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- Niemi, MEK, et al.
(författare)
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- 2021
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swepub:Mat__t
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| 2. |
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| 3. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)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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| 4. |
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| 5. |
- Garcia-Alonso, Luz, et al.
(författare)
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Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro
- 2021
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Ingår i: Nature Genetics. - : Springer Nature. - 1061-4036 .- 1546-1718. ; 53:12, s. 1698-
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Tidskriftsartikel (refereegranskat)abstract
- The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma. Single-cell and spatial transcriptomic profiling of the human endometrium highlights pathways governing the proliferative and secretory phases of the menstrual cycle. Analyses of endometrial organoids show that WNT and NOTCH signaling modulate differentiation into the secretory and ciliated epithelial lineages, respectively.
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| 6. |
- Hao, J., et al.
(författare)
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Culture of human ovarian tissue in xeno-free conditions using laminin components of the human ovarian extracellular matrix
- 2020
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Ingår i: Journal of Assisted Reproduction and Genetics. - : Springer. - 1058-0468 .- 1573-7330. ; 37, s. 2137-2150
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animal-based mixture of ECM components. Methods: Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS. Results: Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, β-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells. Conclusions: LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries.
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| 9. |
- Bulovaite, Edita, et al.
(författare)
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A brain atlas of synapse protein lifetime across the mouse lifespan
- 2022
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Ingår i: Neuron. - : Elsevier BV. - 0896-6273 .- 1097-4199. ; 110:24, s. 4057-
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Tidskriftsartikel (refereegranskat)abstract
- The lifetime of proteins in synapses is important for their signaling, maintenance, and remodeling, and for memory duration. We quantified the lifetime of endogenous PSD95, an abundant postsynaptic protein in excitatory synapses, at single-synapse resolution across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of PSD95 lifetimes extending from hours to several months, with distinct spatial distributions in dendrites, neurons, and brain regions. Synapses with short protein lifetimes are enriched in young animals and in brain regions controlling innate behaviors, whereas synapses with long protein lifetimes accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. Synapse protein lifetime increases throughout the brain in a mouse model of autism and schizophrenia. Protein lifetime adds a further layer to synapse diversity and enriches prevailing concepts in brain development, aging, and disease.
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