Hippo pathway-manipulating neutrophil-mimic hybrid nanoparticles for cardiac ischemic injury via modulation of local immunity and cardiac regeneration

The promise of regeneration therapy for restoration of damaged myocardium after cardiac ischemic injury relies on targeted delivery of proliferative molecules into cardiomyocytes whose healing benefits are still limited owing to severe immune microenvironment due to local high concentration of proinflammatory cytokines. Optimal therapeutic strategies are therefore in urgent need to both modulate local immunity and deliver proliferative molecules. Here, we addressed this unmet need by developing neutrophil-mimic nanoparticles NM@miR, fabricated by coating hybrid neutrophil membranes with artificial lipids onto mesoporous silica nanoparticles (MSNs) loaded with microRNA-10b. The hybrid membrane could endow nanoparticles with strong capacity to migrate into inflammatory sites and neutralize proinflammatory cytokines and increase the delivery efficiency of microRNA-10b into adult mammalian cardiomyocytes (CMs) by fusing with cell membranes and leading to the release of MSNs-miR into cytosol. Upon NM@miR administration, this nanoparticle could home to the injured myocardium, restore the local immunity, and efficiently deliver microRNA-10b to cardiomyocytes, which could reduce the activation of Hippo-YAP pathway mediated by excessive cytokines and exert the best proliferative effect of miR-10b. This combination therapy could finally improve cardiac function and mitigate ventricular remodeling. Consequently, this work offers a combination strategy of immunity modulation and proliferative molecule delivery to boost cardiac regeneration after injury.

Nucleic acid drug delivery strategies without the lysosomal pathway / 中国药科大学学报

@#By regulating gene expression, nucleic acid drugs functioning in the cytoplasm or nucleus are of great significance in the treatment of acquired or inherited diseases and vaccine development.A variety of nucleic acid delivery vectors currently developed are suffering from low transfection efficiency due to endosome/lysosome entrapment.This paper introduces and summarizes the nucleic acid delivery strategies that bypass the endosomal/lysosomal pathway, including membrane translocation, membrane fusion, receptor/transporter-mediated non-endocytic uptake and caveolae-mediated endocytosis, and discusses the problems and challenges facing such strategies, aiming to facilitate the development of intracellular delivery of nucleic acid drugs bypassing lysosomal pathway.

Anti-herpes simplex virus effect of aristolochic acids in vitro and in vivo / 中国药理学通报

Aim To investigate the inhibition effects and mechanisms of aristolochic acids(AAs)against herpes simplex virus(HSV)in vitro and in vivo. Methods The cytopathic effect(CPE), plaque assay, indirect immunofluorescence and others were used to explore the anti-HSV effects and mechanisms of aristolochic acid in Vero cells, and the in vivo anti-HSV activity of AAs was evaluated using HSV-1 infected BALB/c mouse model. Results The IC

Reciprocal regulation between lunapark and atlastin facilitates ER three-way junction formation

Three-way junctions are characteristic structures of the tubular endoplasmic reticulum (ER) network. Junctions are formed through atlastin (ATL)-mediated membrane fusion and stabilized by lunapark (Lnp). However, how Lnp is preferentially enriched at three-way junctions remains elusive. Here, we showed that Lnp loses its junction localization when ATLs are deleted. Reintroduction of ATL1 R77A and ATL3, which have been shown to cluster at the junctions, but not wild-type ATL1, relocates Lnp to the junctions. Mutations in the N-myristoylation site or hydrophobic residues in the coiled coil (CC1) of Lnp N-terminus (NT) cause mis-targeting of Lnp. Conversely, deletion of the lunapark motif in the C-terminal zinc finger domain, which affects the homo-oligomerization of Lnp, does not alter its localization. Purified Lnp-NT attaches to the membrane in a myristoylation-dependent manner. The mutation of hydrophobic residues in CC1 does not affect membrane association, but compromises ATL interactions. In addition, Lnp-NT inhibits ATL-mediated vesicle fusion in vitro. These results suggest that CC1 in Lnp-NT contacts junction-enriched ATLs for proper localization; subsequently, further ATL activity is limited by Lnp after the junction is formed. The proposed mechanism ensures coordinated actions of ATL and Lnp in generating and maintaining three-way junctions.

Research progress in membrane fusion process among Paramyxoviruses / 中华实验和临床病毒学杂志

The family Paramyxoviridae is a group of viruses that significantly affect human and animal health. Paramyxoviruses are generally thought to enter host cells by direct fusion of the viral and host cell membranes. Membrane fusion, essential for syncytium formation, is not only a pathological hallmark of paramyxoviral infections, but also a kind of mechanism of cell-to-cell viral spread. Nevertheless, the process of membrane fusion is highly conserved, only consisting of two-component fusion apparatus: an attachment protein (HN/G/H) and a fusion (F) protein. However, there is a significant knowledge gap on the mechanism(s) by which HN/H/G couples receptor binding to F-triggering, since different attachment proteins possess special triggering process. In this review, we summarize the general property and distinction of the fusion process during paramyxoviral infection for further research on the pathogenic mechanism.

Role of PKCα/HO-1 signaling pathway in endotoxin-induced damage to alveolar macrophages of rats: the relationship with mitofusin-1 / 中华麻醉学杂志

Objective To evaluate the role of protein kinase Cα (PKCα)/heme oxygenase-1 (HO-1) signaling pathway in endotoxin-induced damage to alveolar macrophages and the relationship with mitofusin-1 (Mfn1) in rats.Methods Rat alveolar macrophages NR8383 cells cultured in vitro were seeded in 96-well plates at a density of 1 × 104 cells/ml.NR8383 cells were divided into 5 groups (n =15 each)using a random number table:control group (group C),endotoxin challenge model group (group E),PKCα inhibitor Go6976 group (group G),PKCα agonist PMA group (group P) and dimethyl sulfoxide group (group D).NR8383 cells were stimulated with 10 μg/ml lipopolysaccharide (LPS) to establish the model of endotoxin challenge in alveolar macrophages.In G,P and D groups,cells were pretreated with 5 μmol/L Go6976,100 nmol/L PMA and 0.1％ dimethyl sulfoxide,respectively,for 30 min starting from 30 min before stimulation with LPS,and 10 μg/ml LPS was then given.The cells were collected after 24 h of incubation for measurement of malondialdehyde (MDA) and reactive oxygen species (ROS) contents,superoxide dismutase (SOD) activity and expression of PKCα,HO-1 and Mfn1 protein and mRNA (by fluorescent quantitative polymerase chain reaction or Western blot).Results Compared with group C,MDA and ROS contents were significantly increased,the SOD activity was decreased,the expression of PKCα and HO-1 protein and mRNA was up-regulated,and the expression of Mfn1 protein and mRNA was downregulated in E,G,P and D groups (P＜0.05).Compared with group E,MDA and ROS contents were significantly increased,the SOD activity was decreased,and the expression of PKCα,HO-1 and Mfn1 protein and mRNA was down-regulated in group G,MDA and ROS contents were significantly decreased,the SOD activity was increased,and the expression of PKCα,HO-1 and Mfn1 protein and mRNA was upregulated in group P (P＜0.05),and no significant change was found in the parameters mentioned above in group D (P＞0.05).Conclusion Promotion of Mfn1 expression following PKCα/HO-1 signaling pathway activation is the endogenous protective mechanism of endotoxin-induced damage to alveolar macrophages of rats.

Research progress in interaction mechanism of enveloped glycoproteins of human parainfluenza virus type 3 / 中华实验和临床病毒学杂志

Human parainfluenza viruses (hPIVs), a series of single-stranded RNA viruses of Paramyxoviridae, are the main pathogen of respiratory tract infection. hPIV3 is the main cause of lower respiratory tract infection leading to bronchiolitis and pneumonias in young children under the age of six months, and it is the second major pathogen only next to respiratory syncytial virus (RSV). In this paper, we mainly discuss two kinds of virulence-related surface glycoprotein of hPIV3: hemagglutinin-neuraminidase (HN) protein and fusion protein (F) by briefly introducing the protein structure and physiological functions of HN and F. According to the latest research progress, we focus on the models which have been postulated to explain how F and HN work in concert to bring about membrane fusion.

Relationship between mechanism of SIRT1-mediated mitophagy in myocardium and Mfn2 in diabetic rats / 中华麻醉学杂志

Objective To evaluate the relationship between the mechanism of silent information regulator factor 2-related enzyme 1 (SIRT1)-mediated mitophagy in the myocardium and mitofusin 2 (Mfn2) in diabetic rats.Methods Twenty-four SPF healthy adult male Sprague-Dawley rats,weighing 210-220 g,were allocated into 3 groups (n =8 each) using a random number table:control group (group C),diabetes mellitus group (group DM) and diabetes mellitus plus SIRT1 activator SRT1720 group (group DM+SRT).Diabetes mellitus was induced by intraperitoneal 1％ streptozotocin 60 mg/kg and confirmed by blood glucose ≥ 16.7 mmol/L 3 days later.SRT1720 1 mg/kg was injected via the caudal vein for 7 consecutive days in group DM+SRT.The ultrasonic method was used to measure the parameters of heart function including left ventricular end-diastolic volume (LVEDV),left ventricular end-systolic volume (LVESV),left ventricular ejection fraction (LVEF),heart rate (HR),E wave velocity (E),A wave velocity (A) and E/A ratio.Myocardial specimens were obtained for determination of the interaction between SIRT1 and Mfn2 and acetylation of Mfn2 (by co-immuno-precipitation) and expression of SIRT1,Mfn2,microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ),LC3 Ⅰ,Beclin1 and P62 (by Western blot).The ratio LC3 Ⅱ to LC3 Ⅰ (LC3 Ⅱ / Ⅰ ratio) was calculated.Results Compared with group C,LVEDV,LVEF,HR,E and E/A ratio were significantly decreased,A was increased,LC3 Ⅱ / Ⅰ ratio was decreased,the expression of Beclin1,SIRT1 and Mfn2 was down-regulated,the expression of P62 was up-regulated,and the interaction between SIRT1 and Mfn2 was decreased in group DM (P＜0.05).Compared with group DM,LVEDV,LVEF,E and E/A ratio were significantly increased,A was decreased,LC3 Ⅱ / Ⅰ ratio was increased,the expression of Beclin1 and SIRT1 was up-regulated,the expression of P62 was down-regulated,the interaction between SIRT1 and Mfn2 was increased,and the acetylation of Mfn2 was decreased in group DM+SRT (P＜0.05).Conclusion The mechanism by which SIRT1 mediates mitophagy in the myocardium is related to SIRT1-induced deacetylation of Mfn2 in diabetic rats.

Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.

Materials with lysosomal escape function and its mechanism / 中国药学杂志

OBJECTIVE: To review the progress on materials with lysosomal escape function. METHODS: Based on the original researches in recent years, the structure properties and escape mechanism of materials with lysosomal escape function were introduced in the present paper. RESULTS: The materials which can help the drug/gene to get out of the lysosomes were summarized and classified into two groups in the accordance with their escape mechanism: breaking the lysosomes by raising the osmotic pressure or destabilizing the membrane structure of lysosomes. CONCLUSION: The materials with lysosomal escape function, as a novel drug delivery tool, has promising application for the delivery of drug/gene and need to be studied deeply before the materials get widely applied.

Cellular Localization Analysis of Enhanced Green Fluorescent Protein Tagged hnRNP A1 Under Stress / 天津医药

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.

Strategy for Developing Medical Arsenals by Modulation of Membrane Fusion Activity of Influenza Virus Hemagglutinin

Influenza virus is a serious pathogen that burdens society with health care costs, and can lead to fatality. The virus is dealt with currently by vaccination and anti-influenza drugs. However, vaccines need to be improved towards safer and more efficient production formats, and drugs need to be constantly renewed to cope with resistances. That the neuraminidase inhibitors are only drugs currently available warrants urgent attention to an alternative anti-influenza target. In this paper we introduce studies on fusion activity of influenza virus hemagglutinin (HA), and discuss how to best utilize the knowledge for an improved vaccine development and an anti-influenza drug search. Potential application of mutations resulting in changes in fusion activity to cell culture optimized vaccine virus development and strategies to develop broad spectrum anti-influenza drugs through targeting the conserved fusion domain of the HA are discussed.

Surface Display of Rice Stripe Virus NSvc2 and Analysis of Its Membrane Fusion Activity / 中国病毒学·英文版

Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper.How RSV enters an insect cell to initiate the infection cycle is poorly understood.Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion.To conveniently study the membrane fusion activity of NSvc2,we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses.Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells.When induced by low pH,the membrane fusion was not observed in the cells that expressed NSvc2.Additionally,the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface.Thus,RSV NSvc2 is probably different from the phlebovirus counterparts,which could suggest different functions.RSV might enter insect cells other than by fusion with plasma or endosome membrane.

Mecanismos de sllida de parásitos intracelulares de su célula hospedrra / Exit Mechanisms of Intracellular Parasites from their Host Cells

Algunos parásitos intracelulares durante la infección en hospederos vertebrados se localizan al interior de sus células hospederas en un compartimiento intracelular rodeado por membrana denominado vacuola parasitófora. Para el sostenimiento e incremento de las infecciones causadas por estos parásitos es necesario que se dé un evento de liberación/salida de las formas infectivas, para que estas reinicien la infección en nuevas células. Para dicho fenómeno de liberación se han planteado dos mecanismos básicos: 1. la salida se da por eventos de ruptura de la membrana de la vacuola parasitófora (MVP) y de la membrana plasmática de la célula hospedera y/o 2. la salida se da por un proceso de fusión entre la MVP y la membrana de la célula hospedera, de forma que la luz de la VP y el espacio extracelular se hacen continuos, permitiendo la liberación del parásito. En esta revisión se presenta la evidencia que apoya estos modelos en bacterias y protozoarios intracelulares obligatorios, con especial énfasis en la salida de Leishmania.

Some intracellular parasites inhabit intracellular compartments known as parasitophorous vacuoles. To maintain and amplify infection, infective forms of the parasite must exit from the host cell to infect new cells. During parasite egress, two main mechanisms have been proposed: lysis of the membranes of the parasitophorus vacuole and the plasma membrane of the cell host, or fusion of these two membranes providing continuity between the lumen of the parasitophorous vacuole and the extracellular compartment. In this review we present evidence supporting these models for intracellular parasites of human importance with particular emphasis on Leishmania exit.

Human Herpesvirus 7 Glycoprotein B (gB) , gH, gL, gO Can Mediate Cell Fusion / 生物化学与生物物理进展

Human herpesvirus 7 (HHV-7) infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteins mediate the initial attachment and fusion events that occur between the viral envelope and a host cell membrane, as well as cell to cell spread of the virus. To characterize the HHV-7 glycoproteins that can mediate cell fusion, a cell-based fusion assay was used. 293T cells expressing the HHV-7 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with SupT1 cells transfected with T7 RNA polymerase. HHV-7 glycoproteins gB, gH, gL and gO can mediate the fusion of 293T cells with SupT1 cells, and the fusion can be inhibited by anti-CD4 mAbs. Thus, the coexpression of HHV-7 gB, gO, gH and gL is sufficient and necessary for HHV-7 induced membrane fusion, and one of these glycoproteins or protein complex formed by these glycoproteins might be the ligand(s) of CD4 molecule.

Antitumor effect of GALV membrane fusion glycoprotein on lung adenocarcinoma in vivo and in vitro / 第三军医大学学报

Objective To investigate the antitumor effect of special promoter-controlled Gibbon ape leukemia virus membrane fusion glycoprotein (GALV.fus) mediated by type Ⅰ herpes simplex virus (HSV-Ⅰ) on lung adenocarcinoma. Methods Recombinant HSV-Ⅰ plasmids encoding GALV.fus was introduced into green monkey kidney cells(Vero)by liposome to amplify the virus, and then the virus was transfected into lung adenocarcinoma (A549), human fetal fibroblasts (HFL-Ⅰ GNHu 5) and human lung adenocarcinoma xenografts which were established in nude mice subcutaneously to observe antitumor and cytotoxic effect in vitro and in vivo; Recombined cytomegalovirus (CMV) containing GALV.fus or enhanced green fluorescence protein were served as control. Results Recombinant HSV-Ⅰ virus were packed successfully. Heterotransplantative tumourigenicity of the tumour was 100% in nude mice after A549 cells were inoculated. Recombinant HSV-Ⅰvirus exerted obvious antitumor effects in vitro, with relative survival rate of 23%, while for CMV virus containing GALV. fus, the rate was 20%, and for CMV virus encoding EGFP, the rate was 68%. Recombinant HSV-Ⅰvirus also showed striking antitumor effect on the implanted tumor. Conclusion GALV.fus has powerful effect against lung cancer in vitro and in vivo and maybe a promising candidate for gene therapy.