Efficient evaluation of a gene containment system for poplar through early flowering induction

[EN] Key message The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.Open Access funding provided by Projekt DEAL. This work was funded with a scholarship by the Deutscher Akademischer Austauschdienst (DAAD). We thank S. Bein, D. Ebbinghaus, and A. Worm for helpful technical assistance in the laboratory, and the greenhouse staff (M. Hunger, G. Wiemann, R. Ebbinghaus, and M. Spauszus) for plant cultivation.Briones, MV.; Hoenicka, H.; Cañas Clemente, LA.; Beltran Porter, JP.; Hanelt, D.; Sharry, S.; Fladung, M. (2020). Efficient evaluation of a gene containment system for poplar through early flowering induction. Plant Cell Reports. 39(5):577-587. https://doi.org/10.1007/s00299-020-02515-1S577587395Al-Ahmad H (2018) Biotechnology for bioenergy dedicated trees: meeting future energy demands. Z Naturforsch 73:15–32. https://doi.org/10.1515/znc-2016-0185Beltrán JP, Roque E, Medina M, Madueño F, Gómez MD, Cañas LA (2007) Androesterilidad inducida mediante ingeniería genética. Fundamentos y aplicaciones biotecnológicas. An R Acad Nac Farm 73:1237–1264Böhlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O (2006) CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312:1040–1043Brunner AM, Varkonyi-Gasic E, Jones RC (2017) Phase change and phenology in trees. In: Groover A, Cronk Q (eds) Comparative and evolutionary genomics of Angiosperm trees. Springer, Cham, pp 227–274. https://doi.org/10.1007/7397_2016_30Chang YY, Wang AY, Cronan JE (1993) Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase. An enzyme of the acetohydroxy acid synthase-pyruvate oxidase family. J Biol Chem 268:3911–3919Dumolin S, Demesure B, Petit RJ (1995) Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method. Theor Appl Genet 91:1253–1256Elorriaga E, Meilan R, Ma C, Skinner JS, Etherington E, Brunner AM, Strauss SH (2014) A tapetal ablation transgene induces stable male sterility and slows field growth in Populus. Tree Gen Gen 10:1583–1593Elorriaga E, Klocko AL, Ma C, Strauss SH (2018) Variation in mutation spectra among CRISPR/Cas9 mutagenized poplars. Front Plant Sci 9:594. https://doi.org/10.3389/fpls.2018.00594Fladung M, Ahuja M (1995) Sandwich method for non-radioactive hybridisations. Biotechniques 18:800–802Fladung M, Muhs HJ, Ahuja MR (1996) Morphological changes observed in transgenic Populus carrying the rolC gene from Agrobacterium rhizogenes. Silv Genet 45:349–354Fladung M, Kumar S, Ahuja MR (1997) Genetic transformation of Populus genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis. Transgenic Res 6:111–121Fritsche S, Klocko AL, Boron A, Brunner AM, Thorlby G (2018) Strategies for engineering reproductive sterility in plantation forests. Front Plant Sci 9:1671. https://doi.org/10.3389/fpls.2018.01671García-Sogo B, Pineda B, Castelblanque L, Antón T, Medina M, Roque E, Torresi C, Beltrán JP, Moreno V, Cañas LA (2010) Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation. Plant Cell Rep 29:61–77García-Sogo B, Pineda B, Roque E, Atarés A, Antón T, Beltrán JP, Moreno V, Cañas LA (2012) Production of engineered long-life and male sterile Pelargonium plants. BMC Plant Biol 12:156–162Gardner N, Felsheim R, Smith AG (2009) Production of male- and female-sterile plants through reproductive tissue ablation. J Plant Physiol 166(8):871–881Gómez MD, Beltrán JP, Cañas LA (2004) The pea END1 promoter drives anther-specific gene expression in different plant species. Planta 219:967–981Hartley RW (1988) Barnase and barstar expression of its cloned inhibitor permits expression of a cloned ribonuclease. J Mol Biol 202:913–915Hoenicka H, Fladung M (2006) Biosafety in Populus spp. and other forest trees: from non-native species to taxa derived from traditional breeding and genetic engineering. Trees 20:131–144Hoenicka H, Nowitzki O, Debener T, Fladung M (2006) Faster evaluation of sterility strategies in transgenic trees. Silv Genet 55:285–291Hoenicka H, Lehnhardt D, Polak O, Fladung M (2012) Early flowering and genetic containment studies in transgenic poplar. iForest 5:138–146Hoenicka H, Lehnhardt D, Nilsson O, Hanelt D, Fladung M (2014) Successful crossings with early flowering transgenic poplar: interspecific crossings, but not transgenesis, promoted aberrant phenotypes in offspring. Plant Biotechnol J 12:1066–1074Hoenicka H, Lehnhardt D, Briones V, Nilsson O, Fladung M (2016) Low temperatures are required to induce the development of fertile flowers in transgenic male and female early flowering poplar (Populus tremula L.). Tree Physiol 36:667–677. https://doi.org/10.1093/treephys/tpw015Hsu CY, Liu YX, Luthe DS, Yuceer C (2006) Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell 18:1846–1861Klocko AL, Brunner AM, Huang J, Meilan R, Lu H, Strauss SH (2016) Containment of transgenic trees by suppression of LEAFY. Nat Biotechnol 34:918–922. https://doi.org/10.1038/nbt.3636Klocko AL, Lu H, Magnuson A, Brunner AM, Ma C, Strauss SH (2018) Phenotypic expression and stability in a large-scale field study of genetically engineered poplars containing sexual containment transgenes. Front Bioeng Biotechnol 6:100. https://doi.org/10.3389/fbioe.2018.00100Lännenpää M, Hassinen M, Ranki A, Hölttä-Vuori M, Lemmetyinen J, Keinonen K, Sopanen T (2005) Prevention of flower development in birch and other plants using a BpFULL1:BARNASE construct. Plant Cell Rep 24(2):69–78Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Comb Proc Int Plant Prop Soc 30:421–427Lu S, Yi S, Zhang J, Liu L, Bao M, Liu G (2019) Isolation and functional characterization of the promoter of SEPALLATA3 gene in London plane and its application in genetic engineering of sterility. Plant Cell Tiss Org Cult 136:109–121Mariani C, Gossele V, De Beuckeleer M, De Block M, Goldberg RB, De Greef W, Leemans J (1992) A chimaeric ribonuclease inhibitor gene restores fertility to male sterile plants. Nature 357:384–387Medina M, Roque E, Pineda B, Cañas LA, Rodríguez-Concepción M, Beltrán JP, Gómez-Mena C (2013) Early anther ablation triggers parthenocarpic fruit development in tomato plants. Plant Biotechnol J 11:770–779Meilan R, Brunner A, Skinner J, Strauss SH (2001) Modification of flowering in transgenic trees. In: Komamine A, Morohoshi N (eds) Molecular breeding of woody plants. Elsevier Science BV, Amsterdam, pp 247–256Paddon CJ, Hartley RW (1986) Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase). Gene 40(2–3):231–239. https://doi.org/10.1016/0378-1119(85)90045-9Parmentier-Line CM, Coleman GD (2016) Constitutive expression of the poplar FD-like basic leucine zipper transcription factor alters growth and bud development. Plant Biotechnol J 14:260–270Pistón F, García C, de la Viña G, Beltrán JP, Cañas LA, Barro F (2008) The pea PsEND1 promoter drives the expression of GUS in transgenic wheat at the binucleate microspores stage and during pollen tube development. Mol Breed 21:401–405Rojas-Gracia P, Roque E, Medina M, Rochina M, Hamza R, Angarita-Díaz P, Moremo V, Pérez-Martín F, Lozano R, Cañas LA, Beltrán JP, Gómez-Mena C (2017) The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato. New Phytol 214:1198–1212Rojas-Gracia P, Roque E, Medina M, López-Martín MJ, Cañas LA, Beltrán JP, Gómez-Mena C (2019) The DOF transcription factor SlDOF10 regulates vascular tissue formation during ovary development in tomato. Front Plant Sci 10:216Roque E, Gómez MD, Ellul P, Wallbraun M, Madueño F, Beltrán JP, Cañas LA (2007) The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation. Plant Cell Rep 26:313–325Roque E, Gómez-Mena C, Hamza R, Beltrán JP, Cañas LA (2019) Engineered male sterility by early anther ablation using the Pea anther-specific promoter PsEND1. Front Plant Sci 10:819Rottmann WH, Meilan R, Sheppard LA, Brunner AM, Skinner JS, Cheng S, Jouanin L, Pilate G, Strauss SH (2000) Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis. Plant J 22:235–245Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu YC, Lee PY, Truong MT, Beals TP, Goldberg RB (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11:297–322Shen L, Chen Y, Su X, Zhang S, Pan H, Huang M (2012) Two FT orthologs from Populus simonii Carrière induce early flowering in Arabidopsis and poplar trees. Plant Cell Tissue Org Cult 108:371–379Skinner JS, Meilan R, Ma C, Strauss SH (2003) The Populus PTD promoter imparts floral-predominant expression and enables high levels of floral–organablation in Populus, Nicotiana, and Arabidopsis. Mol Breed 12:119–132Smith EF, Townsend CO (1907) A plant tumor of bacterial origin. Science 25:671473Tränkner C, Lehmann S, Hoenicka H, Hanke M-V, Fladung M, Lehnhardt D, Dunemann F, Gau A, Schlangen K, Malnoy M, Flachowsky H (2010) Over-expression of an FT-homologous gene of apple induces early flowering in annual and perennial plants. Planta 232:1309–1324Weigel D, Nilsson O (1995) A developmental switch sufficient for flower initiation in diverse plants. Nature 377:495–500Wildholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47:189–194WWF (2015) WWF living forests report: chapter 5: saving forests at risk. Gland: WWF- WorldWide Fund for Nature. https://www.worldwildlife.org/publications/living-forests-report-chapter-5-saving-forests-at-risk. Accessed Jan 2020Xiaoming J, Huanling Z (2014) FT gene with a CaMV35S promoter to control early flowering of transgenic poplar. J Zhejiang A&F 31:404–409Xiaoming J, Hualing Z, Jungfeng F (2011) System optimization of precociously flowering of poplar induced by FT gene controlled by a heat shock promoter. Sci Silvae Sin 47:37–43Zhang H, Harry DE, Yuceer C, Hsu CY, Vikram V, Shevchenko O, Etherington E, Strauss SH (2010) Precocious flowering in trees: the FLOWERING LOCUS T gene as a research and breeding tool in Populus. J Exp Bot 61:2549–256

Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of Argentina

Phytolacca tetramera Hauman is an endemic species of the Province of Buenos Aires (Argentina), in danger of extinction. This species has active fungicides. The methanol extract of its berries possess antifungal activity against opportunistic fungal pathogens. The somatic embryogenesis technique relies on the formation of an embryo from a somatic cell, that is, without the need for the fusion of gametes (Tisserat et al, 1979), which facilitates mass production of in vitro plants. Obtaining somatic embryos gives us the ability to automate the production process in short periods of time and in a confined space. The technique is a necessary step to generate synthetic seeds. In order to adjust a protocol of somatic embryogenesis sections young leaves of Phytolacca tetramera obtained in vitro were placed in a Murashige and Skoog medium supplemented with 2, 4-dichlorophenoxyacetic acid at different concentrations. Somatic embryos were obtained directly from the cutting edge of the blade and embryogenic callus (indirect way) from the midrib.Facultad de Ciencias Agrarias y ForestalesCentro Experimental de Propagación Vegetativ

Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of Argentina

Phytolacca tetramera Hauman is an endemic species of the Province of Buenos Aires (Argentina), in danger of extinction. This species has active fungicides. The methanol extract of its berries possess antifungal activity against opportunistic fungal pathogens. The somatic embryogenesis technique relies on the formation of an embryo from a somatic cell, that is, without the need for the fusion of gametes (Tisserat et al, 1979), which facilitates mass production of in vitro plants. Obtaining somatic embryos gives us the ability to automate the production process in short periods of time and in a confined space. The technique is a necessary step to generate synthetic seeds. In order to adjust a protocol of somatic embryogenesis sections young leaves of Phytolacca tetramera obtained in vitro were placed in a Murashige and Skoog medium supplemented with 2, 4-dichlorophenoxyacetic acid at different concentrations. Somatic embryos were obtained directly from the cutting edge of the blade and embryogenic callus (indirect way) from the midrib.Facultad de Ciencias Agrarias y ForestalesCentro Experimental de Propagación Vegetativ

Optimización de un sistema de modificación génetica de álamos para la incorporación de genes inductores de floración temprana y androesterilidad

Las especies de árboles forestales alcanzan la madurez reproductiva después de muchos años o incluso décadas de crecimiento juvenil. Los tratamientos especiales, basados en hormonas vegetales, inhibidores del crecimiento, manipulación del ambiente de crecimiento o métodos físicos, han promovido el desarrollo de flores en plantas juveniles de varias especies de árboles forestales, pero no en álamo. Sólo la ingeniería genética ha permitido que la floración temprana sea inducida en plantas juveniles de álamo. La liberación al medio de álamos genéticamente modificados (GM) tiene limitantes, tanto desde el punto de vista ambiental, como comercial y de percepción pública. La obtención de álamos GM estériles facilitarían la liberación de los mismos al medio de forma segura. El objetivo general de este trabajo fue optimizar un sistema de modificación génetica de Populus sp. para la incorporación, en simultáneo, de genes de floración temprana y androesterilidad, acelerando el tiempo de obtención de líneas transgénicas dobles. Se logró ajustar un método eficiente de regeneración de plantas in vitro de clones recalcitrantes de Populus deltoides y clones del híbrido Populus x canadensis. Se obtuvieron líneas transgénicas dobles de P. tremula L, conteniendo en simultaneo los genes inductores de la floración temprana y la androesterilidad. También se obtuvieron líneas transgénicas simples de P. deltoides y P. x euramericana, portadoras del gen inductor de la floración temprana. Todas las líneas obtenidas regeneraron en plantas completas donde pudo confirmarse, con técnicas moleculares, la presencia de los transgenes. Se evaluó el desarrollo de la floración precoz inducida por el constructo HSP::AtFT , al mismo tiempo que la androesterilidad, inducida por la expresión del constructo XY::barnase en plantas crecidas en invernáculo de Populus tremula L. transgénicas. Este estudio representa un gran avance en la transformación del P. deltoides y P. euramericana siendo éstos los primeros álamos de estas dos especies con la característica de floración temprana. Confirmamos que el sistema HSP::AtFT en álamos combina una inducción de la floración temprana con un normal crecimiento vegetativo y que el sistema XY::barnase es efectivo para obtener alámos masculinos transgénicos estériles. Disponer de este protocolo eficiente de transformación con genes de androesterilidad permitirá en un futuro modificar genéticamente clones de álamo de interés local para introducir, junto con esta característica, caracteres de importancia silvicultural, de forma controlada. También se podrán desarrollar clones de álamos con floración temprana para incorporar a los programas de mejora genética convencional.Instituto de Biotecnología y Biología MolecularLaboratorio de Investigaciones en Mader

Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of Argentina

Phytolacca tetramera Hauman is an endemic species of the Province of Buenos Aires (Argentina), in danger of extinction. This species has active fungicides. The methanol extract of its berries possess antifungal activity against opportunistic fungal pathogens. The somatic embryogenesis technique relies on the formation of an embryo from a somatic cell, that is, without the need for the fusion of gametes (Tisserat et al, 1979), which facilitates mass production of in vitro plants. Obtaining somatic embryos gives us the ability to automate the production process in short periods of time and in a confined space. The technique is a necessary step to generate synthetic seeds. In order to adjust a protocol of somatic embryogenesis sections young leaves of Phytolacca tetramera obtained in vitro were placed in a Murashige and Skoog medium supplemented with 2, 4-dichlorophenoxyacetic acid at different concentrations. Somatic embryos were obtained directly from the cutting edge of the blade and embryogenic callus (indirect way) from the midrib.Facultad de Ciencias Agrarias y ForestalesCentro Experimental de Propagación Vegetativ

Desde el pacífico a la foresta tropical: redes de interacción social en el desierto de atacama durante el pleistoceno final

The social groups that initially inhabited the hyper arid core of the Atacama Desert of northern Chile during the late Pleistocene integrated a wide range of local, regional and supra regional goods and ideas for their social reproduction as suggested by the archaeological evidence contained in several open camps in Pampa del Tamarugal (PdT). Local resources for maintaining their every-day life, included stone raw material, wood, plant and animal fibers, game, and fresh water acquired within a radius of ∼30 km (ca. 1-2 days journey). At a regional scale, some goods were introduced from the Pacific coast (60-80 km to the west, ca. 3-4 days journey), including elongated rounded cobbles used as hammer stones in lithic production, and shells, especially from non-edible species of mollusks. From the Andes (ranging 80-150 km to the east, ca. 5-8 days of journey), they obtained camelid fiber, obsidian and a high-quality chalcedony, in addition to sharing knowledge on projectile point designs (Patapatane and Tuina type forms). Pieces of wood of a tropical forest tree species (Ceiba spp.) from the east Andean lowlands (600 km away, ca. 30 days of journey) were also brought to the PdT. While local goods were procured by the circulation of people within the PdT, the small number of foreign items would have been acquired through some sort of exchange networks that integrated dispersed local communities throughout several ecosystems. These networks may have been a key factor behind the success exhibited by these early huntergatherers in the hyper arid ecosystems of the Atacama Desert at the end of the Pleistocene. Different lines of archaeological evidence including open camps, workshop-quarries, lithic artifacts, archaeofaunal remains, plant and animal fibers and textiles, archaeobotanical remains, and paleoecological data show that people of the PdT managed a wide range of cultural items from the Pacific coast, the Andean highland and the tropical forest, that were integrated with resources gathered locally within the socio-cultural systems established by the end of the Pleistocene. These results are interpreted as material expressions of multi-scalar networking for resource management and other social material and immaterial requirements, which in other words, means that these people were actively connected to regional (coastal and highland), and supra-regional (trans-Andean) exchange networks from and out of the PdT.Los grupos sociales que inicialmente habitaban el núcleo hiperárido del Desierto de Atacama en el norte de Chile durante el Pleistoceno tardío integraron una amplia gama de bienes e ideas, locales, regionales y supra regionales, para su reproducción social, como lo sugieren las evidencias arqueológicas materiales recuperadas en varios campamentos al aire libre en Pampa del Tamarugal (PdT). Los recursos locales para mantener su vida diaria, incluían materias primas líticas, fibras de plantas y animales, presas de caza y agua dulce adquiridos en un radio de ̴30 km (ca. 1-2 días de viaje). A escala regional, se introdujeron algunos elementos desde la costa del Pacífico (60-80 km hacia el oeste, ca. 3 a 4 días de viaje), incluidos rodados redondeados alargados, utilizados como percutores en la producción lítica y conchas, especialmente de especies no comestibles de moluscos. Desde los Andes (80-150 km al este, ca. 5-8 días de viaje), obtuvieron fibra de camélido, obsidiana y una calcedonia de alta calidad, además de compartir conocimientos sobre diseños de puntas de proyectil (tipo Patapatane y Tuina). También se llevaron a la PdT trozos de madera de una especie de árbol de los bosques tropicales (Ceiba spp.) de las tierras bajas al este de los Andes (600 km de distancia, ca. 30 días de viaje). Mientras que los bienes locales fueron adquiridos por la circulación de personas dentro de la PdT, el pequeño número de artículos foráneos se adquirieron a través de redes de intercambio que integraron comunidades locales dispersas en varios ecosistemas; lo que debió ser un factor clave detrás del éxito demostrado por estos primeros cazadores-recolectores en los ecosistemas hiperáridos del Desierto de Atacama hacia el final del Pleistoceno. Diferentes líneas de evidencia arqueológica que incluyen campamentos al aire libre, talleres, canteras, artefactos líticos, restos arqueofaunales, fibras y textiles de plantas y animales, restos arqueobotánicos y datos paleoecológicos, muestran que la gente de la PdT manejaron una amplia gama de elementos culturales desde la costa del Pacífico, el altiplano andino y el bosque tropical, que se integraron a los recursos recolectados localmente dentro de los sistemas socioculturales establecidos al final del Pleistoceno. Estos resultados se interpretan como una expresión material de una red de múltiples escalas para la gestión de recursos y otros requisitos sociales e inmateriales, lo que en otras palabras, significaría que estos grupos sociales estaban conectados activamente con redes de interacción regionales (costa y tierras altas) y supra-regionales (transandinas) desde y hacia la PdT.Fil: Santoro, Calógero M.. Universidad de Tarapaca.; ChileFil: Gayo, Eugenia M.. Centro de Ciencia del Clima y la Resiliencia; Chile. Universidad de Concepción; ChileFil: Capriles Flores, Jose Mariano. State University of Pennsylvania; Estados UnidosFil: Rivadeneira Valenzuela, Marcelo Michel. Comisión Nacional de Investigación Científica y Tecnológica. Centro de Investigación Regional. Centro de Estudios en Zonas Áridas; Chile. Universidad Católica del Norte; Chile. Universidad de La Serena; ChileFil: Herrera, Katherine A.. Universite Paris Ouest Nanterre la Defense; Francia. Universidad de Tarapaca.; ChileFil: Mandakovic, Valentina. No especifíca;Fil: Rallo, Mónica. No especifíca;Fil: Rech, Jason A.. University of Miami; Estados UnidosFil: Cases, Bárbara. Universidad de Tarapaca.; ChileFil: Briones, Luis. Museo Municipalidad de Pica; ChileFil: Olguín, Laura. Universidad Católica del Norte, San Pedro de Atacama; ChileFil: Valenzuela, Daniela. Universidad de Tarapaca.; ChileFil: Borrero, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Saavedra 15. Instituto Multidisciplinario de Historia y Ciencias Humanas; ArgentinaFil: Ugalde, Paula C.. University of Arizona; Estados Unidos. Universidad de Tarapaca.; ChileFil: Rothhammer, Francisco. Universidad de Tarapaca.; ChileFil: Latorre, Claudio. Pontificia Universidad Católica de Chile; Chile. Instituto de Ecología y Biodiversidad; ChileFil: Szpak, Paul. Trent University; Canad

Global disparities in surgeons’ workloads, academic engagement and rest periods: the on-calL shIft fOr geNEral SurgeonS (LIONESS) study

: The workload of general surgeons is multifaceted, encompassing not only surgical procedures but also a myriad of other responsibilities. From April to May 2023, we conducted a CHERRIES-compliant internet-based survey analyzing clinical practice, academic engagement, and post-on-call rest. The questionnaire featured six sections with 35 questions. Statistical analysis used Chi-square tests, ANOVA, and logistic regression (SPSS® v. 28). The survey received a total of 1.046 responses (65.4%). Over 78.0% of responders came from Europe, 65.1% came from a general surgery unit; 92.8% of European and 87.5% of North American respondents were involved in research, compared to 71.7% in Africa. Europe led in publishing research studies (6.6 ± 8.6 yearly). Teaching involvement was high in North America (100%) and Africa (91.7%). Surgeons reported an average of 6.7 ± 4.9 on-call shifts per month, with European and North American surgeons experiencing 6.5 ± 4.9 and 7.8 ± 4.1 on-calls monthly, respectively. African surgeons had the highest on-call frequency (8.7 ± 6.1). Post-on-call, only 35.1% of respondents received a day off. Europeans were most likely (40%) to have a day off, while African surgeons were least likely (6.7%). On the adjusted multivariable analysis HDI (Human Development Index) (aOR 1.993) hospital capacity > 400 beds (aOR 2.423), working in a specialty surgery unit (aOR 2.087), and making the on-call in-house (aOR 5.446), significantly predicted the likelihood of having a day off after an on-call shift. Our study revealed critical insights into the disparities in workload, access to research, and professional opportunities for surgeons across different continents, underscored by the HDI

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old