Tracking hantavirus nucleocapsid protein using intracellular antibodies

<p>Abstract</p> <p>Background</p> <p>Hantavirus nucleocapsid (N) protein is a multifunctional viral macromolecule involved in multiple stages of the viral replication cycle. The intracellular trafficking of N protein during virus assembly remains unclear.</p> <p>Methods</p> <p>We used N protein-specific intracellular expressed antibodies to track the localization and distribution of Hantaan virus and Seoul virus N protein. The N protein-specific antibody single-chain variable antibody fragments (scFvs), which bind an N-terminal linear epitope (L13F3) and C-terminal conformational domain (H34), were intracellularly expressed in the endoplasmic reticulum (ER) by fusion of the SEKDEL retention signal peptide at the carboxyl terminus, and in the cytoplasm (Cyto) by deletion of the ER membrane target signal peptide. Stable Vero-E6 cell lines expressing intracellular scFvs were either infected with hantavirus or transfected with an N protein expression plasmid; virus replication and N protein intracellular localization were determined.</p> <p>Result</p> <p>N protein co-localized with scFvs in the ER and cytoplasm with or without viral membrane glycoproteins. Hantavirus replication was inhibited in both the scFvs-ER- and scFvs-Cyto-expressing stable cell lines.</p> <p>Conclusion</p> <p>N protein may be expressed in the ER retention signal peptide of KDEL circulating region (ER/<it>cis</it>-Golgi) without the assistance of G protein, and so expression of N protein in both the cytoplasm and within the ER/<it>cis</it>-Golgi plays an important role in virus replication.</p

Fusion with extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 leads to enhancement of immunogenicity of Hantaan virus DNA vaccines in C57BL/6 mice

<p>Abstract</p> <p>Background</p> <p>Hantaan virus (HTNV) is the causative agent of the most severe form of a rodent-borne disease known as hemorrhagic fever with renal syndrome (HFRS). A safe and effective HTNV vaccine is needed. Vaccination with DNA constructs expressing fused antigen with bioactive factors, has shown promising improvement of immunogenicity for viral agents in animal models, but the effect of fusion strategy on HTNV DNA vaccine has not been investigated.</p> <p>Results</p> <p>DNA plasmids encoding the HTNV nucleocapsid protein (N) and glycoprotein (Gn and Gc) in fusion to the extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 (eCTLA-4) targeting to antigen presenting cells (APCs) were constructed. Intramuscular immunization of mice with plasmids expressing eCTLA-4-HTNV-N/GP fusion proteins leads to a significant enhancement of the specific antibody response as well as cytotoxic T-lymphocyte (CTL) response in C57BL/6 mice. Moreover, this effect could be further augmented when co-administered with CpG motifs.</p> <p>Conclusions</p> <p>Modification of viral antigen in fusion to bioactive factor will be promising to confer efficient antigen presentation and improve the potency of DNA vaccine in mice.</p

Effect of a 180 mg ticagrelor loading dose on myocardial necrosis in patients undergoing elective percutaneous coronary intervention: A preliminary study

Background: To examine whether a loading dose of ticagrelor on top of clopidogrel reduced postpercutaneous coronary intervention (PCI) myonecrosis. Methods: Seventy seven coronary artery disease patients received a loading dose of 300 mg clopidogrel pre-PCI and were divided into three groups: group TT (n = 36): a loading dose of 180 mg ticagrelor pre-PCI, followed by ticagrelor 90 mg twice daily commencing one day post-PCI; group CT (n = 26): a maintenance dose of ticagrelor 90 mg twice daily; group CC (n = 15): clopidogrel 75 mg daily post- PCI. High sensitivity cardiac troponin T (hs-cTnT) and creatine kinase-MB (CK-MB) were measured pre-PCI and 0 h, 2 h or 24 h post-PCI. Platelet aggregation was measured in a separate cohort of 54 coronary artery disease patients (35 diabetic and 19 non-diabetic patients). Results: There were no significant differences in hs-cTnT and CK-MB concentration among the three groups. In group TT, diabetic patients had significant higher Δhs-cTnT2h–0h than non-diabetic patients. In the second cohort, although baseline platelet aggregation was higher in diabetic than non-diabetic patients, platelet aggregation was comparable between diabetic and non-diabetic patients at 0 and 2 h post-PCI. Conclusions: This study indicates that a loading dose of ticagrelor does not significantly reduce post- PCI myonecrosis. Diabetes is associated with more post-PCI myonecrosis. A loading dose of ticagrelor effectively reduces platelet aggregation in diabetic patients.

Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice

<p>Abstract</p> <p>Background</p> <p>The incidence of dengue, an infectious disease caused by dengue virus (DENV), has dramatically increased around the world in recent decades and is becoming a severe public health threat. However, there is currently no specific treatment for dengue fever, and licensed vaccine against dengue is not available. Vaccination with virus-like particles (VLPs) has shown considerable promise for many viral diseases, but the effect of DENV VLPs to induce specific immune responses has not been adequately investigated.</p> <p>Results</p> <p>By optimizing the expression plasmids, recombinant VLPs of four antigenically different DENV serotypes DENV1-4 were successfully produced in 293T cells. The vaccination effect of dengue VLPs in mice showed that monovalent VLPs of each serotype stimulated specific IgG responses and potent neutralizing antibodies against homotypic virus. Tetravalent VLPs efficiently enhanced specific IgG and neutralizing antibodies against all four serotypes of DENV. Moreover, vaccination with monovalent or tetravalent VLPs resulted in the induction of specific cytotoxic T cell responses.</p> <p>Conclusions</p> <p>Mammalian cell expressed dengue VLPs are capable to induce VLP-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection.</p

β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

Sustained β-adrenergic receptors (βAR) activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The immediate signaling pathways downstream from βAR stimulation, however, have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following βAR stimulation. In adult C57BL/6 mice, acute βAR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in β1/β2AR double knockout mice. We further showed the specificity of β2AR in this crosstalk as treatment with formoterol, a β2AR-selective agonist, but not dobutamine, a predominantly β1AR agonist, activated cardiac Akt and ERK1/2. Acute βAR stimulation also significantly increased the phosphorylation of mTOR (the mammalian target of rapamycin), P70S6K, ribosomal protein S6, GSK-3α/β (glycogen synthase kinase-3α/β), and FOXO1/3a (the forkhead box family of transcription factors 1 and 3a). Moreover, acute βAR stimulation time-dependently decreased the mRNA levels of the muscle-specific E3 ligases atrogin-1 and muscle ring finger protein-1 (MuRF1) in mouse heart. Our results indicate that acute βAR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between βAR and PI3K signaling pathways; 2) confirm the β2AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between β2AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions

Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42

<p>Abstract</p> <p>Background</p> <p>Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spreads rapidly and has a high case-mortality rate. The nucleocapsid protein (NP) of SARS-CoV may be critical for pathogenicity. This study sought to discover the host proteins that interact with SARS-CoV NP.</p> <p>Results</p> <p>Using surface plasmon resonance biomolecular interaction analysis (SPR/BIA) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, we found that only the proteasome subunit p42 from human fetal lung diploid fibroblast (2BS) cells bound to SARS-CoV NP. This interaction was confirmed by the glutathione S-transferase (GST) fusion protein pulldown technique. The co-localization signal of SARS-CoV NP and proteasome subunit p42 in 2BS cells was detected using indirect immunofluorescence and confocal microscopy. p42 is a subunit of the 26S proteasome; this large, multi-protein complex is a component of the ubiquitin-proteasome pathway, which is involved in a variety of basic cellular processes and inflammatory responses.</p> <p>Conclusion</p> <p>To our knowledge, this is the first report that SARS-CoV NP interacts with the proteasome subunit p42 within host cells. These data enhance our understanding of the molecular mechanisms of SARS-CoV pathogenicity and the means by which SARS-CoV interacts with host cells.</p

Ecological engineering projects increased vegetation cover, production, and biomass in semiarid and subhumid Northern China

Multiple ecological engineering projects have been implemented in semiarid and subhumid Northern China since 1978 with the purpose to combat desertification, control dust storms, and improve vegetation cover. Although a plethora of local studies exist, the effectiveness of these projects has not been studied in a systematic and comprehensive way. Here, we used multiple satellite-based time-series data as well as breakpoint analysis to assess shifts in leaf area index (a proxy for green vegetation cover), gross primary production, and aboveground biomass in Northern China. We documented increased vegetation growth in northwest and southeastern parts of the region, despite drought anomalies as documented by the standardized precipitation-evapotranspiration index during 1982–2016. Significant breakpoints in leaf area index were observed for over 72.5% of the southeastern and northwestern regions, and 70.6% of these breakpoints were detected after 1999, which correspond well to the areas with the highest ecological engineering efforts. Areas with negative trends were mainly located in the Inner Mongolian Plateau, Hulun Biur, Horqin Sand Land, and urban areas. The Loess Plateau had the largest increase in vegetation growth, followed by the north parts of Northern China where biomass increased more in the provinces of Shanxi, Liaoning, Shannxi, Hebei, and Beijing than Xinjiang, Inner Mongolia, Tianjin, and Qinghai. Our results show that multiple ecological engineering projects in the region have increased vegetation cover, production, and aboveground biomass that have led to improved environmental conditions in the study area