD/2018/014). Pietro Gramazio is grateful to Japan Society for the Promotion of Science for a Postdoctoral Grant (P19105, FY2019 JSPS Postdoctoral Fellowship for Research in Japan (Standard)). The Spanish Ministerio de Educacion, Cultura y Deporte funded a predoctoral fellowship granted to Edgar Garcia-Fortea (FPU17/02389).Vilanova Navarro, S.; Alonso-Martín, D.; Gramazio, P.; Plazas Ávila, MDLO.; García-Fortea, E.; Ferrante, P.; Schmidt, M.... (2020). SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species. Plant Methods. 16(1):1-11. https://doi.org/10.1186/s13007-020-00652-yS111161Scheben A, Batley J, Edwards D. Genotyping-by-sequencing approaches to characterize crop genomes: choosing the right tool for the right application. Plant Biotechnol J. 2017;15:149–61.Jung H, Winefield C, Bombarely A, Prentis P, Waterhouse P. Tools and strategies for long-read sequencing and de novo assembly of plant genomes. Trends Plant Sci. 2019;24:700–24.Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE. 2011;6:e19379.Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, Lewis ZA, Selker EU, Cresko WA, Johnson EA. Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS ONE. 2008;3:e3376.Scaglione D, Pinosio S, Marroni F, Centa E, Fornasiero A, Magris G, Scalabrin S, Cattonaro F, Taylor G, Morgante M. Single primer enrichment technology as a tool for massive genotyping: a benchmark on black poplar and maize. Ann Bot. 2019;124:543–51.Barchi L, Acquadro A, Alonso D, Aprea G, Bassolino L, Demurtas O, Ferrante P, Gramazio P, Mini P, Portis E, Scaglione D, Toppino L, Vilanova S, Díez MJ, Rotino G, Lanteri S, Prohens J, Giuliano G. Single primer enrichment technology (SPET) for high-throughput genotyping in tomato and eggplant germplasm. Front Plant Sci. 2019;10:1005.Vaillancourt B, Buell CR. High molecular weight DNA isolation method from diverse plant species for use with Oxford Nanopore sequencing. bioRxiv. 2019;1:783159.Anderson CB, Franzmayr BK, Hong SW, Larking AC, van Stijn TC, Tan R, Moraga R, Faville M, Griffiths A. Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing. Plant Methods. 2018;14:75.Rana MM, Aycan M, Takamatsu T, Kaneko K, Mitsui T, Itoh K. Optimized nuclear pellet method for extracting next-generation sequencing quality genomic DNA from fresh leaf tissue. Methods Protoc. 2019;2:54.Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Focus. 1990;12:13–5.Healey A, Furtado A, Cooper T, Henry RJ. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods. 2014;10:21.Martínez-González CR, Ramírez-Mendoza R, Jiménez-Ramírez J, Gallegos-Vázquez C, Luna-Vega I. Improved method for genomic DNA extraction for Opuntia Mill. (Cactaceae). Plant Methods. 2017;13:82.Barbier FF, Chabikwa TG, Ahsan MU, Cook SE, Powell R, Tanurdzic M, Beveridge C. A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing. Plant Methods. 2019;15:62.Souza DC, Teixeira TA. A simple and effective method to obtain high DNA quality and quantity from Cerrado plant species. Mol Biol Rep. 2019;46:4611–5.Kovačević N. Magnetic beads based nucleic acid purification for molecular biology applications. Sample preparation techniques for soil, plant, and animal samples. In: Micic M, editor. Springer Protoc Handb. 2016;53–67.Martin SL, Parent JS, Laforest M, Page E, Kreiner JM, James T. Population genomic approaches for weed science. Plants. 2019;8:354.Zhou Y, Zhang Y, He W, Wang J, Peng F, Huang L, Zhao S, Deng W. Rapid regeneration and reuse of silica columns from PCR purification and gel extraction kits. Sci Rep. 2018;8:12870.Park HJ, Cho H, Jung HS, Cho BH, Lee MY. Development of a DNA isolation device using poly(3,4-dihydroxy-l-phenylalanine)-coated swab for on-site molecular diagnostics. Sci Rep. 2019;9:8144.Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28:495–503.Carter MJ, Milton ID. An inexpensive and simple method for DNA purifications on silica particles. Nucleic Acids Res. 1993;21:1044.Carvalho J, Puertas G, Gaspar J, Azinheiro S, Diéguez L, Garrido-Maestu A, Vázquez M, Barros-Velázquez J, Cardoso S, Padro M. Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device. Anal Chim Acta. 2018;1020:30–40.Branton D, Deamer D, Quick J, Loman NJ. DNA extraction strategies for nanopore sequencing. Nanopore Seq. World Sci. 2019;1:91–105.Cheng H, Zhang K, Libera J, De La Cruz M, Bedzyk M. Polynucleotide adsorption to negatively charged surfaces in divalent salt solutions. Biophys J. 2016;90:1164–74.Shi B, Shin Y, Hassanali A, Singer S. DNA Binding to the Silica Surface. J Phys Chem B. 2015;119:11030–40.Katevatis C, Fan A, Klapperich CM. Low concentration DNA extraction and recovery using a silica solid phase. PLoS ONE. 2017;12:e0176848.Green MR, Sambrook J. Isolation and quantification of DNA. Cold Spring Harb Protoc. 2018;2018:403–14.Toole K, Roffey P, Young E, Cho K, Shaw T, Smith M, Blagojevic N. Evaluation of commercial forensic DNA extraction kits for decontamination and extraction of DNA from biological samples contaminated with radionuclides. Forensic Sci Int. 2019;302:109867.Piskata Z, Servusova E, Babak V, Nesvadbova M, Borilova G. The quality of DNA isolated from processed food and feed via different extraction procedures. Molecules. 2019;24:1188.Xia Y, Chen F, Du Y, Liu C, Bu G, Xin Y, Boye L. A modified SDS-based DNA extraction method from raw soybean. Biosci Rep. 2019;39:2.Akkurt M. Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.). Genet Mol Res. 2012;11:2343–51.Marsal G, Baiges I, Canals JM, Zamora F, Fort F. A Fast, efficient method for extracting DNA from leaves, stems, and seeds of Vitis vinifera L. Am J Enol Vitic. 2011;62:376–81.Abdel-Latif A, Osman G. Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods. 2017;13:1.Huang J, Ge X, Sun M. Modified CTAB protocol using a silica matrix for isolation of plant genomic DNA. Biotechniques. 2000;28:432–4.Rogstad SH. Plant DNA extraction using silica. Plant Mol Biol Report. 2012;21:463.Li J-F, Li L, Sheen J. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology. Plant Methods. 2010;6:1.Li J-F, Sheen J. DNA purification from multiple sources in plant research with homemade silica resins. Humana Press. 2012;862:53–9.Vandeventer PE, Lin JS, Zwang TJ, Nadim A, Johal MS, Niemz A. Multiphasic DNA adsorption to silica surfaces under varying buffer, pH, and ionic strength conditions. J Phys Chem B. 2012;116:5661–70.Boesenberg-Smith KA, Pessarakli MM, Wolk DM. Assessment of DNA yield and purity: an overlooked detail of PCR troubleshooting. Clin Microbiol Newsl. 2012;34:1–6.Emaus MN, Clark KD, Hinners P, Anderson JL. Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification. Anal Bioanal Chem. 2018;410:4135–44.Dumschott K, Schmidt MHW, Chawla HS, Snowdon R, Usadel B. Oxford Nanopore sequencing: new opportunities for plant genomics? J Exp Bot. 2020;eraa263Knapp S, Sagona E, Carbonell AKZ, Chiarini F. A revision of the Solanum elaeagnifolium clade (Elaeagnifolium clade; subgenus Leptostemonum, Solanaceae). PhytoKeys. 2017;84:1–104.García-Fortea E, Gramazio P, Vilanova S, Fita A, Mangino G, Villanueva G, Arrones A, Knapp S, Prohens J, Plazas M. First successful backcrossing towards eggplant (Solanum melongena) of a New World species, the silverleaf nightshade (S. elaeagnifolium), and characterization of interspecific hybrids and backcrosses. Sci Hort. 2019;246:563–73.Ihaka R, Gentleman R. R: a language for data analysis and graphics. J Comput Graph Stat. 1996;5:3299–314.Wickham H. ggplot2: Elegant graphics for data analysis. New York: Springer-Verlag; 2016.Ponti G, Maccaferri M, Manfredini M, Kaleci S, Mandrioli M, Pellacani G, Ozben T, Depenni R, Bianchi G, Pirola G, Tomasi A. The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients. Clin Chim Acta. 2018;479:14–9.Lakshmi R, Baskar V, Ranga U. Extraction of superior-quality plasmid DNA by a combination of modified alkaline lysis and silica matrix. Anal Biochem. 1999;272:109–12.Taylor JI, Hurst CD, Davies MJ, Sachsinger N, Bruce IJ. Application of magnetite and silica–magnetite composites to the isolation of genomic DNA. J Chromatogr A. 2000;890:159–66.Prodělalová J, Rittich B, Španová A, Petrová K, Beneš MJ. Isolation of genomic DNA using magnetic cobalt ferrite and silica particles. J Chromatogr A. 2004;1056:43–8.Shan Z, Jiang Y, Guo M, Bennett JC, Li X, Tian H, Oakes K, Zhang, Zhou Y, Huang Q, Chen H. Promoting DNA loading on magnetic nanoparticles using a DNA condensation strategy. Colloids Surfaces B Biointerfaces. 2015;125:247–54.Greco M, Sáez C, Brown M, Bitonti M. A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga. PLoS ONE. 2014;9:e96470.Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitor – occurrence, properties and removal. J Appl Microbiol. 2012;113:1014–26.Demeke T, Adams RP. The effects of plant polysaccharides and buffer additives on PCR. Biotechniques. 1992;12:332–4.Asami DK, Hong YJ, Barrett DM, Mitchell AE. Comparison of the total phenolic and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn grown using conventional, organic, and sustainable agricultural practices. J Agric Food Chem. 2003;51:1237–41.Schmidt M, Vogel A, Denton A, Istace B, Wormit A, van de Geest H, Bolger M, Alseekh S, Maß J, Pfaff C, Schurr U, Chetelat R, Maumus F, Aury J, Koren S, Fernie A, Zamir D, Bolger A, Usadel B. De novo assembly of a new Solanum pennellii accession using nanopore sequencing. Plant cell. 2017;29:2336–48

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone