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Nanopulse Stimulation Therapy
Published in Marko S. Markov, James T. Ryaby, Erik I. Waldorff, Pulsed Electromagnetic Fields for Clinical Applications, 2020
The first indication that applying pulsed electric fields in the nanosecond domain could influence tumor growth came from the pioneering work of Beebe and Schoenbach (Beebe et al., 2002) who treated subdermal murine fibrosarcoma allografts. Subsequent to their published work, more than 60 papers have been published describing various aspects of tumor ablation using nanosecond pulsed electric fields in murine models. Collectively, these studies suggest that NPS treatment of tumors results in a slower cell death process in treated tissue over a period of days, unlike IRE which generally triggers necrotic death within hours. In a subset of these studies treating malignant tumors, the authors identified that the treatment with NPS initiated immunogenic cell death (ICD), a subset of regulated cell death (RCD) in which tumor-associated antigens are released and presented to the immune system to generate an adaptive immune response. The ICD process involves releasing danger-associated molecular patterns (DAMPs) such as the translocation of calreticulin from the endoplasmic reticulum to the cell surface, as well as the release of both ATP and HMGB1 (Guo et al., 2018; Nuccitelli et al., 2017). This collective body of preclinical research suggests a link between NPS’s release of key DAMPs and the subsequent generation of a CD8+-dependent adaptive immune response. This adaptive immune response may prevent growth of rechallenge tumor cells. (Beebe et al., 2018; Chen et al., 2014; Guo et al., 2018; Lassiter et al., 2018; Nuccitelli et al., 2012, 2015; Skeate et al., 2018).
Cell Biology for Bioprocessing
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Throughout the process of protein secretion, N-glycosylation takes place on the protein, starting in the ER and continuing into the Golgi (Figure 2.8, also Figure 3.21). It is called N-glycosylation because the oligoglycan is linked to the asparagine residue of specific sequences on the protein. As the protein is being synthesized and folded, a preassembled oligoglycan containing three terminal glucose residues is transferred and covalently linked to the asparagine (Figure 2.9). The addition of glycan to the protein increases its stability in a soluble form. It is also an important marker of protein folding. While the protein undergoes folding, the glucose residues are cleaved. A glucosyl transferase adds a glucose to the aglycosylated glycan on any protein that is not correctly folded, leaving the completely folded protein with an N-glycan that does not have any terminal glucose. Calnexin and calreticulin are lectins (glycan-binding proteins). They bind to the glycan on the protein that has only one glucose residue and retain them in the ER for further folding. Once the protein is completely folded and has no terminal glucose, it is ready for transfer to the Golgi apparatus. The protein molecules that are improperly folded are then exported to the cytosol and degraded in a proteasome.
Immunotherapy and Nanovaccines
Published in Sourav Bhattacharjee, Principles of Nanomedicine, 2019
Tumors undergo necrotic changes due to the death of cells, resulting from a scarcity of adequate oxygen and nutrient supply because of the uncontrolled cell proliferation associated with rapid angiogenesis and metastasis [11, 12]. The necrotic cancer cells release various molecules as tumor-associated antigens (TAAs), such as nucleotides, calreticulin, high-mobility-group box 1 protein, and filamentous actin, which activate the antigen presenting cells (APCs), for example, dendritic cells (DCs) and macrophages (Fig. 12.1). As a result, the activated CD8+ T-cells destroy the cancer cells carrying major histocompatibility complex-I (MHC-I) with the release of perforins and granzymes. The CD4+ T-cells secrete various proinflammatory markers, for example, interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α), which catalyze the recognition by CD8+ T-cells due to the expression of MHC-I [13, 14]. The natural killer (NK) cells can also exhibit cytotoxic effects toward cancer cells [15]. However, enhanced infiltration of pleura and peritoneum with the NK cells (e.g., in metastatic ovarian carcinoma) is taken as poor prognosis. The NK T-cells with overexpressed T-cell receptors (TCRs) can also contribute in killing the cancer cells via proinflammatory cytokines, for example, perforins, Fas-FasL, and IFN-γ [16]. Interestingly, a few tumoral immune cells are recognized to actually promote the growth of tumors rather than restricting it, as mentioned below.
L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression
Published in Preparative Biochemistry and Biotechnology, 2019
Brian Effer, Guilherme Meira Lima, Sindy Cabarca, Adalberto Pessoa, Jorge G. Farías, Gisele Monteiro
According to the model in Figure 3, the 6 histidine residues (green in Figure 3(B)), as well as the rhinovirus sequence (orange in Figure 3(B)) (used as a site for proteases to remove the histidine residues after protein purification), were localized inside the tridimensional protein monomer, which could adversely affect its native conformation and folding, blocking its passage to the extracellular medium by failing cell quality control mechanisms, represented by calnexin and calreticulin, and promoting its degradation via ERAD.[18] Similar findings have been observed by Chant et al.,[11] where conformational changes appeared when they used a 6 His-tag in the HZFB AreA protein (transcription factor) compared to the ZFB AreaA protein (without 6 His-tag); however, significant functional changes were not found in this protein. This is in contrast to the results reported by Fonda et al.,[12] where they produced TNF-alpha recombinant with 7 His-tag attached to the N-terminal portion (His7-(ΔN6)TNF), which resulted in significant reduction of biological activity compared to wild type. In relation to the tridimensional model of His7-(ΔN6)TNF, the 7 histidine was not inside structure itself as in our study. Goel et al.[13] expressed scFV (monoclonal antibody) in P. pastoris, fused to 6 His-tag either C-(scFv-His6) or N-terminus (His6-scFv) of the polypeptide. They found the lowest antigen binding (20–25%) in the scFv-His6 construction because 6 His-tag partially covered the antigen binding site of the protein. In our case, the 6 His-tag does not cover the active site from L-ASNase (red in Figure 3(A,B)); instead, the 6 His-tag goes next to the active site and penetrates the monomeric structure, which can be detrimental to correct protein folding or oligomerization.