| 1. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 2. |
- 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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| 3. |
- Vinel, Claire, et al.
(författare)
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Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma
- 2021
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM. The identification of patient-specific disease mechanisms and druggable targets is crucial for precision medicine in glioblastoma. Here, the authors show that comparing patients-matched glioma-initiating cells with neural stem cells enables the discovery of patient-specific mechanisms of disease and the identification of effective drugs
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| 4. |
- Holley, Rebecca J., et al.
(författare)
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Mucopolysaccharidosis Type I, Unique Structure of Accumulated Heparan Sulfate and Increased N-Sulfotransferase Activity in Mice Lacking alpha-L-iduronidase
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:43, s. 37515-37524
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Tidskriftsartikel (refereegranskat)abstract
- Mucopolysaccharide (MPS) diseases are characterized by accumulation of glycosaminoglycans (GAGs) due to deficiencies in lysosomal enzymes responsible for GAG breakdown. Using a murine model of MPSI Hurler (MPSIH), we have quantified the heparan sulfate (HS) accumulation resulting from alpha-L-iduronidase (Idua) deficiency. HS levels were significantly increased in liver and brain tissue from 12-week-old Idua(-/-) mice by 87- and 20-fold, respectively. In addition, HS chains were shown to contain significantly increased N-, 2-O-, and 6-O-sulfation. Disaccharide compositional analyses also uncovered an HS disaccharide uniquely enriched in MPSIH, representing the terminal iduronic acid residue capping the non-reducing end of the HS chain, where no further degradation can occur in the absence of Idua. Critically, we identified that excess HS, some of which is colocalized to the Golgi secretory pathway, acts as a positive regulator of HS-sulfation, increasing the N-sulfotransferase activity of HS-modifying N-deacetylase/N-sulfotransferase enzymes. This mechanism may have severe implications during disease progression but, now identified, could help direct improved therapeutic strategies.
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| 5. |
- Johnson, Benjamin B., et al.
(författare)
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Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes
- 2024
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Ingår i: Cell Reports. - : Elsevier. - 2211-1247. ; 43:1
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Tidskriftsartikel (refereegranskat)abstract
- Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2(+/-)) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late -stage HSPG2(+/-) hPSC-CMs have immature features, including reduced alpha-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecanpeptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.
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| 6. |
- Deligny, Audrey, et al.
(författare)
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NDST2 (N-Deacetylase/N-Sulfotransferase-2) Enzyme Regulates Heparan Sulfate Chain Length
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:36, s. 18600-18607
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of heparan sulfate synthesized by HEK 293 cells overexpressing murine NDST1 and/or NDST2 demonstrated that the amount of heparan sulfate was increased in NDST2-but not in NDST1-overexpressing cells. Altered transcript expression of genes encoding other biosynthetic enzymes or proteoglycan core proteins could not account for the observed changes. However, the role of NDST2 in regulating the amount of heparan sulfate synthesized was confirmed by analyzing heparan sulfate content in tissues isolated from Ndst2(-/-) mice, which contained reduced levels of the polysaccharide. Detailed disaccharide composition analysis showed no major structural difference between heparan sulfate from control and Ndst2(-/-) tissues, with the exception of heparan sulfate from spleen where the relative amount of trisulfated disaccharides was lowered in the absence of NDST2. In vivo transcript expression levels of the heparan sulfate-polymerizing enzymes Ext1 and Ext2 were also largely unaffected by NDST2 levels, pointing to a mode of regulation other than increased gene transcription. Size estimation of heparan sulfate polysaccharide chains indicated that increased chain lengths in NDST2-overexpressing cells alone could explain the increased heparan sulfate content. A model is discussed where NDST2-specific substrate modification stimulates elongation resulting in increased heparan sulfate chain length.
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| 7. |
- Mendonca, Tania, et al.
(författare)
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OptoRheo : Simultaneous in situ micro-mechanical sensing and imaging of live 3D biological systems
- 2023
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- A new instrument named OptoRheo combines light sheet fluorescence microscopy and particle tracking microrheology for live imaging and micromechanical sensing of extracellular matrix-cell interactions. Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named 'OptoRheo' that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.
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| 8. |
- Pijuan-Galito, Sara, et al.
(författare)
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Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-alpha-inhibitor (I alpha I), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IaI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IaI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.
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| 9. |
- Smeds, Emanuel, 1975-
(författare)
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In Vitro Studies of the Substrate Specificities of Heparan Sulfate 2-O- and 6-O-sulfotransferases
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-O-sulfotransferase (2OST) and HS glucosaminyl 6-O-sulfotransferases (6OSTs) were investigated. Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-O-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-O-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (35S-labeled PAPS). All three enzymes catalyzed 6-O-sulfation of both N-acetylated (GlcNAc) as well as N-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted.To understand how pre-existing 2-O-sulfates affects 6-O-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-O-sulfates. The specificities for substrates with two or three 2-O-sulfates were higher compared to octasaccharides with no or one 2-O-sulfate indicating that 2-O-sulfate groups substantially promote the subsequent 6-O-sulfation. Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-O-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. The 2OST catalyzes 2-O-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-O-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and 35S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-O-sulfation of GlcA occurs before IdoA is formed.
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