Guidelines for the use and interpretation of assays for monitoring autophagy (2nd edition)

In 2008 we published the first set of guidelines for standardiz- ing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes..

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth

Defects in Mitochondrial Dynamics and Metabolomic Signatures of Evolving Energetic Stress in Mouse Models of Familial Alzheimer's Disease

The identification of early mechanisms underlying Alzheimer's Disease (AD) and associated biomarkers could advance development of new therapies and improve monitoring and predicting of AD progression. Mitochondrial dysfunction has been suggested to underlie AD pathophysiology, however, no comprehensive study exists that evaluates the effect of different familial AD (FAD) mutations on mitochondrial function, dynamics, and brain energetics.We characterized early mitochondrial dysfunction and metabolomic signatures of energetic stress in three commonly used transgenic mouse models of FAD. Assessment of mitochondrial motility, distribution, dynamics, morphology, and metabolomic profiling revealed the specific effect of each FAD mutation on the development of mitochondrial stress and dysfunction. Inhibition of mitochondrial trafficking was characteristic for embryonic neurons from mice expressing mutant human presenilin 1, PS1(M146L) and the double mutation of human amyloid precursor protein APP(Tg2576) and PS1(M146L) contributing to the increased susceptibility of neurons to excitotoxic cell death. Significant changes in mitochondrial morphology were detected in APP and APP/PS1 mice. All three FAD models demonstrated a loss of the integrity of synaptic mitochondria and energy production. Metabolomic profiling revealed mutation-specific changes in the levels of metabolites reflecting altered energy metabolism and mitochondrial dysfunction in brains of FAD mice. Metabolic biomarkers adequately reflected gender differences similar to that reported for AD patients and correlated well with the biomarkers currently used for diagnosis in humans.Mutation-specific alterations in mitochondrial dynamics, morphology and function in FAD mice occurred prior to the onset of memory and neurological phenotype and before the formation of amyloid deposits. Metabolomic signatures of mitochondrial stress and altered energy metabolism indicated alterations in nucleotide, Krebs cycle, energy transfer, carbohydrate, neurotransmitter, and amino acid metabolic pathways. Mitochondrial dysfunction, therefore, is an underlying event in AD progression, and FAD mouse models provide valuable tools to study early molecular mechanisms implicated in AD

Notch and Presenilin Regulate Cellular Expansion and Cytokine Secretion but Cannot Instruct Th1/Th2 Fate Acquisition

Recent reports suggested that Delta1, 4 and Jagged1, 2 possessed the ability to instruct CD4+ T cell into selection of Th1 or Th2 fates, respectively, although the underlying mechanism endowing the cleaved Notch receptor with memory of ligand involved in its activation remains elusive. To examine this, we prepared artificial antigen-presenting cells expressing either DLL1 or Jag1. Although both ligands were efficient in inducing Notch2 cleavage and activation in CD4+ T or reporter cells, the presence of Lunatic Fringe in CD4+ T cells inhibited Jag1 activation of Notch1 receptor. Neither ligand could induce Th1 or Th2 fate choice independently of cytokines or redirect cytokine-driven Th1 or Th2 development. Instead, we find that Notch ligands only augment cytokine production during T cell differentiation in the presence of polarizing IL-12 and IL-4. Moreover, the differentiation choices of naïve CD4+ T cells lacking γ-secretase, RBP-J, or both in response to polarizing cytokines revealed that neither presenilin proteins nor RBP-J were required for cytokine-induced Th1/Th2 fate selection. However, presenilins facilitate cellular proliferation and cytokine secretion in an RBP-J (and thus, Notch) independent manner. The controversies surrounding the role of Notch and presenilins in Th1/Th2 polarization may reflect their role as genetic modifiers of T-helper cells differentiation

Notch and Presenilin Regulate Cellular Expansion and Cytokine Secretion but Cannot Instruct Th1/Th2 Fate Acquisition

Recent reports suggested that Delta1, 4 and Jagged1, 2 possessed the ability to instruct CD4+ T cell into selection of Th1 or Th2 fates, respectively, although the underlying mechanism endowing the cleaved Notch receptor with memory of ligand involved in its activation remains elusive. To examine this, we prepared artificial antigen-presenting cells expressing either DLL1 or Jag1. Although both ligands were efficient in inducing Notch2 cleavage and activation in CD4+ T or reporter cells, the presence of Lunatic Fringe in CD4+ T cells inhibited Jag1 activation of Notch1 receptor. Neither ligand could induce Th1 or Th2 fate choice independently of cytokines or redirect cytokine-driven Th1 or Th2 development. Instead, we find that Notch ligands only augment cytokine production during T cell differentiation in the presence of polarizing IL-12 and IL-4. Moreover, the differentiation choices of naïve CD4+ T cells lacking γ-secretase, RBP-J, or both in response to polarizing cytokines revealed that neither presenilin proteins nor RBP-J were required for cytokine-induced Th1/Th2 fate selection. However, presenilins facilitate cellular proliferation and cytokine secretion in an RBP-J (and thus, Notch) independent manner. The controversies surrounding the role of Notch and presenilins in Th1/Th2 polarization may reflect their role as genetic modifiers of T-helper cells differentiation

First measurement of the |t|-dependence of coherent J/ψ photonuclear production

The first measurement of the cross section for coherent J/ψ photoproduction as a function of |t|, the square of the momentum transferred between the incoming and outgoing target nucleus, is presented. The data were measured with the ALICE detector in ultra-peripheral Pb–Pb collisions at a centre-of-mass energy per nucleon pair sNN=5.02TeV with the J/ψ produced in the central rapidity region |y|<0.8, which corresponds to the small Bjorken-x range (0.3−1.4)×10−3. The measured |t|-dependence is not described by computations based only on the Pb nuclear form factor, while the photonuclear cross section is better reproduced by models including shadowing according to the leading-twist approximation, or gluon-saturation effects from the impact-parameter dependent Balitsky–Kovchegov equation. These new results are therefore a valid tool to constrain the relevant model parameters and to investigate the transverse gluonic structure at very low Bjorken-x.publishedVersio

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference