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

A Floristical and Ecological Study of the Medicinal Flora Used by the Local Population of the Haouz-Rehamna Region (Middle Atlantic Morocco-4)

To carry out a floristical inventory of plants used in traditional medicine in the HaouzRehamna region, a series of ethnobotanical surveys were conducted during five campaigns (2012–2017) with a representative sample of 1,700 people. These researches are completed by the determination of species collected in the field. It is worth mentioning that, taking into account the recent changes at the international level on taxonomy, the results obtained allowed us to elaborate a catalogue of 415 plant species (bryophytes (2 species); lichens (1), superior mushrooms (1), pteridophytes (4), gymnosperms (8), chlamydosperms (2) and angiosperms (397)) belonging to 291 genera and 99 botanical families, of which nine are the most representative and total 53.49%, namely: Asteraceae (11.33%), Lamiaceae (10.12%), Fabaceae (8.43%), Apiaceae (6.50%), Solanaceae (4.34%), Poaceae (3.86%), Rosaceae (3.37%), Brassicaceae (3.13%) and Cucurbitaceae (2.41%). On the contrary, the other 90 families represent a specific number less than or equal to 1.93%. The data also reflect a high degree of monotypic, where a single species represented 47.48% of the recorded families, and 79.03% of the genera were monotypic. The spontaneous plants occupy the first place with 241 species (58.07%). In addition, the classification by genus showed that the genus Mentha is the most used by its number of species (8 species). Moreover, we noted the use of 12 hybrid species. The chorological analysis revealed the domination of taxa with Mediterranean distribution for spontaneous species. Therophytes (27%) and phanerophytes (23.36%) are the most represented life forms. The results of this study could serve as a basis for future research in the field of floristics and ecology for the conservation of biodiversity

Taxonomy, Ethnobotany, Phytochemistry and Biological Activities of Thymus Saturejoides: a Review

Thymus saturejoides is an endemic species of the Lamiaceae family, native to Morocco and Algeria with a restricted distribution to the High Atlas, Middle Atlas, Anti-Atlas, Middle Atlantic Morocco, and the Saharan Atlas regions of Morocco, and the Aures Mountains in Algeria. This research focused on taxonomy, ethnobotany, chemical compounds, and biological and pharmacological actions of T. saturejoides . Folk medicine has documented continued use of this plant species. The review summarises the scientific literature and experimental research from the databases including Google Scholar, Semantic Scholar, ResearchGate, Academia.edu, PubMed, and PubFacts. Finally, we have provided a complete document on ethnobotany, phytochemistry, and biological properties fields of T. saturejoides

The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response

Item does not contain fulltextBACKGROUND AND PURPOSE: Lysosome-associated membrane protein 3 (LAMP3) is induced by the PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the unfolded protein response (UPR) during hypoxia. LAMP3 has prognostic value in breast cancer patients treated with radiotherapy. Here, we specifically investigated the role of the PERK/ATF4/LAMP3-arm in the radiation response of breast cancer cells. MATERIAL AND METHODS: Radiosensitivity of breast cancer cells was examined after siRNA-mediated knockdown of PERK, ATF4 and LAMP3. Activation of DNA damage repair proteins was evaluated by Western blotting and immunocytochemistry. RESULTS: Knockdown of the PERK/ATF4/LAMP3-arm and chemical inhibition of PERK could radiosensitise MDA-MB-231 cells significantly. Western blot analysis of several DNA damage repair proteins showed that LAMP3 knockdowns had an attenuated DNA damage response after radiation compared to controls. gamma-H2AX foci analysis revealed that LAMP3 knockdowns had a reduced number of positive cells after irradiation, indicating that their DNA damage repair signalling response is decreased. In addition, the effect of autophagy inhibition was examined and revealed a radiosensitising effect and the presence of residual gamma-H2AX foci. CONCLUSIONS: The PERK/ATF4/LAMP3-arm causes radioresistance of breast cancer cells by increasing DNA damage repair signalling. Inhibition of PERK and/or autophagy may sensitise tumours to radiotherapy