Sesamin Induces the Transdifferentiation of Type II Alveolar Epithelial Cells via AnnexinA1 and TRPV1.

PURPOSE: Acute lung injury (ALI) with high rates of morbidity is often accompanied by the apoptosis in the type I alveolar epithelial cells (ATIs). Thus, the transdifferentiation of type II alveolar epithelial cells (ATIIs) into ATIs is crucial for the maintenance of alveolar epithelial functions. We aimed to elucidate the role of sesamin in the transdifferentiation of ATIIs to ATIs and the involvement of the TRPV1/AKT pathway. METHODS: In vivo, the mouse model of ALI was simulated by intraperitoneal and intratracheal injections of lipopolysaccharide (LPS), respectively. The protective effects of sesamin on ALI were investigated using the survival rate, lung/body weight ratio, histological analysis of lung with HE staining, and mRNA levels of inflammatory factors. Western blot analysis and immunofluorescence detection of ATIs marker AQP5 were used to evaluate the protective effect of sesamin on ATIs. Western blot, EdU, and qPCR analyses were applied to detect changes in apoptosis, proliferation, and transdifferentiation markers of ATII A549 cell lines. Small interfering RNA (siRNA) was used to detect the involvement and relationships between the sesamin receptors (ANXA1 and TRPV1) and the AKT pathway in transdifferentiation. RESULTS: Sesamin (200 mg/kg) significantly improved LPS-induced ALI and inhibited LPS-induced ATIs reduction. A low concentration of sesamin (20 µM) promoted the transdifferentiation of ATIIs to ATIs. Both ANXA1 and TRPV1 were involved in sesamin-promoted transdifferentiation, while the P-AKT (S473) level was down-regulated by TRPV1 siRNA. CONCLUSION: Sesamin may promote transdifferentiation of ATII to ATI to ultimately rescue ALI, with TRPV1/AKT pathway involved in this transdifferentiation. This study revealed a novel role of sesamin in promoting the transdifferentiation of ATIIs to ATIs, providing experimental supports for the potential targets of ALI therapy.

Zebrafish: A smart tool for heart disease research.

The increasing prevalence of heart disease poses a significant threat to human survival and safety. However, the current treatments available for heart disease are quite limited. Therefore, it is of great importance to utilize suitable animal models that can accurately simulate the physiological characteristics of heart disease. This would help improve our understanding of this disease and aid in the development of new treatment methods and drugs. Zebrafish hearts not only exhibit similarities to mammalian hearts, but they also share ~70% of homologous genes with humans. Utilizing zebrafish as an alternative to costly and time-consuming mammalian models offers numerous advantages. Zebrafish models can be easily established and maintained, and compound screening and genetic methods allow for the creation of various economical and easily controlled zebrafish and zebrafish embryonic heart disease models in a short period of time. Consequently, zebrafish have become a powerful tool for exploring the pathological mechanisms of heart disease and identifying new effective genes. In this review, we summarize recent studies on different zebrafish models of heart disease. We also describe the techniques and protocols used to develop zebrafish models of myocardial infarction, heart failure, and congenital heart disease, including surgical procedures, forward and reverse genetics, as well as drug and combination screening. This review aims to promote the utilization of zebrafish models in investigating diverse pathological mechanisms of heart disease, enhancing our knowledge and comprehension of heart disease, and offering novel insights and objectives for exploring the prevention and treatment of heart disease.

JNK-dependent phosphorylation and nuclear translocation of EGR-1 promotes cardiomyocyte apoptosis.

Myocardial apoptosis induced by myocardial ischemia and hyperlipemia are the main causes of high mortality of cardiovascular diseases. It is not clear whether there is a common mechanism responsible for these two kinds of cardiomyocyte apoptosis. Previous studies demonstrated that early growth response protein 1 (EGR-1) has a pro-apoptotic effect on cardiomyocytes under various stress conditions. Here, we found that EGR-1 is also involved in cardiomyocyte apoptosis induced by both ischemia and high-fat, but how EGR-1 enters the nucleus and whether nuclear EGR-1 (nEGR-1) has a universal effect on cardiomyocyte apoptosis are still unknown. By analyzing the phosphorylation sites and nucleation information of EGR-1, we constructed different mutant plasmids to confirm that the nucleus location of EGR-1 requires Ser501 phosphorylation and regulated by JNK. Furthermore, the pro-apoptotic effect of nEGR-1 was further explored through genetic methods. The results showed that EGR-1 positively regulates the mRNA levels of apoptosis-related proteins (ATF2, CTCF, HAND2, ELK1), which may be the downstream targets of EGR-1 to promote the cardiomyocyte apoptosis. Our research announced the universal pro-apoptotic function of nEGR-1 and explored the mechanism of its nucleus location in cardiomyocytes, providing a new target for the "homotherapy for heteropathy" to cardiovascular diseases.

Sphingosylphosphorylcholine ameliorates doxorubicin-induced cardiotoxicity in zebrafish and H9c2 cells by reducing excessive mitophagy and mitochondrial dysfunction.

Doxorubicin (DOX, C27H29NO11), is an anthracycline tumor chemotherapy drug, which has significant side effects on many organs including the heart. In recent years, mitochondrial dysfunction caused by DOX was identified as an important reason for cardiotoxic injury. Sphingosylphosphorylcholine (SPC) is essential for mitochondrial homeostasis in our previous report, however, its role in DOX-caused cardiomyopathy has remained elusive. Herein, DOX treated zebrafish embryos (90 µM) and adult fish (2.5 µM/g) were used to simulate DOX-induced cardiotoxic damage. Histopathological and ultrastructural observations showed that SPC (2.5 µM) significantly ameliorated DOX-induced pericardial edema, myocardial vacuolization and apoptosis. Furthermore, SPC (2.5 µM) can significantly inhibit DOX-induced apoptosis and promote cell proliferation in DOX treated H9c2 cells (1 µM), which is dependent on the restoration of mitochondrial homeostasis, including restored mitochondrial membrane potential, mitochondrial superoxide and ATP levels. We finally confirmed that SPC restored mitochondrial homeostasis through ameliorating DOX-induced excessive mitophagy. Mechanistically, SPC reduced calmodulin (CaM) levels and thus inhibiting Parkin activation and Parkin-dependent mitophagy. These results suggest that reducing the cardiotoxicity of chemotherapeutic drugs by targeting SPC may be a new solution to rescue chemotherapy injury.

Regulatory effects of chicken TRIM25 on the replication of ALV-A and the MDA5-mediated type I interferon response.

This study focuses on the immunoregulatory effects of chicken TRIM25 on the replication of subgroup A of avian leukosis virus (ALV-A) and the MDA5-mediated type I interferon response. The ALV-A-SDAU09C1 strain was inoculated into DF1 cells and 1-day-old SPF chickens, and the expression of TRIM25 was detected at different time points after inoculation. A recombinant overexpression plasmid containing the chicken TRIM25 gene (TRIM25-GFP) was constructed and transfected into DF1 cells to analyse the effects of the overexpression of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-ß. A small interfering RNA targeting chicken TRIM25 (TRIM25-siRNA) was prepared and transfected into DF1 cells to assess the effects of the knockdown of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-ß. The results showed that chicken TRIM25 was significantly upregulated at all time points both in ALV-A-infected cells and in ALV-A-infected chickens. Overexpression of chicken TRIM25 in DF1 cells dramatically decreased the antigenic titres of ALV-A in the cell supernatant and upregulated the relative expression of MDA5, MAVS and IFN-ß induced by ALV-A or by poly(I:C); in contrast, knockdown of chicken TRIM25 significantly increased the antigenic titres of ALV-A and downregulated the relative expression of MDA5, MAVS and IFN-ß. It can be concluded that chicken TRIM25 can inhibit the replication of ALV-A and upregulate the MDA5 receptor-mediated type I interferon response in chickens. This study can help improve the understanding of the antiviral activities of chicken TRIM25 and enrich the knowledge of antiviral responses in chickens.

The tail segments are required by the performance but not the accomplishment of various modes of Drosophila larval locomotion.

The tail plays important roles in locomotion control in many animals. But in animals with multiple body segments, the roles of the hind body segments and corresponding innervating neurons in locomotion control are not clear. Here, using the Drosophila larva as the model animal, we investigated the roles of the posterior terminal segments in various modes of locomotion and found that they participate in all of them. In forward crawling, paralysis of the larval tail by blocking the Abdb-Gal4 labeled neurons in the posterior segments of VNC led to a slower locomotion speed but did not prevent the initiation of forward peristalsis. In backward crawling, larvae with the Abdb-Gal4 neurons inhibited were unable to generate effective displacement although waves of backward peristalsis could be initiated and persist. In head swing where the movement of the tail is not obvious, disabling the larval tail by blocking Abdb-Gal4 neurons led to increased bending amplitude upon touching the head. In the case of larval lateral rolling, larval tail paralysis by inhibition of Abdb-Gal4 neurons did not prevent the accomplishment of rolling, but resulted in slower rolling speed. Our work reveals that the contribution of Drosophila larval posterior VNC segments and corresponding body segments in the tail to locomotion is comprehensive but could be compensated at least partially by other body segments. We suggest that the decentralization in locomotion control with respect to animal body parts helps to maintain the robustness of locomotion in multi-segment animals.

TRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J infection in chickens by targeting 14-3-3σ protein.

ALV-J-SD1005 strain was subcutaneously inoculated into the necks of 1-day-old HY-Line Brown chickens and caused severe growth retardation, viremia and subcutaneous fibrosarcomas in the necks of all infected chickens from 14 days post inoculation (DPI) to 21 DPI, and also significantly increased the expressions of TRIM25, P53, etc., but significantly decreased the expressions of 14-3-3σ, etc. Overexpression of chicken TRIM25 (chTRIM25) significantly promoted cell proliferation and improved the expressions of P53, CDC2, and CDK2 tumor factors; and significantly inhibited the expression of 14-3-3σ in ALV-J-SD1005-infected DF1 cells; but knockdown of chTRIM25 caused the opposite effects. The results of co-immunoprecipitation (Co-IP) and confocal microscopy confirmed that chTRIM25 can recognize and bind 14-3-3σ protein in ALV-J-SD1005-infected cells, and they were co-located in the cytoplasm. It can be concluded that chTRIM25 participates in the fibrous tissue hyperplasia induced by ALV-J-SD1005 infections in chickens by binding 14-3-3σ protein and regulating the expressions of 14-3-3σ, P53, CDC2, and CDK2.

MORN4 protects cardiomyocytes against ischemic injury via MFN2-mediated mitochondrial dynamics and mitophagy.

The imbalance of mitochondrial fission and fusion dynamics causes ischemic cardiomyocyte apoptosis and heart injury by affecting mitophagy. Regulation of mitochondrial dynamics is an important therapeutic strategy for ischemic heart diseases. Considering the important roles of MORN motifs in heart diseases and chloroplast fission, we aimed to investigate the possible role of MORN repeat-containing protein 4 (MORN4) in the progression of myocardial infarction (MI), ischemic cardiomyocyte apoptosis, mitochondrial dynamics, and mitophagy. We found that in the MI mouse, MORN4 knockdown remarkably accelerated cardiac injury and fibrosis with deteriorating cardiac dysfunction. Sphingosylphosphorylcholine (SPC) alleviated ischemic cardiomyocyte apoptosis and heart injury through increased level of MORN4, indicating a vital function of MORN4 in heart with SPC used to clarify the molecular mechanisms underlying the functions of MORN4. Mechanistically, we found that MORN4 directly binds to MFN2 and promotes the phosphorylation of MFN2 S442 through Rho-associated protein kinase 2 (ROCK2), which mediates beneficial mitophagy induced by mitochondrial dynamics, while SPC promoted the binding of MORN4 and MFN2 and the process. Taken together, our data reveal a new perspective role of MORN4 in ischemic heart injury, and report that SPC could regulate myocardial mitochondrial homeostasis by activating the MORN4-MFN2 axis during the ischemic situation, this finding provides novel targets for improving myocardial ischemia tolerance and rescue of acute myocardial infarction.

Membrane occupation and recognition nexus (MORN) motif controls protein localization and function.

The membrane occupation and recognition nexus (MORN) motif was first defined in 2000, when it was identified in the junctophilin protein family. Dozens of studies have been published ever since, mainly focusing on the function of a given MORN motif-containing protein in parasites, plants or animal cells. Proteins with MORN motifs are not only expressed in most animal and plant cell types, but also significantly differ in their intracellular localization, suggesting that the MORN motifs may fulfill multiple physiological functions. Recent studies have found that MORN motif-containing proteins junctophilin-1/2 and MORN3 play a role in cardiac hypertrophy, skeletal muscle fiber stability and cancer. Hence, MORN motif-containing proteins may be exploited to develop improved treatments for various pathological conditions, such as cardiovascular diseases. Here, we review current research on MORN motif-containing proteins in different organisms and provide both ideas and approaches for follow-up exploration of their functions and applications.

MicroRNA-155-5p/EPAS1/interleukin 6 pathway participated in the protection function of sphingosylphosphorylcholine to ischemic cardiomyocytes.

AIM: Previous research in our laboratory found that a biologically active sphingomyelin metabolite, sphingosylphosphorylcholine (SPC), can inhibit myocardial cell apoptosis caused by ischemia with an unknown mechanism. Here, we aimed to study the possible participation of EPAS1 in the protection process of SPC. METHODS: The rat cardiomyocytes deprived of serum were used to mimic ischemic-caused apoptosis, then treated with or without SPC. The expression and nuclear shift of EPAS1 were detected by western blot and immunofluorescence, and its function was studied using its siRNA. KEY FINDING: Our research shows that SPC inhibited serum starvation caused cardiomyocyte apoptosis, accompanied by the up-regulation and nucleus translocation of EPAS1. EPAS1 levels did not change when its transcript was blocked by Actinomycin D, which prompted us to search for a post-transcription mechanism for its increased expression, and finally found that miR-155-5p, regulated by STAT3, was a new post-transcription regulator to EPAS1. Further investigation found that EPAS1 participated in the protective effect of SPC is mainly achieved by activating the downstream target gene, interleukin-6 (IL-6). SIGNIFICANCE: Our results expand our understanding of the biological functions of SPC, and bring a new pathway as a potential therapeutic target to the treatment of cardiovascular diseases caused by myocardial apoptosis.

Design and Application of a Rotatory Device for Detecting Transient Ca2+ Signals in Response to Mechanical Stimulation Using an Aequorin-Based Ca2+ Imaging System.

Elevation of the cytosolic free calcium ion (Ca2+ ) concentration ([Ca2+ ]cyt ) is one of the earliest responses to biotic and abiotic stress in plant cells. Among the various Ca2+ detection systems available, aequorin-based luminescence Ca2+ imaging systems provide a relatively amenable and robust method that facilitates large-scale genetic-mutant screening based on [Ca2+ ]cyt responses. Compared to that mediated by chemical elicitors, mechanical stimulation-induced elevation of [Ca2+ ]cyt is considerably more rapid, occurring within 10 s following stimulation. Therefore, its assessment using aequorin-based Ca2+ imaging systems represents a notable challenge, given that a time interval of ≥1 min is required to reduce the background light before operating the photon imaging detector. In this context, we designed a device that can rotate automatically within the confines of an enclosed dark box, and using this, we can record [Ca2+ ]cyt dynamics immediately after plants had been rotated to induce mechanical stimulation. This tool can facilitate the study of perception and early signal transduction in response to mechanical stimulation on a large scale based on [Ca2+ ]cyt responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of background luminance signals in aequorin-transformed Arabidopsis seedlings using a photon imaging detector Basic Protocol 2: Construction of the rotatory device Basic Protocol 3: Calcium measurement in Arabidopsis seedlings after rotatory stimulation Basic Protocol 4: Data analysis and processing.

Preparation of the Secretory Recombinant ALV-J gp85 Protein Using Pichia pastoris and Its Immunoprotection as Vaccine Antigen Combining with CpG-ODN Adjuvant.

This study focuses on preparing the secretory recombinant J subgroup of avian leukosis virus (ALV-J) gp85 protein using Pichia pastoris and evaluating its immunoprotection as vaccine antigen combining with CpG-ODN adjuvant. The secretory recombinant plasmid pPIC9-gp85 containing ALV-J gp85 gene was designed and was transfected into the genome of P. pastoris (GS115) cells. The recombinant plasmid was expressed under the induction of methanol. The expressed products in the medium of the cells were purified and identified with endoglycosidase digestion assay and western blot mediated with monoclonal antibody (MAb) JE9. The purified product combining with CpG-ODN adjuvant was inoculated intramuscularly into 7-day-old chickens and three booster inoculations were performed on 21 days post first inoculation (dpfi), 42, and 56 dpfi. The antibody responses and cellular immune responses were detected, and the protective effects were analyzed after challenge with ALV-J. The results showed that the secretory pPIC9-gp85 plasmid was successfully constructed and could be stably expressed in GS115 cells. The expressed products were N-acetylglucosylated and could specifically combine with MAb (JE9). The secreted gp85 protein combining with CpG-ODN adjuvant could induce higher antibody response and spleen lymphocyte proliferation response and IFN-γ-inducing response, and could protect all the inoculated chickens against the viremia and the immunosuppressive lesions caused by ALV-J challenge. The results of neutralizing test in vitro suggested that the antisera with some ALV-J antibody titers could neutralize ALV-J strain and inhibit the growth of virus in vitro. The result of IFA showed that IgG antibody in the antisera could specifically combine with ALV-J strain in cells. It can be concluded that the secretory recombinant gp85 protein, as a new acetylglucosylated gp85 protein, was successfully prepared and combining with CpG-ODN adjuvant could protect the inoculated chickens against ALV-J infection. This study first reported the methods on preparing the secretory recombinant ALV-J gp85 protein using P. pastoris and evaluated its immunoprotection.

miR­199a­3p/Sp1/LDHA axis controls aerobic glycolysis in testicular tumor cells.

Aerobic glycolysis is one of the characteristics of tumor metabolism and contributes to the development of tumors. Studies have identified that microRNA (miRNA/miR) serves an important role in glucose metabolism of tumors. miR­199a­3p is a member of the miR­199a family that controls the outcomes of cell survival and death processes, and previous studies have indicated that the expression of miR­199a­3p is low and may be an inhibitor in several cancer types, including testicular tumors. The present study discussed the role and underlying mechanism of miR­199a­3p in aerobic glycolysis of Ntera­2 cells and identified its downstream factors. Firstly, miR­199a­3p exhibited an inhibitory effect on lactic acid production, glucose intake, and reactive oxygen species (ROS) and adenosine 5'­triphosphate (ATP) levels in Ntera­2 cells. Then, using bioinformatics, recombinant construction and a dual luciferase reporter gene system, transcription factor Specificity protein 1 (Sp1) was determined as the direct target of miR­199a­3p. Also, downregulation of Sp1 by RNA interference decreased lactic acid production, glucose intake, and ROS and ATP levels in Ntera­2 cells. Subsequently, through a functional rescue experiment, it was identified that the overexpression of Sp1 may abate the inhibition of miR­199a­3p on glucose metabolism, with the exception of ATP level, suggesting a reciprocal association between Sp1 and miR­199a­3p. Finally, it was determined that miR­199a­3p overexpression and Sp1 knockdown decreased lactate dehydrogenase A (LDHA) protein expression, which indicated that LDHA is a downstream target of the miR­199a­3p/Sp1 signaling pathway. To additionally verify the regulation of LDHA expression by 199a­3p/Sp1, a LDHA promoter reporter plasmid was generated and the high activity of the promoter, which contained 3 potential Sp1 binding elements, was confirmed. In addition, the overexpression of Sp1 led to the increased activity of the LDHA promoter, whereas knockdown of Sp1 exhibited the opposite effect. Therefore, the results of the present study demonstrated that miR­199a­3p can inhibit LDHA expression by downregulating Sp1, and provided mechanistic evidence supporting the existence of a novel miR­199a­3p/Sp1/LDHA axis and its critical contribution to aerobic glycolysis in testicular cancer cells.