Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: a neutron reflectometry, AFM and QCM-D structural study

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.publishe

Evaluation of hydrogen sulfide supply to biostimulate the nutritive and phytochemical quality and the antioxidant capacity of Cabbage (<i>Brassica oleracea</i> L. ‘Bronco’)

The potential effects of the hydrogen sulfide on shoot biomass, nutritional quality and antioxidant capacity of Brassica oleracea, were investigated through the application of increasing doses of NaHS (H2S donor NaHS; 0.5, 1, 2.5, and 5 mM). The results showed that the 0.5 and 1 mM NaHS treatments increased biomass and the quality composition of ‘Bronco’ cabbage (i.e. chlorophylls, carotenoids, anthocyanins, flavonols, total phenolics and sinigrin). On the other hand, there was an increase in lipid peroxidation and hydrogen peroxide content with the application of doses higher than 2.5 mM NaHS. Therefore, we selected the 0.5 and 1 mM NaHS dosages as optimal for cabbage. The 2.5 and 5 mM NaHS produced an excessive lipid peroxidation, decreases in plant biomass and losses of chlorophylls, being all considered negative effects, and clear evidences of stressful situation for the plants. For practical purposes, this study suggested that exogenous application of H2S donor NaHS at 0.5 and 1 mM may be useful as bio-stimulant to boost the yield and the health-promoting composition of ‘Bronco’ cabbage (Brassica oleracea L.).   The online version of theis article (doi: 10.5073/JABFQ.2016.089.038) contains a supplementary file

In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study

BackgroundIbrexafungerp (SCY-078) is the newest oral and intravenous antifungal drug with broad activity, currently undergoing clinical trials for invasive candidiasis. ObjectiveThe aim of this study was to assess the in vitro activity of ibrexafungerp and comparators against a collection of 434 European blood isolates of Candida. MethodsIbrexafungerp, caspofungin, fluconazole, and micafungin minimum inhibitory concentrations (MICs) were collected from 12 European laboratories for 434 blood isolates, including 163 Candida albicans, 108 Candida parapsilosis, 60 Candida glabrata, 40 Candida tropicalis, 29 Candida krusei, 20 Candida orthopsilosis, 6 Candida guilliermondii, 2 Candida famata, 2 Candida lusitaniae, and 1 isolate each of Candida bracarensis, Candida catenulata, Candida dubliniensis, and Candida kefyr. MICs were determined by the EUCAST broth microdilution method, and isolates were classified according to recommended clinical breakpoints and epidemiological cutoffs. Additionally, 22 Candida auris from different clinical specimens were evaluated. ResultsIbrexafungerp MICs ranged from 0.016 to >= 8 mg/L. The lowest ibrexafungerp MICs were observed for C. albicans (geometric MIC 0.062 mg/L, MIC range 0.016-0.5 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (geometric MIC 0.517 mg/L, MIC range 0.06->= 8 mg/L). Modal MICs/MIC(50)s (mg/L) against Candida spp. were 0.125/0.06 for C. albicans, 0.5/0.5 for C. parapsilosis, 0.25/0.25 for C. glabrata, 0.5/0.5 for C. tropicalis, 1/1 for C. krusei, 4/2 for C. orthopsilosis, and 0.5/0.5 for C. auris. Ibrexafungerp showed activity against fluconazole- and echinocandin-resistant isolates. If adopting wild-type upper limits, a non-wild-type phenotype for ibrexafungerp was only observed for 16/434 (3.7%) isolates: 11 (4.6%) C. parapsilosis, 4 (5%) C. glabrata, and 1 (2.5%) C. tropicalis. ConclusionIbrexafungerp showed a potent in vitro activity against Candida.This study received funding from SCYNEXIS. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of the article, or the decision to submit it for publication. CM-A is a recipient of a grant from Fundació n ONCE (Oportunidad al Talento). EE, AG, NJ, CM-A, and GQ have received grant support from Consejerı́a de Educación, Universidades e Investigación del Gobierno Vasco (GIC15 IT-990-16), Fondo de Investigación Sanitaria del Gobierno de España (FIS PI11/00203), and UPV/EHU (UFI 11/25). All authors declare no other competing interests

Acute leukemia arising from myeloproliferative or myelodysplastic/myeloproliferative neoplasms: A series of 372 patients from the PETHEMA AML registry

PETHEMA group.Treatment of acute myeloid leukemia (AML) evolving from myeloproliferative (MPN) or myelodysplastic/myeloproliferative neoplasms (MDS/MPN) is challenging. We evaluated disease characteristics, treatment patterns and outcomes in 372 patients diagnosed with AML after MPN or MDS/MPN over a 27-year period. Frontline treatment was intensive chemotherapy (38%), hypomethylating agents [HMAs] (17%), non-intensive chemotherapy (14%), and supportive care (31%). Median overall survival was 4.8 months, with a 5-year survival rate of 4%. Median survival was 2.8, 3.9 and 8.3 months for the 1992-2010, 2011-2015 and 2016-2019 periods, respectively (test for trend p < 0.001). Complete response (CR) rate was higher with intensive chemotherapy (43%) than with non-intensive chemotherapy (12%) or HMAs (8.5%) [p < 0.001], but responses were short-lived without allogeneic hematopoietic cell transplantation. Patients treated with intensive chemotherapy or HMAs had superior survival than those receiving non-intensive chemotherapy (median: 8.5 vs. 8.6 vs. 4.2 months, respectively). No differences in treatment response or survival were observed according to prior disease subtypes. Patients undergoing transplantation in CR had better survival than those transplanted in other response categories (3-year survival rate of 64% vs. 22%, p = 0.002). Our results support the use of intensive chemotherapy followed by transplant whenever possible, and the preferential use of HMAs over attenuated chemotherapy regimens in unfit patients. In spite of the survival improvement in recent years, this subset of AML constitutes an unmet medical need and deserves systematic incorporation in clinical trials.Peer reviewe

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Estudio de diferentes aspectos agronómicos, fisiológicos y moleculares de la aplicación de H2S en plantas de Brassica oleracea CV. BRONCO

En el capítulo 1, se investigaron los efectos potenciales del sulfuro de hidrógeno en la biomasa de la parte aérea, la calidad nutricional y la capacidad antioxidante de Brassica oleracea, mediante la aplicación de dosis crecientes de NaHS (NaHS como donador de H2S; 0,5, 1, 2,5 y 5 mM). Los resultados mostraron que los tratamientos 0,5 y 1 mM de NaHS aumentaron la biomasa y la calidad nutricional de la col 'Bronco' (es decir, clorofilas, carotenoides, antocianinas, flavonoles, fenoles totales y sinigrina). Por otra parte, hubo un aumento en la peroxidación de lípidos y el contenido de peróxido de hidrógeno tras la aplicación de dosis superiores a 2,5 mM NaHS. Por lo tanto, seleccionamos las dosis de 0,5 y 1 mM NaHS como óptimas para las plantas de Col. La aplicación de 2,5 y 5 mM de NaHS produjo una excesiva peroxidación de lípidos, disminuciones en la biomasa de las plantas y pérdidas de clorofilas, siendo todos ellos considerados efectos negativos, y claras evidencias de estrés en las plantas. En cuanto a aplicaciones prácticas, este estudio sugiere que la aplicación exógena de NaHS como donador de H2S a 0,5 y 1 mM puede ser útil como bioestimulante para aumentar el rendimiento y la composición promotora de la salud de la planta de Col (Brassica oleracea L. 'Bronco'). Dada la relación íntima que guardan los procesos de asimilación de N y S en plantas, en el capítulo 2, se estudiaron los efectos de diferentes dosis de sulfuro de hidrógeno en forma de NaSH (0, 0,5, 1,2,5 y 5 mM) sobre la formación y asimilación del NH4 + en plantas de Brassica oleracea L. 'Bronco'. Según nuestros resultados, los tratamientos 0,5 y 1 mM de NaSH aumentaron la biomasa mientras disminuían la concentración de NO3 -. En el tratamiento 0,5 mM de NaSH, la acumulación de NH4 + disminuyó con la estimulación de la actividad GS, resultando en un mayor contenido en ciertos aminoácidos (AAs) y proteínas solubles, lo que podría estar relacionado con la mayor biomasa encontrada en este tratamiento. Sin embargo, los tratamientos 2,5 y 5 mM de NaSH indujeron la formación y acumulación de NH4 +, así como la fotorrespiración. Esta acumulación excesiva de NH4 + pudo ser responsable de la menor biomasa en estos tratamientos (2,5 y 5 mM de NaSH). Por ello, en este capítulo concluimos que la acumulación excesiva de NH4 + puede ser responsable de la disminución de la biomasa en los tratamientos 2,5 y 5 mM de NaSH. Por otro lado, concluimos que la aplicación de 0,5 mM de NaSH podría ser una estrategia beneficiosa para mejorar los procesos involucrados en la asimilación del N, acompañada de un aumento de la biomasa de los cultivos de col. A continuación, en el capítulo 3, se investigó cómo la aplicación de sulfuro de hidrógeno (0,5 mM de NaHS) en Brassica oleracea L. 'Bronco' influye en los procesos implicados en la homeostasis del glutatión y la tolerancia al estrés alcalino (50mM NaHCO3:Na2CO3). Según nuestros resultados, el estrés alcalino aumenta el contenido en O2 .-, la peroxidación de lípidos y las actividades de las enzimas glioxalasa I (Gly I) y glioxalasa II (Gly II) que desintoxican el metilglioxal (MG) al tiempo que disminuyen la biomasa, la actividad de la superóxido dismutasa (SOD), la actividad de las enzimas que intervienen en la síntesis de glutatión (GSH) y en el ciclo AsA-GSH, así como el contenido en glutatión reducido y las diferentes formas de ascorbato (AsA). Por otro lado, la aplicación del NaHS mejoró la respuesta antioxidante, induciendo la actividad SOD y mejorando los procesos involucrados en la homeostasis del glutatión, impulsando el contenido en glutatión reducido (GSH) así como la actividad de las enzimas clave en la síntesis del glutatión y en el ciclo de AsAGSH. En consecuencia, la aplicación de H2S en forma de NaHS a una concentración de 0,5 mM podría fortalecer la tolerancia de Brassica oleracea L. 'Bronco' frente a estrés alcalino. Por último, en el capítulo 4 se investigó como la aplicación de sulfuro de hidrógeno (50 μM de NaHS) en plantas de Arabidopsis thaliana L. influyó sobre la expresión relativa de los genes implicados en la asimilación del azufre, la homeostasis del glutatión y la implicación de éstos en la tolerancia al estrés alcalino (50mM NaHCO3:Na2CO3). De acuerdo con nuestros resultados la aplicación de H2S (NaHS) mitigó el efecto nocivo del estrés alcalino evidenciado por la reducción en la perdida de biomasa del 31 % tras aplicar H2S a plantas sometidas a estrés alcalino. Por otro lado, no se observaron incrementos significativos específicos en respuesta a la aplicación de H2S sobre la expresión relativa de los genes implicados en la biosíntesis de las enzimas e isoenzimas claves en la regulación de los procesos de asimilación de SO4 -2 (Serat 1,1; OAS TL, OAS TL B; OAS TL C; ATPS ; ATPS 2, ATPS 3; ATPS 4 y APR 1) y la homeostasis del glutatión (GMPasa; GDPME; L-GAL PPasa; LGALDH; L-GLDH; SOD 1;SOD 2; APX; MDHAR; GR2; GR 1; gsh 1; gsh 2 y GPX) bajo condiciones de estrés alcalino.In chapter 1, the potential effects of hydrogen sulfide on the biomass of the aerial part, the nutritional quality and the antioxidant capacity of Brassica oleracea were investigated, through the application of increasing doses of NaHS (NaHS as a donor of H2S; 0.5, 1, 2.5 and 5 mM). The results showed that the treatments 0.5 and 1 mM of NaHS increased the biomass and the nutritional quality of the 'Bronco' cabbage (that is, chlorophylls, carotenoids, anthocyanins, flavonols, total phenols and sinigrin). On the other hand, there was an increase in lipid peroxidation and hydrogen peroxide content after the application of doses above 2.5 mM NaHS. Therefore, we selected the doses of 0.5 and 1 mM NaHS as optimal for the plants of Col. The application of 2.5 and 5 mM NaHS produced an excessive peroxidation of lipids, decreases in the biomass of the plants and losses of chlorophylls, being all of them considered negative effects, and clear evidence of stress in the plants. In terms of practical applications, this study suggests that the exogenous application of NaHS as a donor of H2S at 0.5 and 1 mM can be useful as a biostimulant to increase the yield and healthpromoting composition of the cabbage plant (Brassica oleracea L. 'Bronco'). Given the intimate relationship between the processes of assimilation of N and S in plants, in chapter 2, the effects of different doses of hydrogen sulfide in the form of NaSH (0, 0.5, 1,2,5 and 5 mM) on the formation and assimilation of NH4 + in plants of Brassica oleracea L. 'Bronco' were studied. According to our results, the 0.5 and 1 mM NaSH treatments increased the biomass while decreasing the NO3 - concentration. In the 0.5 mM NaSH treatment, NH4 + accumulation decreased with the stimulation of GS activity, resulting in a higher content of certain amino acids (AAs) and soluble proteins, which could be related to the higher biomass found in this treatment. However, the 2.5 and 5 mM NaSH treatments induced the formation and accumulation of NH4 +, as well as photorespiration. This excessive accumulation of NH4 + could be responsible for the lower biomass in these treatments (2.5 and 5 mM NaSH). Therefore, in this chapter we conclude that the excessive accumulation of NH4 + may be responsible for the decrease in biomass in the 2.5 and 5 mM NaSH treatments. On the other hand, we conclude that the application of 0.5 mM of NaSH could be a beneficial strategy to improve the processes involved in the assimilation of N, accompanied by an increase in the biomass of cabbage crops. Next, in Chapter 3, we investigated how the application of hydrogen sulfide (0.5 mM NaHS) in Brassica oleracea L. 'Bronco' influences the processes involved in glutathione homeostasis and alkali stress tolerance (50 mM NaHCO3:Na2CO3). According to our results, alkaline stress increases the O2 .- content, lipid peroxidation and the activities of the enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II) which detoxify methylglyoxal (MG) while decreasing the biomass, superoxide dismutase (SOD) activity, the activity of the enzymes involved in glutathione (GSH) synthesis and in the AsA-GSH cycle, as well as the reduced glutathione content and the different forms of ascorbate (AsA) On the other hand, the application of NaHS improved the antioxidant response, inducing SOD activity and improving the processes involved in glutathione homeostasis, boosting the reduced glutathione (GSH) content as well as the activity of key enzymes in glutathione synthesis and the AsA-GSH cycle. Consequently, the application of H2S in the form of NaHS at a concentration of 0.5 mM could strengthen the tolerance of Brassica oleracea L. 'Bronco' to alkaline stress. Finally, in Chapter 4 it was investigated how the application of hydrogen sulfide (50 μM NaHS) in Arabidopsis thaliana L. plants influenced the relative expression of genes involved in sulfur assimilation, glutathione homeostasis and the involvement of these genes in alkali stress tolerance (50 mM NaHCO3:Na2CO3). According to our results, the application of H2S (NaHS) mitigated the harmful effect of alkaline stress evidenced by the reduction in biomass loss of 31% after applying H2S to plants subjected to alkaline stress. On the other hand, no significant specific increases were observed in response to the application of H2S on the relative expression of genes involved in the biosynthesis of enzymes and isoenzymes key to the regulation of SO4 2- (Serat 1,1) assimilation processes; OAS TL, OAS TL B; OAS TL C; ATPS ; ATPS 2, ATPS 3; ATPS 4 and APR 1) and glutathione homeostasis (GMPase; GDPME; L-GAL PPase; LGALDH; L-GLDH; SOD 1;SOD 2; APX; MDHAR; GR2; GR 1; gsh 1; gsh 2 and GPX) under alkaline stress conditions.Tesis Univ. Granada.Grupo de investigación “Fisiología y fitotecnia de cultivos para el desarrollo de una agricultura sostenible” (AGR-282, Plan andaluz de investigación, Junta de Andalucía)Programa de Movilidad Internacional para Estudiantes de Doctorado de la Universidad de Granad

Engineering Phosphatidylinositol-4,5-bisphosphate model membranes enriched 1 in endocytic cargo: a neutron reflectometry, AFM and QCM-D structural study

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological mem-branes with the incorporation of inositol phospholipids and synthetic endocytic motifs