Astragaloside IV- and nesfatin-1-encapsulated phosphatidylserine liposomes conjugated with wheat germ agglutinin and leptin to activate anti-apoptotic pathway and block phosphorylated tau protein expression for Parkinson's disease treatment.

Heap-up of α-synuclein (α-Syn) and its association with tau protein are esteemed to trigger the onset of Parkinson's disease (PD). The purpose of this study was to develop multi-functional liposomes incorporated with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-dimyristoyl-sn-glycero-3-phosphocholine and phosphatidylserine (PS) to load astragaloside IV (AS-IV) and nestifin-1 (NF-1), followed by grafting with wheat germ agglutinin (WGA) and leptin (Lep) (WGA-Lep-AS-IV-NF-1-PS-liposomes) to protect dopaminergic neurons from apoptosis. Experimental results showed that increasing the mole percentage of DSPC and PS enhanced the particle size, particle stability and entrapment efficiency of AS-IV and NF-1, and reduced the drug releasing rate. Strong affinity of NF-1 to PS was evidenced by nuclear magnetic resonance spectroscopy. WGA-Lep-AS-IV-NF-1-PS-liposomes diminished transendothelial electrical resistance and improved the capacity of propidium iodide, AS-IV and NF-1 to penetrate the blood-brain barrier (BBB). Immunocytochemical staining exhibited the ability of functionalized liposomes to target Lep receptor and α-Syn in MPP+-insulted SH-SY5Y cells. Western blots revealed a substantial reduction of α-Syn and phosphorylated tau protein in the anti-oxidative pathway through interaction with PS. During the course of treatment with WGA-Lep-AS-IV-NF-1-PS-liposomes, the combined activity of AS-IV and NF-1 and recognition capability simultaneously decreased the expression of Bax, and increased the expressions of Bcl-2, tyrosine hydroxylase and dopamine transporter. The liposomes carrying AS-IV and NF-1 can rescue degenerated neurons and are a promising formulation to achieve better PD management.

Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome.

Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1ß) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1ß secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1ß secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K+ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

Protection against Neurodegeneration in the Hippocampus Using Sialic Acid- and 5-HT-Moduline-Conjugated Lipopolymer Nanoparticles.

Significant involvement of oxidative stress in the brain can develop Alzheimer's disease (AD); however, a great number of clinical trials explains the limited success of antioxidant therapy in dealing with this neurodegenerative disease. Here, we established a lipopolymer system of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) incorporated with phosphatidic acid (PA) and modified with sialic acid (SA) and 5-hydroxytryptamine-moduline (5HTM) to improve quercetin (QU) activity against oxidative stress induced by amyloid-ß (Aß) deposits. Morphological studies revealed a uniform exterior of QU-SA-5HTM-PA-PLGA NPs with a spherical structure and enhanced aggregation with inclusion of PA in the formulation. A better brain-targeted delivery of the lipopolymeric NPs was verified from the high blood-brain barrier (BBB) permeability of QU through strong interactions of surface SA and 5HTM with O-linked N-acetylglucosamine and 5-HT1B receptors, respectively. Immunofluorescence staining images also supported QU-SA-5HTM-PA-PLGA NPs to traverse the microvessels of AD rat brain. Western blot analysis showed that QU-loaded PA-PLGA NPs suppressed caspase-3 expression. The ability of the nanocarriers to recognize Aß fibrils was demonstrated from the reduced senile plaque formation and the attenuated acetylcholinesterase and malondialdehyde activity in the hippocampus. Hence, the medication of QU-SA-5HTM-PA-PLGA NPs can facilitate the BBB penetration and prevent Aß accumulation, lipid peroxidation, and neuronal apoptosis for the AD management.

The RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1ß Secretion.

UNLABELLED: Inflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1ß (IL-1ß) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1ß secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1ß secretion. However, the precise mechanism by which NS1 inhibits IL-1ß secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1ß secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1ß secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1ß secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1ß secretion. IMPORTANCE: Innate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1ß (IL-1ß) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1ß secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1ß secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1ß secretion. These results will facilitate the development of new anti-inflammatory drugs.

Surface vimentin is critical for the cell entry of SARS-CoV.

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. RESULTS: A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. CONCLUSIONS: A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.

Response of host inflammasomes to viral infection.

Inflammasomes are multiprotein complexes that induce downstream immune responses to specific pathogens, environmental stimuli, and host cell damage. Components of specific viruses activate different inflammasomes; for example, the influenza A virus M2 protein and encephalomyocarditis virus (EMCV) 2B protein activate the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain (PYD)-containing 3 (NLRP3) inflammasome, whereas viral double-stranded RNA (dsRNA) activates the retinoic acid inducible gene-I (RIG-I) inflammasome. Once activated in response to viral infection, inflammasomes induce the activation of caspases and the release of mature forms of interleukin-1ß (IL-1ß) and IL-18. Here we review the association between viral infection and inflammasome activation. Identifying the mechanisms underlying virus-induced inflammasome activation is important if we are to develop novel therapeutic strategies to target viruses.

Hepatitis C virus NS5A protein enhances gluconeogenesis through upregulation of Akt-/JNK-PEPCK signalling pathways.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is highly associated with the type 2 diabetes mellitus, but the detailed mechanisms by which the viral proteins are involved in the clinical outcome remain unclear. METHODS: A cDNA microarray analysis was performed following introducing an NS5A-encoding plasmid or a control vector into a mouse system by hydrodynamics- based transfection. Differentially expressed genes that are associated with gluconeogenesis were selected and their expression levels in HCV patients, in NS5A-expressing systems, and in the viral subgenomic replicon system were further examined by real-time quantitative polymerase chain reaction and Western blot analysis. RESULTS: Differential gene expression including an upregulation of the gluconeogenic rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK) compared with controls was detected in mouse hepatocytes expressing HCV NS5A and in HCV patients with diabetes. In addition, an NS5A-dependent increase in glucose production was demonstrated in human primary hepatocytes. The upregulation of PEPCK and peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a) were also detected in NS5A-expressing cells and in the viral genotype 1b subgenomic replicon system. Further studies demonstrated that the NS5A-mediated upregulation of PEPCK and PGC-1a genes were resulted from the activation of PI3K-Akt and JNK signalling pathways. In addition, the expression levels of the forkhead transcription factor FoxO1 and the liver-enriched transcription factor HNF-4a were increased in HCV NS5A expressing cells. CONCLUSIONS: By upregulating the expression of PEPCK gene via its transactivators FoxO1 and HNF-4a, and the coactivator PGC-1a, the NS5A promotes the production of hepatic glucose which may contribute to the development of HCV-associated type 2 diabetes mellitus.

Upregulation of the chemokine (C-C motif) ligand 2 via a severe acute respiratory syndrome coronavirus spike-ACE2 signaling pathway.

Severe acute respiratory syndrome coronavirus (SARS-CoV) was identified to be the causative agent of SARS with atypical pneumonia. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV. It is not clear whether ACE2 conveys signals from the cell surface to the nucleus and regulates expression of cellular genes upon SARS-CoV infection. To understand the pathogenesis of SARS-CoV, human type II pneumocyte (A549) cells were incubated with the viral spike protein or with SARS-CoV virus-like particles containing the viral spike protein to examine cytokine modulation in lung cells. Results from oligonucleotide-based microarray, real-time PCR, and enzyme-linked immunosorbent assays indicated an upregulation of the fibrosis-associated chemokine (C-C motif) ligand 2 (CCL2) by the viral spike protein and the virus-like particles. The upregulation of CCL2 by SARS-CoV spike protein was mainly mediated by extracellular signal-regulated kinase 1 and 2 (ERK1/2) and AP-1 but not the IkappaBalpha-NF-kappaB signaling pathway. In addition, Ras and Raf upstream of the ERK1/2 signaling pathway were involved in the upregulation of CCL2. Furthermore, ACE2 receptor was activated by casein kinase II-mediated phosphorylation in cells pretreated with the virus-like particles containing spike protein. These results indicate that SARS-CoV spike protein triggers ACE2 signaling and activates fibrosis-associated CCL2 expression through the Ras-ERK-AP-1 pathway.

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent.

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.