Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens

Calpain mobilizes Atg9/Bif-1 vesicles from Golgi stacks upon autophagy induction by thapsigargin

CAPNS1 is essential for stability and function of the ubiquitous calcium-dependent proteases micro- and milli-calpain. Upon inhibition of the endoplasmic reticulum Ca2+ ATPase by 100 nM thapsigargin, both micro- calpain and autophagy are activated in human U2OS osteosarcoma cells in a CAPNS1- dependent manner. As reported for other autophagy triggers, thapsigargin treatment induces Golgi fragmentation and fusion of Atg9/Bif-1-containing vesicles with LC3 bodies in control cells. By contrast, CAPNS1 depletion is coupled with an accumulation of LC3 bodies and Rab5 early endosomes. Moreover, Atg9 and Bif-1 remain in the GM130positive Golgi stacks and Atg9 fails to interact with the endocytic route marker transferrin receptor and with the core autophagic protein Vps34 in CAPNS1-depleted cells. Ectopic expression of a Bif-1 point mutant resistant to calpain processing is coupled to endogenous p62 and LC3-II accumulation. Altogether, these data indicate that calpain allows dynamic flux of Atg9/Bif-1 vesicles from the Golgi toward the budding autophagosome

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Dissection of the role of calpain in the regulation of autophagy

Lo scopo principale del progetto di dottorato \ue8 stato quello di studiare il ruolo delle calpaine ubiquitarie nella regolazione del processo autofagico. Differenti tecniche biochimiche, di microscopia confocale e elettronica, ci hanno portato all'identificazione di una proteina target della micro-calpaina: Bif-1. L'azione proteasica su Bif-1 sembra sia indispensabile per la fissione e il trasporto di porzioni del Golgi verso il sito di formazione dell'autofagosome

L'abitato di Cà Spadolino di Coccanile (Copparo- FE) e il popolamento lungo i rami meridionali del delta del Po nell'età del Bronzo

I lavori di realizzazione del metanodotto tra Minerbio (BO) e Cavarzere (VE), condotti nel 2006-2007, hanno permesso di individuare nel comune di Copparo (FE) le tracce di un abitato dell'età del Bronzo recente. In questo lavoro si prende in esame la documentazione archeologica dello scavo e si presenta una prima valutazione dell'insediamento all'interno di un contesto paleoambientale di estremo interesse per la prossimità all'antico ambiente deltizio, ai margini dell’antica frangia lagunare. Il sito localizzato a Ca’ Spadolino ad alcuni Km a NE di Coccanile, infatti, rappresenta una delle rare testimonianze archeologiche associate al percorso di uno dei rami meridionali del Po nell'età del Bronzo, ben evidenziato dall'analisi geomorfologica e dalla valutazione geoarcheologica della stratigrafia. Le analisi dei dati archeozoologici e dei resti archeobotanici permettono di completare la ricostruzione paleoambientale del contesto e di valutare l'impatto antropico nell'area in prossimità del Delta. L'attribuzione cronologica al Bronzo recente è supportata dallo studio dei reperti, prevalentemente ceramici, che includono tipologie ben attribuibili alle facies padane adriatiche e parzialmente da due datazioni al C14, effettuate su carboni.Recent excavations for laying a pipeline at Coccanile, near Copparo (Ferrara) allowed to discover a Bronze Age settlement at a depth of -1,5 and -2,5/-3 m under the ground level, related to an ancient branch of Po river. The archaeological deposit included several anthropic layers alternated with alluvial sediments. An enlarged excavation of 20 x 6 m allowed to locate the oldest archaeological level (US 821) above natural sediments composed of silt and sand with convex surface (FIG. 4). This was covered by alluvial deposit that could have also weared the upper part of the earlier anthropic occupation. The sequence followed with another archaeological layer (US 896) with posts structure (USS 1049/1052/-1050) suggesting a wooden feature bounding the settled area (FIG. 6). A third layer linked to human activities (US 892), was very thick (up to 60 cm ca.) and excavated in two levels, the lower connected to several features (post holes and an hypothetical embankment, the higher corresponding to the phase of abandonment. The geomorphological analysis based on geomorphological data and on the stratigraphy led to recognize dynamics of alluvial aggradatio

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway