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

Cornelia de Lange syndrome: description of the orofacial features and case report.

Cornelia de Lange Syndrome (CdLS) is a very rare syndrome characterised by multiple congenital anomaly affecting various organs and severe mental retardation. Incidence has been reported to be 1: 10.000-20.000 among the general population, with no racial predilection. The aetiology is still unknown but researchers, in 2004, discovered a mutation of the NIPBL gene located on chromosome 5 which is considered to be responsible of the disease. The main clinical features of the syndrome regard distinctive facial features, severe growth retardation, developmental and mental delay, hirsutism, structural limb abnormalities. The authors describe the main features of the syndrome focusing on oral and facial malformations and report a case of a three years old patient with CdLS

Lesioni dentali di origine traumatica in soggetti disabili in et\ue0 evolutiva.

I soggetti disabili rappresentano una categoria di pazienti ad elevato rischio di lesioni dentali di origine traumatica. Queste lesioni sono di frequente riscontro all\u2019anamnesi, sono potenziali responsabili di danno di tipo estetico e funzionale, comportano problematiche terapeutiche, in molti casi sono sottostimate, in altri hanno importanti ripercussioni psicologiche sulla famiglia. Fattori predisponenti locali di frequente riscontro sono patologie ortopedico-ortodontiche (in particolare aumento dell\u2019overjet), lesioni cariose, parodontopatie profonde. Fattori predisponenti sistemici sono handicap motori, ritardi mentali, epilessie, difetti visivi. Obiettivo di questa indagine epidemiologica \ue8 descrivere la gestione delle lesioni dentali di origine traumatica in pazienti disabili in et\ue0 pediatrica afferiti al Servizio di assistenza odontoiatrica per disabili dell\u2019Universit\ue0 di Bologna. Sono state esaminati i dati relativi a 283 pazienti disabili giunti ad osservazione in un periodo di due anni. L\u2018analisi dei dati evidenzia come le lesioni dentali presentino alcune specificit\ue0 rispetto alla restante popolazione.Per quanto concerne le terapie, sulla base di motivazioni etiche, le linee guida sono le stesse da utilizzare nella restante popolazione, con particolare attenzione agli elementi del settore frontale, in considerazione dell\u2019importanza dell\u2019estetica nel favorire l\u2019integrazione sociale di questi soggetti

Pain and anxiety control in Down syndrome

Authors describe most recent techniques in anxiety and pain control in Down Syndrome patients. Physical and psychological considerations are factors that determine the choice of methods. Down Syndrome represents one of the most frequent genetic diseases causing mental and physical impairment and because of crescent deinstitutionalisation and social opportunities these patients often require dental treatment. Pain control represents one of the most important aspects in therapeutical approach to obtain patient compliance, considering that oral health is the result of continuing relationship between dentist and patient. Congenital heart and neurological diseases are to be carefully considered in choosing a pain control technique. Local anaesthesia together with psychological approach is always the first choice and other techniques are reserved to non collaborative patients and to cases needing one step therapy. However conscious and deep sedation and general anaesthesia required hospital environment and the presence of an anaesthesiologist to monitorize vital parameters

Two cases of familial hypomagnesemia with hypercalciuria and nephrocalcinosis: dental findings.

The authors describe dental and periodontal conditions of two Chinese sisters affected by familial hypomagnesemia with hypercalciuria and nefrocalcinosis (FHHNC). FHHNC is a rare syndrome, genetically investigated since 1999, transmitted as an autosomal recessive disease. It is related to a mutation of PCN-1 gene which encodes for a tight junction protein named paracellin. CASE REPORT: Oral examination of two Chinese patients showed marked enamel hypoplasia, acute gingivitis and periodontal bone loss and severe malocclusion. The literature does not report previous investigations about the dental conditions of FHHNC patients. The syndrome is not definitely described from the clinical point of view. CONCLUSION: Further researches are necessary to understand the linkage between bone loss and enamel structure anomalies in FHHNC and to discover the relationships between nefrocalcinosis, hypomagnesemia, hypercalciuria and amelogenesis imperfecta.Background The authors describe dental and periodontal conditions of two Chinese sisters affected by familial hypomagnesemia with hypercalciuria and nefrocalcinosis (FHHNC). FHHNC is a rare syndrome, genetically investigated since 1999, transmitted as an autosomal recessive disease. It is related to a mutation of PCN-1 gene which encodes for a tight junction protein named paracellin. Case report Oral examination of two Chinese patients showed marked enamel hypoplasia, acute gingivitis and periodontal bone loss and severe malocclusion. The literature does not report previous investigations about the dental conditions of FHHNC patients. The syndrome is not definitely described from the clinical point of view. Conclusion Further researches are necessary to understand the linkage between bone loss and enamel structure anomalies in FHHNC and to discover the relationships between nefrocalcinosis, hypomagnesemia, hypercalciuria and amelogenesis imperfecta