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

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Decreased expression of KGF/FGF7 and its receptor in pathological hypopigmentation.

[No abstract available

High quality process of care increases one-year survival after acute myocardial infarction (AMI): A cohort study in Italy

Background The relationship between guideline adherence and outcomes in patients with acute myocardial infarction (AMI) has been widely investigated considering the emergency, acute, post-acute phases separately, but the effectiveness of the whole care process is not known. Aim The study aim was to evaluate the effect of the multicomponent continuum of care on 1-year survival after AMI. Methods We conducted a cohort study selecting all incident cases of AMI from health information systems during 2011–2014 in the Lazio region. Patients’ clinical history was defined by retrieving previous hospitalizations and drugs prescriptions. For each subject the probability to reach the hospital and the conditional probabilities to survive to 30 days from admission and to 31–365 days post discharge were estimated through multivariate logistic models. The 1-year survival probability was calculated as the product of the three probabilities. Quality of care indicators were identified in terms of emergency timeliness (time between residence and the nearest hospital), hospital performance in treatment of acute phase (number/timeliness of PCI on STEMI) and drug therapy in post-acute phase (number of drugs among antiplatelet, β-blockers, ACE inhibitors/ARBs, statins). The 1-year survival Probability Ratio (PR) and its Bootstrap Confidence Intervals (BCI) between who were exposed to the highest level of quality of care (timeliness85 years. For patients with diabetes and COPD a slight increase in PRs was also observed. Conclusions The 1-year survival probability post AMI depends strongly on the quality of the whole multicomponent continuum of care. Improving the performance in the different phases, taking into account the relationship among these, can lead to considerable saving of lives, in particular for the elderly and for subjects with chronic diseases

Characteristics and effectiveness of smoking cessation programs in Italy. Results of a multicentric longitudinal study

AIM: to describe the characteristics and effectiveness of various smoking cessation programs offered by Italian treatment services operating within the National Health Service. DESIGN: prospective longitudinal multicentre study involving 41 smoking cessation services in 16 Italian regions. Study population: the study population includes patients entering smoking cessation programs between April 2003 and June 2004. The "study population" includes 1226 patients (54.2% males and 45.4% females), mean age 47 years. Patients have a middle/high level of education and a long history of smoking; most are highly dependent on nicotine and report previous attempts to quit smoking. METHODS: treatment effectiveness in smoking cessation is assessed six months after entering treatment service. Logistic Regression Model was used to determine the predictors of successfiul cessation, independent of treatment typology. The predictors were included as confounding variables in the logistic regression model that was used to evaluate the effectiveness of treatments. Besides the effect of treatment completion on smoking cessation was estimated. RESULTS: predictors of successful smoking cessation are: being male, presence of a partner, strong motivation to quit, previous attempts to give up smoking, mild nicotine dependence, and not suffering from mood disturbances. All treatments are effective in helping people to stop smoking: cessation rate ranges between 25.00% for patients receiving a single session of motivational counselling and 65.3% for those receiving nicotine replacement therapy combined to group therapy. Compared to a single session of motivational counseling, nicotine replacement therapy combined to group therapy is the most effective therapeutic program (OR 5.4; 95%CI 12.5-12.0). Treatment completion is a strong determinant ofsuccess (OR 4.8; 95%CI 3.5-6.4). CONCLUSION: enrolling people in any type of therapeutic program, in particular nicotine replacement therapy combined with group therapy increases the probability of successfully quitting smoking; moreover, patients that begin a smoking cessation program should be encouraged to complete the therap