41 research outputs found

    THE CHALLENGED STATE SOVEREIGNTY: A REVIEW OF ELI CASE

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    Normally the intellectual property is defined as “asset” (Frankel, 2016, p. 21) in FTAs that allows the investors to protect their rights and interests when disputes arise. In 2010 and 2011, the Canadian courts made decisions that invalidated two patents protection on Strattera and Zyprexa, respectively. To protect its interests, Eli Lilly and Company brought a patent right dispute to ICSID in the late of 2012. According to the claims of Eli, the decisions of the Canadian courts can be deemed as the violation of Article 1110 (Expropriation) and Article 1105 (Minimum Standard of Treatment) of North American Free Trade Agreement (NAFTA). Although the Tribunal dismissed the claims of Eli in March 2017 eventually, the actions of Eli de facto challenged state sovereignty and decreased the discretion of Canada to define and regulate its internal intellectual property system (Billingsley, 2015, p. 27). This essay will first present a brief introduction of the Eli Lilly and Company v. Canada and will discuss the evaluation of the Tribunal’s decision in Part 2

    BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF BACTERIAL RNA-GUIDED DNA TARGETING SYSTEMS

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    The CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) immune systems found in many prokaryotes rely on small guide CRISPR RNAs (crRNAs) to destroy invading viruses and plasmids. This RNA-guided adaptive immune response is mediated by numerous diverse Cas proteins, several of which form complexes with crRNAs and function to silence foreign nucleic acids. Current understanding of the molecular basis of these proteins is limited. Here, we present biochemical and structural characterization of two sets of such proteins: Cascade from Escherichia coli and Cas9 from Streptococcus thermophilus. E. coli Cascade, a large multimeric ribonucleoprotein complex, uses crRNA to base pair with complementary DNA (protospacer) at sites adjacent to a signature sequence termed the protospacer adjacent motif (PAM). The bound structure, known as an R-loop, propagates from PAM to the other end of the protospacer. A crystal structure of Cascade bound to a ssDNA target, previously determined by our laboratory, reveals a potential pocket for binding of the displaced strand in the R-loop. Here we provide experimental evidence that this pocket serves as a docking site for the displaced strand, and this binding facilitates DNA strand separation during R-loop formation. Structure-guided mutagenesis of the basic residues in the pocket confirms their importance for double strand DNA binding. Single-molecule experiments reveal that these mutations kinetically hinder R-loop formation. We further show that Cascade exerts a strong conformational “lock” upon completion of an R-loop, and this locked conformation is sufficient for recruiting the trans-acting Cas3 helicase/nuclease for target destruction. Cas9 from S. thermophilus LMG18311 is 1122 amino acid protein harboring a HNH nuclease domain and a RuvC-like nuclease domain. We demonstrate that LMG18311 Cas9 utilizes a crRNA in conjunction with a trans-acting crRNA (tracrRNA) to cleave double strand DNA in vivo and in vitro. The cleavage is dependent on the presence of PAM as well as the position of the PAM. We further show that the HNH and RuvC-like nuclease domains of Cas9 select the location of their cleavage sites via different mechanisms. The HNH domain catalyzes cleavage of the target strand at a fixed position, whereas the RuvC-like domain catalyzes cleavage of the non-target strand using a ruler mechanism

    Metabolites changes of a low-temperature and low-salt fermented Chinese kohlrabi during fermentation based on non-targeted metabolomic analysis

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    A low-temperature and low-salt industrially fermented Chinese kohlrabi (LSCK) was developed in this study, with the salt usage decreased by approximately 70% compared to the traditional high-salt fermented Chinese kohlrabi (HSCK). The differences in physicochemical properties, metabolites and overall flavors during LSCK fermented for 0, 45 and 90 days (d) were analyzed by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS), electronic nose (E-nose) and other techniques. The results showed that the total acid content increased significantly from 3.68 to 8.59 g/kg. However, the protein content significantly decreased from 2.52/100 to 0.66 g/100 g. The number of lactic acid bacteria cells increased significantly from 3.69 to 4.46 log10CFU/g. Based on multivariate statistical analysis, 21, 14, and 15 differential metabolites were identified in the three treatment groups A1 (0 and 45 days), A2 (45 and 90 days), and A3 (0 and 90 days) respectively (VIP > 1, p < 0.05, |log2FC| ≥ 1.1). Carbohydrates, sugar alcohols, amino acids and their derivatives were the main differential metabolites in the LSCKs fermented for different periods. Aminoacyl−tRNA biosynthesis and glycine, serine and threonine metabolism pathways significantly correlated with the differential metabolites based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (p < 0.05). Furthermore, the overall odors were significantly different among the LSCKs with different fermentation periods, as detected by E-nose. The present study describes the change trend of metabolites during LSCK fermentation and elucidates important metabolic pathways in LSCK, providing a theoretical basis for the target regulation of functional metabolites in kohlrabi and the optimization of LSCK processing

    A generic coordination assembly-enabled nanocoating of individual tumor cells for personalized immunotherapy

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    A generic and effective tumor cells encapsulation strategy enabled by metal–organic coordination is developed to prepare a vaccine for personalized immunotherapy. Specifically, an epigallocatechin‐3‐gallate (EGCG)‐Al(III) coordination layer is in situ formed onto individual living cells in aqueous phase and the process can be completed within an hour. 98% of proteins in the cells are entrapped within the microparticles, which are endowed with high antigens loading capacity. The microparticles enhance the uptake efficiency of antigens, protect antigens from degradation in vivo, and delay the retention time of antigens in the lymph nodes. Moreover, dendritic cells (DCs) activation is triggered by the microparticles, and simultaneously, the expression of costimulation marker on DCs and the production of Th1‐related cytokines are significantly upregulated. Moreover, six kinds of tumor cells are utilized and successfully coated with the EGCG/Al(III) layer, suggesting the generalization of this strategy. More importantly, the microparticles exhibit a comparative antitumor effect with polyinosinic–polycytidylic acid (PolyI:C) in B16 pulmonary metastasis model. Overall, the encapsulation strategy enabled by metal–organic coordination can be potentially useful for personalized immunotherapy customized to individual patient's tumor cells

    Angiogenic Role of Mesothelium-Derived Chemokine CXCL1 During Unfavorable Peritoneal Tissue Remodeling in Patients Receiving Peritoneal Dialysis as Renal Replacement Therapy

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    Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy

    Expanded Hemodialysis Therapy Ameliorates Uremia-Induced Systemic Microinflammation and Endothelial Dysfunction by Modulating VEGF, TNF-α and AP-1 Signaling

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    Systemic chronic microinflammation and altered cytokine signaling, with adjunct cardiovascular disease (CVD), endothelial maladaptation and dysfunction is common in dialysis patients suffering from end-stage renal disease and associated with increased morbidity and mortality. New hemodialysis filters might offer improvements. We here studied the impact of novel improved molecular cut-off hemodialysis filters on systemic microinflammation, uremia and endothelial dysfunction. Human endothelial cells (ECs) were incubated with uremic serum obtained from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation (PERCI-II) crossover clinical trial, comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes, and then assessed for their vascular endothelial growth factor (VEGF) production and angiogenesis. Compared to HF membranes, dialysis with MCO membranes lead to a reduction in proinflammatory mediators and reduced endothelial VEGF production and angiogenesis. Cytokine multiplex screening identified tumor necrosis factor (TNF) superfamily members as promising targets. The influence of TNF-alpha and its soluble receptors (sTNF-R1 and sTNF-R2) on endothelial VEGF promoter activation, protein release, and the involved signaling pathways was analyzed, revealing that this detrimental signaling was indeed induced by TNF-alpha and mediated by AP-1/c-FOS signaling. In conclusion, uremic toxins, in particular TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel MCO membranes

    Control of neutrophil influx during peritonitis by transcriptional cross‐regulation of chemokine CXCL1 by IL‐17 and IFN‐γ

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    Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1–STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis

    Functional analysis of the C-reactive protein (CRP) gene -717A>G polymorphism associated with coronary heart disease

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    <p>Abstract</p> <p>Background</p> <p>Atherosclerosis underlies the major pathophysiological mechanisms of coronary heart disease (CHD), and inflammation contributes to all phases of atherosclerosis. C-reactive protein (CRP), a sensitive, but nonspecific marker of inflammation has been shown to play proatherogenic roles in the process of atherosclerosis. Our previous report showed that rs2794521 (-717A>G), located in the promoter of the CRP gene, was independently associated with CHD in Chinese subjects. In the present study, we tried to investigate the biological significance of this genetic variation <it>in vitro</it>.</p> <p>Methods</p> <p>The influence of G to A substitution at the site of rs2794521 on the transcriptional activity of the promoter of the CRP gene was assessed by luciferase reporter assay, and protein binding to the site of rs2794521 was detected by EMSA assay.</p> <p>Results</p> <p>The G to A exchange at the site of rs2794521 resulted in an increased transcriptional activity of the promoter of CRP gene, and glucocorticoid receptor (GR) protein factor bound drastically differently to the A and G alleles at the site of rs2794521.</p> <p>Conclusion</p> <p>These results provided functional evidence supporting the association of the SNP rs2794521 of the CRP gene with CHD probably through regulating the expression level of CRP by different variations of rs2794521.</p

    A Novel Docetaxel-Loaded Poly (ε-Caprolactone)/Pluronic F68 Nanoparticle Overcoming Multidrug Resistance for Breast Cancer Treatment

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    Multidrug resistance (MDR) in tumor cells is a significant obstacle to the success of chemotherapy in many cancers. The purpose of this research is to test the possibility of docetaxel-loaded poly (ε-caprolactone)/Pluronic F68 (PCL/Pluronic F68) nanoparticles to overcome MDR in docetaxel-resistance human breast cancer cell line. Docetaxel-loaded nanoparticles were prepared by modified solvent displacement method using commercial PCL and self-synthesized PCL/Pluronic F68, respectively. PCL/Pluronic F68 nanoparticles were found to be of spherical shape with a rough and porous surface. The nanoparticles had an average size of around 200 nm with a narrow size distribution. The in vitro drug release profile of both nanoparticle formulations showed a biphasic release pattern. There was an increased level of uptake of PCL/Pluronic F68 nanoparticles in docetaxel-resistance human breast cancer cell line, MCF-7 TAX30, when compared with PCL nanoparticles. The cytotoxicity of PCL nanoparticles was higher than commercial Taxotere®in the MCF-7 TAX30 cell culture, but the differences were not significant (p > 0.05). However, the PCL/Pluronic F68 nanoparticles achieved significantly higher level of cytotoxicity than both of PCL nanoparticles and Taxotere®(p < 0.05), indicating docetaxel-loaded PCL/Pluronic F68 nanoparticles could overcome multidrug resistance in human breast cancer cells and therefore have considerable potential for treatment of breast cancer

    Autoantibodies from patients with kidney allograft vasculopathy stimulate a proinflammatory switch in endothelial cells and monocytes mediated via GPCR-directed PAR1-TNF-α signaling

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    Non-HLA-directed regulatory autoantibodies (RABs) are known to target G-protein coupled receptors (GPCRs) and thereby contribute to kidney transplant vasculopathy and failure. However, the detailed underlying signaling mechanisms in human microvascular endothelial cells (HMECs) and immune cells need to be clarified in more detail. In this study, we compared the immune stimulatory effects and concomitant intracellular and extracellular signaling mechanisms of immunoglobulin G (IgG)-fractions from kidney transplant patients with allograft vasculopathy (KTx-IgG), to that from patients without vasculopathy, or matched healthy controls (Con-IgG). We found that KTx-IgG from patients with vasculopathy, but not KTx-IgG from patients without vasculopathy or Con-IgG, elicits HMEC activation and subsequent upregulation and secretion of tumor necrosis factor alpha (TNF-α) from HMECs, which was amplified in the presence of the protease-activated thrombin receptor 1 (PAR1) activator thrombin, but could be omitted by selectively blocking the PAR1 receptor. The amount and activity of the TNF-α secreted by HMECs stimulated with KTx-IgG from patients with vasculopathy was sufficient to induce subsequent THP-1 monocytic cell activation. Furthermore, AP-1/c-FOS, was identified as crucial transcription factor complex controlling the KTx-IgG-induced endothelial TNF-α synthesis, and mircoRNA-let-7f-5p as a regulatory element in modulating the underlying signaling cascade. In conclusion, exposure of HMECs to KTx-IgG from patients with allograft vasculopathy, but not KTx-IgG from patients without vasculopathy or healthy Con-IgG, triggers signaling through the PAR1-AP-1/c-FOS-miRNA-let7-axis, to control TNF-α gene transcription and TNF-α-induced monocyte activation. These observations offer a greater mechanistic understanding of endothelial cells and subsequent immune cell activation in the clinical setting of transplant vasculopathy that can eventually lead to transplant failure, irrespective of alloantigen-directed responses
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