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

Orbital/ocular inflammatory involvement in VEXAS syndrome: Data from the international AIDA network VEXAS registry

VEXAS syndrome is a recently described monogenic autoinflammatory disease capable of manifesting itself with a wide array of organs and tissues involvement. Orbital/ocular inflammatory manifestations are frequently described in VEXAS patients. The objective of this study is to further describe orbital/ocular conditions in VEXAS syndrome while investigating potential associations with other disease manifestations. In the present study, twenty-seven out of 59 (45.8 %) VEXAS patients showed an inflammatory orbital/ocular involvement during their clinical history. The most frequent orbital/ocular affections were represented by periorbital edema in 8 (13.6 %) cases, episcleritis in 5 (8.5 %) patients, scleritis in 5 (8.5 %) cases, uveitis in 4 (6.8 %) cases, conjunctivitis in 4 (6.8 %) cases, blepharitis in 3 (5.1 %) cases, orbital myositis in 2 (3.4 %) cases. A diagnosis of systemic immune-mediated disease was observed in 15 (55.6 %) cases, with relapsing polychondritis diagnosed in 12 patients. A significant association was observed between relapsing polychondritis and orbital/ocular involvement in VEXAS syndrome (Relative Risk: 2.37, 95 % C.I. 1.03-5.46, p = 0.048). Six deaths were observed in the whole cohort of patients after a median disease duration of 1.2 (IQR=5.35) years, 5 (83.3 %) of which showed orbital/ocular inflammatory involvement. In conclusion, this study confirms that orbital/ocular inflammatory involvement is a common finding in VEXAS patients, especially when relapsing polychondritis is diagnosed. This makes ophthalmologists a key figure in the diagnostic process of VEXAS syndrome. The high frequency of deaths observed in this study seems to suggest that patients with orbital/ocular involvement may require increased attention and more careful follow-up

Probing the chiral magnetic wave with charge-dependent flow measurements in Pb-Pb collisions at the LHC

The Chiral MagneticWave (CMW) phenomenon is essential to provide insights into the strong interaction in QCD, the properties of the quark-gluon plasma, and the topological characteristics of the early universe, offering a deeper understanding of fundamental physics in high-energy collisions. Measurements of the charge-dependent anisotropic flow coefficients are studied in Pb-Pb collisions at center-of-mass energy per nucleon-nucleon collision v sNN = 5.02TeV to probe the CMW. In particular, the slope of the normalized difference in elliptic (v2) and triangular (v3) flow coefficients of positively and negatively charged particles as a function of their event-wise normalized number difference, is reported for inclusive and identified particles. The slope rNorm 3 is found to be larger than zero and to have a magnitude similar to rNorm 2, thus pointing to a large background contribution for these measurements. Furthermore, rNorm 2 can be described by a blast wave model calculation that incorporates local charge conservation. In addition, using the event shape engineering technique yields a fraction of CMW (fCMW) contribution to this measurement which is compatible with zero. This measurement provides the very first upper limit for fCMW, and in the 10-60% centrality interval it is found to be 26% (38%) at 95% (99.7%) confidence level

Accessing the strong interaction between Λ baryons and charged kaons with the femtoscopy technique at the LHC

The interaction between Lambda baryons and kaons/antikaons is a crucial ingredient for the strangeness S= 0 and S= -2 sector of the meson-baryon interaction at low energies. In particular, the Lambda(K) over bar might help in understanding the origin of states such as the Xi(1620), whose nature and properties are still under debate. Experimental data on Lambda-K and Lambda-(K) over bar systems are scarce, leading to large uncertainties and tension between the available theoretical predictions constrained by such data. In this Letter we present the measurements of Lambda-K+ circle plus (Lambda) over bar -K- and Lambda-K- circle plus (Lambda) over bar -K+ correlations obtained in the high-multiplicity triggered data sample in pp collisions at root s= 13TeV recorded by ALICE at the LHC. The correlation function for both pairs is modeled using the Lednicky-Lyuboshits analytical formula and the corresponding scattering parameters are extracted. The Lambda-K- circle plus (Lambda) over bar -K+ correlations show the presence of several structures at relative momenta k* above 200 MeV/c, compatible with the Omega baryon, the Xi(1690), and Xi(1820) resonances decaying into Lambda-K- pairs. The low k* region in the Lambda-K- circle plus (Lambda) over bar -K+ also exhibits the presence of the Xi(1620) state, expected to strongly couple to the measured pair. The presented data allow to access the Lambda K+ and Lambda K- strong interaction with an unprecedented precision and deliver the first experimental observation of the Xi(1620) decaying into Lambda K-. (c) 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3

Measurement of non-prompt D0-meson elliptic flow in Pb–Pb collisions at sqrt{s_NN} = 5.02 TeV

The elliptic flow (v2) of D mesons from beauty-hadron decays (non-prompt D) was measured in midcentral (30–50%) Pb–Pb collisions at a centre-of-mass energy per nucleon pair sNN=5.02 TeV with the ALICE detector at the LHC. The D mesons were reconstructed at midrapidity (| y| < 0.8) from their hadronic decay D → K -π + , in the transverse momentum interval 2 < pT< 12 GeV/c. The result indicates a positive v2 for non-prompt D mesons with a significance of 2.7 σ . The non-prompt D -meson v2 is lower than that of prompt non-strange D mesons with 3.2 σ significance in 2<8GeV/c , and compatible with the v2 of beauty-decay electrons. Theoretical calculations of beauty-quark transport in a hydrodynamically expanding medium describe the measurement within uncertainties

Higher-order correlations between different moments of two flow amplitudes in Pb-Pb collisions at sqrt(sNN) = 5.02 TeV

The correlations between different moments of two flow amplitudes, extracted with the recently developed asymmetric cumulants, are measured in Pb-Pb collisions at sNN=5.02TeV recorded by the ALICE detector at the CERN Large Hadron Collider. The magnitudes of the measured observables show a dependence on the different moments as well as on the collision centrality, indicating the presence of nonlinear response in all even moments up to the eighth. Furthermore, the higher-order asymmetric cumulants show different signatures than the symmetric and lower-order asymmetric cumulants. Comparisons with state-of-the-art event generators using two different parametrizations obtained from Bayesian optimization show differences between data and simulations in many of the studied observables, indicating a need for further tuning of the models behind those event generators. These results provide new and independent constraints on the initial conditions and transport properties of the system created in heavy-ion collision

Proton reconstruction with the CMS-TOTEM Precision Proton Spectrometer

The Precision Proton Spectrometer (PPS) of the CMS and TOTEM experiments collected 107.7 fb-1 in proton-proton (pp) collisions at the LHC at 13 TeV (Run 2). This paper describes the key features of the PPS alignment and optics calibrations, the proton reconstruction procedure, as well as the detector efficiency and the performance of the PPS simulation. The reconstruction and simulation are validated using a sample of (semi)exclusive dilepton events. The performance of PPS has proven the feasibility of continuously operating a near-beam proton spectrometer at a high luminosity hadron collider

Observation of τ Lepton Pair Production in Ultraperipheral Pb-Pb Collisions at sqrt[s_{NN}]=5.02 TeV

: We present an observation of photon-photon production of τ lepton pairs in ultraperipheral lead-lead collisions. The measurement is based on a data sample with an integrated luminosity of 404 μb^{-1} collected by the CMS experiment at a center-of-mass energy per nucleon pair of sqrt[s_{NN}]=5.02 TeV. The γγ→τ^{+}τ^{-} process is observed for τ^{+}τ^{-} events with a muon and three charged hadrons in the final state. The measured fiducial cross section is σ(γγ→τ^{+}τ^{-})=4.8±0.6(stat)±0.5(syst) μb, where the second (third) term corresponds to the statistical (systematic) uncertainty in σ(γγ→τ^{+}τ^{-}) in agreement with leading-order QED predictions. Using σ(γγ→τ^{+}τ^{-}), we estimate a model-dependent value of the anomalous magnetic moment of the τ lepton of a_{τ}=0.001_{-0.089}^{+0.055}