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

2009

ARTICLES 913 Acute Myeloid Leukemia Introduction The NPM1 gene is frequently altered in hematologic malignancies. 2 NPM1-mutated AML has distinctive clinicopathological and molecular features. Most notably, the mutation is closely associated with normal karyotype and relatively favorable prognosis in the absence of FLT3-internal tandem duplication (ITD). 3 While genotyping the NPM1 mutations, a polymorphic nucleotide T deletion (delT) at position 1,146 of the NPM1 3'-untranslated region (UTR) was reported in 60%-70% of AML patients. 6,7 Here, we revealed that the homozygous state of the NPM1 delT polymorphism had important clinical and biological implications in AML involving an illegitimate microRNA (miRNA) regulation. Design and Methods Patients We analyzed 149 adult and 70 childhood patients with de novo AML; therapy-related AML or AML arising from a prior myelodysplastic syndrome were excluded. Patients' characteristics are shown in Online Supplementary Table S1. All patients gave informed consent for the study, which was approved by the Joint CUHK-NTEC Clinical Research Ethics Committee and was conducted in accordance with the Declaration of Helsinki. Patients with acute promyelocytic leukemia (APL) were excluded for survival analysis. Overall, complete survival data were available from 93 adult and 61 childhood patients with non-APL who had received treatment. All the 93 adult patients were treated with the standard cytarabine plus daunorubicin '7+3' induction chemotherapy regimen. Normal bone marrow (BM) and peripheral blood samples were obtained from healthy donors who had no prior history of malignancy. Cell culture Cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Nucleophosmin, encoded by NPM1, is a haploinsufficient suppressor in hematologic malignancies. NPM1 mutations are mostly found in acute myeloid leukemia patients with normal karyotype and associated with favorable prognosis. A polymorphic nucleotide T deletion with unknown significance is present in the NPM1 3'-untranslated region. Here, we showed that the homozygous nucleotide T deletion was associated with adverse outcomes and could independently predict shortened survival in patients with de novo acute myeloid leukemia. Mechanistically, we demonstrated that the nucleotide T deletion created an illegitimate binding NPM1 for miR-337-5p, which was widely expressed in different acute myeloid leukemia subtypes and inhibited NPM1 expression. Accordingly, NPM1 levels were found to be significantly reduced and correlated with miR-337-5p levels in patients carrying a homozygous nucleotide T-deletion genotype. Together, our findings uncover a microRNAmediated control of NPM1 expression that contributes to disease heterogeneity and suggest additional prognostic values of NPM1 in acute myeloid leukemia. A polymorphism in the 3'-untranslated region of the ABSTRACT Cytogenetic and mutational studies Cytogenetics were classified into favorable, intermediate, and adverse according to the UK MRC classification

Inhibiting Interleukin 11 Signaling Reduces Hepatocyte Death and Liver Fibrosis, Inflammation, and Steatosis in Mouse Models of Non-Alcoholic Steatohepatitis.

10.1053/j.gastro.2019.05.002Gastroenterolog

The Clickable Guard Cell, Version II: Interactive Model of Guard Cell Signal Transduction Mechanisms and Pathways

Guard cells are located in the leaf epidermis and pairs of guard cells surround and form stomatal pores, which regulate CO2 influx from the atmosphere into leaves for photosynthetic carbon fixation. Stomatal guard cells also regulate water loss of plants via transpiration to the atmosphere. Signal transduction mechanisms in guard cells integrate a multitude of different stimuli to modulate stomatal apertures. Stomata open in response to light. Stomata close in response to drought stress, elevated CO2, ozone and low humidity. In response to drought, plants synthesize the hormone abscisic acid (ABA) that triggers closing of stomatal pores. Guard cells have become a highly developed model system for dissecting signal transduction mechanisms in plants and for elucidating how individual signaling mechanisms can interact within a network in a single cell. Many new findings have been made in the last few years. This chapter is an update of an electronic interactive chapter in the previous edition of The Arabidopsis Book (Mäser et al. 2003). Here we focus on mechanisms for which genes and mutations have been characterized, including signaling components for which there is substantial signaling, biochemical and genetic evidence. Ion channels have been shown to represent targets of early signal transduction mechanisms and provide functional signaling and quantitative analysis points to determine where and how mutations affect branches within the guard cell signaling network. Although a substantial number of genes and proteins that function in guard cell signaling have been identified in recent years, there are many more left to be identified and the protein-protein interactions within this network will be an important subject of future research. A fully interactive clickable electronic version of this publication can be accessed at the following web site: http://www-biology.ucsd.edu/labs/schroeder/clickablegc2/. The interactive clickable version includes the following features: Figure 1. Model for the roles of ion channels in ABA signaling.Figure 2. Blue light signaling pathways in guard cells.Figure 3. ABA signaling pathways in guard cells. Figure 1 is linked to explanations that appear upon mouse-over. Figure 2 and Figure 3 are clickable and linked to info boxes, which in turn are linked to TAIR, to relevant abstracts in PubMed, and to updated background explanations from Schroeder et al (2001), used with permission of Annual Reviews of Plant Biology

Homologous recombination DNA repair defects in PALB2-associated breast cancers

Abstract Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD

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

International audienceIn 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