The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants

[EN] CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in Nicotiana tabacum. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free SPL edited T-1 plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the Solanum lycopersicum Mtb promoter or the Agrobacterium tumefaciens nopaline synthase promoter in transient expression in Nicotiana benthamiana. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering.This work had been funded by EU Horizon 2020 Project Newcotiana Grant 760331 and PID2019-108203RB-100 Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness. MV-V was recipient of aGeneralitat Valenciana and Fondo Social Europeo post-doctoral grant. JB-O and SS were recipients of FPI fellowships. CP was recipient of a Santiago Grisolia fellowship (Generalitat Valenciana).Vazquez-Vilar, M.; Garcia-Carpintero, V.; Selma, S.; Bernabé-Orts, JM.; Sánchez-Vicente, J.; Salazar-Sarasua, B.; Ressa, A.... (2021). The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants. Frontiers in Plant Science. 12:1-14. https://doi.org/10.3389/fpls.2021.6899371141

Obstetric outcomes of sars-cov-2 infection in asymptomatic pregnant women

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER)Around two percent of asymptomatic women in labor test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Spain. Families and care providers face childbirth with uncertainty. We determined if SARS-CoV-2 infection at delivery among asymptomatic mothers had different obstetric outcomes compared to negative patients. This was a multicenter prospective study based on universal antenatal screening for SARS-CoV-2 infection. A total of 42 hospitals tested women admitted for delivery using polymerase chain reaction, from March to May 2020. We included positive mothers and a sample of negative mothers asymptomatic throughout the antenatal period, with 6-week postpartum follow-up. Association between SARS-CoV-2 and obstetric outcomes was evaluated by multivariate logistic regression analyses. In total, 174 asymptomatic SARS-CoV-2 positive pregnancies were compared with 430 asymptomatic negative pregnancies. No differences were observed between both groups in key maternal and neonatal outcomes at delivery and follow-up, with the exception of prelabor rupture of membranes at term (adjusted odds ratio 1.88, 95% confidence interval 1.13-3.11; p = 0.015). Asymptomatic SARS-CoV-2 positive mothers have higher odds of prelabor rupture of membranes at term, without an increase in perinatal complications, compared to negative mothers. Pregnant women testing positive for SARS-CoV-2 at admission for delivery should be reassured by their healthcare workers in the absence of symptoms

Multi-ancestry genome-wide association study of asthma exacerbations

Altres ajuts: European Regional Development Fund "ERDF A way of making Europe"; Allergopharma-EAACI award 2021; SysPharmPedia grant from the ERACoSysMed 1st Joint Transnational Call from the European Union under the Horizon 2020; Sandler Family Foundation; American Asthma Foundation; RWJF Amos Medical Faculty Development Program; National Heart, Lung, and Blood Institute of the National Institutes of Health (R01HL117004, R01HL128439, R01HL135156, X01HL134589, R01HL141992, R01HL141845); National Institute of Health and Environmental Health Sciences (R01ES015794, R21ES24844); National Institute on Minority Health and Health Disparities (NIMHD) (P60MD006902, R01MD010443, R56MD013312); National Institute of General Medical Sciences (NIGMS) (RL5GM118984); Tobacco-Related Disease Research Program (24RT-0025, 27IR-0030); National Human Genome Research Institute (NHGRI) (U01HG009080); GlaxoSmithKline and Utrecht Institute for Pharmaceutical Sciences; Slovenian Research Agency (P3-0067); SysPharmPediA grant, co-financed by the Ministry of Education, Science and Sport Slovenia (MIZS) (C3330-16-500106); NHS Research Scotland; Wellcome Trust Biomedical Resource (099177/Z/12/Z); Genotyping National Centre (CeGEN) CeGen-PRB3-ISCIII (AC15/00015); UK Medical Research Council and Wellcome (102215/2/13/2); University of Bristol; Swedish Heart-Lung Foundation, Swedish Research Council; Region Stockholm (ALF project and database maintenance); NHS Chair of Pharmacogenetics via the UK Department of Health; Innovative Medicines Initiative (IMI) (115010); European Federation of Pharmaceutical Industries and Associations (EFPIA); Spanish National Cancer Research Centre; Fundación Canaria Instituto de Investigación Sanitaria de Canarias (PIFIISC19/17); Erasmus Medical Center; Erasmus University Rotterdam; Netherlands Organization for the Health Research and Development (ZonMw); the Research Institute for Diseases in the Elderly (RIDE); Ministry of Education, Culture and Science; Ministry for Health, Welfare and Sports; European Commission (DG XII); Municipality of Rotterdam; German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF); U.S. National Institutes of Health (HL07966); European Social Fund "ESF Investing in your future"; Ministerio de Ciencia, Innovación y Universidades; Universidad de La Laguna (ULL); European Academy of Allergy and Clinical Immunology (EAACI); European Respiratory Society (ERS) (LTRF202101-00861); Ministry of Education, Science and Sport of the Republic of Slovenia (C3330-19-252012); Singapore Ministry of Education Academic Research Fund; Singapore Immunology Network (SIgN); National Medical Research Council (NMRC Singapore); Biomedical Research Council (BMRC Singapore); Agency for Science Technology and Research (A*STAR Singapore, N-154-000-038-001, R-154-000-191-112, R-154-000-404-112, R-154-000-553-112, R-154-000-565-112, R-154-000-630-112, R-154-000-A08-592, R-154-000-A27-597, R-154-000-A91-592, R-154-000-A95-592, R-154-000-B99-114, BMRC/01/1/21/18/077, BMRC/04/1/21/19/315, SIgN-06-006, SIgN-08-020, NMRC/1150/2008, H17/01/a0/008); Sime Darby Technology Centre; First Resources Ltd; Genting Plantation; Olam International; U.S. National Institutes of Health (HL138098).Background: Asthma exacerbations are a serious public health concern due to high healthcare resource utilization, work/school productivity loss, impact on quality of life, and risk of mortality. The genetic basis of asthma exacerbations has been studied in several populations, but no prior study has performed a multi-ancestry meta-analysis of genome-wide association studies (meta-GWAS) for this trait. We aimed to identify common genetic loci associated with asthma exacerbations across diverse populations and to assess their functional role in regulating DNA methylation and gene expression. Methods: A meta-GWAS of asthma exacerbations in 4989 Europeans, 2181 Hispanics/Latinos, 1250 Singaporean Chinese, and 972 African Americans analyzed 9.6 million genetic variants. Suggestively associated variants (p ≤ 5 × 10) were assessed for replication in 36,477 European and 1078 non-European asthma patients. Functional effects on DNA methylation were assessed in 595 Hispanic/Latino and African American asthma patients and in publicly available databases. The effect on gene expression was evaluated in silico. Results: One hundred and twenty-six independent variants were suggestively associated with asthma exacerbations in the discovery phase. Two variants independently replicated: rs12091010 located at vascular cell adhesion molecule-1/exostosin like glycosyltransferase-2 (VCAM1/EXTL2) (discovery: odds ratio (OR) = 0.82, p = 9.05 × 10 and replication: OR = 0.89, p = 5.35 × 10) and rs943126 from pantothenate kinase 1 (PANK1) (discovery: OR = 0.85, p = 3.10 × 10 and replication: OR = 0.89, p = 1.30 × 10). Both variants regulate gene expression of genes where they locate and DNA methylation levels of nearby genes in whole blood. Conclusions: This multi-ancestry study revealed novel suggestive regulatory loci for asthma exacerbations located in genomic regions participating in inflammation and host defense

Letter, Albacete, Spain, to Victoriano Manteiga

Last page of letter in Spanish that was probably written to Victoriano Manteiga, the editor of the Tampa newspaper La Gaceta. This page is the only one extant. The text reads: ... I am bound to the cause of the Democratic Spanish Republic and only Spain can give me orders. I live to serve her, and I am happy serving her and knowing that as long as the Republic needs me, I\u27ll be at her service until death, now and forever. Handwritten on stationery with letterhead Comisariado General de Guerra, Ministerio de Defensa Nacional, Casa del Ejercito Popular.https://digitalcommons.usf.edu/span_civil_war/1036/thumbnail.jp

Orius laevigatus strengthens its role as a biological control agent by inducing plant defenses

[EN] Orius laevigatus is a generalist predator that is widely used in augmentative strategies against the key pest of sweet pepper, Frankliniella occidentalis. Despite being a zoophytophagous predator, the phytophagous behavior of O. laevigatus has not been previously explored in depth nor has the impact of phytophagy on plant physiology. Here, the hierarchical significance of O. laevigatus feeding on sweet pepper is compared with other behaviors. O. laevigatus spends the majority of its time (38%) feeding on apical meristems and apical fresh leaves, which were also preferred residence locations. Here, the phytophagous feeding behavior of O. laevigatus on sweet pepper is shown to trigger defensive responses in the plant. These O. laevigatus plant-induced defenses are then shown to contribute to the repellence or attraction of pests or natural enemies, respectively. Specifically, O. laevigatus-punctured sweet pepper plants induce repellency for the whitefly Bemisia tabaci and the thrips species F. occidentalis. In contrast, the whitefly parasitoid Encarsia formosa was significantly attracted to O. laevigatus-punctured plants. The plant responses to O. laevigatus punctures include the release of an altered blend of volatiles and activation of the jasmonic acid and salicylic acid signalling pathways. These results highlight an interesting facet to the biology of O. laevigatus, in which the ability of the predator to induce defensive responses in sweet pepper plants may serve to improve the biological control of both thrips and whiteflies.The research leading to these results was partially funded by the Spanish Ministry of Economy and Competitiveness (AGL2014-55616-C3) and the Conselleria d'Agricultura, Pesca i Alimentacio de la Generalitat Valenciana. The authors thank Dr. Javier Calvo (KOPPERT BS, Spain) for supplying the insects, Dr. Pablo Bielza (Universidad Politecnica de Cartagena, Spain) for thrips colonies, Dimitrios Afentoulis, Zoi Thanou and Konstantinos Birmpilis (Erasmus + from Agricultural University of Athens) for providing support in the behavior experiments and Alice Mockford (University of Worcester) and two anonymous reviewers for their constructive review of this manuscript. MP-H was the recipient of a postdoctoral fellowship from the MEC (Juan de la Cierva program-FPDI-2013-17968) and SB was the recipient of a grant from Russell IPM Ltd.Bouagga, S.; Urbaneja Garcia, A.; Rambla Nebot, JL.; Granell Richart, A.; Pérez-Hedo, M. (2018). Orius laevigatus strengthens its role as a biological control agent by inducing plant defenses. Journal of Pest Science. 91(1):55-64. https://doi.org/10.1007/s10340-017-0886-45564911Ardanuy A, Albajes R, Turlings TC (2016) Innate and learned prey-searching behavior in a generalist predator. J Chem Ecol 42:497–507Armer CA, Wiedenmann RN, Bush DR (1998) Plant feeding site selection on soybean by the facultatively phytophagous predator Orius insidiosus. Entomol Exp Appl 86:109–118Arnó J, Roig J, Riudavets J (2008) Evaluation of Orius majusculus and O. laevigatus as predators of Bemisia tabaci and estimation of their prey preference. Biol Control 44:1–6Calvo J, Bolckmans K, Belda JE (2009) Development of a biological control-based integrated pest management method for Bemisia tabaci for protected sweet pepper crops. Entomol Exp Appl 133:9–18Chambers R, Long S, Helyer NL (1993) Effectiveness of Orius laevigatus (Hem, Anthocoridae) for the control of Frankliniella occidentalis on cucumber and pepper in the UK. Biocontrol Sci Technol 3:295–307Cocuzza G, De Clercq P, Veire M, de Cock A, Degheele D, Vacante V (1997) Reproduction of Orius laevigatus and Orius albidipennis on pollen and Ephestia kuehniella eggs. Entomol Exp Appl 82:101–104De Puysseleyr V, Hofte M, De Clercq P (2011) Ovipositing Orius laevigatus increase tomato resistance against Frankliniella occidentalis feeding by inducing the wound response. Arth-Plant Int 5:71–80Evans E, Dixon AFG (1986) Cues for oviposition by ladybird beetles (Coccinellidae): response to aphids. J Anim Ecol 55:1027–1034Frescata C, Mexia A (1996) Biological control of thrips (Thysanoptera) by Orius laevigatus (Heteroptera: Anthocoridae) in organically grown strawberries. Biol Agric Hortic 13:141–148Fritshe ME, Tamó M (2000) Influence of thrips prey species on the life-history and behaviour of Orius albidipennis. Entomol Exp Appl 96:111–118Gerling D, Alomar O, Arnó J (2001) Biological control of Bemisia tabaci using predators and parasitoids. Crop Prot 20:779–799Gill R, Gupta K, Taggar GK, Taggar MS (2010) Role of oxidative enzymes in plant defenses against herbivory. Acta Phytopathol Entomol Hung 45:277–290Hemptinne J, Dixon AFG, Coffin J (1992) Attack strategy of ladybird beetles (Coccinellidae): factors shaping their numercial response. Oecol 90:238–245Hernández LM, Stonedahl GM (1999) A review of the economically important species of the genus Orius (Heteroptera: anthocoridae) in East Africa. J Nat Hist 33:543–568Jonathan GL, Fergen J (2006) The oviposition behavior of the predator Orius insidiosus: acceptability and preference for different plants. Biocontrol 51:217–227Kessler A, Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science 291:2141–2144Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53:299–328Kigathi RN, Unsicker SB, Reichelt M, Kesselmeier J, Gershenzon J, Weisser WW (2009) Emission of volatile organic compounds after herbivory from Trifolium pratense (L.) Under laboratory and field conditions. J Chem Ecol 35:1335–1348Lee DH, Nyrop JP, Sanderson JP (2014) Non-consumptive effects of the predatory beetle Delphastus catalinae (Coleoptera: coccinellidae) on habitat use patterns of adult whitefly Bemisia argentifolii (Hemiptera: Aleyrodidae). Appl Entomol Zool 49:599–606Lundgren JG, Fergen JK, Riedell WE (2008) Influence of plant anatomy on oviposition and reproductive success of the omnivorous bug Orius insidiosus. Anim Behav 75:1495–1502McCormick AC, Irmisch S, Reiecke A, Boeckler AG, Veit D, Reichelt M, Köllner TG, Hansson BS, Gershenzon J, Unsicker SB (2014) Herbivore-induced volatile emission in black poplar: regulation and role in attracting herbivore enemies. Plant Cell Environ 37:1909–1923Nakashima Y, Hirose Y (2002) Sex differences in foraging behaviour and oviposition site preference in an insect predator, Orius sauteri. Entomol Exp Appl 106:79–86Naselli M, Urbaneja A, Siscaro G, Jaques JA, Zappalà L, Flors V, Pérez-Hedo M (2016) Stage-related defense response induction in tomato plants by Nesidiocoris tenuis. Int J Mol Sci 17:1210Ninkovic V, Feng Y, Olsson U, Pettersson J (2013) Ladybird footprints induce aphid avoidance behavior. Biol Control 65:63–71Nomikou M, Janssen A, Sabelis MW (2003) Herbivore host plant selection: whitefly learns to avoid host plants that harbor predators of her offspring. Oecologia 136:484–488Pappas M, Steppuhn A, Geuss D, Topalidou N, Zografou A, Sabelis MW, Broufas GD (2015) Beyond Predation: the Zoophytophagous predator Macrolophus pygmaeus induces tomato resistance against spider mites. PLoS ONE 10(5):e0127251Pappas M, Steppuhn A, Broufas GD (2016) The role of phytophagy by predators in shaping plant interactions with their pests. Commun Integr Biol 9(2):e1145320Pérez-Hedo M, Urbaneja A (2015) Prospects for predatory mirid bugs as biocontrol agents of aphids in sweet peppers. J Pest Sci 88:65–73Pérez-Hedo M, Urbaneja-Bernat P, Jaques JA, Flors V, Urbaneja A (2015a) Defensive plant responses induced by Nesidiocoris tenuis (Hemiptera: miridae) on tomato plants. J Pest Sci 88:543–554Pérez-Hedo M, Bouagga S, Jaques JA, Flors V, Urbaneja A (2015b) Tomato plant responses to feeding behavior of three zoophytophagous predators (Hemiptera: miridae). Biol Control 86:46–51Ponzio C, Gols R, Weldegergis BT, Dicke M (2014) Caterpillar-induced plant volatiles remain a reliable signal for foraging wasps during dual attack with a plant pathogen or non-host insect herbivore. Plant Cell Environ 37:1924–1935Riudavets J (1995) Predators of Frankliniella occidentalis (Perg.) and Thrips tabaci Lind: a review. Wag Ag Un P 95:43–87Sanchez JA, Lacasa A (2002) Modelling population dynamics of Orius laevigatus and O. albidipennis (Hemiptera: anthocoridae) to optimize their use as biological control agents of Frankliniella occidentalis (Thysanoptera: Thripidae). Bull Entomol Res 92:77–88Sanchez JA, Alcazar A, Lacasa A, Llamas A, Bielza P (2000) Integrated pest management strategies in sweet pepper plastic houses in the Southeast of Spain. IOBC/WPRS Bull 23:21–27Sendoya F, Freitas VL, Oliveira PS (2009) Egg-laying butterflies distinguish predaceous ants by sight. Am Nat 174:134–140Shivaji R, Camas A, Ankala A, Engelberth J, Tumlinson JH, Williams WP, Wilkinson JR, Luthe DS (2010) Plants on constant alert: elevated levels of jasmonic acid and jasmonate-induced transcripts in caterpillar resistant maize. 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Arch Int Physiol Biochim 44:9–13

Rationale and study design for an individualized perioperative open lung ventilatory strategy (iPROVE) : study protocol for a randomized controlled trial

Background: Postoperative pulmonary and non-pulmonary complications are common problems that increase morbidity and mortality in surgical patients, even though the incidence has decreased with the increased use of protective lung ventilation strategies. Previous trials have focused on standard strategies in the intraoperative or postoperative period, but without personalizing these strategies to suit the needs of each individual patient and without considering both these periods as a global perioperative lung-protective approach. The trial presented here aims at comparing postoperative complications when using an individualized ventilatory management strategy in the intraoperative and immediate postoperative periods with those when using a standard protective ventilation strategy in patients scheduled for major abdominal surgery. Methods: This is a comparative, prospective, multicenter, randomized, and controlled, four-arm trial that will include 1012 patients with an intermediate or high risk for postoperative pulmonary complications. The patients will be divided into four groups: (1) individualized perioperative group: intra-and postoperative individualized strategy; (2) intraoperative individualized strategy + postoperative continuous positive airway pressure (CPAP); (3) intraoperative standard ventilation + postoperative CPAP; (4) intra-and postoperative standard strategy (conventional strategy). The primary outcome is a composite analysis of postoperative complications. Discussion: The Individualized Perioperative Open-lung Ventilatory Strategy (iPROVE) is the first multicenter, randomized, and controlled trial to investigate whether an individualized perioperative approach prevents postoperative pulmonary complications

Prognostic utility of serum free light chain ratios and heavy-light chain ratios in multiple myeloma in three PETHEMA/GEM phase III clinical trials.

We investigated the prognostic impact and clinical utility of serum free light chains (sFLC) and serum heavy-light chains (sHLC) in patients with multiple myeloma treated according to the GEM2005MENOS65, GEM2005MAS65, and GEM2010MAS65 PETHEMA/GEM phase III clinical trials. Serum samples collected at diagnosis were retrospectively analyzed for sFLC (n = 623) and sHLC (n = 183). After induction or autologous transplantation, 309 and 89 samples respectively were available for sFLC and sHLC assays. At diagnosis, a highly abnormal (HA) sFLC ratio (sFLCr) (32) was not associated with higher risk of progression. After therapy, persistence of involved-sFLC levels >100 mg/L implied worse survival (overall survival [OS], P = 0.03; progression-free survival [PFS], P = 0.007). Among patients that achieved a complete response, sFLCr normalization did not necessarily indicate a higher quality response. We conducted sHLC investigations for IgG and IgA MM. Absolute sHLC values were correlated with monoclonal protein levels measured with serum protein electrophoresis. At diagnosis, HA-sHLCrs (73) showed a higher risk of progression (P = 0.006). Additionally, involved-sHLC levels >5 g/L after treatment were associated with shorter survival (OS, P = 0.001; PFS, P = 0.018). The HA-sHLCr could have prognostic value at diagnosis; absolute values of involved-sFLC >100 mg/L and involved-sHLC >5 g/L could have prognostic value after treatment

Multi-ancestry genome-wide association study of asthma exacerbations

BACKGROUND: Asthma exacerbations are a serious public health concern due to high healthcare resource utilization, work/school productivity loss, impact on quality of life, and risk of mortality. The genetic basis of asthma exacerbations has been studied in several populations, but no prior study has performed a multi-ancestry meta-analysis of genome-wide association studies (meta-GWAS) for this trait. We aimed to identify common genetic loci associated with asthma exacerbations across diverse populations and to assess their functional role in regulating DNA methylation and gene expression. METHODS: A meta-GWAS of asthma exacerbations in 4989 Europeans, 2181 Hispanics/Latinos, 1250 Singaporean Chinese, and 972 African Americans analyzed 9.6 million genetic variants. Suggestively associated variants (p ≤ 5 × 10-5 ) were assessed for replication in 36,477 European and 1078 non-European asthma patients. Functional effects on DNA methylation were assessed in 595 Hispanic/Latino and African American asthma patients and in publicly available databases. The effect on gene expression was evaluated in silico. RESULTS: One hundred and twenty-six independent variants were suggestively associated with asthma exacerbations in the discovery phase. Two variants independently replicated: rs12091010 located at vascular cell adhesion molecule-1/exostosin like glycosyltransferase-2 (VCAM1/EXTL2) (discovery: odds ratio (ORT allele ) = 0.82, p = 9.05 × 10-6 and replication: ORT allele  = 0.89, p = 5.35 × 10-3 ) and rs943126 from pantothenate kinase 1 (PANK1) (discovery: ORC allele  = 0.85, p = 3.10 × 10-5 and replication: ORC allele  = 0.89, p = 1.30 × 10-2 ). Both variants regulate gene expression of genes where they locate and DNA methylation levels of nearby genes in whole blood. CONCLUSIONS: This multi-ancestry study revealed novel suggestive regulatory loci for asthma exacerbations located in genomic regions participating in inflammation and host defense