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

Autophagy, Journal Year: 2016, Volume and Issue: 12(1), P. 1 - 222

Published: Jan. 2, 2016

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

Language: Английский

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Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018

Cell Death and Differentiation, Journal Year: 2018, Volume and Issue: 25(3), P. 486 - 541

Published: Jan. 23, 2018

Over the past decade, Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for definition and interpretation of cell death from morphological, biochemical, functional perspectives. Since field continues to expand novel mechanisms that orchestrate multiple pathways are unveiled, we propose an updated classification subroutines focusing mechanistic essential (as opposed correlative dispensable) aspects process. As provide molecularly oriented definitions terms including intrinsic apoptosis, extrinsic mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic death, NETotic lysosome-dependent autophagy-dependent immunogenic cellular senescence, mitotic catastrophe, discuss utility neologisms refer highly specialized instances these processes. The mission NCCD is a widely accepted nomenclature in support continued development field.

Language: Английский

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Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis

Journal of Innate Immunity, Journal Year: 2016, Volume and Issue: 8(4), P. 330 - 349

Published: Jan. 1, 2016

R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well the identification of principal metabolites hepatic microsomes, plasma, urine. Following microsomal incubation at 10 microg/mL (28 micromol/L) for 60 minutes, 86.7% parent drug was metabolized 60% this loss due to formation one particular metabolite. This identified carboxylic acid resulting from oxidation hydroxymethyl group amino alcohol substituent C2 purine core present R-roscovitine. Identification confirmed by chemical synthesis comparison an authentic sample R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor were C8-oxo-R-roscovitine N9-desisopropyl-R-roscovitine; these accounted 4.9% 2.6% parent, respectively. The same metabolic pattern observed vivo, 4.5-fold lower AUC(infinity) (38 micromol/L/h) than COOH-R-roscovitine (174 micromol/L/h). Excretion urine up 24 hours post-dosing average only 0.02% administered dose 50 mg/kg, whereas represented 65% 68% irrespective route administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) synthesized investigate if could be inhibited replacement metabolically labile protons deuterium. After minutes R-roscovitine-d9 liver COOH-R-roscovitine-d9 decreased approximately 24% compared production COOH-R-roscovitine. In addition, levels remaining 33% higher those However, several enhanced R-roscovitine-d9, suggesting that switching major carbinol pathway had occurred. Synthetic tested functional assays shown less active R-roscovitine, confirming reactions are deactivation pathways.

Language: Английский

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Elements of cancer immunity and the cancer–immune set point

Nature, Journal Year: 2017, Volume and Issue: 541(7637), P. 321 - 330

Published: Jan. 1, 2017

Language: Английский

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The selectivity of protein kinase inhibitors: a further update

Biochemical Journal, Journal Year: 2007, Volume and Issue: 408(3), P. 297 - 315

Published: Nov. 28, 2007

The specificities of 65 compounds reported to be relatively specific inhibitors protein kinases have been profiled against a panel 70–80 kinases. On the basis this information, effects that we studied in cells and other data literature, recommend use following small-molecule inhibitors: SB 203580/SB202190 BIRB 0796 used parallel assess physiological roles p38 MAPK (mitogen-activated kinase) isoforms, PI-103 wortmannin inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 with Src-I1 (Src inhibitor-1) Src family members; PD 184352 0325901 MKK1 (MAPK kinase-1) plus MKK5, Akt-I-1/2 activation PKB (protein kinase B/Akt), rapamycin TORC1 [mTOR (mammalian target rapamycin)–raptor (regulatory associated mTOR) complex], CT 99021 GSK3 (glycogen synthase 3), BI-D1870 SL0101 FMK (fluoromethylketone) RSK (ribosomal S6 kinase), D4476 CK1 (casein 1), VX680 Aurora kinases, roscovitine as pan-CDK (cyclin-dependent inhibitor. We also identified harmine potent inhibitor DYRK1A (dual-specificity tyrosine-phosphorylated -regulated 1A) vitro. results further emphasized need for considerable caution using these enzymes. Despite being widely, many analysed were too non-specific useful conclusions made, than exclude involvement particular cellular processes.

Language: Английский

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Neutrophil extracellular traps in immunity and disease

Nature reviews. Immunology, Journal Year: 2017, Volume and Issue: 18(2), P. 134 - 147

Published: Oct. 9, 2017

Language: Английский

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The molecular machinery of regulated cell death

Cell Research, Journal Year: 2019, Volume and Issue: 29(5), P. 347 - 364

Published: April 4, 2019

Cells may die from accidental cell death (ACD) or regulated (RCD). ACD is a biologically uncontrolled process, whereas RCD involves tightly structured signaling cascades and molecularly defined effector mechanisms. A growing number of novel non-apoptotic forms have been identified are increasingly being implicated in various human pathologies. Here, we critically review the current state art regarding types RCD, including necroptosis, pyroptosis, ferroptosis, entotic death, netotic parthanatos, lysosome-dependent autophagy-dependent alkaliptosis oxeiptosis. The in-depth comprehension each these lethal subroutines their intercellular consequences uncover therapeutic targets for avoidance pathogenic loss.

Language: Английский

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

Autophagy, Journal Year: 2021, Volume and Issue: 17(1), P. 1 - 382

Published: Jan. 2, 2021

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

Language: Английский

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Genetic and epigenetic fine mapping of causal autoimmune disease variants

Nature, Journal Year: 2014, Volume and Issue: 518(7539), P. 337 - 343

Published: Oct. 29, 2014

Language: Английский

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Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity

Cell, Journal Year: 2020, Volume and Issue: 183(4), P. 996 - 1012.e19

Published: Sept. 16, 2020

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches adaptive immunity at level SARS-CoV-2-specific CD4

Language: Английский

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