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

Conducting Expeditionary Operations in the Contested Littorals Final Review [video]

Conducting Expeditionary Operations in the Contested Littorals Final Review, Systems Engineering Analysis Integrated Project Team 21B. Brief presented by Major Juan Carleton (USA)

High-resolution infrared spectrum of the ν1 band of OClO

The infrared absorption spectrum of the ν1 band of OClO (care must be taken to distinguish it from ClOO, which also exists) has been recorded in the 950 cm-1 region, with a Fourier transform infrared spectrometer with an instrumental resolution of ∼0.004 cm-1. Most lines appear as doublets owing to the spin-rotation interaction present in this molecule. Around 2800 lines have been assigned for the 35ClO2 species and ∼800 for the 37ClO2 species. In addition, a number of lines of the >hot band>, ν1 + ν2 - ν2, have been assigned for the 35ClO2 species. Effective rotational and spin-rotational spectroscopic constants have been obtained for the ground and the v1 = 1 vibrational states of 35ClO2 and 37ClO2 and the corresponding band origins have also been determined. Fermi resonance between the 2ν2 and ν1 bands has been found to be negligible; however, a weak resonance between the Ka = 7 levels of v1 = 1 and the Ka = 9 levels of v2 = 2 has been observed. The measured integrated ν1 band strength is 87 ± 6 cm-2 atm-1 at 300 K. © 1991.supported by the DGICYT, Project PB87-0273; supported in part by the NASA Upper Atmospheric Division Program; Atmospheric Science Division of NASA ; PFPI grant, Spanish GovernmentPeer Reviewe

Solvent-dependent switch of ligand donor ability and catalytic activity of ruthenium(II) complexes containing pyridinylidene amide (PYA) n-heterocyclic carbene hybrid ligands

Chelating ligands incorporating both N-[1-alkylpyridin-4(1H)-ylidene]amide (PYA) and N-heterocyclic carbene (NHC) donor sites were prepared and used for the synthesis of ruthenium(II) complexes. Cyclic voltammetry, NMR, and UV–vis spectroscopy of the complexes indicate a solvent-dependent contribution of the limiting resonance structures associated with the ligand in solution. The neutral pyridylidene imine structure is more pronounced in apolar solvents (CH2Cl2), while the mesoionic pyridinium amide form is predominant in polar solvents (MeOH, DMSO). The distinct electronic properties of these hybrid PYA-NHC ligands in different solvents have a direct influence on the catalytic activity of the ruthenium center, e.g., in the dehydrogenation of benzyl alcohol to benzaldehyde. The activity in different solvents qualitatively correlates with the solvent permittivity.European Research CouncilUniversity of Aucklan

Branching out to gain control: how the pre-TCR is linked to multiple functions

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRβ locus, and further differentiation

Morphogenetic action through flux-limited spreading

A central question in biology is how secreted morphogens act to induce different cellular responses within a group of cells in a concentration-dependent manner. Modeling morphogenetic output in multicellular systems has so far employed linear diffusion, which is the normal type of diffusion associated with Brownian processes. However, there is evidence that at least some morphogens, such as Hedgehog (Hh) molecules, may not freely diffuse. Moreover, the mathematical analysis of such models necessarily implies unrealistic instantaneous spreading of morphogen molecules, which are derived from the assumptions of Brownian motion in its continuous formulation. A strict mathematical model considering Fick's diffusion law predicts morphogen exposure of the whole tissue at the same time. Such a strict model thus does not describe true biological patterns, even if similar and attractive patterns appear as results of applying such simple model. To eliminate non-biological behaviors from diffusion models we introduce flux-limited spreading (FLS), which implies a restricted velocity for morphogen propagation and a nonlinear mechanism of transport. Using FLS and focusing on intercellular Hh-Gli signaling, we model a morphogen gradient and highlight the propagation velocity of morphogen particles as a new key biological parameter. This model is then applied to the formation and action of the Sonic Hh (Shh) gradient in the vertebrate embryonic neural tube using our experimental data on Hh spreading in heterologous systems together with published data. Unlike linear diffusion models, FLS modeling predicts concentration fronts and the evolution of gradient dynamics and responses over time. In addition to spreading restrictions by extracellular binding partners, we suggest that the constraints imposed by direct bridges of information transfer such as nanotubes or cytonemes underlie FLS. Indeed, we detect and measure morphogen particle velocity in such cell extensions in different systems. © 2013 Elsevier B.V.Projects MTM2008-05271, MTM2011-23384; BFU2008-03320/BMC,BFU2011-25987; Consolider Program CSD2007-00008 MICINN; MarieCurie FP6 (RTN035528-2); FP7 (ITN238186) Fundación Areces; Junta de Andalucía ProjectP08-FQM-4267; Swiss National Science Foundation (SNFprofessorgrantnum-berPP0033-119169andSinergiagrantCRSII3-127456); University of Geneva; the European Research Council (contractnumberERC-2009-StG-243344-NEUROCHEMS); Leenaards foundationand; the European Molecular Biology Organization (younginvestigatorprogram)Peer Reviewe

On flux-limited morphogenesis. Reply to the comments on >Morphogenetic action through flux-limited spreading>.

Spanish MINECO; Marie Curie FP6 (RTN035528-2); FP7(ITN238186); Fundación Areces; Juntad e Andalucía (Project P08-FQM-4267); Swiss National Science Foundation; the University of Geneva; European Research Council; Leenaards foundation; European Molecular Biology Organization (younginvestigatorprogram); Cantonet Republique de GenèvetoPeer Reviewe

Effect of Nivolumab vs Bevacizumab in patients with recurrent glioblastoma: the checkmate 143 phase 3 randomized clinical trial

Importance: Clinical outcomes for glioblastoma remain poor. Treatment with immune checkpoint blockade has shown benefits in many cancer types. To our knowledge, data from a randomized phase 3 clinical trial evaluating a programmed death-1 (PD-1) inhibitor therapy for glioblastoma have not been reported. Objective: To determine whether single-agent PD-1 blockade with nivolumab improves survival in patients with recurrent glioblastoma compared with bevacizumab. Design, Setting, and Participants: In this open-label, randomized, phase 3 clinical trial, 439 patients with glioblastoma at first recurrence following standard radiation and temozolomide therapy were enrolled, and 369 were randomized. Patients were enrolled between September 2014 and May 2015. The median follow-up was 9.5 months at data cutoff of January 20, 2017. The study included 57 multicenter, multinational clinical sites. Interventions: Patients were randomized 1:1 to nivolumab 3 mg/kg or bevacizumab 10 mg/kg every 2 weeks until confirmed disease progression, unacceptable toxic effects, or death. Main Outcomes and Measures: The primary end point was overall survival (OS). Results: A total of 369 patients were randomized to nivolumab (n = 184) or bevacizumab (n = 185). The MGMT promoter was methylated in 23.4% (43/184; nivolumab) and 22.7% (42/185; bevacizumab), unmethylated in 32.1% (59/184; nivolumab) and 36.2% (67/185; bevacizumab), and not reported in remaining patients. At median follow-up of 9.5 months, median OS (mOS) was comparable between groups: nivolumab, 9.8 months (95% CI, 8.2-11.8); bevacizumab, 10.0 months (95% CI, 9.0-11.8); HR, 1.04 (95% CI, 0.83-1.30); P = .76. The 12-month OS was 42% in both groups. The objective response rate was higher with bevacizumab (23.1%; 95% CI, 16.7%-30.5%) vs nivolumab (7.8%; 95% CI, 4.1%-13.3%). Grade 3/4 treatment-related adverse events (TRAEs) were similar between groups (nivolumab, 33/182 [18.1%]; bevacizumab, 25/165 [15.2%]), with no unexpected neurological TRAEs or deaths due to TRAEs. Conclusions and Relevance: Although the primary end point was not met in this randomized clinical trial, mOS was comparable between nivolumab and bevacizumab in the overall patient population with recurrent glioblastoma. The safety profile of nivolumab in patients with glioblastoma was consistent with that in other tumor types. Trial Registration: ClinicalTrials.gov Identifier: NCT02017717

Conducting expeditionary operations in the contested littorals

Reissued 21 Oct 2015 to revise figure cross references in body text.The United States armed services have identified capability gaps in the areas of company-sized raid and sustainment operations in contested littoral environments. Multiple joint platform packages can be employed to provide the required mission capabilities to fill the gap. This thesis identifies the operational, functional, and physical architecture and effectiveness of mission packages necessary to provide capabilities associated with littoral sustainment operations. Physical architecture configurations are evaluated using discrete event modeling. Cost and performance estimates for the mission packages are presented in order to provide the decision maker tools for identifying which alternative provides the most cost-effective solution for the needs of a scenario’s stakeholders. This thesis report concludes by identifying potential assets that would provide cost-effective support of littoral operations. Feasible alternatives provide varying levels of effectiveness in terms of average deployment time and percentage of threats successfully affected.http://archive.org/details/conductingexpedi1094546910Approved for public release; distribution is unlimited