Nicotiana benthamiana protein, NbPCIP1, interacting with Potato virus X coat protein plays a role as susceptible factor for viral infection

AbstractThe interactions of viral coat protein (CP) and host factors play an important role in viral replication and/or host defense mechanism. In this study, we constructed Nicotiana benthamiana cDNA library to find host factors interacting with Potato virus X (PVX) CP. Using yeast two-hybrid assay, we screened 3.3×106 independent yeast transformants from N. benthamiana cDNA library and identified six positive clones. One positive clone, named PVX CP-interacting protein 1 (NbPCIP1), is a plant-specific protein with homologue in N. tabacum (GenBank accession no. AB04049). We confirmed the PVX CP–NbPCIP1 interaction using yeast-two hybrid assay in yeast, protein–protein binding assay in vitro, and bimolecular fluorescent complementation assay in planta. Quantitative real-time RT-PCR analysis showed that the mRNA level of NbPCIP1 increased in PVX-infected N. benthamiana plants as compared to that of healthy plants. The green fluorescent protein (sGFP)-fused NbPCIP1 (NbPCIP1-sGFP) was localized in ER or ER-associated granular-like structure of cells. When we co-express NbPCIP1-sGFP and red fluorescent protein (RFP)-fused PVX CP (PVX CP-RFP), which were introduced by transiently expressing these proteins in N. benthamiana protoplasts and epidermal cells, however, we observed the co-localization of these proteins in the inclusion body-like complex in areas surrounding nucleus. Transient over-expression and transgene silencing of NbPCIP1 assay analysis indicated that NbPCIP1 plays a critical role in viral replication during PVX infection in host plant

Development of a high yield purification process for the production of influenza virus vaccines

Production of influenza virus in animal cells has emerged as an alternative to conventional platforms such as egg-based production system. Animal cells, especially MDCK and VERO cell lines, are widely used as the primary production cell for influenza virus vaccine because of their high susceptibility to infection with various influenza viruses. Recently, a robust and reliable purification process was successfully developed for the production of quadri-valent HA proteins (from two strains of the type A virus and two strains of the type B virus) by using animal cell-based production system in Green Cross Corp., Korea. The UF/DF process, Benzonase treatment at high temperature as well as column chromatography strategy was optimized to maximize the final HA production yields. Benzonase treatment was conducted to reduce in hcDNA (host cell DNA) because hcDNA was main impurity for cell-based influenza virus vaccine. A simple and stable UF/DF process has been tested with membrane molecular weight cutoffs of 100 and 300 kDa as well as 0.2 and 0.45 um microfiltration membrane. Anion exchange chromatography (AEC) and size exclusion chromatography (SEC) were selected for acceptable reduction in hcDNA and HCP. AEC was used to separate hcDNA from virus at a salt concentration of 0.5 M sodium chloride. The HA yield through AEC & SEC combination process was sufficiently achieved under specific purification process condition. Overall, the amount of residual hcDNA was reduced to an acceptable level (10ng/dose) and the increased HA yield was maintained throughout the whole process. The performance, productivity and scalability of the purification process were successfully demonstrated in over 30 GMP batches using 4 different influenza virus strains

Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Studies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs.</p> <p>Results</p> <p>From hESCs, we derived NESs, the <it>in-vitro </it>version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of <it>in-vivo</it>-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their <it>in-vivo </it>counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a γ-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons.</p> <p>Conclusion</p> <p>Our results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an <it>in-vitro </it>model for <it>in-vivo </it>neurogenesis.</p

Global estimate of submarine groundwater discharge based on an observationally constrained radium isotope model

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Geophysical Research Letters 41 (2014): 8438–8444, doi:10.1002/2014GL061574.Along the continental margins, rivers and submarine groundwater supply nutrients, trace elements, and radionuclides to the coastal ocean, supporting coastal ecosystems and, increasingly, causing harmful algal blooms and eutrophication. While the global magnitude of gauged riverine water discharge is well known, the magnitude of submarine groundwater discharge (SGD) is poorly constrained. Using an inverse model combined with a global compilation of 228Ra observations, we show that the SGD integrated over the Atlantic and Indo-Pacific Oceans between 60°S and 70°N is (12 ± 3) × 1013 m3 yr−1, which is 3 to 4 times greater than the freshwater fluxes into the oceans by rivers. Unlike the rivers, where more than half of the total flux is discharged into the Atlantic, about 70% of SGD flows into the Indo-Pacific Oceans. We suggest that SGD is the dominant pathway for dissolved terrestrial materials to the global ocean, and this necessitates revisions for the budgets of chemical elements including carbon.This work was supported by the Ministry of Oceans and Fisheries, Korea, through the Korea Institute of Marine Science and Technology (KIMST) (20120176) and National Research Foundation (NRF) of Korea (2013R1A2A1A05004343 and 2013R1A1A1058203). Charette and Moore's contributions were supported by the US National Science Foundation through the GEOTRACES project

A Herbal Medicine, Gongjindan

This study protocol aims to explore the effectiveness, safety, and cost-effectiveness of a herbal medication, Gongjindan (GJD), in patients with chronic dizziness. This will be a prospective, multicenter, randomized, double-blind, placebo-controlled, parallel-group, clinical trial. Seventy-eight patients diagnosed with Meniere’s disease, psychogenic dizziness, or dizziness of unknown cause will be randomized and allocated to either a GJD or a placebo group in a 1 : 1 ratio. Participants will be orally given 3.75 g GJD or placebo in pill form once a day for 56 days. The primary outcome measure will be the Dizziness Handicap Inventory score. Secondary outcome measures will be as follows: severity (mean vertigo scale and visual analogue scale) and frequency of dizziness, balance function (Berg Balance Scale), fatigue (Fatigue Severity Scale) and deficiency pattern/syndrome (qi blood yin yang-deficiency questionnaire) levels, and depression (Korean version of Beck’s Depression Inventory) and anxiety (State-Trait Anxiety Inventory) levels. To assess safety, adverse events, including laboratory test results, will be monitored. Further, the incremental cost-effectiveness ratio will be calculated based on quality-adjusted life years (from the EuroQoL five dimensions’ questionnaire) and medical expenses. Data will be statistically analyzed at a significance level of 0.05 (two-sided). This trial is registered with ClinicalTrials.gov NCT03219515, in July 2017

Clinical Impact of Prophylactic Antibiotic Treatment for Self-Expandable Metallic Stent Insertion in Patients with Malignant Colorectal Obstruction

Purpose. The aim of this study was to determine the efficacy of prophylactic antibiotics (PA) for reducing the infectious complications and the potential risk factors responsible for the infectious complications after stent insertion for malignant colorectal obstruction. Methods. We performed a retrospective review of 224 patients who underwent self-expandable metallic stent (SEMS) insertion for malignant colorectal obstruction from May 2004 to December 2012. Results. There were 145 patients in the PA group and 79 in non-PA group. The CRP level in PA group was significantly higher than that in non-PA. Abdominal tenderness and mechanical ileus were significantly more frequent in PA group than those in non-PA. The frequency of post-SEMS insertion fever, systemic inflammatory response syndrome (SIRS), and bacteremia was not significantly different between PA and non-PA groups. In multivariate analysis, the CRP level was risk factor related to post-SEMS insertion SIRS. However, in propensity score matching analysis, there was no independent risk factor related to post-SEMS insertion fever, SIRS, and bacteremia. Conclusion. The use of PA in patients with malignant colorectal obstruction may be not effective to prevent the development of infectious complications after SEMS insertion

Effects of Hyul-Bu-Chuke-Tang on Erythrocyte Deformability and Cerebrovascular CO2 Reactivity in Normal Subjects

Aim. Hyul-bu-chuke-tang (HCEt) is a well-known traditional herbal medicine that is used for the treatment of ischemic cerebrovascular disorders. We investigated the acute effects of HCEt on erythrocyte deformability and cerebrovascular CO2 reactivity (CVR) in healthy male subjects. Materials and Methods. We examined erythrocyte deformability in an HCEt group (n = 14) and a control group (n = 10). CVR was measured using hyperventilation-induced CO2 reactivity of the middle cerebral artery and transcranial Doppler (TCD) in the HCEt group (n = 11). A historical control group (n = 10) of CVR measurements was also created from our previous study. All measurements were performed prior to and 1, 2, and 3 hours after HCEt administration. Results. HCEt significantly improved erythrocyte deformability 1 hour after administration compared to the control group (2.9 ± 1.1% versus −0.6 ± 1.0%, P = 0.034). HCEt significantly improved the CVR 2 hours after administration compared to the historical control group (9.1 ± 4.0% versus −8.1 ± 4.1%, P = 0.007). The mean blood pressure and pulse rate did not vary from baseline values in either group. Conclusions. We demonstrated that HCEt improved erythrocyte deformability and CVR. Our findings suggest that an improvement in erythrocyte deformability contributes to HCEt's effect on cerebral microcirculation

The Therapeutic Effect of STAT3 Signaling-Suppressed MSC on Pain and Articular Cartilage Damage in a Rat Model of Monosodium Iodoacetate-Induced Osteoarthritis

Osteoarthritis (OA) is a degenerative disease that induces pain, cartilage deformation, and joint inflammation. Mesenchymal stem cells (MSCs) are potential therapeutic agents for treatment of OA. However, MSC therapy can cause excessive inflammation. Signal transducer and activator of transcription 3 (STAT3) modulates secretion of many proinflammatory cytokines. Experimental OA was induced by intra-articular (IA) injection of monosodium iodoacetate (MIA) to the right knee of rats. MSCs from OA patients (OA-MSCs) were treated with STA21, a small molecule that blocks STAT3 signaling, by IA or intravenous (IV) injection after MIA injection. Pain severity was quantified by assessment of secondary tactile allodynia using the von Frey assessment test. Cartilage degradation was measured by microcomputed tomography image analysis, histological analysis, and the Mankin score. Protein and gene expression was evaluated by enzyme-linked immunosorbent assay, immunohistochemistry, and real-time polymerase chain reaction. MSCs increased production of proinflammatory cytokines under inflammatory conditions. STA21 significantly decreased expression of these proinflammatory molecules via inhibition of STAT3 activity but increased gene expression of molecules related to migration potential and immunomodulation in OA-MSCs. STAT3-inhibited OA-MSCs administrated by IV or IA injection decreased pain severity and cartilage damage in rats with MIA-induced OA rats by decreasing proinflammatory cytokines in the joints. Combined IA and IV-injected STAT3-inhibited OA-MSCs had an additive effect of pain relief in MIA-induced OA rats. STAT3 inhibition may optimize the therapeutic activities of MSCs for treating OA by attenuating pain and progression of MIA by inhibiting inflammation and cartilage damage