Assessing Tolerance to Heavy-Metal Stress in Arabidopsis thaliana Seedlings

The deposited book chapter is a post-print version and has been submitted to peer review.The deposited book chapter version contains attached the supplementary materials within the pdf.This publication hasn't any creative commons license associated.The deposited book chapter is part of the book series: "Environmental Responses in Plants: Methods and Protocols" (pp.197-208) published by Springer.Heavy-metal soil contamination is one of the major abiotic stress factors that, by negatively affecting plant growth and development, severely limit agricultural productivity worldwide. Plants have evolved various tolerance and detoxification strategies in order to cope with heavy-metal toxicity while ensuring adequate supply of essential micronutrients at the whole-plant as well as cellular levels. Genetic studies in the model plant Arabidopsis thaliana have been instrumental in elucidating such mechanisms. The root assay constitutes a very powerful and simple method to assess heavy-metal stress tolerance in Arabidopsis seedlings. It allows the simultaneous determination of all the standard growth parameters affected by heavy-metal stress (primary root elongation, lateral root development, shoot biomass, and chlorophyll content) in a single experiment. Additionally, this protocol emphasizes the tips and tricks that become particularly useful when quantifying subtle alterations in tolerance to a given heavy-metal stress, when simultaneously pursuing a large number of plant lines, or when testing sensitivity to a wide range of heavy metals for a single line.Fundação para a Ciência e a Tecnologia grants: (EXPL/AGR-PRO/1013/2013, SFRH/BPD/44640/2008); GREEN-it "Bioresources for Sustainability": (UID/Multi/04551/2013).info:eu-repo/semantics/publishedVersio

RNA helicase EIF4A1-mediated translation is essential for the GC response

EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5' untranslated regions. However, very little is known of their roles in nonmalignant cells. Using mouse models to delete Eif4a1, Eif4b or Eif4h in B cells, we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. After B cell activation in vitro, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression, and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable, whereas inhibition of EIF4A1 and EIF4A2 by Hippuristanol treatment induces cell death.</p

RNA helicase EIF4A1-mediated translation is essential for the GC response

EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5' untranslated regions. However, very little is known of their roles in nonmalignant cells. Using mouse models to delete Eif4a1, Eif4b or Eif4h in B cells, we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. After B cell activation in vitro, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression, and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable, whereas inhibition of EIF4A1 and EIF4A2 by Hippuristanol treatment induces cell death.</p

Mapping interactions between the sustainable development goals: lessons learned and ways forward

Pursuing integrated research and decision-making to advance action on the sustainable development goals (SDGs) fundamentally depends on understanding interactions between the SDGs, both negative ones (“trade-offs”) and positive ones (“co-benefits”). This quest, triggered by the 2030 Agenda, has however pointed to a gap in current research and policy analysis regarding how to think systematically about interactions across the SDGs. This paper synthesizes experiences and insights from the application of a new conceptual framework for mapping and assessing SDG interactions using a defined typology and characterization approach. Drawing on results from a major international research study applied to the SDGs on health, energy and the ocean, it analyses how interactions depend on key factors such as geographical context, resource endowments, time horizon and governance. The paper discusses the future potential, barriers and opportunities for applying the approach in scientific research, in policy making and in bridging the two through a global SDG Interactions Knowledge Platform as a key mechanism for assembling, systematizing and aggregating knowledge on interactions

Morbidity from in-hospital complications is greater than treatment failure in patients with Staphylococcus aureus bacteraemia

Background: Various studies have identified numerous factors associated with poor clinical outcomes in patients with Staphylococcus aureus bacteraemia (SAB). A new study was created to provide deeper insight into in-hospital complications and risk factors for treatment failure. Methods: Adult patients hospitalised with Staphylococcus aureus bacteraemia (SAB) were recruited prospectively into a multi-centre cohort. The primary outcome was treatment failure at 30 days (composite of all-cause mortality, persistent bacteraemia, or recurrent bacteraemia), and secondary measures included in-hospital complications and mortality at 6- and 12-months. Data were available for 222 patients recruited from February 2011 to December 2012. Results: Treatment failure at 30-days was recorded in 14.4% of patients (30-day mortality 9.5%). Multivariable analysis predictors of treatment failure included age > 70 years, Pitt bacteraemia score ≥ 2, CRP at onset of SAB > 250 mg/L, and persistent fevers after SAB onset; serum albumin at onset of SAB, receipt of appropriate empiric treatment, recent healthcare attendance, and performing echocardiography were protective. 6-month and 12-month mortality were 19.1% and 24.2% respectively. 45% experienced at least one in-hospital complication, including nephrotoxicity in 19.5%. Conclusions: This study demonstrates significant improvements in 30-day outcomes in SAB in Australia. However, we have identified important areas to improve outcomes from SAB, particularly reducing renal dysfunction and in-hospital treatment-related complications

Prediction Model to Identify Patients at Risk of 30-Day Treatment Failure in Patients With Staphylococcus aureus Bacteremia

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Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation

Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model syste

Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR

Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen

microRNA-132 regulates gene expression programs involved in microglial homeostasis

microRNA-132 (miR-132), a known neuronal regulator, is one of the most robustly downregulated microRNAs (miRNAs) in the brain of Alzheimer's disease (AD) patients. Increasing miR-132 in AD mouse brain ameliorates amyloid and Tau pathologies, and also restores adult hippocampal neurogenesis and memory deficits. However, the functional pleiotropy of miRNAs requires in-depth analysis of the effects of miR-132 supplementation before it can be moved forward for AD therapy. We employ here miR-132 loss- and gain-of-function approaches using single-cell transcriptomics, proteomics, and in silico AGO-CLIP datasets to identify molecular pathways targeted by miR-132 in mouse hippocampus. We find that miR-132 modulation significantly affects the transition of microglia from a disease-associated to a homeostatic cell state. We confirm the regulatory role of miR-132 in shifting microglial cell states using human microglial cultures derived from induced pluripotent stem cells