Evolution of Class IITCPgenes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia

[EN] Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.We thank Anny Garces Palacio, Sarita Munoz, Pablo Perez-Mesa (Universidad de Antioquia, Colombia), Cecilia Zumajo-Cardona (The New York Botanical Garden), Ana Berbel and Clara Ines Ortiz-Ramirez (Instituto de Biologia Molecular y Celular de Plantas, CSIC-UVP, Valencia, Spain) for photographs and assistance during laboratory work. We also thank Sebastian Gonzalez (Massachusetts College of Art and Design) for taking some of the photographs in Figs 1 and 2. Thanks are also due to the Dresden Junior Fellowship for allowing the visiting professor fellowship of NPM to the Technishe Universitat Dresden during 2019. This research was funded by Estrategia de Sostenibilidad 2018-2019 the Convocatoria Programaticas 2017-2018 (code 2017-16302), and the 2018-2019 Fondo de Internacionalizacion (code 201926230) from the Universidad de Antioquia, the iCOOP + 2016 grant COOPB20250 from Centro Superior de Investigacion Cientifica, CSIC and the ExpoSEED (H2020.MSCA-RISE2015-691109) EU grant.Pabon-Mora, N.; Madrigal, Y.; Alzate, JF.; Ambrose, BA.; Ferrandiz Maestre, C.; Wanke, S.; Neinhuis, C.... (2020). Evolution of Class IITCPgenes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia. New Phytologist. 228(2):752-769. https://doi.org/10.1111/nph.16719S7527692282Aguilar-Martínez, J. A., Poza-Carrión, C., & Cubas, P. (2007). Arabidopsis BRANCHED1Acts as an Integrator of Branching Signals within Axillary Buds. The Plant Cell, 19(2), 458-472. doi:10.1105/tpc.106.048934Almeida, J., Rocheta, M., & Galego, L. (1997). Genetic control of flower shape in Antirrhinum majus. Development, 124(7), 1387-1392. doi:10.1242/dev.124.7.1387Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. doi:10.1016/s0022-2836(05)80360-2Ambrose, B. A., Lerner, D. R., Ciceri, P., Padilla, C. M., Yanofsky, M. F., & Schmidt, R. J. (2000). Molecular and Genetic Analyses of the Silky1 Gene Reveal Conservation in Floral Organ Specification between Eudicots and Monocots. Molecular Cell, 5(3), 569-579. doi:10.1016/s1097-2765(00)80450-5Ballester, P., Navarrete-Gómez, M., Carbonero, P., Oñate-Sánchez, L., & Ferrándiz, C. (2015). Leaf expansion in Arabidopsis is controlled by a TCP-NGA regulatory module likely conserved in distantly related species. Physiologia Plantarum, 155(1), 21-32. doi:10.1111/ppl.12327Bartlett, M. 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Evolution of Class IITCPgenes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia

[EN] Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.We thank Anny Garces Palacio, Sarita Munoz, Pablo Perez-Mesa (Universidad de Antioquia, Colombia), Cecilia Zumajo-Cardona (The New York Botanical Garden), Ana Berbel and Clara Ines Ortiz-Ramirez (Instituto de Biologia Molecular y Celular de Plantas, CSIC-UVP, Valencia, Spain) for photographs and assistance during laboratory work. We also thank Sebastian Gonzalez (Massachusetts College of Art and Design) for taking some of the photographs in Figs 1 and 2. Thanks are also due to the Dresden Junior Fellowship for allowing the visiting professor fellowship of NPM to the Technishe Universitat Dresden during 2019. This research was funded by Estrategia de Sostenibilidad 2018-2019 the Convocatoria Programaticas 2017-2018 (code 2017-16302), and the 2018-2019 Fondo de Internacionalizacion (code 201926230) from the Universidad de Antioquia, the iCOOP + 2016 grant COOPB20250 from Centro Superior de Investigacion Cientifica, CSIC and the ExpoSEED (H2020.MSCA-RISE2015-691109) EU grant.Pabon-Mora, N.; Madrigal, Y.; Alzate, JF.; Ambrose, BA.; Ferrandiz Maestre, C.; Wanke, S.; Neinhuis, C.... (2020). Evolution of Class IITCPgenes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia. New Phytologist. 228(2):752-769. https://doi.org/10.1111/nph.167197527692282Aguilar-Martínez, J. A., Poza-Carrión, C., & Cubas, P. (2007). Arabidopsis BRANCHED1Acts as an Integrator of Branching Signals within Axillary Buds. The Plant Cell, 19(2), 458-472. doi:10.1105/tpc.106.048934Almeida, J., Rocheta, M., & Galego, L. (1997). Genetic control of flower shape in Antirrhinum majus. Development, 124(7), 1387-1392. doi:10.1242/dev.124.7.1387Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. doi:10.1016/s0022-2836(05)80360-2Ambrose, B. A., Lerner, D. R., Ciceri, P., Padilla, C. M., Yanofsky, M. F., & Schmidt, R. J. (2000). Molecular and Genetic Analyses of the Silky1 Gene Reveal Conservation in Floral Organ Specification between Eudicots and Monocots. Molecular Cell, 5(3), 569-579. doi:10.1016/s1097-2765(00)80450-5Ballester, P., Navarrete-Gómez, M., Carbonero, P., Oñate-Sánchez, L., & Ferrándiz, C. (2015). Leaf expansion in Arabidopsis is controlled by a TCP-NGA regulatory module likely conserved in distantly related species. Physiologia Plantarum, 155(1), 21-32. doi:10.1111/ppl.12327Bartlett, M. E., & Specht, C. D. (2011). Changes in expression pattern of the teosinte branched1- like genes in the Zingiberales provide a mechanism for evolutionary shifts in symmetry across the order. American Journal of Botany, 98(2), 227-243. doi:10.3732/ajb.1000246Bliss, B. J., Wanke, S., Barakat, A., Ayyampalayam, S., Wickett, N., Wall, P. K., … dePamphilis, C. W. (2013). Characterization of the basal angiosperm Aristolochia fimbriata: a potential experimental system for genetic studies. BMC Plant Biology, 13(1), 13. doi:10.1186/1471-2229-13-13Busch, A., & Zachgo, S. (2007). Control of corolla monosymmetry in the Brassicaceae Iberis amara. Proceedings of the National Academy of Sciences, 104(42), 16714-16719. doi:10.1073/pnas.0705338104Citerne, H. L., Reyes, E., Le Guilloux, M., Delannoy, E., Simonnet, F., Sauquet, H., … Damerval, C. (2016). Characterization ofCYCLOIDEA-like genes in Proteaceae, a basal eudicot family with multiple shifts in floral symmetry. 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Assessing the Flowering Genetic Regulatory Network in Neotropical Orchids

During the reproductive transition in flowering plants, a vegetative apical meristem (SAM) transforms into an inflorescence meristem (IM) that forms bracts and flowers. In grasses such as rice, a genetic regulatory network (GRN) controlling reproductive transitions has been identified. It includes the integration of promoters and repressors from different gene lineages with active duplication events during angiosperm diversification. With the objective to understand the evolution and expression of flowering GRN in Orchidaceae, we performed comprehensive phylogenetic analyses of all genes from the flowering GRN and analyzed by RT-PCR the expression of targeted homologs in key developmental stages. Our ML results indicate that the FT/TFL1, FD, FLC/FUL, SOC1 and AGL24/SVP gene lineages have been subject to multiple duplications in monocots, as well as in Orchidaceae. Conversely, FLC genes are lost in Orchidaceae, suggesting major changes in the repression of flowering. Our studies also show active expression of many target genes in Elleanthus aurantiacus (Orchidoideae) in the SAM and in IM, indicating important functions in the reproductive transition. We describe how the flowering GRN in orchids has significant variations in copy number and expression patterns when compared to the canonical rice flowering GRN

The effect of a therapeutic regimen of Traditional Chinese Medicine rehabilitation for post-stroke cognitive impairment: study protocol for a randomized controlled trial

Background Post-stroke cognitive impairment (PSCI) lessens quality of life, restricts the rehabilitation of stroke, and increases the social and economic burden stroke imposes on patients and their families. Therefore effective treatment is of paramount importance. However, the treatment of PSCI is very limited. The primary aim of this protocol is to propose a lower cost and more effective therapy, and to confirm the long-term effectiveness of a therapeutic regimen of Traditional Chinese Medicine (TCM) rehabilitation for PSCI. Methods/Design A prospective, multicenter, large sample, randomized controlled trial will be conducted. A total of 416 eligible patients will be recruited from seven inpatient and outpatient stroke rehabilitation units and randomly allocated into a therapeutic regimen of TCM rehabilitation group or cognitive training (CT) control group. The intervention period of both groups will last 12 weeks (30 minutes per day, five days per week). Primary and secondary outcomes will be measured at baseline, 12 weeks (at the end of the intervention), and 36 weeks (after the 24-week follow-up period). Discussion This protocol presents an objective design of a multicenter, large sample, randomized controlled trial that aims to put forward a lower cost and more effective therapy, and confirm the long-term effectiveness of a therapeutic regimen of TCM rehabilitation for PSCI through subjective and objective assessments, as well as highlight its economic advantages

Weaning from mechanical ventilation in intensive care units across 50 countries (WEAN SAFE): a multicentre, prospective, observational cohort study

Background Current management practices and outcomes in weaning from invasive mechanical ventilation are poorly understood. We aimed to describe the epidemiology, management, timings, risk for failure, and outcomes of weaning in patients requiring at least 2 days of invasive mechanical ventilation. Methods WEAN SAFE was an international, multicentre, prospective, observational cohort study done in 481 intensive care units in 50 countries. Eligible participants were older than 16 years, admitted to a participating intensive care unit, and receiving mechanical ventilation for 2 calendar days or longer. We defined weaning initiation as the first attempt to separate a patient from the ventilator, successful weaning as no reintubation or death within 7 days of extubation, and weaning eligibility criteria based on positive end-expiratory pressure, fractional concentration of oxygen in inspired air, and vasopressors. The primary outcome was the proportion of patients successfully weaned at 90 days. Key secondary outcomes included weaning duration, timing of weaning events, factors associated with weaning delay and weaning failure, and hospital outcomes. This study is registered with ClinicalTrials.gov, NCT03255109. Findings Between Oct 4, 2017, and June 25, 2018, 10 232 patients were screened for eligibility, of whom 5869 were enrolled. 4523 (77·1%) patients underwent at least one separation attempt and 3817 (65·0%) patients were successfully weaned from ventilation at day 90. 237 (4·0%) patients were transferred before any separation attempt, 153 (2·6%) were transferred after at least one separation attempt and not successfully weaned, and 1662 (28·3%) died while invasively ventilated. The median time from fulfilling weaning eligibility criteria to first separation attempt was 1 day (IQR 0–4), and 1013 (22·4%) patients had a delay in initiating first separation of 5 or more days. Of the 4523 (77·1%) patients with separation attempts, 2927 (64·7%) had a short wean (≤1 day), 457 (10·1%) had intermediate weaning (2–6 days), 433 (9·6%) required prolonged weaning (≥7 days), and 706 (15·6%) had weaning failure. Higher sedation scores were independently associated with delayed initiation of weaning. Delayed initiation of weaning and higher sedation scores were independently associated with weaning failure. 1742 (31·8%) of 5479 patients died in the intensive care unit and 2095 (38·3%) of 5465 patients died in hospital. Interpretation In critically ill patients receiving at least 2 days of invasive mechanical ventilation, only 65% were weaned at 90 days. A better understanding of factors that delay the weaning process, such as delays in weaning initiation or excessive sedation levels, might improve weaning success rates

Weaning from mechanical ventilation in intensive care units across 50 countries (WEAN SAFE): a multicentre, prospective, observational cohort study

Background: Current management practices and outcomes in weaning from invasive mechanical ventilation are poorly understood. We aimed to describe the epidemiology, management, timings, risk for failure, and outcomes of weaning in patients requiring at least 2 days of invasive mechanical ventilation. Methods: WEAN SAFE was an international, multicentre, prospective, observational cohort study done in 481 intensive care units in 50 countries. Eligible participants were older than 16 years, admitted to a participating intensive care unit, and receiving mechanical ventilation for 2 calendar days or longer. We defined weaning initiation as the first attempt to separate a patient from the ventilator, successful weaning as no reintubation or death within 7 days of extubation, and weaning eligibility criteria based on positive end-expiratory pressure, fractional concentration of oxygen in inspired air, and vasopressors. The primary outcome was the proportion of patients successfully weaned at 90 days. Key secondary outcomes included weaning duration, timing of weaning events, factors associated with weaning delay and weaning failure, and hospital outcomes. This study is registered with ClinicalTrials.gov, NCT03255109. Findings: Between Oct 4, 2017, and June 25, 2018, 10 232 patients were screened for eligibility, of whom 5869 were enrolled. 4523 (77·1%) patients underwent at least one separation attempt and 3817 (65·0%) patients were successfully weaned from ventilation at day 90. 237 (4·0%) patients were transferred before any separation attempt, 153 (2·6%) were transferred after at least one separation attempt and not successfully weaned, and 1662 (28·3%) died while invasively ventilated. The median time from fulfilling weaning eligibility criteria to first separation attempt was 1 day (IQR 0-4), and 1013 (22·4%) patients had a delay in initiating first separation of 5 or more days. Of the 4523 (77·1%) patients with separation attempts, 2927 (64·7%) had a short wean (≤1 day), 457 (10·1%) had intermediate weaning (2-6 days), 433 (9·6%) required prolonged weaning (≥7 days), and 706 (15·6%) had weaning failure. Higher sedation scores were independently associated with delayed initiation of weaning. Delayed initiation of weaning and higher sedation scores were independently associated with weaning failure. 1742 (31·8%) of 5479 patients died in the intensive care unit and 2095 (38·3%) of 5465 patients died in hospital. Interpretation: In critically ill patients receiving at least 2 days of invasive mechanical ventilation, only 65% were weaned at 90 days. A better understanding of factors that delay the weaning process, such as delays in weaning initiation or excessive sedation levels, might improve weaning success rates. Funding: European Society of Intensive Care Medicine, European Respiratory Society

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

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present