41 research outputs found
Genome editing methods in animal models
No full text© 2020 The Author(s). Genetically engineered animal models that reproduce human diseases are very important for the pathological study of various conditions. The development of the clustered regularly interspaced short palindromic repeats (CRISPR) system has enabled a faster and cheaper production of animal models compared with traditional gene-targeting methods using embryonic stem cells. Genome editing tools based on the CRISPR-Cas9 system are a breakthrough technology that allows the precise introduction of mutations at the target DNA sequences. In particular, this accelerated the creation of animal models, and greatly contributed to the research that utilized them. In this review, we introduce various strategies based on the CRISPR-Cas9 system for building animal models of human diseases and describe various in vivo delivery methods of CRISPR-Cas9 that are applied to disease models for therapeutic purposes. In addition, we summarize the currently available animal models of human diseases that were generated using the CRISPR-Cas9 system and discuss future directions11Nsciescopuskc
The Intermolecular Interaction of Ephexin4 Leads to Autoinhibition by Impeding Binding of RhoG
No full textEphexin4 is a guanine nucleotide-exchange factor (GEF) for RhoG and is involved in various RhoG-related cellular processes such as phagocytosis of apoptotic cells and migration of cancer cells. Ephexin4 forms an oligomer via an intermolecular interaction, and its GEF activity is increased in the presence of Elmo, an Ephexin4-interacting protein. However, it is uncertain if and how Ephexin4 is autoinhibited. Here, using an Ephexin4 mutant that abrogated the intermolecular interaction, we report that this interaction impeded binding of RhoG to Ephexin4 and thus inhibited RhoG activation. Mutation of the glutamate residue at position 295, which is a highly conserved residue located in the region of Ephexin4 required for the intermolecular interaction, to alanine (Ephexin4E295A) disrupted the intermolecular interaction and increased binding of RhoG, resulting in augmented RhoG activation. In addition, phagocytosis of apoptotic cells and formation of membrane ruffles were increased more by expression of Ephexin4E295A than by expression of wild-type Ephexin4. Taken together, our data suggest that Ephexin4 is autoinhibited through its intermolecular interaction, which impedes binding of RhoG
A scaffold for signaling of Tim-4-mediated efferocytosis is formed by fibronectin
No full textAn essential step during clearance of apoptotic cells is the recognition of phosphatidylserine (PS) exposed on apoptotic cells by its receptors on phagocytes. Tim-4 directly binding to PS and functioning as a tethering receptor for phagocytosis of apoptotic cells has been extensively studied over the past decade. However, the molecular mechanisms by which Tim-4 collaborates with other engulfment receptors during efferocytosis remain elusive. By comparing efferocytosis induced by Tim-4 with that by Anxa5-GPI, an artificial tethering receptor, we found that Tim-4 possesses auxiliary machinery to induce a higher level of efferocytosis than Anxa5-GPI. To search for that, we performed a yeast two-hybrid screen and identified Fibronectin (Fn1) as a novel Tim-4-associating protein. Tim-4 directly associated with Fn1 and formed a complex with integrins via the association of Fn1. Through Tim-4(-/-) mice and cell-based assays, we found that modulation of the Fn1 level affected efferocytosis induced by Tim-4 and disruption of the interaction between Tim-4 and Fn1 abrogated Tim-4-mediated efferocytosis. In addition, Tim-4 depletion attenuated integrin signaling activation and perturbation of integrin signaling suppressed Tim-4-promoted efferocytosis. Taken together, the data suggest that Fn1 locates Tim-4 and integrins in close proximity by acting as a scaffold, resulting in synergistic cooperation of Tim-4 with integrins for efficient efferocytosis.
Apoptotic Cells Trigger Calcium Entry in Phagocytes by Inducing the Orai1-STIM1 Association
No full textSwift and continuous phagocytosis of apoptotic cells can be achieved by modulation of calcium flux in phagocytes. However, the molecular mechanism by which apoptotic cells modulate calcium flux in phagocytes is incompletely understood. Here, using biophysical, biochemical, pharmaceutical, and genetic approaches, we show that apoptotic cells induced the Orai1-STIM1 interaction, leading to store-operated calcium entry (SOCE) in phagocytes through the Mertk-phospholipase C (PLC) γ1-inositol 1,4,5-triphosphate receptor (IP3R) axis. Apoptotic cells induced calcium release from the endoplasmic reticulum, which led to the Orai1-STIM1 association and, consequently, SOCE in phagocytes. This association was attenuated by masking phosphatidylserine. In addition, the depletion of Mertk, which indirectly senses phosphatidylserine on apoptotic cells, reduced the phosphorylation levels of PLCγ1 and IP3R, resulting in attenuation of the Orai1-STIM1 interaction and inefficient SOCE upon apoptotic cell stimulation. Taken together, our observations uncover the mechanism of how phagocytes engulfing apoptotic cells elevate the calcium level
The Peroxisomal Localization of Hsd17b4 Is Regulated by Its Interaction with Phosphatidylserine
No full textPhosphatidylserine (PS), a negatively charged phospholipid exclusively located in the inner leaflet of the plasma membrane, is involved in various cellular processes such as blood coagulation, myoblast fusion, mammalian fertilization, and clearance of apoptotic cells. Proteins that specifically interact with PS must be identified to comprehensively understand the cellular processes involving PS. However, only a limited number of proteins are known to associate with PS. To identify PS-associating proteins, we performed a pulldown assay using streptavidin-coated magnetic beads on which biotin-linked PS was immobilized. Using this approach, we identified Hsd17b4, a peroxisomal protein, as a PS-associating protein. Hsd17b4 strongly associated with PS, but not with phosphatidylcholine or sphingomyelin, and the Scp-2-like domain of Hsd17b4 was responsible for this association. The association was disrupted by PS in liposomes, but not by free PS or the components of PS. In addition, translocation of PS to the outer leaflet of the plasma membrane enriched Hsd17b4 in peroxisomes. Collectively, this study suggests an unexpected role of PS as a regulator of the subcellular localization of Hsd17b4.
Phagocyte Chemoattraction Is Induced through the Mcp-1–Ccr2 Axis during Efferocytosis
No full textApoptotic cells generated during development and for tissue homeostasis are swiftly and continuously removed by phagocytes via a process called efferocytosis. Efficient efferocytosis can be achieved via transcriptional modulation in phagocytes that have engulfed apoptotic cells. However, such modulation and its effect on efferocytosis are not completely understood. Here, we report that phagocytes are recruited to apoptotic cells being cleared through the Mcp-1–Ccr2 axis, which facilitates clearance of apoptotic cells. We identified Mcp-1 as a modulated transcript using a microarray and found that Mcp-1 secretion was augmented in phagocytes engulfing apoptotic cells. This augmented Mcp-1 secretion was impaired by blocking phagolysosomal degradation of apoptotic cells. Conditioned medium from wild type (WT) phagocytes promoted cell migration, but that from Mcp-1−/− phagocytes did not. In addition, blockade of Ccr2, the receptor for Mcp-1, abrogated cell migration to conditioned medium from phagocytes incubated with apoptotic cells. The intrinsic efferocytosis activity of Mcp-1−/− and Ccr2−/− phagocytes was unaltered, but clearance of apoptotic cells was less efficient in the peritoneum of Mcp-1−/− and Ccr2−/− mice than in that of WT mice because fewer Ccr2-positive phagocytes were recruited. Taken together, our findings demonstrate a mechanism by which not only apoptotic cells but also phagocytes induce chemoattraction to recruit phagocytes to sites where apoptotic cells are cleared for efficient efferocytosis
Crbn modulates calcium influx by regulating Orai1 during efferocytosis
No full textCalcium flux regulating intracellular calcium levels is essential and modulated for efficient efferocytosis. However, the molecular mechanism by which calcium flux is modulated during efferocytosis remains elusive. Here, we report that Orai1, a Crbn substrate, is upregulated via its attenuated interaction with Crbn during efferocytosis, which increases calcium influx into phagocytes and thereby promotes efferocytosis. We found that Crbn deficiency promoted phagocytosis of apoptotic cells, which resulted from facilitated phagocytic cup closure and was nullified by a CRAC channel inhibitor. In addition, Orai1 associated with Crbn, resulting in ubiquitination and proteasomal degradation of Orai1 and alteration of SOCE-mediated calcium influx. The association of Orai1 with Crbn was attenuated during efferocytosis, leading to reduced ubiquitination of Orai1 and consequently upregulation of Orai1 and calcium influx. Collectively, our study reveals a regulatory mechanism by which calcium influx is modulated by a Crbn-Orai1 axis to facilitate efferocytosis. Calcium flux must be carefully controlled during the phagocytosis of apoptotic cells (efferocytosis), although how this occurs is not fully understood. Here, the authors show that the Cereblon E3 ligase regulates Orai1 degradation and subsequently SOCE-mediated calcium influx.
Tim-4 functions as a scavenger receptor for phagocytosis of exogenous particles
No full textThe phosphatidylserine (PS) receptor Tim-4 mediates phagocytosis of apoptotic cells by binding to PS exposed on the surface of these cells, and thus functions as a PS receptor for apoptotic cells. Some of PS receptors are capable of recognizing other molecules, such as LPS on bacteria, besides PS on apoptotic cells. However, it is unclear whether Tim-4 perceives other molecules like the PS receptors. Here, we report that Tim-4 facilitates the phagocytosis of exogenous particles as well as apoptotic cells. Similar to the process that occurs during Tim-4-mediated efferocytosis, the uptake of exogenous E. coli and S. aureus bioparticles was promoted by overexpression of Tim-4 on phagocytes, whereas phagocytosis of the bioparticles was reduced in Tim-4-deficient cells. A truncation mutant of Tim-4 lacking the cytoplasmic tail promoted phagocytosis of the particles, but a mutant lacking the IgV or the mucin domain failed to enhance phagocytosis. However, expression of Tim-4(AAA) (a mutant form of Tim-4 that does not bind phosphatidylserine and does not promote efferocytosis) still promoted phagocytosis. Tim-4-mediated phagocytosis was not blocked by expression of the phosphatidylserine-binding protein Anxa5. Furthermore, binding of lipopolysaccharide (LPS), which is found in the outer membrane of Gram-negative bacteria, was higher in Tim-4-overexpressing cells than in Tim-4-deficient cells. In summary, our study suggests that Tim-4 acts as a scavenger receptor and mediates phagocytosis of exogenous particles in a phosphatidylserine-independent manner.
Hierarchical spline for time series prediction: An application to naval ship engine failure rate
No full textWhen seasonality meets Bayesian: Decomposing seasonalities in Stan
No full textMultiple seasonalities play a key role in time series forecasting, especially for business time series where seasonal effects are often dramatic. Previous approaches including Fourier decomposition, exponential smoothing, and Seasonal ARIMA do not reflect distinct characteristics of each period in seasonal patterns such as unique behavior of specific day of the week in business data. We propose a multi-dimensional hierarchical model. Intermediate parameters for each seasonal period are first estimated, then mixture of intermediate parameters are then taken, resulting in the model which successfully reflects interactions between multiple seasonalities. Although this process leads to the reduction of data available for each parameter, a robust estimation can be obtained through a hierarchical Bayesian model. Consideration of not only the characteristics of each seasonal periods but also the interactions between characteristics from multiple seasonalities becomes possible through this model. Our new model is implemented in Stan and considerable improvements in prediction accuracy compared to previous models are achieved. Previous models include Fourier decomposition which Prophet uses to model seasonalities. Comparison has been performed on real-world dataset from a nation-scale logistic network
