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Mcconnell Crowell posted an update 4 days, 10 hours ago
nidulans conidia.Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA120) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. BAY-293 The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.This work studied the impact of three types of styrene-butadiene (SB and SBS) block copolymers on the morphology, electrical, and rheological properties of immiscible blends of polypropylenepolystyrene (PPPS)/multi-walled carbon nanotubes (MWCNT) with a fixed blend ratio of 7030 vol.%. The addition of block copolymers to PPPS/MWCNT blend nanocomposites produced a decrease in the droplet size. MWCNTs, known to induce co-continuity in PPPS blends, did not interfere with the copolymer migration to the interface and, thus, there was morphology refinement upon addition of the copolymers. Interestingly, the addition of the block copolymers decreased the electrical resistivity of the PPPS/1.0 vol.% MWCNT system by 5 orders of magnitude (i.e., increase in electrical conductivity). This improvement was attributed to PS Droplets-PP-Copolymer-Micelle assemblies, which accumulated MWCNTs, and formed an integrated network for electrical conduction. Molecular simulation and solubility parameters were used to predict the MWCNT localization in the immiscible blend. The simulation results showed that diblock copolymers favorably interact with the nanotubes in comparison to the triblock copolymer, PP, and PS. However, the interaction between the copolymers and PP or PS is stronger than the interaction of the copolymers and MWCNTs. Hence, the addition of copolymer also changed the localization of MWCNT from PS to PS-PP-Micelles-Interface, as observed by TEM images. In addition, in the last step of this work, we investigated the effect of the addition of copolymers on inter- and intra-cycle viscoelastic behavior of the MWCNT incorporated polymer blends. It was found that addition of the copolymers not only affects the linear viscoelasticity (e.g., increase in the value of the storage modulus) but also dramatically impacts the nonlinear viscoelastic behavior under large deformations (e.g., higher distortion of Lissajous-Bowditch plots).].Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.Immunogenic cell death (ICD) is a type of death, which has the hallmarks of necroptosis and apoptosis, and is best characterized in malignant diseases. Chemotherapeutics, radiotherapy and photodynamic therapy induce intracellular stress response pathways in tumor cells, leading to a secretion of various factors belonging to a family of damage-associated molecular patterns molecules, capable of inducing the adaptive immune response. One of them is calreticulin (CRT), an endoplasmic reticulum-associated chaperone. Its presence on the surface of dying tumor cells serves as an “eat me” signal for antigen presenting cells (APC). Engulfment of tumor cells by APCs results in the presentation of tumor’s antigens to cytotoxic T-cells and production of cytokines/chemokines, which activate immune cells responsible for tumor cells killing. Thus, the development of ICD and the expression of CRT can help standard therapy to eradicate tumor cells. Here, we review the physiological functions of CRT and its involvement in the ICD appearance in malignant disease. Moreover, we also focus on the ability of various anti-cancer drugs to induce expression of surface CRT on ovarian cancer cells. The second aim of this work is to discuss and summarize the prognostic/predictive value of CRT in ovarian cancer patients.