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

Excited-state dynamics of size-dependent colloidal TiO\u3csub\u3e2\u3c/sub\u3e-Au nanocomposites

© 2016 AIP Publishing LLC. The ultrafast excited-state dynamics of size-dependent TiO2-Au nanocomposites synthesized by reducing gold nanoclusters to the surface of colloidal TiO2 nanoparticles are studied using pump-probe transient absorption spectroscopy with 400 nm excitation pulses. The results show that the relaxation processes of the plasmon depletion band, which are described by electron-phonon and phonon-phonon scattering lifetimes, are independent of the gold nanocluster shell size surrounding the TiO2 nanoparticle core. The dynamics corresponding to interfacial electron transfer between the gold nanoclusters and the TiO2 bandgap are observed to spectrally overlap with the gold interband transition signal, and the electron transfer lifetimes are shown to significantly decrease as the nanocluster shell size increases. Additionally, size-dependent periodic oscillations are observed and are attributed to acoustic phonons of a porous shell composed of aggregated gold nanoclusters around the TiO2 core, with frequencies that decrease and damping times that remain constant as the nanocluster shell size increases. These results are important for the development of improved catalytic nanomaterial applications

Monitoring the growth dynamics of colloidal gold-silver core-shell nanoparticles using in situ

© 2019 Author(s). The growth dynamics of gold-silver core-shell (Au@Ag) nanoparticles are studied using in situ time-dependent second harmonic generation (SHG) and extinction spectroscopy to investigate the nanoparticle shell formation. The silver shell is grown by reduction of silver cations onto a 14 nm gold core using ascorbic acid in colloidal aqueous solution under varying reaction concentrations producing Au@Ag nanoparticles of final sizes ranging from 51 to 78 nm in diameter. The in situ extinction spectra show a rapid increase in intensity on the timescale of 5-6 s with blue shifting and narrowing of the plasmonic peak during the silver shell formation. The in situ SHG signals show an abrupt rise at early times of the reaction, followed by a time-dependent biexponential decrease, where the faster SHG lifetime corresponds to the timescale of the shell growth, and where the slower SHG lifetime is attributed to changes in the nanoparticle surface charge density. A large enhancement in the SHG signal at early stages of the reaction is caused by plasmonic hot spots due to the nanoparticle surface morphology, which becomes smoother as the reaction proceeds. The final extinction spectra are compared to finite-difference time-domain (FDTD) calculations, showing general agreement with experiment, where the plasmon peak red shifts and increases in spectral width as the silver shell thickness increases. These in situ SHG and extinction spectroscopy results, combined with FDTD calculations, help characterize the complicated processes involved in colloidal nanoparticle shell formation in real time for developing potential plasmon-enhanced nanomaterial applications

Monitoring the Seed-Mediated Growth of Gold Nanoparticles Using in Situ Second Harmonic Generation and Extinction Spectroscopy

© 2018 American Chemical Society. In situ second harmonic generation (SHG) coupled with extinction spectroscopy is used for real-time monitoring of seed-mediated growth dynamics of colloidal citrate-stabilized gold nanoparticles in water. The time-dependent in situ SHG results capture an early stage of the growth process where a large enhancement in the SHG signal is observed, which is attributed to the formation of plasmonic hot spots from a rough and uneven nanoparticle surface. The temporal peak in the SHG signal is followed by a decay that is fit to an exponential function to characterize the size-dependent nanoparticle growth lifetime, which varies from 0.45 to 1.7 min for final nanoparticle sizes of 66 and 94 nm, respectively. This early growth stage also corresponds to a broadening of the plasmon spectra, as monitored using time-dependent in situ extinction spectroscopy. Over the course of the seed-mediated growth reaction, the nanoparticle becomes more thermodynamically stable through surface reconstruction resulting in a smoother, more uniform surface, corresponding to lower, stable SHG signals and narrower plasmon spectra. With real-time monitoring of nanoparticle formation, in situ SHG spectroscopy combined with in situ extinction spectroscopy provides an important insight for controlling nanoparticle synthesis and surface morphology for potential nanoscale engineering of different colloidal nanomaterials

Role of p38 MAPK and Tie2 in the Pathogenesis of MDS and Their Inhibition by Dual Inhibitor ARRY-614

Abstract Abstract 2825 Myelodysplastic syndromes (MDS) are a heterogeneous collection of disorders characterized by dysfunctional bone marrow progenitors leading to peripheral cytopenias. The molecular mechanisms underlying MDS pathophysiology are unclear, but emerging data support a role for both p38 MAPK (p38) and TEK (Tie2). Over-activation of the p38 pathway and increased apoptosis have been reported in the bone marrow of MDS patients, and dysregulation of Tie2 signaling, a potential regulator of hematopoiesis via maintenance of normal hematopoietic stems cell (HSC) quiescence, is associated with worse outcome. To better understand the role of Tie2 in hematopoiesis, CD34+ cells from human cord blood were separated by multi-parameter flow cytometry and cell sorted into HSCs (i.e., CD34+, CD38−) or the more differentiated common myeloid progenitors (CMPs; i.e., CD38+, IL-3Rαlo, CD45RA−), myeloid erythroid progenitors (MEPs; i.e., CD38+, IL-3Rα−, CD45RA−), and granulocyte myeloid progenitors (GMPs; i.e., CD38+, IL-3Rαlo, CD45RA+). Expression of Tie2, Tie1, Ang-1 (Tie2 agonist) and Ang-2 (Tie2 antagonist) was determined by qPCR. The data showed that Tie2, Tie1, and Ang-1 were expressed in CD34+ cells and appeared to be regulated during differentiation, with reduced Tie2 expression observed in the GMP population. To further understand the roles of p38 and Tie2 in MDS, bone marrow and plasma samples were analyzed from a Phase 1 clinical study conducted with ARRY-614, a p38/Tie2 inhibitor, in patients with IPSS Low/Int-1 Risk MDS. At baseline, 65% (13/20) of the MDS patients showed aberrant p38 activation (≥5% phospho-p38 positive cells). Following treatment with ARRY-614, the median percent of cells positive for phospho-p38 was decreased by 85% through up to 4 cycles of treatment (∼112 days). Apoptosis in patient bone marrow samples was reduced as well (monitored by cleaved caspase-3). In cell-based assays, ARRY-614 inhibits both p38-mediated HSP27 phosphorylation (IC50 = 1 nM) and Tie2-dependent AKT phosphorylation (IC50 = 13 nM). Cellular IC50 values, corrected for plasma protein binding, and preliminary pharmacokinetic parameters were used to predict inhibition of these targets in patients. Analysis of the highest administered dose (1200 mg QD) of ARRY-614 as a powder-in-capsule (PiC) formulation in the Phase 1 clinical study predicted robust suppression of phospho-p38 (≥ 50% for 24 hours), consistent with bone marrow and plasma biomarker findings. However, partial inhibition of Tie2 (≥ 50% for 17.1 hours) was predicted. In a second Phase 1 clinical study, an optimized ARRY-614 formulation has demonstrated decreased intra- and inter-patient variability and increased peak plasma concentrations. With this new formulation, peak plasma concentrations of the 400 mg QD cohort were ∼50% higher than those of the 1200 mg QD PiC formulation cohort, possibly affording more extensive Tie2 inhibition. In summary, these observations suggest that inhibition of both p38 and Tie2 may be important for the effects of ARRY-614 in MDS patients. The ongoing Phase 1 dose escalation trial of the optimized ARRY-614 formulation may further our understanding of the contributions of these targets to the pathogenesis of MDS. Disclosures: Winski: Array Biopharma Inc.: Employment. Cable:Array Biopharma Inc.: Employment. Hogeland:Array Biopharma Inc.: Employment. Brown:Array Biopharma Inc.: Employment. Weaver:Array Biopharma Inc.: Employment. Garrus:Array Biopharma Inc.: Employment. Rhodes:Array Biopharma Inc.: Employment. Maloney:Array Biopharma Inc.: Employment. Ptaszynski:Array BioPharma: Consultancy. Chantry:Array Biopharma Inc.: Employment

Phase 1 Dose-Escalation/Expansion Study Of ARRY-614 In Patients With IPSS Low/Int-1 Risk Myelodysplastic Syndromes

Abstract Background Emerging data support a role for both p38 MAPK (p38) and Tie2 in the molecular mechanisms underlying MDS pathophysiology and suggest that inhibition of p38/Tie2 has the potential to improve hematopoiesis. ARRY-614 is an orally bioavailable, dual inhibitor of p38 and Tie2 that has demonstrated multi-lineage hematologic improvement (HI) and acceptable tolerability with a powder-in-capsule (PIC) formulation. However, due to suboptimal PK properties of the PIC, a Phase 1 study in patients (pts) with MDS was initiated to evaluate an optimized formulation of ARRY-614. Methods The aims of this study were to determine the MTD, establish a recommended Phase 2 dose and evaluate the safety, PK profile and PD effects of a formulated capsule of ARRY-614 administered once daily (QD) and twice daily (BID) in continuous 28-day cycles. Pts with IPSS low/intermediate-1 risk MDS, ≥ 1 cytopenia (per IWG 2006 criteria) and adequate ECOG performance status, hepatic and renal function were eligible. Prior therapy for MDS was permitted; CMML per FAB criteria and secondary MDS were allowed. The study used a 3+3 dose escalation design modified to allow additional pt accrual at lower dose levels already determined to be tolerable. Additional expansions were permitted at selected dose levels to determine the recommended Phase 2 dose. Response was assessed using IWG 2006 criteria. In addition, platelet transfusion dependent pts (TD, ≥ 1 platelet transfusion within 8 weeks prior to starting treatment) were assessed for transfusion independence (TI) or transfusion reduction (TR, 50% decrease). Results At the time of this analysis (01 May 2013), 62 pts with IPSS low (n = 16) or intermediate-1 (n = 46) risk MDS were enrolled (median age 72 years, range 46-85). Patients had received a median of 3 prior regimens (range 0-6); 5 pts had no prior therapy. Prior regimens included: hypomethylating agents (HMAs; 85%), erythropoiesis-stimulating agents (65%) and lenalidomide (32%). ARRY-614 was administered at doses of 200-1000 mg QD (n=50) and 100-200 mg BID (n=12), with a median treatment duration of 13 weeks (range 1 day to 65 weeks). The MTD of the QD schedule was determined to be 800 mg QD after 2/6 pts experienced dose limiting toxicities (DLTs) of atrial fibrillation at the 1000 mg QD dose. The 200 mg BID dose was deemed not tolerable after 3/8 pts developed DLTs of Grade 3 rash (2 pts) and Grade 3 ECG QT prolongation (1 pt). The most common treatment-related adverse events (> 5% of total pts) included rash, nausea, atrial fibrillation, decreased appetite, fatigue, asthenia and vomiting. The majority of these events were mild or moderate. Nineteen pts remain on study (median time on treatment of 10 weeks). To date, 12 of 54 (22%) evaluable pts and 9 of 31 (29%) pts on treatment ≥ 16 weeks experienced HI per IWG 2006 criteria and/or achieved platelet TR or TI. Responses were observed at all dose levels. HI-E, HI-P, and HI-N responses were observed in 6% (3/49), 21% (7/34) and 31% (5/16) of the pts, respectively. In addition, of the 12 pts who were platelet TD, a total of 50% (6/12) achieved at least a TR, and 33% (4/12) achieved complete TI. All pts who experienced a response had received prior treatment with an HMA. ARRY-614 and its metabolite AR00451575 demonstrated increasing exposure with increasing dose, which was approximately dose proportional based on Cmax and AUC. At doses ≥ 400 mg QD, the plasma concentrations exceeded the predicted p38 and Tie2 IC50 values (113 ng/mL and 1150 ng/mL, respectively, plasma protein binding corrected). Preliminary biomarker analyses demonstrated primary target inhibition with persistent reduction in phospho-p38 levels in the bone marrow as compared to baseline during the course of treatment. In addition to primary target inhibition, observed decreases in plasma chemokines during the course of treatment were consistent with functional inhibition of p38. Conclusions The ARRY-614 formulated capsule demonstrates good tolerability and on-target activity at doses up to 800 mg QD, as well as a more favorable PK profile than the previous formulation. In addition, HI responses were seen across dose levels and across lineages in pts previously treated with HMAs, suggesting ARRY-614 may provide a treatment option for pts with lower-risk disease failing standard therapies. Next steps will be to explore the 400 mg and 800 mg dosing schedules further. Disclosures: Garcia-Manero: Array BioPharma: steering committee participation Other. Sekeres:Amgen: data safety monitoring board, data safety monitoring board Other; Celgene: advisory board Other. List:Array BioPharma: Member of advisory board on MDS Other. Khoury:Array BioPharma: steering committee participation Other. Kantarjian:Array BioPharma: Research Funding. Cable:Array BioPharma: Employment. Guthrie:Array BioPharma: Employment. Hogeland:Array BioPharma: Employment. Ptaszynski:Array BioPharma: Employment. Maloney:Array BioPharma: Employment. Corson:Array BioPharma: Employment. Komrokji:Array BioPharma: steering committee participation Other