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- 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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- Colbourne, JK, et al.
(författare)
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The Precision Toxicology initiative
- 2023
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Ingår i: Toxicology letters. - 1879-3169. ; 383, s. 33-42
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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- Wei, A., et al.
(författare)
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Differentiation of rodent bone marrow mesenchymal stem cells into intervertebral disc-like cells following coculture with rat disc tissue
- 2009
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Ingår i: Tissue Engineering Part A. - 1937-335X. ; 15:9, s. 2581-95
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Tidskriftsartikel (refereegranskat)abstract
- This study aimed to evaluate whether rat mesenchymal stem cells (rMSCs) could be differentiated in vitro into disc-like cells by coculturing with intervertebral disc tissue. rMSCs were cultured with rodent intervertebral disc for up to 30 days in transwell plates. The differentiation of rMSCs was evaluated by immunostaining, Western blot, real-time RT-PCR, Northern blot, and electron microscopy. The potentials of multilineage differentiation and proteoglycan and collagen synthesis were also investigated. rMSCs underwent morphological changes to form three-dimensional micromasses and expressed collagen-2, aggrecan, and sox-9 at RNA and protein levels after 14 days of coculture. These changes were not detected in the samples of rMSCs cultured alone. Cocultured rMSCs also showed other characteristic features of disc-like cells, including the extracellular matrix formation, and proteoglycan and collagen synthesis. In addition, cellular contact between cocultured rMSCs and disc tissue was observed by electron microscopy. Committed rMSCs still retained their differentiation ability into mesoderm lineages of adipocytes or osteocytes when the local environment was altered. This study supports that MSCs are a promising source for cell therapy and tissue engineering in disc regeneration, and highlights that rMSCs can be induced into nucleus pulposus-like cells in vitro under the direct influence of intact disc tissue.
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| 7. |
- Wei, A., et al.
(författare)
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The fate of transplanted xenogeneic bone marrow-derived stem cells in rat intervertebral discs
- 2009
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Ingår i: Journal of Orthopaedic Research. - 1554-527X. ; 27:3, s. 374-9
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Tidskriftsartikel (refereegranskat)abstract
- Intervertebral disc degeneration is a major cause and a risk factor for chronic low back pain. The potential of using stem cells to treat disc degeneration has been raised. The aims of our study were to assess whether xenogeneic bone-marrow derived stem cells could survive in a rat disc degeneration model and to determine which cell types, if any, survived and differentiated into disc-like cells. Human bone-marrow derived CD34(+) (hematopoietic progenitor cells) and CD34(-) (nonhematopoietic progenitor cells, including mesenchymal stem cells) cells were isolated, fluorescent-labeled, and injected into rat coccygeal discs. The rats were sacrificed at day 1, 10, 21, and 42. Treated discs were examined by histological and immunostaining techniques and compared to control discs. The survival of transplanted cells was further confirmed with a human nuclear specific marker. Fluorescent labeled CD34(-) cells were detected until day 42 in the nucleus pulposus of the injected discs. After 3 weeks these cells had differentiated into cells expressing chondrocytic phenotype (Collagen II and Sox-9). In contrast, the fluorescent labeled CD34(+) cells could not be detected after day 21. No fluorescence-positive cells were detected in the noninjected control discs. Further, no inflammatory cells infiltrated the nucleus pulposus, even though these animals had not received immunosuppressive treatment. Our data provide evidence that transplanted human BM CD34(-) cells survived and differentiated within the relative immune privileged nucleus pulposus of intervertebral disc degeneration.
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- Brisby, Helena, 1965, et al.
(författare)
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The effect of running exercise on intervertebral disc extracellular matrix production in a rat model
- 2010
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Ingår i: Spine. - 1528-1159. ; 35:15, s. 1429-1436
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Tidskriftsartikel (refereegranskat)abstract
- STUDY DESIGN: Using a running rat model, the effects of physical exercise on cellular function and intervertebral disc (IVD) extracellular matrix were studied. OBJECTIVE: To investigate whether 3-weeks treadmill running exercise can stimulate matrix production and cellular proliferation of the IVD. SUMMARY OF BACKGROUND DATA: Appropriate physical exercise plays an important role in the treatment of patients with low back pain-associated IVD disorder. However, it is unknown how regular exercise affects the disc at the cellular level. METHODS: Twelve Sprague-Dawley rats underwent a daily treadmill exercise regime for a total of 3 weeks. Twelve nonexercised rats served as controls. The spinal lumbar IVD were collected and paraffin embedded for histologic analysis. Cell counts were determined on hematoxylin-eosin- and Masson-Trichrome-stained paraffin sections. Protein expression of collagen-I, collagen-II, aggrecan, Sox-9, and Sox-6 was evaluated with immunohistochemical staining. mRNA expression of Sox-9 and collagen-2 were studied by in situ hybridization. Proteoglycans were visualized with Alcian blue. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. RESULTS: The cell numbers in the anulus fibrosus (AF) increased by 25% (P < 0.05) after 3 weeks of exercise. Collagen-2 and Sox-9 mRNA were strongly expressed in the nucleus pulposus (NP) samples of the running group, but weakly expressed in the controls. An increase in collagen-II, aggrecan, and Sox-9 protein expression in NP and AF regions of the disc was detected in the exercised rats compared with controls. Quantification of Alcian blue staining demonstrated increased proteoglycan in both NP (8-fold) and AF (7-fold) in the exercised group compared with controls (P < 0.05). In addition, no significant differences were observed between the experimental groups in cellular apoptosis, collagen-I, or Sox-6 expression. CONCLUSION: In this study, increased extracellular matrix production and cell proliferation with no induction of disc cell apoptosis was observed in the lumbar IVD after a 3-week running regimen in rats, suggesting that regular exercise may have an augmentative effect on cells and matrix production.
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- Brisby, Helena, 1965, et al.
(författare)
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Cell therapy for disc degeneration--potentials and pitfalls
- 2004
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Ingår i: Orthop Clin North Am. - 0030-5898. ; 35:1, s. 85-93
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Tidskriftsartikel (refereegranskat)abstract
- Disc degeneration is considered a major source of pain in patients with chronic low back pain. Novel strategies to cure or decrease the symptoms and increase the patient's quality of life and function are under development. Until recently conservative treatment and fusion surgery were the main therapeutic options. Disc prostheses are undergoing clinical evaluation. The potential for cell transplantation to the intervertebral disc with mature autologous disc cells, chondrocytes, or stem cells is in early stages of investigation. Cell transplantation potentially can increase proteoglycan production and induce disc regeneration or slow down the degeneration process. In animal models, transplantation of autologous disc cells and chondrocytes (derived from costal cartilage) has been demonstrated to be feasible and may slow disc degeneration.
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- Gorman, D. J., et al.
(författare)
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When is spinal pain "neuropathic"?
- 2004
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Ingår i: Orthop Clin North Am. - 0030-5898. ; 35:1, s. 73-84
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Tidskriftsartikel (refereegranskat)abstract
- Although one cannot always determine when a patient's persistent pain is neuropathic, the issues are becoming clearer. Patients with spinal pain need to be examined carefully and the persistence of pain should not necessarily be considered to indicate a continuing search for a nociceptive focus. Similarly, continuing pain following successful surgery may be caused by persistent spinal or central neurologic changes. New methods for characterizing the site of neural changes will develop--functional MRI and PET scanning are now characterizing brain activity and will possibly yield results soon that will be helpful in the clinic. Further work needs to be done to identify which clinical features lead to better responses to agents for the treatment of neuropathic pain. New, more specific agents with actions focused on the specific parts of neural transmission are being developed. These include agents (such as growth factors) that will cure, not just suppress, the pathologic pain generators and pathways in patients with persistent pain.
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