Optimización de la regeneración in vitro del olmo utilizando explantes de hoja y evaluación del proceso en experimentos de transformación

We set up a regeneration process for the elm hybrid ‘Sapporo’ resistant to Dutch Elm Disease, using leaf discs from in vitro grown plants and tested it in transformation experiments. Two steps were needed : bud induction (3 weeks) followed by bud elongation (4 weeks). Bud meristems initiated near the cut ribs, from several tissues (phloem, procambium, rib upper parenchyma) except epidermis. Regeneration depended on the use of agarose (6 g/l LSM) and diluted MS medium (1/2). The best results (11-14 buds/explant) were obtained in presence of 0.1 μM TDZ and 0.06 μM IAA and when maltose (55-110 mM) or sorbitol (110 mM) were used as carbohydrate sources. During shoot elongation TDZ should be decreased to 0.01 μM or replaced by 1-2 μM BAP and 1.4 μM of GA3 added. At this stage, a mixture of 55 mM sorbitol and 27.5 mM maltose was required. Up to 7 shoots / explant developed and were easily rooted (96%) and acclimatized when sorbitol (27.5 mM) and active charcoal (2 g/l) were added to the rooting medium. Transformation experiments were performed with Agrobacterium tumefaciens disarmed strain GV3101-pMP90 (pKyGUSintron). Regeneration zones exhibited stable GUS expression. However, all shoots grown in vitro died on a selective rooting medium (50 mg/l kanamycin). When attempts for in vitro selection were made (12.5 mg/l kanamycin), some buds were initiated but elongation was prevented. Susceptibility to neomycin seemed very high, therefore, selection markers and selection steps must be revised carefully.Se ha desarrollado un procedimiento para la regeneración del olmo híbrido «Sapporo» resistente a la grafiosis mediante el uso de discos foliares procedentes de plantas cultivadas in vitro y examinadas en ensayos de transformación. Para ello se requirieron dos pasos: inducción de las yemas (3 semanas), seguido de la elongación de las yemas (4 semanas). Los meristemas que dieron origen a las yemas se formaron en la cercanía de las costillas de corte a partir de diversos tejidos (floema, procámbium, parénquima) excepto la epidermis. La regeneración dependió del uso de agarosa (6 g/l LSM) y de medio MS diluido (1/2). Los mejores resultados (11-14 yemas por explante) se obtuvieron en presencia de 0,1 μM de TDZ y 0,06 μM de IAA y cuando la maltosa (55-110 mM) o el sorbitol (110 mM) se usaron como fuente de carbohidratos. Durante la elongación del tallo, el TDZ hubo de ser disminuido a 0,01 μM o sustituido por 1-2 μM de BAP y 1,4 μM de GA3. En esta fase fue necesaria una mezcla de 55 mM de sorbitol y 27,5 mM de maltosa. Se desarrollaron al menos 7 tallos por explante que fácilmente enraizaron (96%) y se aclimataron cuando se añadió sorbitol (27,5 mM) y carbón activo (2 g/l) al medio de enraizamiento. Los ensayos de transformación se desarrollaron con las cepas GV3101-pMP90 (pKyGUSintron) de Agrobacterium tumefaciens. Las zonas de regeneración presentaron una expresión GUS estable. No obstante, todas las yemas cultivadas in vitro murieron en un medio selectivo de enraizamiento (50 mg/l de kanamicina). Cuando se intentó la selección in vitro (12,5 mg/l de kanamicina), algunas yemas se iniciaron pero la elongación fue impedida. La susceptibilidad a la neomicina parece ser muy alta, por lo que los marcadores y los pasos de selección deben ser cuidadosamente revisados

Optimization of elm regeneration in vitro using leaf explants and evaluation of the process in transformation experiments

Optimización de la regeneración in vitro del olmo utilizando explantes de hoja y evaluación del proceso en experimentos de transformación Se ha desarrollado un procedimiento para la regeneración del olmo híbrido "Sapporo" resistente a la grafiosis mediante el uso de discos foliares procedentes de plantas cultivadas in vitro y examinadas en ensayos de transformación. Para ello se requirieron dos pasos: inducción de las yemas (3 semanas), seguido de la elongación de las yemas (4 semanas). Los meristemas que dieron origen a las yemas se formaron en la cercanía de las costillas de corte a partir de diversos tejidos (floema, procámbium, parénquima) excepto la epidermis. La regeneración dependió del uso de agarosa (6 g /1 LSM) y de medío MS diluido (1 /2). Los mejores resultados (11-14 yemas por explante) se obtuvieron en presencia de 0,1 UM de TDZ y 0,06 UM de IAA y cuando la maltosa (55-110 mM) a el sorbitol (110 mM) se usaron como fuente de carbohidratos. Durante la elongación del tallo, el TDZ hubo de ser disminuido a 0,01 UM o sustituido por 1-2 UM de BAP y 1,4 UM de GA3. En esta fase fue necesaria una mezcla de 55 mM de sorbítol y 27,5 mM de maltosa. Se desarrollaron al menos 7 tallos por explante que fácilmente enraizaron (96%) y se aclimataron cuando se añadió sorbítol (27,5 mM) y carbón activo (2 g/l) al medio de enraizamiento. Los ensayos de transformación se desarrollaron con las cepas GV3101-pMP9O (pKyGUSintron) de Agrobacterium tumefaciens. Las zonas de regeneración presentaron una expresión GUS estable. No obstante, todas las yemas cultivadas in vitro murieron en un medío selectivo de enraizamiento (50 mg /1 de kanamícina). Cuando se intentó la selección in vitro (12,5 mg/l de kanamicína), algunas yemas se iniciaron pero la elongación fue impedida. La susceptibilidad a la neomicina parece ser muy alta, por lo que los marcadores y los pasos de selección deben ser cuidadosamente revisado

Cancer cell-derived long pentraxin 3 (PTX3) promotes melanoma migration through a toll-like receptor 4 (TLR4)/NF-kappa B signaling pathway

Cutaneous melanoma is one of the most aggressive cancers characterized by a high plasticity, a propensity for metastasis, and drug resistance. Melanomas are composed of phenotypically diverse subpopulations of tumor cells with heterogeneous molecular profiles that reflect intrinsic invasive abilities. In an attempt to identify novel factors of the melanoma invasive cell state, we previously investigated the nature of the invasive secretome by using a comparative proteomic approach. Here, we have extended this analysis to show that PTX3, an acute phase inflammatory glycoprotein, is one such factor secreted by invasive melanoma to promote tumor cell invasiveness. Elevated PTX3 production was observed in the population of MITFlow invasive cells but not in the population of MITFhigh differentiated melanoma cells. Consistently, MITF knockdown increased PTX3 expression in MITFhigh proliferative and poorly invasive cells. High levels of PTX3 were found in tissues and blood of metastatic melanoma patients, and in BRAF inhibitor-resistant melanoma cells displaying a mesenchymal invasive MITFlow phenotype. Genetic silencing of PTX3 in invasive melanoma cells dramatically impaired migration and invasion in vitro and in experimental lung extravasation assay in xenografted mice. In contrast, addition of melanoma-derived or recombinant PTX3, or expression of PTX3 enhanced motility of low migratory cells. Mechanistically, autocrine production of PTX3 by melanoma cells triggered an IKIC/NF kappa B signaling pathway that promotes migration, invasion, and expression of the EMT factor TWIST1. Finally, we found that TLR4 and MYD88 knockdown inhibited PTX3-induced melanoma cell migration, suggesting that PTX3 functions through a TLR4-dependent pathway. Our work reveals that tumor-derived PTX3 contributes to melanoma cell invasion via targetable inflammation-related pathways. In addition to providing new insights into the biology of melanoma invasive behavior, this study underscores the notion that secreted PTX3 represents a potential biomarker and therapeutic target in a subpopulation of MITFlow invasive and/or refractory melanoma

Effects of pre-operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care. We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care