Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples

Biorefining of wheat straw:accounting for the distribution of mineral elements in pretreated biomass by an extended pretreatment–severity equation

BACKGROUND: Mineral elements present in lignocellulosic biomass feedstocks may accumulate in biorefinery process streams and cause technological problems, or alternatively can be reaped for value addition. A better understanding of the distribution of minerals in biomass in response to pretreatment factors is therefore important in relation to development of new biorefinery processes. The objective of the present study was to examine the levels of mineral elements in pretreated wheat straw in response to systematic variations in the hydrothermal pretreatment parameters (pH, temperature, and treatment time), and to assess whether it is possible to model mineral levels in the pretreated fiber fraction. RESULTS: Principal component analysis of the wheat straw biomass constituents, including mineral elements, showed that the recovered levels of wheat straw constituents after different hydrothermal pretreatments could be divided into two groups: 1) Phosphorus, magnesium, potassium, manganese, zinc, and calcium correlated with xylose and arabinose (that is, hemicellulose), and levels of these constituents present in the fiber fraction after pretreatment varied depending on the pretreatment-severity; and 2) Silicon, iron, copper, aluminum correlated with lignin and cellulose levels, but the levels of these constituents showed no severity-dependent trends. For the first group, an expanded pretreatment-severity equation, containing a specific factor for each constituent, accounting for variability due to pretreatment pH, was developed. Using this equation, the mineral levels could be predicted with R(2) > 0.75; for some with R(2) up to 0.96. CONCLUSION: Pretreatment conditions, especially pH, significantly influenced the levels of phosphorus, magnesium, potassium, manganese, zinc, and calcium in the resulting fiber fractions. A new expanded pretreatment-severity equation is proposed to model and predict mineral composition in pretreated wheat straw biomass

Global, regional, and national burden of chronic kidney disease, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

© 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access Article under the CC BY 4.0 license. Background: Health system planning requires careful assessment of chronic kidney disease (CKD) epidemiology, but data for morbidity and mortality of this disease are scarce or non-existent in many countries. We estimated the global, regional, and national burden of CKD, as well as the burden of cardiovascular disease and gout attributable to impaired kidney function, for the Global Burden of Diseases, Injuries, and Risk Factors Study 2017. We use the term CKD to refer to the morbidity and mortality that can be directly attributed to all stages of CKD, and we use the term impaired kidney function to refer to the additional risk of CKD from cardiovascular disease and gout. Methods: The main data sources we used were published literature, vital registration systems, end-stage kidney disease registries, and household surveys. Estimates of CKD burden were produced using a Cause of Death Ensemble model and a Bayesian meta-regression analytical tool, and included incidence, prevalence, years lived with disability, mortality, years of life lost, and disability-adjusted life-years (DALYs). A comparative risk assessment approach was used to estimate the proportion of cardiovascular diseases and gout burden attributable to impaired kidney function. Findings: Globally, in 2017, 1·2 million (95% uncertainty interval [UI] 1·2 to 1·3) people died from CKD. The global all-age mortality rate from CKD increased 41·5% (95% UI 35·2 to 46·5) between 1990 and 2017, although there was no significant change in the age-standardised mortality rate (2·8%, −1·5 to 6·3). In 2017, 697·5 million (95% UI 649·2 to 752·0) cases of all-stage CKD were recorded, for a global prevalence of 9·1% (8·5 to 9·8). The global all-age prevalence of CKD increased 29·3% (95% UI 26·4 to 32·6) since 1990, whereas the age-standardised prevalence remained stable (1·2%, −1·1 to 3·5). CKD resulted in 35·8 million (95% UI 33·7 to 38·0) DALYs in 2017, with diabetic nephropathy accounting for almost a third of DALYs. Most of the burden of CKD was concentrated in the three lowest quintiles of Socio-demographic Index (SDI). In several regions, particularly Oceania, sub-Saharan Africa, and Latin America, the burden of CKD was much higher than expected for the level of development, whereas the disease burden in western, eastern, and central sub-Saharan Africa, east Asia, south Asia, central and eastern Europe, Australasia, and western Europe was lower than expected. 1·4 million (95% UI 1·2 to 1·6) cardiovascular disease-related deaths and 25·3 million (22·2 to 28·9) cardiovascular disease DALYs were attributable to impaired kidney function. Interpretation: Kidney disease has a major effect on global health, both as a direct cause of global morbidity and mortality and as an important risk factor for cardiovascular disease. CKD is largely preventable and treatable and deserves greater attention in global health policy decision making, particularly in locations with low and middle SDI. Funding: Bill & Melinda Gates Foundation

The global burden of childhood and adolescent cancer in 2017: an analysis of the Global Burden of Disease Study 2017

© 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license Background: Accurate childhood cancer burden data are crucial for resource planning and health policy prioritisation. Model-based estimates are necessary because cancer surveillance data are scarce or non-existent in many countries. Although global incidence and mortality estimates are available, there are no previous analyses of the global burden of childhood cancer represented in disability-adjusted life-years (DALYs). Methods: Using the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 methodology, childhood (ages 0–19 years) cancer mortality was estimated by use of vital registration system data, verbal autopsy data, and population-based cancer registry incidence data, which were transformed to mortality estimates through modelled mortality-to-incidence ratios (MIRs). Childhood cancer incidence was estimated using the mortality estimates and corresponding MIRs. Prevalence estimates were calculated by using MIR to model survival and multiplied by disability weights to obtain years lived with disability (YLDs). Years of life lost (YLLs) were calculated by multiplying age-specific cancer deaths by the difference between the age of death and a reference life expectancy. DALYs were calculated as the sum of YLLs and YLDs. Final point estimates are reported with 95% uncertainty intervals. Findings: Globally, in 2017, there were 11·5 million (95% uncertainty interval 10·6–12·3) DALYs due to childhood cancer, 97·3% (97·3–97·3) of which were attributable to YLLs and 2·7% (2·7–2·7) of which were attributable to YLDs. Childhood cancer was the sixth leading cause of total cancer burden globally and the ninth leading cause of childhood disease burden globally. 82·2% (82·1–82·2) of global childhood cancer DALYs occurred in low, low-middle, or middle Socio-demographic Index locations, whereas 50·3% (50·3–50·3) of adult cancer DALYs occurred in these same locations. Cancers that are uncategorised in the current GBD framework comprised 26·5% (26·5–26·5) of global childhood cancer DALYs. Interpretation: The GBD 2017 results call attention to the substantial burden of childhood cancer globally, which disproportionately affects populations in resource-limited settings. The use of DALY-based estimates is crucial in demonstrating that childhood cancer burden represents an important global cancer and child health concern. Funding: Bill & Melinda Gates Foundation, American Lebanese Syrian Associated Charities (ALSAC), and St. Baldrick's Foundation

In vivo efficacy of artemether-lumefantrine against uncomplicated Plasmodium falciparum malaria in Central Ethiopia

<p>Abstract</p> <p>Background</p> <p><it>In vivo </it>efficacy assessments of the first-line treatments for <it>Plasmodium falciparum </it>malaria are essential for ensuring effective case management. In Ethiopia, artemether-lumefantrine (AL) has been the first-line treatment for uncomplicated <it>P. falciparum </it>malaria since 2004.</p> <p>Methods</p> <p>Between October and November 2009, we conducted a 42-day, single arm, open label study of AL for <it>P. falciparum </it>in individuals >6 months of age at two sites in Oromia State, Ethiopia. Eligible patients who had documented <it>P. falciparum </it>mono-infection were enrolled and followed according to the standard 2009 World Health Organization <it>in vivo </it>drug efficacy monitoring protocol. The primary and secondary endpoints were PCR uncorrected and corrected cure rates, as measured by adequate clinical and parasitological response on days 28 and 42, respectively.</p> <p>Results</p> <p>Of 4426 patients tested, 120 with confirmed falciparum malaria were enrolled and treated with AL. Follow-up was completed for 112 patients at day 28 and 104 patients at day 42. There was one late parasitological failure, which was classified as undetermined after genotyping. Uncorrected cure rates at both day 28 and 42 for the per protocol analysis were 99.1% (95% CI 95.1-100.0); corrected cure rates at both day 28 and 42 were 100.0%. Uncorrected cure rates at day 28 and 42 for the intention to treat analysis were 93.3% (95% CI 87.2-97.1) and 86.6% (95% CI 79.1-92.1), respectively, while the corrected cure rates at day 28 and 42 were 94.1% (95% CI 88.2-97.6) and 87.3% (95% CI 79.9-92.7), respectively. Using survival analysis, the unadjusted cure rate was 99.1% and 100.0% adjusted by genotyping for day 28 and 42, respectively. Eight <it>P. falciparum </it>patients (6.7%) presented with <it>Plasmodium vivax </it>infection during follow-up and were excluded from the per protocol analysis. Only one patient had persistent parasitaemia at day 3. No serious adverse events were reported, with cough and nausea/vomiting being the most common adverse events.</p> <p>Conclusions</p> <p>AL remains a highly effective and well-tolerated treatment for uncomplicated falciparum malaria in the study setting after several years of universal access to AL. A high rate of parasitaemia with <it>P. vivax </it>possibly from relapse or new infection was observed.</p> <p>Trial Registration</p> <p><a href="http://www.clinicaltrials.gov/ct2/show/NCT01052584">NCT01052584</a></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

Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts

Background Consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which contains xylose as a major component in concentrations that can reach up to 40% of the total biomass in hardwoods and agricultural residues. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiaethe most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. Results These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 g/g of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. Conclusions These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.This work has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)-LISBOA-01-0145-FEDER-022059, supported by Operational Programme for Competitiveness and Internationalization (PORTUGAL2020), by Lisbon Portugal Regional Operational Programme (Lisboa 2020) and by North Portugal Regional Operational Programme (Norte 2020) under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and has been supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020, the “Contrato-Programa” UIDB/04050/2020, the MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to Joana T. Cunha) and through Project FatVal (POCI-01-0145-FEDER-032506) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Past, present, and future of global health financing: a review of development assistance, government, out-of-pocket, and other private spending on health for 195 countries, 1995–2050

© 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license Background: Comprehensive and comparable estimates of health spending in each country are a key input for health policy and planning, and are necessary to support the achievement of national and international health goals. Previous studies have tracked past and projected future health spending until 2040 and shown that, with economic development, countries tend to spend more on health per capita, with a decreasing share of spending from development assistance and out-of-pocket sources. We aimed to characterise the past, present, and predicted future of global health spending, with an emphasis on equity in spending across countries. Methods: We estimated domestic health spending for 195 countries and territories from 1995 to 2016, split into three categories—government, out-of-pocket, and prepaid private health spending—and estimated development assistance for health (DAH) from 1990 to 2018. We estimated future scenarios of health spending using an ensemble of linear mixed-effects models with time series specifications to project domestic health spending from 2017 through 2050 and DAH from 2019 through 2050. Data were extracted from a broad set of sources tracking health spending and revenue, and were standardised and converted to inflation-adjusted 2018 US dollars. Incomplete or low-quality data were modelled and uncertainty was estimated, leading to a complete data series of total, government, prepaid private, and out-of-pocket health spending, and DAH. Estimates are reported in 2018 US dollars, 2018 purchasing-power parity-adjusted dollars, and as a percentage of gross domestic product. We used demographic decomposition methods to assess a set of factors associated with changes in government health spending between 1995 and 2016 and to examine evidence to support the theory of the health financing transition. We projected two alternative future scenarios based on higher government health spending to assess the potential ability of governments to generate more resources for health. Findings: Between 1995 and 2016, health spending grew at a rate of 4·00% (95% uncertainty interval 3·89–4·12) annually, although it grew slower in per capita terms (2·72% [2·61–2·84]) and increased by less than $1 per capita over this period in 22 of 195 countries. The highest annual growth rates in per capita health spending were observed in upper-middle-income countries (5·55$8·0 trillion (7·8–8·1) in 2016 (comprising 8·6% [8·4–8·7] of the global economy and $10·3 trillion [10·1–10·6] in purchasing-power parity-adjusted dollars), with a per capita spending of US$5252 (5184–5319) in high-income countries, $491 (461–524) in upper-middle-income countries,$81 (74–89) in lower-middle-income countries, and $40 (38–43) in low-income countries. In 2016, 0·4$9·5 billion, 24·3% of total DAH), although spending on other infectious diseases (excluding tuberculosis and malaria) grew fastest from 2010 to 2018 (6·27% per year). The leading sources of DAH were the USA and private philanthropy (excluding corporate donations and the Bill & Melinda Gates Foundation). For the first time, we included estimates of China's contribution to DAH ($644·7 million in 2018). Globally, health spending is projected to increase to$15·0 trillion (14·0–16·0) by 2050 (reaching 9·4% [7·6–11·3] of the global economy and $21·3 trillion [19·8–23·1] in purchasing-power parity-adjusted dollars), but at a lower growth rate of 1·84% (1·68–2·02) annually, and with continuing disparities in spending between countries. In 2050, we estimate that 0·6% (0·6–0·7) of health spending will occur in currently low-income countries, despite these countries comprising an estimated 15·7% of the global population by 2050. The ratio between per capita health spending in high-income and low-income countries was 130·2 (122·9–136·9) in 2016 and is projected to remain at similar levels in 2050 (125·9 [113·7–138·1]). The decomposition analysis identified governments’ increased prioritisation of the health sector and economic development as the strongest factors associated with increases in government health spending globally. Future government health spending scenarios suggest that, with greater prioritisation of the health sector and increased government spending, health spending per capita could more than double, with greater impacts in countries that currently have the lowest levels of government health spending. Interpretation: Financing for global health has increased steadily over the past two decades and is projected to continue increasing in the future, although at a slower pace of growth and with persistent disparities in per-capita health spending between countries. Out-of-pocket spending is projected to remain substantial outside of high-income countries. Many low-income countries are expected to remain dependent on development assistance, although with greater government spending, larger investments in health are feasible. In the absence of sustained new investments in health, increasing efficiency in health spending is essential to meet global health targets. Funding: Bill & Melinda Gates Foundation