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

<i>In vitro</i> and <i>in planta</i> antagonistic effects of plant growth-promoting rhizobacteria consortium against soilborne plant pathogens of <i>Solanum tuberosum</i> and <i>Solanum lycopersicum</i>

ABSTRACT Potatoes (Solanum tuberosum L.) and tomatoes (Solanum lycopersicum L.), among the main crops belonging to the Solanaceae family, are attacked by several pathogens. Among them Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani are very common and cause significant losses. Four plant growth-promoting rhizobacteria, Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Burkholderia ambifaria were tested against these pathogens. In vitro antagonistic activities of single strains were assessed through dual culture plates. Strains showing antagonistic activity (G. diazotrophicus, H. seropedicae and B. ambifaria) were combined and, after an in vitro confirmation, the consortium was applied on S. lycopersicum and S. tuberosum in a greenhouse pot experiment. The bioprotection was assessed in pre-emergence (infection before germination) and post-emergence (infection after germination). The consortium was able to successfully counteract the infection of both F. oxysporum and R. solani, allowing a regular development of plants. The biocontrol of the fungal pathogens was highlighted both in pre-emergence and post-emergence conditions. This selected consortium could be a valid alternative to agrochemicals and could be exploited as biocontrol agent to counteract losses due to these pathogenic fungi.</jats:p

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis

Geomorphological analysis of San Nicola Island (Tremiti Islands, Southern Adriatic Sea). Results from the 2021 and 2022 Environmental Geomorphology field camps of the MSc in Geological Sciences and Technologies of Earth and Planets (University ‘G. d’Annunzio’ of Chieti-Pescara)

This paper presents the results of the 2021 and 2022 field activities carried out at San Nicola Island (Tremiti Islands, Southern Adriatic Sea) by two groups of students as part of the Environmental Geomorphology Field Mapping course held within the Master's Degree in Geological Sciences and Technologies of Earth and Planets at University ‘G. d'Annunzio’ of Chieti-Pescara. Field activities were carried out following an integrated approach that involved morphometric, geological, and geomorphological analyses, supported by the combination of traditional methods with modern survey instruments and techniques. The Main Map (1:2,500 scale) comprehensively depicts the landscape of San Nicola Island, which is affected by both long- and short-term evolutionary processes, as witnessed by widespread slope landforms. The cartographic products presented in this work can represent a useful tool in territorial planning and management, as well as a valuable base for further scientific studies.</p

Drug prescription appropriateness in hospitalized older patients: 15-year results and lessons from a countrywide register.

The global increase of aging with the related increase of multiple noncommunicable diseases is inevitably accompanied by the associated issue of multimorbidity and polypharmacy. The latter is not without peculiar consequences on health, because it has been shown to be associated with drug-related adverse events, mainly due to poor prescription appropriateness and drug-drug interactions. To contribute to tackle this gigantic problem, a registry of drug dispensation in hospitalized older patient has been initiated in Italy in 2008. Through the last 15 years, data on nearly 11,000 older people have been accrued during their hospital stay in internal medicine and geriatric wards. This review article summarizes the main findings obtained, and how these data contribute to tackle the issue of appropriateness of drug prescription and the need of deprescribing in hospitalized older people affected by the most common noncommunicable diseases

Proton pump inhibitors and 1-year risk of adverse outcomes after discharge from internal medicine wards: an observational study in the REPOSI cohort.

Proton pump inhibitors are widely prescribed at hospital discharge from internal medicine wards and inappropriate use is common. We retrospectively conducted a survival analysis on data collected from the Registro Politerapie SIMI (REPOSI) registry to evaluate the 1-year risk of hospitalization or mortality associated with the use of PPI, with a particular focus on the appropriateness of use and newly initiated prescriptions at discharge. 7280 patients were discharged from hospital and 4579 (62.9%) had a PPI prescription. The use of PPI was significantly associated with 1-year risk of mortality in the univariate model (hazard ratio (HR) 1.33, p = 0.0012) and also when adjusted for confounders (adjusted HR 1.47, p = 0.0009). In the sensitivity analysis, new PPI prescription use at discharge was associated with an increased risk of mortality (adjusted HR of 1.53, p = 0.006). Inappropriate use was also linked to a nearly 60% higher risk of 1-year mortality and 27% increased risk of 1-year re-hospitalization. Among new PPI users, inappropriate use was associated with nearly 70% increased risk of 1-year mortality (HR 1.69). PPI use was associated with an increased risk of 1-year mortality and re-hospitalization in older adults discharged from hospitals. A higher risk of mortality was observed among new inappropriate PPI users, underscoring the importance of carefully evaluating the unnecessary initiation of new medications at discharge to maintain a favorable benefit-risk ratio.Impact of findings on practice statements. Proton pump inhibitors are among the most commonly prescribed medications. Use of proton pump inhibitors at hospital discharge was associated with a risk of 1-year mortality. Unnecessary PPI use was associated with higher risk of mortality. Patients discharged from internal medicine wards had high rates of inappropriate PPI use. The unnecessary initiation of new drugs at discharge for a favorable benefit-risk ratio was evaluated

Prescription of ACE-Is/ARBs in patients with cardio-renal disease: a multicenter retrospective cohort study from the REPOSI registry

Cardio-renal disease is a common clinical condition leading to increased morbidity and mortality. Angiotensin-converting enzyme inhibitors (ACE-Is) and angiotensin II receptor blockers (ARBs) are the cornerstone of treatment of chronic cardio-renal disease. Using data from the REPOSI register, we performed a multicenter, observational, retrospective study to determine which factors are associated with the non-prescription or discontinuation of ACE-Is/ARBS in a cohort of 889 cardio-renal patients hospitalized in 109 Italian internal medicine and geriatric wards. Only 55% of the patients with cardio-renal disease of the investigated cohort were on treatment with ACE-Is or ARBs at admission. The primary end point was ACE-Is/ARBs use at discharge. Patients with lower probability of receiving ACE-Is/ARBs at discharge were older and hospitalized for longer periods. Furthermore, patients with advanced chronic kidney disease (advanced CKD: eGFR ≤ 29 mL/min/1.73m2) had a much lower (54%) probability of being discharged or continuing ACE-Is/ARBs treatment than those with eGFR ≥ 60 mL/min/1.73m2. A more prominent lower probability was found comparing advanced CKD patients with G3 stage CKD patients (eGFR: 59-30 mL/min/1.73m2) in multivariate analyses (OR and 95%CI: 0.37, 0.24-0.57. multivariate p-value &lt; 0.001). The probability of stopping treatment in patients already on treatment with ACE-Is/ARBs at hospital admission (secondary end point) almost reached a threefold increase (OR and 95%CI: 2.82, 1.69-4.71. multivariate p-value &lt; 0.001) when the advanced CKD group was compared with G3 CKD patients. The data of our study are not in line with the recently published updated KDIGO 2024 Guidelines, which recommend patients with advanced CKD to continue treatment with ACE-Is/ARBs. (REPOSI investigators

Comparison of Anticholinergic Burden Scales and Their Association with Cognitive and Functional Impairment in Older Adults: A Cross-Sectional Study Using the REPOSI Database.

Background: The increasing use of anticholinergic medications in older adults with multiple chronic conditions raises significant concerns regarding their cumulative anticholinergic burden, which is linked to several adverse outcomes. This study aimed to compare existing anticholinergic burden scales to identify those most effective at correlating drug-induced anticholinergic load with cognitive and functional impairment. In addition, we proposed a new classification system on the basis of published scales to optimally correlate total anticholinergic burden with observed clinical deficits. Methods: This cross-sectional study analyzed data from the REPOSI registry, which collects clinical and therapeutic information on patients aged 65 years and older admitted to internal medicine and geriatric wards across Italy. Anticholinergic exposure was assessed using 20 established anticholinergic burden scales from literature. In addition, seven experimental scales were developed on the basis of published scales and various mathematical functions (maximum, mode, median, and mean) to evaluate potential differences in measuring anticholinergic load. Outcomes included cognitive impairment, assessed using the Short Blessed Test (SBT), and functional independence, measured by the Barthel Index (BI). A zero-inflated negative binomial model was applied to analyze associations between anticholinergic burden and each outcome. Given the variability in drug scoring across published scales, we developed seven experimental scales using different mathematical functions (maximum, mode, median, and mean) to standardize scoring. Three versions included a null-score adjustment to account for drugs omitted in some scales, ensuring a more comprehensive measure of anticholinergic burden. Results: Among 7735 patients, higher anticholinergic burden was consistently associated with increased cognitive impairment (SBT) and physical dependency (BI) across all existing and proposed scales. The modified Anticholinergic Risk Scale (mARS) scale showed the strongest associations with cognitive (rate ratio [RR] 1.10, 95% confidence interval [CI] 1.06, 1.13; P &lt; 0.0001) and physical impairment (RR 1.18, 95% CI 1.11, 1.24; P &lt; 0.0001), indicating an 18% higher risk of dependency per unit increase, while the CRIDECO Anticholinergic Load Scale (CALS) scale exhibited the best model fit. Our newly developed scales confirmed these associations, with the median with null score and the mean with null score scale showing the strongest link to cognitive impairment (RR 1.07, 95% CI 1.05, 1.09; P &lt; 0.0001) and the strongest association with physical dependency (RR 1.11, 95% CI 1.08, 1.15; P &lt; 0.0001). Conclusions: Scales that identify a greater proportion of patients with at least one anticholinergic drug may provide a more comprehensive assessment of anticholinergic burden in clinical practice. While no single scale demonstrated a definitive advantage across all outcomes, these scales may identify patients at risk. Prioritizing the use of scales with broader coverage could enhance clinical decision-making and optimize management of polypharmacy in older adults and recognize its potential impact on cognitive and functional outcomes