Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing 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 technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different 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. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Correction: Need for ICU and outcome of critically ill patients with COVID-19 and haematological malignancies: results from the EPICOVIDEHA survey

Peer reviewe

Gold-Rhodium Nanoflowers for the Plasmon-Enhanced Hydrogen Evolution Reaction under Visible Light

State of the art electrocatalysts for the hydrogen evolution reaction (HER) are based on metal nanoparticles (NPs). It has been shown that the localized surface plasmon resonance (LSPR) excitation in plasmonic NPs can be harvested to accelerate a variety of molecular transformations. This enables the utilization of visible light as an energy input to enhance HER performances. However, most metals that are active toward the HER do not support LSPR excitation in the visible or near-IR ranges. We describe herein the synthesis of gold-rhodium core-shell nanoflowers (Au@Rh NFs) that are composed of a core made up of spherical Au NPs and shells containing Rh branches. The Au@Rh NFs were employed as a model system to probe how the LSPR excitation from Au NPs can lead to an enhancement in the HER performance for Rh. Our data demonstrate that the LSPR excitation at 533 nm (and 405 nm) leads to an improvement in the HER performance of Rh, which depends on the morphological features of the Au Rh NFs, offering opportunities for optimization of the catalytic performance. Control experiments indicate that this improvement originates from the stronger interaction of Au@Rh NFs with H2O molecules at the surface, leading to an icelike configuration, which facilitated the HER under LSPR excitation.Peer reviewe

Embolism resistance drives the distribution of Amazonian rainforest tree species along hydro-topographic gradients

Species distribution is strongly driven by local and global gradients in water availability but the underlying mechanisms are not clear. Vulnerability to xylem embolism (P-50) is a key trait that indicates how species cope with drought and might explain plant distribution patterns across environmental gradients. Here we address its role on species sorting along a hydro-topographical gradient in a central Amazonian rainforest and examine its variance at the community scale. We measured P-50 for 28 tree species, soil properties and estimated the hydrological niche of each species using an indicator of distance to the water table (HAND). We found a large hydraulic diversity, covering as much as 44% of the global angiosperm variation in P-50. We show that P-50: contributes to species segregation across a hydro-topographic gradient in the Amazon, and thus to species coexistence; is the result of repeated evolutionary adaptation within closely related taxa; is associated with species tolerance to P-poor soils, suggesting the evolution of a stress-tolerance syndrome to nutrients and drought; and is higher for trees in the valleys than uplands. The large observed hydraulic diversity and its association with topography has important implications for modelling and predicting forest and species resilience to climate change221314571465CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES403764/2012-2078-201

Advanced prostate cancer with ATM Loss: PARP and ATR inhibitors

BACKGROUND: Deleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. OBJECTIVE: To characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. DESIGN, SETTING, AND PARTICIPANTS: We studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: ATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. RESULTS AND LIMITATIONS: Overall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. CONCLUSIONS: ATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. PATIENT SUMMARY: Of aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR

International evaluation of CLSI MIC/MEC distributions for definition of epidemiological cutoff values (ECVs) for species of Sporothrix identified by molecular methods

VCU Med Ctr, Richmond, VA USAUniv Fed Rural Rio de Janeiro, Seropedica, BrazilInst Nacl Infectol, Fundação Oswaldo Cruz Fiocruz, Rio De Janeiro, BrazilUniv Fed Ceara, Specialized Med Mycol Ctr, Fortaleza, Ceara, BrazilPostgrad Inst Med Educ, Chandigarh, IndiaUniv Delhi, Vallabhbhai Patel Chest Inst, Delhi, IndiaCanisius Wilhelmina Hosp, Ctr Expertise Mycol, Nijmegen, NetherlandsInst Nacl Enfermedades Infecciosas Dr C, Dept Micol, Buenos Aires, DF, ArgentinaUniv Autonoma Nuevo Leon, Monterrey, MexicoNatl Inst Communicable Dis, Johannesburg, South AfricaUniv Witwatersra, Johannesburg, South AfricaPubl Hlth England, Mycol Reference Lab, Bristol, Avon, EnglandSA Pathol, Natl Mycol Reference Ctr, Adelaide, SA, AustraliaInst Nacl Infectol Evandro Chagas, Fundação Oswaldo Cruz Fiocruz, Rio De Janeiro, BrazilUniv Fed São Paulo, São Paulo, BrazilInst Biofis, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis, Rio De Janeiro, BrazilRio Claro Labs, Inst Adolfo Lutz, São Paulo, BrazilHosp Clin Dr M Quintela, Dept Lab Clin, Montevideo, UruguayHosp Gen Mexico City, Mexico City, DF, MexicoUniv Rovira & Virgili, Mycol Unit, Sch Med, Reus, SpainInst Nacl Higiene Rafael Rangel, Caracas, VenezuelaUniv Antioquia, Grp Invest Dermatol, Medellin, ColombiaUniv Adelaide, Adelaide, SA, AustraliaUniv Fed São Paulo, São Paulo, BrazilWeb of Scienc