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Archivio istituzionale della ricerca dell'Università degli Studi di Palermo
In 2008 we published the
fi
rst set of guidelines for standardiz-
ing research in autophagy. Since then, research on this topic
has continued to accelerate, and many new scientists have
entered the
fi
eld. Our knowledge base and relevant new tech-
nologies have also been expanding. Accordingly, it is important
to update these guidelines for monitoring autophagy in differ-
ent 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 meas
urements that monitor the num-
bers 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 com-
plete process including the amoun
t and rate of cargo sequestered
and degraded). In particular, a block in macroautophagy that
results in autophagosome accu
mulation must be differentiated
from stimuli that increas
e autophagic activity, de
fi
ned as increased
autophagy induction coupled with in
creased delivery to, and degra-
dation within, lysosomes (in most higher eukaryotes and some pro-
tists such as
Dictyostelium
) or the vacuole (in plants and fungi). In
otherwords,itisespeciallyimportantthatinvestigatorsnewtothe
fi
eld understand that the appearance of more autophagosomes
does not necessarily equate with mo
re autophagy. In fact, in many
cases, autophagosomes accumu
late because of a block in traf
fi
cking
to lysosomes without a concomitant change in autophagosome
biogenesis, whereas an increase in autolysosomes may re
fl
ect a
reductionindegradativeactivity
.Itisworthemphasizingherethat
lysosomal digestion is a stage of au
tophagy and evaluating its com-
petence is a crucial part of the evaluation of autophagic
fl
ux, 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 appro-
priate assays depend in part on the question being asked and
the system being used. In addition, we emphasize that no indi-
vidual 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 target by gene
knockout or RNA interference more than one autophagy-
related protein. In addition, some individual Atg proteins, or
groups of proteins, are involved in other cellular pathways
implying that not all Atg proteins can be used as a speci
fi
c
marker for an autophagic process. In these guidelines, we con-
sider 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 assays, we hope to
encourage technical innovation in the
fi
eld
Klionsky, D., Abdelmohsen, K., Abe, A., Abedin, M., Abeliovich, H., Acevedo Arozena, A., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) [10.1080/15548627.2015.1100356].
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
In 2008 we published the
fi
rst set of guidelines for standardiz-
ing research in autophagy. Since then, research on this topic
has continued to accelerate, and many new scientists have
entered the
fi
eld. Our knowledge base and relevant new tech-
nologies have also been expanding. Accordingly, it is important
to update these guidelines for monitoring autophagy in differ-
ent 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 meas
urements that monitor the num-
bers 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 com-
plete process including the amoun
t and rate of cargo sequestered
and degraded). In particular, a block in macroautophagy that
results in autophagosome accu
mulation must be differentiated
from stimuli that increas
e autophagic activity, de
fi
ned as increased
autophagy induction coupled with in
creased delivery to, and degra-
dation within, lysosomes (in most higher eukaryotes and some pro-
tists such as
Dictyostelium
) or the vacuole (in plants and fungi). In
otherwords,itisespeciallyimportantthatinvestigatorsnewtothe
fi
eld understand that the appearance of more autophagosomes
does not necessarily equate with mo
re autophagy. In fact, in many
cases, autophagosomes accumu
late because of a block in traf
fi
cking
to lysosomes without a concomitant change in autophagosome
biogenesis, whereas an increase in autolysosomes may re
fl
ect a
reductionindegradativeactivity
.Itisworthemphasizingherethat
lysosomal digestion is a stage of au
tophagy and evaluating its com-
petence is a crucial part of the evaluation of autophagic
fl
ux, 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 appro-
priate assays depend in part on the question being asked and
the system being used. In addition, we emphasize that no indi-
vidual 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 target by gene
knockout or RNA interference more than one autophagy-
related protein. In addition, some individual Atg proteins, or
groups of proteins, are involved in other cellular pathways
implying that not all Atg proteins can be used as a speci
fi
c
marker for an autophagic process. In these guidelines, we con-
sider 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 assays, we hope to
encourage technical innovation in the
fi
eld
Klionsky, D., Abdelmohsen, K., Abe, A., Abedin, M., Abeliovich, H., Acevedo Arozena, A., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) [10.1080/15548627.2015.1100356].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10447/166323
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simulazione ASN
Il report seguente simula gli indicatori relativi alla propria produzione scientifica in relazione alle soglie ASN 2023-2025 del proprio SC/SSD. Si ricorda che il superamento dei valori soglia (almeno 2 su 3) è requisito necessario ma non sufficiente al conseguimento dell'abilitazione. La simulazione si basa sui dati IRIS e sugli indicatori bibliometrici alla data indicata e non tiene conto di eventuali periodi di congedo obbligatorio, che in sede di domanda ASN danno diritto a incrementi percentuali dei valori. La simulazione può differire dall'esito di un’eventuale domanda ASN sia per errori di catalogazione e/o dati mancanti in IRIS, sia per la variabilità dei dati bibliometrici nel tempo. Si consideri che Anvur calcola i valori degli indicatori all'ultima data utile per la presentazione delle domande.
La presente simulazione è stata realizzata sulla base delle specifiche raccolte sul tavolo ER del Focus Group IRIS coordinato dall’Università di Modena e Reggio Emilia e delle regole riportate nel DM 589/2018 e allegata Tabella A. Cineca, l’Università di Modena e Reggio Emilia e il Focus Group IRIS non si assumono alcuna responsabilità in merito all’uso che il diretto interessato o terzi faranno della simulazione. Si specifica inoltre che la simulazione contiene calcoli effettuati con dati e algoritmi di pubblico dominio e deve quindi essere considerata come un mero ausilio al calcolo svolgibile manualmente o con strumenti equivalenti.