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
Nanotechnology is expected to open new avenues to fight and prevent disease using atomic scale tailoring of materials. Among the most promising nanomaterials with antibacterial properties are metallic nanoparticles, which exhibit increased chemical activity due to their large surface to volume ratios and crystallographic surface structure. The study of bactericidal nanomaterials is particularly timely considering the recent increase of new resistant strains of bacteria to the most potent antibiotics. This has promoted research in the well known activity of silver ions and silver-based compounds, including silver nanoparticles. The present work studies the effect of silver nanoparticles in the range of 1-100 nm on Gram-negative bacteria using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM). Our results indicate that the bactericidal properties of the nanoparticles are size dependent, since the only nanoparticles that present a direct interaction with the bacteria preferentially have a diameter of approximately 1-10 nm.
Evidence has accumulated in recent years that programmed cell death (PCD) is not necessarily synonymous with the classical apoptosis, as defined by Kerr and Wyllie, but that cells use a variety of pathways to undergo cell death, which are reflected by different morphologies. Although chondrocytes with the hallmark features of classical apoptosis have been demonstrated in culture, such cells are extremely rare in vivo. The present review focuses on the morphological differences between dying chondrocytes and classical apoptotic cells. We propose the term 'chondroptosis' to reflect the fact that such cells are undergoing apoptosis in a non-classical manner that appears to be typical of programmed chondrocyte death in vivo. Unlike classical apoptosis, chondroptosis involves an initial increase in the endoplasmic reticulum and Golgi apparatus, reflecting an increase in protein synthesis. The increased ER membranes also segment the cytoplasm and provide compartments within which cytoplasm and organelles are digested. In addition, destruction occurs within autophagic vacuoles and cell remnants are blebbed into the lacunae. Together these processes lead to complete self-destruction of the chondrocyte as evidenced by the presence of empty lacunae. It is speculated that the endoplasmic reticulum pathway of apoptosis plays a greater role in chondroptosis than receptor-mediated or mitochondrial pathways and that lysosomal proteases are at least as important as caspases. Because chondroptosis does not depend on phagocytosis, it may be more advantageous in vivo, where chondrocytes are isolated within their lacunae. At present the initiation factors or the molecular pathways involved in chondroptosis remain unclear.
The death of chondrocytes and the loss of extracellular matrix are the central features in cartilage degeneration during Osteoarthritis (OA) pathogenesis. The mechanism by which chondrocytes are removed in OA cartilage are still not totally defined, although previous reports support the presence of apoptotic as well as non apoptotic signals. In addition, in 2004 Roach and co-workers suggested the term "Chondroptosis" to design the type of cell death present in articular cartilage, which include the presence of some apoptotic and autophagic processes. To identify the mechanisms, as well as the chronology by which chondrocytes are eliminated during OA pathogenesis, we decided to evaluate apoptosis (by active caspase 3 and TUNEL signal) and autophagy (by LC3II molecule and cytoplasmic vacuolization) using Immunohistochemistry and Western blot techniques in an animal OA model. During OA pathogenesis, chondrocytes exhibit modifications in their death process in each zone of the cartilage. At early stages of OA, the death of chondrocytes starts with apoptosis in the superficial and part of the middle zones of the cartilage, probably as a consequence of a constant mechanical damage in the joint. As the degenerative process progresses, high incidence of active caspase 3 as well as LC3II expression are observed in the same cell, which indicate a combination of both death processes. In contrast, in the deep zone, due the abnormal subchondral bone ossification during the OA pathogenesis, apoptosis is the only mechanism observed.
Knee articular cartilage samples obtained by arthroscopy from ten patients with well defined knee osteoarthritis (OA) were studied by light and transmission electron microscopy. The morphological phenotype of cells from fibrillated and non-fibrillated regions of OA cartilage was characterized. Three different cell sub-populations were identified. Type 1 cells were found in the superficial and upper middle zones and comprised single chondrocytes and cells organized in aggregates or "clones' that showed a typical chondrocyte phenotype. Type 2 cells displayed a secretory phenotype. Type 3 cells comprised chondrocytes undergoing a degenerative process and were distributed throughout all zones of the cartilage. Changes in the cytoskeletal arrangement, presence of abundant filopodia, peripheral localization of centrioles, and presence of primary cilia were found in many chondrocytes suggesting that they are active motile cells. No mitotic figures were found in this study. Morphometrical analysis was performed to determine the total number of cells and the number of chondrocytes per lacuna in the superficial and upper middle zones of fibrillated and non-fibrillated OA cartilage. There were no statistically significant differences in the total number of cells. In contrast, fibrillated OA cartilage contained a statistically significantly higher percentage of lacunae containing four of more chondrocytes than non-fibrillated OA cartilage samples. The absence of mitotic figures and the presence of motile elements in many chondrocytes raise the possibility that cell aggregates or "clones' in damaged OA cartilage originate by an active process of cell migration rather than by cellular division.
The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.
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