Down-regulation of a pectin acetylesterase gene modifies strawberry fruit cell wall pectin stracture and increases fruit firmness

Antisense-mediated down-regulation of several fruit-specific genes has previously demonstrated how the cell wall disassembly in strawberry fruit is mediated by a series of enzymes that act sequentially (Posé et al. 2011). An interesting example, the silencing of the polygalacturonase gene FaPG1, was also related with a significant increase of the post-harvest strawberry fruit firmness (Posé et al. 2013). Our research group has isolated a pectin acetylesterase gene, FaPAE1, which expression is enhanced during strawberry ripening. The main goal of this work was to elucidate the role of the degree of acetylation in cell wall integrity and fruit firmness through the antisense-mediated down-regulation of FaPAE1 in strawberry plants. Several transgenics lines were generated and 5 of them produced fruits 5-15% firmer than controls. Cell wall from ripe fruits was isolated from two independent transgenic lines and a control line, and sequentially extracted with different solvents (PAW, H2O, CDTA, Na2CO3). Modifications in fraction yield, its sugar composition and the degree of acetylation in each fraction were determined. Higher amounts of CDTA and Na2CO3 fractions were obtained in transgenic fruits, suggesting a decreased pectin solubilization as results of FaPAE1 silencing. Accordingly, the degree of acetylation of the Na2CO3-soluble pectins was greater in the transgenic lines than the control, but the opposite result was found in pectins from the CDTA fraction. These results suggest that PAE is preferentially active in pectis that are tightly bound to the cellulose-hemicellulose network and its activity could reduce the complexity of the cell wall structure, allowing that other hydrolytic enzymes could access the pectin chains. Thus, the increased fruit firmness observed in the transgenic FaPAE1 lines could be attributed to the direct effect of the silencing of the PAE enzyme and also to the indirect effect that the increase of the degree of acetylation of pectins has on the activity of other enzymes involved in the cell wall degradation. * Posé et al. (2011). Genes, Genomes and Genomics, 5 (Special Issue 1):40-48 * Posé et al. (2013). Plant Physiology, 150: 1022-1032 We acknowledge support from the Spanish Ministry of Economy and competitivity and Feder EU Funds (grant reference AGL2011-24814), FPI fellowships support for SP (BES-2006-13626) and CP (BES-2009027985), and grant "Ramón y Cajal" support for AJMA (RYC-2011-08839).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Rhamnogalacturonase lyase gene downregulation in strawberry and its potential on mechanical fruit properties

Strawberry softening is one of the main factors that reduces fruit quality and leads to economically important losses. Textural changes during fruit ripening are mainly due to the dissolution of middle lamellae, a reduction in cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Functional studies of genes encoding pectinase enzymes (polygalacturonase, pectate lyase and -galactosidase) support a key role of pectin disassembly in strawberry softening. Evidence that RG-I may play an important role in strawberry texture has been obtained from the transient silencing of a RG-lyase gene. Pectins are major components of fruit cell walls and highly dynamic polysaccharides, but due to their heterogeneity the precise relation between the structures and functions is incomplete. In this work, stable transgenic strawberry lines with a rhamnogalacturonate lyase gene (FaRGLyase1) down-regulated have been analyzed. Several transgenic lines showing more than 95% silencing of FaRGLyase1 displayed fruit firmness values higher than control. Cell walls from these lines were extracted and analyzed by ELISA and Epitope Detection Chromatography (EDC). This last technique is based on the detection of specific cell wall oligosaccharide epitopes and provides information on sub-populations of pectins containing homogalacturonan and RG-I domains, but also reveals potential links with other cell wall polysaccharides such as xyloglucan. The results obtained indicate that the silencing of FaRGLyase1 reduces degradation of RG-I backbones, but also homogalacturonan, in cell walls, especially in pectin fractions covalently bound to the cell wall. These changes contribute to the increased firmness of transgenic fruits.This research was supported by FEDER EU Funds and the Ministerio de Economía y Competitividad of Spain (grant reference AGL2014-55784-C2), a Marie Curie IEF within the 7th European Community Framework Programme (reference: PIEF-2013-625270) for SP and a FPI fellowship (BES-2015-073616) to support PR-V. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Perceptions of Parents’ Attitudes towards Activism and its Impacts on the Political Behavior of Undergraduate Student Activists

This paper seeks to study student activism by focusing on how parents\u27 perception of their activism affects their political actions and decisions. Six students from three undergraduate student activist organizations across Metro Manila were purposely sampled to participate individually in a semi-structured interview developed around the concept of attitudes categorized into three components: the behavioral, the cognitive, and the affective. Thematic analysis reveals that parents generally tend to negatively perceive activism, which does not necessarily affect students\u27 decision to continue activism despite accompanying emotional burdens. The findings ascertain that parents employ various parenting styles from rational to authoritarian and emotions such as worry and rage in reaction to their child’s activism. Moreover, the participants were found to apply values such as tireless persuasion and discretion to negotiate conflicting perceptions of their activism with their parents

Isolation and transfection of strawverry protoplasts for gene editing

Strawberry is the most economically important soft fruit. The improvement of the organoleptic qualities of ripe fruit and the postharvest shelf life are main objectives of strawberry breeding programs. Fruit softening is mainly due to the disassembly of cell walls and the dissolution of middle lamella. In strawberry, functional analyses of genes encoding polygalacturonases (PGs) indicate that these enzymes play a key role in fruit softening, i.e. the antisense downregulation of PG genes FaPG1 or FaPG2 increased fruit firmness and postharvest shelf life (Paniagua et al., 2020). These results suggest that PG encoding genes are excellent targets for gene editing to improve strawberry fruit quality. Transfection of protoplasts with CRISPR/Cas9 ribonucleoprotein complexes is currently being explored in many species to produce DNA-free edited plants. In this research, a protocol for strawberry protoplasts transfection has been optimized with the final goal of producing non-transgenic strawberry plants with the FaPG1 gene edited. Protoplasts were isolated from 9 weeks old in vitro grown plants of Fragaria x ananassa, cv. ‘Chandler’, micropropagated in Murashige and Skoog (MS) medium supplemented with 2 mg/L of BA. Protoplast extraction and purification was performed as described by Barceló et al. (2019). Using this protocol, a yield of 1 x 105 protoplast/g fresh tissue was obtained and nearly 50-70% of them were viable. Protoplasts were transfected with the plasmid pHBT-sGFP(S65T)-NOS using a PEG-mediated transformation system, as reported by Yoo et al. (2007). To improve the efficiency of protoplast transfection, different variables were evaluated: PEG concentration, time of incubation on PEG and DNA concentration. At 48 h after transfection, the highest percentage of protoplasts showing GFP expression, 18%, was obtained with 15 minutes incubation in 20% of PEG and 5 µg of DNA

Right versus left radial artery access for coronary procedures: an international collaborative systematic review and meta-analysis including 5 randomized trials and 3210 patients

BACKGROUND: Radial artery access is a mainstay in the diagnosis and treatment of coronary artery disease. However, there is uncertainty on the comparison of right versus left radial access for coronary procedures. We thus undertook a systematic review and meta-analysis comparing right versus left radial access for coronary diagnostic and interventional procedures. METHODS: Pertinent studies were searched in CENTRAL, Google Scholar, MEDLINE/PubMed, and Scopus, together with international conference proceedings. Randomized trials comparing right versus left radial (or ulnar) access for coronary diagnostic or interventional procedures were included. Risk ratios (RR) and weighted mean differences (WMD) were computed to generate point estimates (95% confidence intervals). RESULTS: A total of 5 trials (3210 patients) were included. No overall significant differences were found comparing right versus left radial access in terms of procedural time (WMD=0.99 [-0.53; 2.51]min, p=0.20), contrast use (WMD=1.71 [-1.32; 4.74]mL, p=0.27), fluoroscopy time (WMD=-35.79 [-3.54; 75.12]s, p=0.07) or any major complication (RR=2.00 [0.75; 5.31], p=0.49). However, right radial access was fraught with a significantly higher risk of failure leading to cross-over to femoral access (RR=1.65 [1.18; 2.30], p=0.003) in comparison to left radial access. CONCLUSIONS: Right and left radial accesses appear largely similar in their overall procedural and clinical performance during transradial diagnostic or interventional procedures. Nonetheless, left radial access can be recommended especially during the learning curve phase to reduce femoral cross-overs

Continuum limit of amorphous elastic bodies: A finite-size study of low frequency harmonic vibrations

The approach of the elastic continuum limit in small amorphous bodies formed by weakly polydisperse Lennard-Jones beads is investigated in a systematic finite-size study. We show that classical continuum elasticity breaks down when the wavelength of the sollicitation is smaller than a characteristic length of approximately 30 molecular sizes. Due to this surprisingly large effect ensembles containing up to N=40,000 particles have been required in two dimensions to yield a convincing match with the classical continuum predictions for the eigenfrequency spectrum of disk-shaped aggregates and periodic bulk systems. The existence of an effective length scale \xi is confirmed by the analysis of the (non-gaussian) noisy part of the low frequency vibrational eigenmodes. Moreover, we relate it to the {\em non-affine} part of the displacement fields under imposed elongation and shear. Similar correlations (vortices) are indeed observed on distances up to \xi~30 particle sizes.Comment: 28 pages, 13 figures, 3 table

The BRENDA Tissue Ontology (BTO): the first all-integrating ontology of all organisms for enzyme sources

BTO, the BRENDA Tissue Ontology (http://www.BTO.brenda-enzymes.org) represents a comprehensive structured encyclopedia of tissue terms. The project started in 2003 to create a connection between the enzyme data collection of the BRENDA enzyme database and a structured network of source tissues and cell types. Currently, BTO contains more than 4600 different anatomical structures, tissues, cell types and cell lines, classified under generic categories corresponding to the rules and formats of the Gene Ontology Consortium and organized as a directed acyclic graph (DAG). Most of the terms are endowed with comments on their derivation or definitions. The content of the ontology is constantly curated with ∼1000 new terms each year. Four different types of relationships between the terms are implemented. A versatile web interface with several search and navigation functionalities allows convenient online access to the BTO and to the enzymes isolated from the tissues. Important areas of applications of the BTO terms are the detection of enzymes in tissues and the provision of a solid basis for text-mining approaches in this field. It is widely used by lab scientists, curators of genomic and biochemical databases and bioinformaticians. The BTO is freely available at http://www.obofoundry.org

PARE0009 COMMUNITY ADVISORY BOARD INPUT CAN MAKE LAY SUMMARIES OF CLINICAL TRIAL RESULTS MORE UNDERSTANDABLE

Background:Under European Union (EU) Clinical Trial regulations,1clinical research sponsors (CRSs) must ensure all studies performed in the EU are accompanied by a trial summary for laypersons, published within 1 year of study completion. These lay summaries should disseminate clinical trial results in an easy-to-understand way for trial participants, patient and caregiver communities, and the general public. The European Patients Forum (EPF)2and European Patients' Academy on Therapeutic Innovation (EUPATI)3encourage CRSs to engage with patient organisations (POs) in the development of lay summaries. This recognises the patients' contribution to clinical research and supports the development of patient-focused material.Objectives:We share learnings from a collaboration between scleroderma POs and a CRS to create the SENSCIS® trial (NCT02597933) written and video lay summaries.Methods:A community advisory board (CAB), comprising representatives from 11 scleroderma POs covering a range of countries/regions, was formed based on the EURORDIS charter for collaboration in clinical research.4Through three structured meetings, over a seven-month period, the CAB provided advice on lay summary materials (written and video) drafted by the CRS' Lay Summary Group (Fig. 1). At each review cycle, the CAB advice was addressed to make content more understandable and more relevant for patients and the general public.Results:The CAB advised that the existence of lay summaries is not well known in the patient community and also recommended the development of trial-specific lay summary videos to further improve understandability of the clinical trial results for the general public. Videos are a key channel of communication, enabling access to information for people with specific health needs and lower literacy levels. Following CAB advice, the CRS developed a stand-alone video entitled"What are lay summaries?"and a trial-specific lay summary video. Revisions to lay summary content (written and video) included colour schemes, iconography and language changes to make content more understandable. For videos, adjustments to animation speed, script and voiceover were implemented to improve clarity and flow of information (Fig. 2). Approved final versions of lay summary materials are publicly available on the CRS website. Translation into languages representing trial-site countries is in progress to widen access to non-English speakers and, where possible, local versions are being reviewed by the patient community.Conclusion:Structured collection and implementation of CAB advice can make lay summary materials more understandable for the patient community and wider general public.References:[1]EU. Summaries of clinical trial results for laypersons. 2018[2]EPF. EPF position: clinical trial results – communication of the lay summary. 2015[3]EUPATI. Guidance for patient involvement in ethical review of clinical trials. 2018[4]EURORDIS. Charter for Collaboration in Clinical Research in Rare Diseases. 2009Disclosure of Interests:Joep Welling Speakers bureau: Four times as a patient advocate for employees of BII and BI MIDI with a fixed amount of € 150,00 per occasion., Annelise Roennow: None declared, Maureen Sauvé Grant/research support from: Educational grants from Boehringer Ingelheim and Janssen., EDITH BROWN: None declared, Ilaria Galetti: None declared, Alex Gonzalez Consultant of: Payment made to the patient organisation (Scleroderma Research Foundation) for participation in advisory boards, Alexandra Paula Portales Guiraud: None declared, Ann Kennedy Grant/research support from: AS FESCA aisbl, Catarina Leite: None declared, Robert J. Riggs: None declared, Alison Zheng Grant/research support from: We get grants from Lorem Vascular; BI China,; Jianke Pharmaceutical Co., Ltd.; Kangjing Biological Co., Ltd.; COFCO Coca-Cola to organize national scleroderma meetings, offer patients service, holding academic meetings and other public activities, there is also a small part of the grants used to pay the workers in our organization., Consultant of: I worked as a paid consultant for BI. Pay-per-job., Speakers bureau: I was invited once to be a speaker at BI China's internal meeting and they paid me., Matea Perkovic Popovic: None declared, Annie Gilbert Consultant of: I have worked as a paid consultant with BI International for over 3 years, since Sept 2016., Lizette Moros Employee of: Lizette Moros is an employee of Boehringer Ingelheim, Kamila Sroka-Saidi Employee of: Paid employee of Boehringer Ingelheim., Thomas Schindler Employee of: Employee of Boehringer Ingelheim Pharma, Henrik Finnern Employee of: Paid employee of Boehringer Ingelheim