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

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Mouse myeloid leukemia cells can be induced to differentiate into macrophages in vitro by la,25-dihydroxyvi. tamin D3, the active form of vitamin D3. The minimal concentration of la,25-dihydroxyvitamin D3 to induce the cell differentiation was 0.12 nM. The degree of cell differentiation in various markers induced by 12 nM la,25-dihydroxyvitamin D3 was nearly equivalent to that induced by 1 ,M dexamethasone, the most potent known stimulator. Among several markers of the differentiation by la,25-dihydroxyvitamin D3, phagocytic activity was induced within 24 hr, and this was followed by induction oflysozyme and locomotive activities. Similar changes were also induced by 0.01-1 ,uM la-hydroxyvitamin D3. 25-Hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D.3 showed only weak inducing activity. These results suggest the possibility that, in addition to its wellknown biological activities in enhancing intestinal calcium transport and bone mineral mobilization, la,25-dihydroxyvitamin D3 is involved in the differentiation of bone marrow cells.The myeloid leukemia cell line (Ml), originally established by Ichikawa (1) from an SL mouse with myeloid leukemia, is known to differentiate into mature macrophages and granulocytes in vitro when treated with conditioned media from various cell cultures (2), ascitic fluid of tumor-bearing animals (3), bacterial lipopolysaccharides (4), polyribonucleotides (5), or glucocorticoids (6, 7). Among various inducers, dexamethasone has been found to be the most potent stimulator (8). The differentiation can be detected by changes in cell morphology, adhesion ofcells to the dish surface, increase in lysosomal enzyme activity, induction of phagocytic and locomotive activities, and the appearance of Fc and C3 receptors on the cell surface (2-8).It C02/95% air in Eagle's minimal essential medium supplemented with twice the normal concentrations ofamino acids and vitamins and 10% heat-inactivated calf serum (Chiba Serum Institute, Chiba, Japan). The cells were transferred every 2 to 3 days. Except for the study of cell fractionation, all cells (both adherent and nonadherent) were used for determining parameters of differentiation.Hormone and Vitamin D Derivatives. Dexamethasone was purchased from Sigma and 25(OH)D3 was from Philips-Duphar (Amsterdam). la,25(OH)2D3, 24R,25(OH)2D3, and la-hydroxyvitamin D3 [la(OH)D3] were kindly donated by I. Matsunaga, Chugai Pharmaceutical, Tokyo.Fractionation of the Cells by Discontinuous Density Gradient Centrifugation. For the study of cell fractionation, only adherent cells were used in the cultures treated with dexamethasone, la,25(OH)2D3, or la(OH)D3 whereas all cells were used in the control culture. After the nonadherent cells and loosely adherent cells had been removed by gently rinsing three times with prewarmed culture medium, the cells attached to dishes were collected as adherent cells by pipetting with phosphate-buffered saline lacking Ca2+ and Mg2-(PJNaCl) on ice. Determination of Lysozyme Activity. Lysozyme activity was determined by a mo...

Recently, the SARS-CoV-2 induced disease COVID-19 has spread all over the world. Nearly 20% of the patients have severe or critical conditions. SARS-CoV-2 exploits ACE2 for host cell entry. ACE2 plays an essential role in the renin-angiotensin-aldosterone system (RAAS), which regulates blood pressure and fluid balance. ACE2 also protects organs from inflammatory injuries and regulates intestinal functions. ACE2 can be shed by two proteases, ADAM17 and TMPRSS2. TMPRSS2-cleaved ACE2 allows SARS-CoV-2 cell entry, whereas ADAM17-cleaved ACE2 offers protection to organs. SARS-CoV-2 infection-caused ACE2 dysfunction worsens COVID-19 and could initiate multi-organ failure. Here, we will explain the role of ACE2 in the pathogenesis of severe and critical conditions of COVID-19 and discuss auspicious strategies for controlling the disease.Viruses 2020, 12, 491 2 of 10 Profile of ACE2Human angiotensin-converting enzyme-related carboxypeptidase ACE2 is encoded by the ACE2 gene which maps to chromosome Xp22 [6]. ACE2 is a type I transmembrane protein, comprised of an extracellular heavily N-glycosylated N-terminal domain containing the carboxypeptidase site and a short intracellular C-terminal cytoplasmic tail [7]. The N-terminal peptidase domain is also the SARS-CoV binding site [8]. There are two forms of ACE2 protein: cellular (membrane-bound) form and circulating (soluble) form. Cellular ACE2 protein is the full-length protein which is expressed abundantly in pneumocytes and enterocytes of the small intestine [9]. ACE2 is also expressed in vascular endothelial cells of the heart, the kidneys, and other organs, such as the brain. However, ACE2 is absent in the spleen, thymus, lymph nodes, bone marrow, and cells of the immune system (including B and T lymphocytes, and macrophages) [10,11].Circulating ACE2 (with the N-terminal peptidase domain) is cleaved from the full-length ACE2 on the cell membrane by the metalloprotease ADAM17 and then released into the extracellular environment [7]. The type II transmembrane serine protease, TMPRSS2 was found to compete with ADAM17 for ACE2 shedding but cleaves ACE2 differently. Both ADAM17 and TMPRSS2 remove a short C-terminal fragment from ACE2. Arginine and lysine residues within amino acids 652 to 659 are critical for ADAM17 shedding, whereas arginine and lysine residues within amino acids 697 to 716 are essential for TMPRSS2 shedding. Only cleavage by TMPRSS2 results in augmented SARS-CoV cell entry [12][13][14][15]. There are two ways for SARS-CoV to enter the target cell: endocytosis, and fusion of the viral membrane with a membrane of the target cell, which is 100 times more efficient than endocytosis for viral replication [16]. With the help of TMPRSS2, ADAM17-regulated ectodomain shedding of ACE2 could induce SARS-CoV cell entry through endocytosis [7,12]; however, ADAM17 activity is not required for SARS-CoV cell entry through fusion [12]. As the N-terminal domain is the coronavirus binding site, circulating ACE2 also binds to the virus. Iwata-Yoshikawa et al. infected both ...

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