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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- 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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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. 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. 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.
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- Bernard-Marissal, N, et al.
(author)
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Altered interplay between endoplasmic reticulum and mitochondria in Charcot-Marie-Tooth type 2A neuropathy
- 2019
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 116:6, s. 2328-2337
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Journal article (peer-reviewed)abstract
- Interactions between mitochondria and the endoplasmic reticulum (ER) at the level of mitochondria-associated membranes (MAM) constitute a key signaling hub, emerging as a shared target altered in multiple neurodegenerative diseases. We use both in vivo and in vitro models of Charcot–Marie–Tooth type 2A, an axonal form of neuropathy, to demonstrate that the presence of mutated Mitofusin 2 leads to altered MAM. In neurons, these modifications occur concomitantly with activation of ER stress response, dysregulated calcium handling, and alterations in mitochondrial morphology and transport, collectively contributing to axonopathy. Importantly, the reported results indicate that the pathological consequences of mutated Mitofusin 2 may be targeted with drugs reinforcing the ER–mitochondria cross-talk and/or reducing ER stress.
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- Stepniewska, Kasia, et al.
(author)
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Safety of single-dose primaquine as a Plasmodium falciparum gametocytocide : a systematic review and meta-analysis of individual patient data
- 2022
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In: BMC Medicine. - : Springer Nature. - 1741-7015. ; 20:1
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Research review (peer-reviewed)abstract
- BackgroundIn 2012, the World Health Organization (WHO) recommended single low-dose (SLD, 0.25 mg/kg) primaquine to be added as a Plasmodium (P.) falciparum gametocytocide to artemisinin-based combination therapy (ACT) without glucose-6-phosphate dehydrogenase (G6PD) testing, to accelerate malaria elimination efforts and avoid the spread of artemisinin resistance. Uptake of this recommendation has been relatively slow primarily due to safety concerns.MethodsA systematic review and individual patient data (IPD) meta-analysis of single-dose (SD) primaquine studies for P. falciparum malaria were performed. Absolute and fractional changes in haemoglobin concentration within a week and adverse effects within 28 days of treatment initiation were characterised and compared between primaquine and no primaquine arms using random intercept models.ResultsData comprised 20 studies that enrolled 6406 participants, of whom 5129 (80.1%) had received a single target dose of primaquine ranging between 0.0625 and 0.75 mg/kg. There was no effect of primaquine in G6PD-normal participants on haemoglobin concentrations. However, among 194 G6PD-deficient African participants, a 0.25 mg/kg primaquine target dose resulted in an additional 0.53 g/dL (95% CI 0.17-0.89) reduction in haemoglobin concentration by day 7, with a 0.27 (95% CI 0.19-0.34) g/dL haemoglobin drop estimated for every 0.1 mg/kg increase in primaquine dose. Baseline haemoglobin, young age, and hyperparasitaemia were the main determinants of becoming anaemic (Hb < 10 g/dL), with the nadir observed on ACT day 2 or 3, regardless of G6PD status and exposure to primaquine. Time to recovery from anaemia took longer in young children and those with baseline anaemia or hyperparasitaemia. Serious adverse haematological events after primaquine were few (9/3, 113, 0.3%) and transitory. One blood transfusion was reported in the primaquine arms, and there were no primaquine-related deaths. In controlled studies, the proportions with either haematological or any serious adverse event were similar between primaquine and no primaquine arms.ConclusionsOur results support the WHO recommendation to use 0.25 mg/kg of primaquine as a P. falciparum gametocytocide, including in G6PD-deficient individuals. Although primaquine is associated with a transient reduction in haemoglobin levels in G6PD-deficient individuals, haemoglobin levels at clinical presentation are the major determinants of anaemia in these patients.
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- Anastacio, A, et al.
(author)
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Ovarian Follicles Rescued 3 Days after Cyclophosphamide Treatment in Adolescent Mice: An Experimental Study Aiming at Maximizing Methods for Fertility Preservation through In Vitro Follicle Culture
- 2019
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In: International journal of molecular sciences. - : MDPI AG. - 1422-0067. ; 20:24
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Journal article (peer-reviewed)abstract
- There is currently a lack of knowledge about the feasibility of performing procedures for fertility preservation after chemotherapy treatment has been initiated. In this experimental controlled study using adolescent mice, we aimed to investigate if the chance of rescuing and growing in vitro secondary follicles (SeF) would be affected three days after a single injection of cyclophosphamide (CPA). The main outcomes included were: (1) The number of SeF with good morphologic quality obtained per ovary 3 days after CPA injection, (2) SeF development in culture, (3) small follicle density (SFD) on histology, and (4) apoptosis markers, including terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL), mRNA expression, and distribution of p 53 upregulated modulator of apoptosis (Puma) and phosphatase and tensin homolog (Pten). We found a 60% reduction of SeF obtained per ovary in all CPA-treated groups vs. controls. However, in vitro survival rates at culture day 12 and antrum formation were similar among all groups. On histology, SFD was only significantly reduced in the high CPA dose group. Apoptotic cells were mainly found in large growing follicles of CPA groups. Our study indicates the feasibility of SeF isolation and in vitro follicle culture 3 days following CPA treatment and a still preserved SFD, particularly following a low-dose CPA treatment.
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- Morris, Ulrika, et al.
(author)
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A cluster randomised controlled trial of two rounds of mass drug administration in Zanzibar, a malaria pre-elimination setting-high coverage and safety, but no significant impact on transmission.
- 2018
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In: BMC Medicine. - : Springer Science and Business Media LLC. - 1741-7015. ; 16:1
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Journal article (peer-reviewed)abstract
- BACKGROUND: Mass drug administration (MDA) has the potential to interrupt malaria transmission and has been suggested as a tool for malaria elimination in low-endemic settings. This study aimed to determine the effectiveness and safety of two rounds of MDA in Zanzibar, a pre-elimination setting.METHODS: A cluster randomised controlled trial was conducted in 16 areas considered as malaria hotspots, with an annual parasite index of > 0.8%. The areas were randomised to eight intervention and eight control clusters. The intervention included two rounds of MDA with dihydroartemisinin-piperaquine and single low-dose primaquine 4 weeks apart in May-June 2016. Primary and secondary outcomes were cumulative confirmed malaria case incidences 6 months post-MDA and parasite prevalences determined by PCR 3 months post-MDA. Additional outcomes included intervention coverage, treatment adherence, occurrence of adverse events, and cumulative incidences 3, 12, and 16 months post-MDA.RESULTS: Intervention coverage was 91.0% (9959/10944) and 87.7% (9355/10666) in the first and second rounds, respectively; self-reported adherence was 82.0% (881/1136) and 93.7% (985/1196). Adverse events were reported in 11.6% (147/1268) and 3.2% (37/1143) of post-MDA survey respondents after both rounds respectively. No serious adverse event was reported. No difference in cumulative malaria case incidence was observed between the control and intervention arms 6 months post-MDA (4.2 and 3.9 per 1000 population; p = 0.94). Neither was there a difference in PCR-determined parasite prevalences 3 months post-MDA (1.4% and 1.7%; OR = 1.0, p = 0.94), although having received at least the first MDA was associated with reduced odds of malaria infection (aOR = 0.35; p = 0.02). Among confirmed malaria cases at health facilities, 26.0% and 26.3% reported recent travel outside Zanzibar in the intervention and control shehias (aOR ≥ 85; p ≤ 0.001).CONCLUSIONS: MDA was implemented with high coverage, adherence, and tolerability. Despite this, no significant impact on transmission was observed. The findings suggest that two rounds of MDA in a single year may not be sufficient for a sustained impact on transmission in a pre-elimination setting, especially when the MDA impact is restricted by imported malaria. Importantly, this study adds to the limited evidence for the use of MDA in low transmission settings in sub-Saharan Africa.TRIAL REGISTRATION: ClinicalTrials.gov, NCT02721186 (registration date: March 29, 2016).
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