Metabolic Adaptation in Transplastomic Plants Massively Accumulating Recombinant Proteins

BACKGROUND: Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Here we used proteomics to characterize tobacco (Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO(2) metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. CONCLUSIONS/SIGNIFICANCE: The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation

Identification of Host Genes Involved in Geminivirus Infection Using a Reverse Genetics Approach

Geminiviruses, like all viruses, rely on the host cell machinery to establish a successful infection, but the identity and function of these required host proteins remain largely unknown. Tomato yellow leaf curl Sardinia virus (TYLCSV), a monopartite geminivirus, is one of the causal agents of the devastating Tomato yellow leaf curl disease (TYLCD). The transgenic 2IRGFP N. benthamiana plants, used in combination with Virus Induced Gene Silencing (VIGS), entail an important potential as a tool in reverse genetics studies to identify host factors involved in TYLCSV infection. Using these transgenic plants, we have made an accurate description of the evolution of TYLCSV replication in the host in both space and time. Moreover, we have determined that TYLCSV and Tobacco rattle virus (TRV) do not dramatically influence each other when co-infected in N. benthamiana, what makes the use of TRV-induced gene silencing in combination with TYLCSV for reverse genetic studies feasible. Finally, we have tested the effect of silencing candidate host genes on TYLCSV infection, identifying eighteen genes potentially involved in this process, fifteen of which had never been implicated in geminiviral infections before. Seven of the analyzed genes have a potential anti-viral effect, whereas the expression of the other eleven is required for a full infection. Interestingly, almost half of the genes altering TYLCSV infection play a role in postranslational modifications. Therefore, our results provide new insights into the molecular mechanisms underlying geminivirus infections, and at the same time reveal the 2IRGFP/VIGS system as a powerful tool for functional reverse genetics studies

Genome-wide association analysis of dementia and its clinical endophenotypes reveal novel loci associated with Alzheimer's disease and three causality networks : The GR@ACE project

Introduction: Large variability among Alzheimer's disease (AD) cases might impact genetic discoveries and complicate dissection of underlying biological pathways. Methods: Genome Research at Fundacio ACE (GR@ACE) is a genome-wide study of dementia and its clinical endophenotypes, defined based on AD's clinical certainty and vascular burden. We assessed the impact of known AD loci across endophenotypes to generate loci categories. We incorporated gene coexpression data and conducted pathway analysis per category. Finally, to evaluate the effect of heterogeneity in genetic studies, GR@ACE series were meta-analyzed with additional genome-wide association study data sets. Results: We classified known AD loci into three categories, which might reflect the disease clinical heterogeneity. Vascular processes were only detected as a causal mechanism in probable AD. The meta-analysis strategy revealed the ANKRD31-rs4704171 and NDUFAF6-rs10098778 and confirmed SCIMP-rs7225151 and CD33-rs3865444. Discussion: The regulation of vasculature is a prominent causal component of probable AD. GR@ACE meta-analysis revealed novel AD genetic signals, strongly driven by the presence of clinical heterogeneity in the AD series

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Multiancestry analysis of the HLA locus in Alzheimer’s and Parkinson’s diseases uncovers a shared adaptive immune response mediated by HLA-DRB1*04 subtypes

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Development of molecular markers linkaged to Sharka ( Plum pox virus , PPV) resistance in Prunus

Sharka ( Plum pox virus , PPV), is one of the most serious viral diseases affecting apricot production in those areas that are affected. The development and cultivation of new resistant cultivars could be the definitive solution to the disease. In this process of development and introduction of new resistant cultivars, the availability of a reliable method is of great interest for the evaluation of resistance. The development of molecular (genomic or proteomic) markers linkage to this trait could greatly accelerate this evaluation process in apricot. In this work, a double strategy for the development of molecular markers linkage to PPV resistance in apricot is presented: search for genomic (DNA) markers and study the protein expression through 2D electrophoresis (proteomic markers). The search of DNA markers linkage to the resistance is focused on the application of SSR (simple sequence repeat) markers located in the linkage group I, in which PPV resistance has been located by different authors. On the other hand, studies of activity of antioxidant enzymes and protein expression by 2D electrophoresis have been performed in resistant and susceptible apricot cultivars

Plum pox virus (PPV) infection produces an imbalance on the antioxidative systems in Prunus species

The effect of Plum pox virus (PPV) infection on the response of some antioxidant enzymes was studied in Prunus species showing different susceptibility to PPV. PPV infection produced changes in the antioxidant systems from the Prunus species at subcellular level. In general, in PPV susceptible plants, the infection produced a decrease in some antioxidant enzymes, mainly in chloroplasts. However, in peach cv GF305 (PPV-susceptible) also an increase in class I APX in the three compartments studied as well as an increase in POX took place. In contrast, the PPV-resistant plants showed a general increase of antioxidant enzymes in soluble, chloroplast and apoplastic fractions. Long-term PPV infection produced an oxidative stress only in susceptible plants, as observed by the increase in leaf lipid peroxidation, protein oxidation and electrolyte leakage as well as in apoplastic H2O2. This H2O2 increase was much higher in the PPV-susceptible than in the PPV-resistant plants. In peach, the increase in apoplastic H2O2 correlated with the rise in NADH-POX and PPO activities. However, in apricot leaves, no increases in apoplastic NADH-POX were observed, and the increases in apoplastic H2O2 correlated with increase in PPO activity in both cv. Real Fino (100%) and cv. SEO (40%), where a rise in SOD was detected, too. The specific inhibitor of NADPH oxidase, diphenylene iodonium (DPI), had only small effects on NADH oxidation, whereas it was inhibited almost completely by 1 mM KCN. Therefore, most of the NADH oxidation in the apoplastic space of Prunus species is due to NADH-POX and not NAD(P)H oxidase. In previous studies and by means of 2D electrophoresis, we showed that PPV infection produced the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase (MDL) in peach apoplast. Mandelonitrile lyase catalyses the breakdown of mandelonitrile into benzaldehyde and cyanide. Benzaldehyde can be oxidised easily in vivo to produce benzoic acid, the precursor of salicylic acid. It was suggested that decreased MDL activity may result in a decreased SA biosynthesis in PPV-infected peach plants. It is also possible that a decline in antioxidant enzymes and an increase in ROS could be necessary for the establishment of infection and the replication and spread of some plant viruses, in agreement with results described by other authors. Results suggest that the higher antioxidant capacity showed by resistant SEO plants in comparison to PPV-susceptible plants could be associated, among other factors with their resistance to PPV

Plant Journal

Mesophyll conductance (gm), the diffusion of CO2 from substomatal cavities to the carboxylation sites in the chloroplasts, is a highly complex trait driving photosynthesis (net CO2 assimilation, AN). However, little is known concerning the mechanisms by which it is dynamically regulated. The apoplast is considered as a ‘key information bridge’ between the environment and cells. Interestingly, most of the environmental constraints affecting gm also cause apoplastic responses, cell wall (CW) alterations and metabolic rearrangements. Since CW thickness is a key determinant of gm, we hypothesize that other changes in this cellular compartiment should also influence gm. We study the relationship between the antioxidant apoplastic system and CW metabolism and the gm responses in tobacco plants (Nicotiana sylvestris L.) under two abiotic stresses (drought and salinity), combining in vivo gas‐exchange measurements with analyses of antioxidant activities, CW composition and primary metabolism. Stress treatments imposed substantial reductions in AN (58–54%) and gm (59%), accompanied by a strong antioxidant enzymatic response at the apoplastic and symplastic levels. Interestingly, apoplastic but not symplastic peroxidases were positively related to gm. Leaf anatomy remained mostly stable; however, the stress treatments significantly affected the CW composition, specifically pectins, which showed significant relationships with AN and gm. The treatments additionally promoted a differential primary metabolic response, and specific CW‐related metabolites including galactose, glucosamine and hydroxycinnamate showed exclusive relationships with gm independent of the stress. These results suggest that gm responses can be attributed to specific changes in the apoplastic antioxidant system and CW metabolism, opening up more possibilities for improving photosynthesis using breeding/biotechnological strategiesThe research leading to these results has been supported by the project CTM2014‐53902‐C2‐1‐P from the Ministerio de Economía y Competitividad (MINECO, Spain) and the ERDF (FEDER). MJCM and JG acknowledge their postdoctoral contracts 2013 (FPDI‐2013‐18167 and FPDI‐2013‐17135 respectively) from MINECO (Spain). In addition, the work by Alisdair Fernie was supported by the Deutsche Forschungsgemeinschaft (DFG) within the TRR175 The Green Hub‐Central Coordinator of Acclimation in Plants.Peer reviewe

Tratamiento endovascular en el ictus isquémico agudo. Plan de Atención al Ictus en la Comunidad de Madrid

Resumen: Introducción: El tratamiento endovascular (trombólisis farmacológica intraarterial y trombectomía mecánica) en el ictus isquémico agudo se está implantando en la práctica clínica cotidiana, aunque continúa en desarrollo su investigación. En ausencia de datos suficientes que permitan fundamentar sólidamente las recomendaciones para su uso, es necesario elaborar protocolos de actuación basados en el conocimiento acumulado, así como monitorizar las actuaciones. Desarrollo: Tras revisión bibliográfica, en reuniones de trabajo de expertos para llegar a un consenso, se ha elaborado un protocolo de actuación que incluye indicaciones y contraindicaciones para la aplicación de tratamiento endovascular y recomendaciones referentes a la metodología de diagnóstico y selección de los pacientes, de los procedimientos de revascularización y manejo posterior, con el objetivo de incrementar las probabilidades de eficacia y beneficio del tratamiento y minimizar los riesgos de complicaciones y de recanalización fútil. En función del análisis de las necesidades asistenciales y los recursos disponibles se ha elaborado un sistema organizativo de colaboración interhospitalaria, para asegurar la accesibilidad al tratamiento garantizando el menor tiempo de respuesta y una relación coste/eficacia favorable. Incluye un registro prospectivo común con fines de monitorización para detectar oportunidades de mejora. Conclusiones: Para la implantación de técnicas endovasculares de tratamiento del ictus isquémico agudo es imprescindible la elaboración de protocolos de actuación basados en las evidencias disponibles y el establecimiento de sistemas adecuados de organización asistencial para garantizar el rigor y la eficacia de las actuaciones. Es necesario monitorizar los procedimientos con el fin de optimizar la metodología. Abstract: Introduction: Endovascular therapies (intra-arterial thrombolysis and mechanical thrombectomy) after acute ischaemic stroke are being implemented in the clinical setting even as they are still being researched. Since we lack sufficient data to establish accurate evidence-based recommendations for use of these treatments, we must develop clinical protocols based on current knowledge and carefully monitor all procedures. Development: After review of the literature and holding work sessions to reach a consensus among experts, we developed a clinical protocol including indications and contraindications for endovascular therapies use in acute ischaemic stroke. The protocol includes methodology recommendations for diagnosing and selecting patients, performing revascularisation procedures, and for subsequent patient management. Its objective is to increase the likelihood of efficacy and treatment benefit and minimise risk of complications and ineffective recanalisation. Based on an analysis of healthcare needs and available resources, a cooperative inter-hospital care system has been developed. This helps to ensure availability of endovascular therapies to all patients, a fast response time, and a good cost-to-efficacy ratio. It includes also a prospective register which serves to monitor procedures in order to identify any opportunities for improvement. Conclusions: Implementation of endovascular techniques for treating acute ischaemic stroke requires the elaboration of evidence-based clinical protocols and the establishment of appropriate cooperative healthcare networks guaranteeing both the availability and the quality of these actions. Such procedures must be monitored in order to improve methodology. Palabras clave: Ictus isquémico, Tratamiento endovascular, Neurointervencionismo, Trombectomía, Trombólisis, Activador tisular del plasminógeno recombinante, Keywords: Ischaemic stroke, Endovascular treatment, Neurointerventional surgery, Mechanical thrombectomy, Thrombolysis, rtP