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RNA-seq Analysis
Published in Altuna Akalin, Computational Genomics with R, 2020
In a cell, there are many layers of quality controls and modifications that act upon a gene’s product until the end-product attains a particular function. These layers of regulation include epigenetic, transcriptional, post-transcriptional, translational, and post-translational control mechanisms, the latter two applying only to protein-coding genes. A protein or RNA molecule, is only functional if it is produced at the right time, at the right cellular compartment, with the necessary base or amino-acid modifications, with the correct secondary/tertiary structure (or unstructure wherever applicable), among the availability of other metabolites or molecules, which are needed to form complexes to altogether accomplish a certain cellular function. However, traditionally, the number of copies of a gene’s products is considered a quantitative measure of a gene’s activity. Although this approach does not reflect all of the complexity that defines a functional molecule, quantification of the abundance of transcripts from a gene has proven to be a cost-effective method of understanding genes’ functions.
Molecular Genetics and Diagnostic Testing
Published in Merlin G. Butler, F. John Meaney, Genetics of Developmental Disabilities, 2019
Additional protein processings including glycosylation, hydroxylation, proteolytic cleavage, and phosphorylation may occur after synthesis. Proteins are then either localized to a particular cellular compartment or extruded into the extracellular space.
Red Cell Aggregation and Yield Stress
Published in Gordon D. O. Lowe, Clinical Blood Rheology, 2019
The effects of hematocrit on viscosity are complex. In a nonaggregating suspension, there is a rapid rise in viscosity at all shear rates as hematocrit increases. This is due to the increased protein content and to membrane effects of the cellular compartment. Beyond hematocrits of about 40% cellular contact must occur, and the resultant cell-cell interaction enhances shear resistance and viscosity further. If now aggregating agents are included, they will superimpose the sorts of effects discussed earlier, that is to say the increased effective cell size due to aggregation will further enhance viscosity. However at high hematocrits, where intense cell-cell interactions occur irrespective of aggregation, due to the density of their packing, the rouleaux effect becomes less significant. This has been shown very clearly by Chien et al.,28 who found that the shear dependence of whole blood increases very rapidly with hematocrit to reach a maximum at about 70%. This has clear relevance to hemodilution therapy, the net result of which is to reduce aggregation, by diluting the aggregating proteins and reducing hematocrit, and also to reduce cell-cell interaction (Chapter 8, Volume II).
M2 macrophage-derived exosomes suppress tumor intrinsic immunogenicity to confer immunotherapy resistance
Published in OncoImmunology, 2023
Naisheng Zheng, Tingting Wang, Qin Luo, Yi Liu, Junyao Yang, Yunlan Zhou, Guohua Xie, Yanhui Ma, Xiangliang Yuan, Lisong Shen
The clustering heat map indicated that highly ApoE enrichment through M2-exosomal transfer or genetic overexpression altered protein expression profiling in MC38 cells (Figure 3D). The overlapped downregulated proteins were subjected to GO classification to investigate biological processes, molecular function, and cellular compartment. Overall, it displayed a striking resemblance that a high overall overlap between the enriched categories in M2-exo-treated MC38 (Figure 3E, Table S3) and ApoE-OE MC38 cells (Figure 3F, Table S4). As expected, Apoe−/− M2-exo rescued protein changes caused by WT M2-exo (Figure S6A-C, Table S5). Remarkably, abundance-decreased proteins in MC38 cells with high ApoE were involved in the biological process such as cellular response to interferon-beta/gamma and innate immune response. These proteins were classified into cellular components such as the MHC class I protein complex and MHC class I peptides loading complex. Proteins associated with the molecular function were involved in antigen presentation-related proteins, as well as T-cell receptor binding, and B2M binding, all of which play important roles in the resistance to immunotherapy response. Downregulation of antigen presentation and IFN signaling categories in highly ApoE enrichment through M2-exosomal transfer concurred with ApoE genetic overexpression MC38, which indicated that M2-exosomal ApoE likely mediated T-cell killing through this pathway.
Design of artificial cells: artificial biochemical systems, their thermodynamics and kinetics properties
Published in Egyptian Journal of Basic and Applied Sciences, 2022
Adamu Yunusa Ugya, Lin Pohan, Qifeng Wang, Kamel Meguellati
Artificial cells are made up of three parts, a shell (cellular compartment), an engine (transcription and translation machinery) and finally the information (genetic components). The constructed circuits in cell-free systems are tested in a second step to make sure that the synthetic circuits and machinery are active inside the designed artificial cells. In the final step, the cell-free systems are encapsulated inside the membrane made up of phospholipids or fatty acids [12]. The different methods by which shell can be constructed are extrusion methods, lyophilization methods, water-in-oil methods and microfluidic devices. Artificial cells with uniform size, multi-lamellar membranes and controlled cellular diameter were generated by extrusion processes. Large artificial cells with heterogeneous size and lamellarity were prepared by lyophilization methods. Large unilamellar artificial cells with heterogenous size artificial cells were produced by water-in-oil methods. Artificial cells with a controllable size by adjusting the diameter of the channels and the flow speed were generated with microfluidic devices [12]. Two different methods were used in the construction of artificial cells namely: Top-down approach and bottom-up approach.
Full-length recombinant antibodies from Escherichia coli: production, characterization, effector function (Fc) engineering, and clinical evaluation
Published in mAbs, 2022
E. coli has unique features compared to most other production hosts. While recombinant proteins are usually secreted into the culture media in other hosts (e.g., mammalian or fungal systems), in E. coli, they are expressed in the cytoplasm, targeted to the periplasmic space, or secreted into the culture media41 (Figure 2). Each cellular compartment has unique properties and, based on the protein to be produced, a strategic decision can be made as to where to direct the recombinant protein. In the reducing cytoplasm, proteins can be produced in soluble form or as inclusion bodies (IBs), which can be resolubilized and refolded into functional forms. Proteins that require an oxidizing environment, for disulfide bond formation, can be secreted into the oxidizing periplasmic compartment in soluble and active forms. In some instances, proteins can also be secreted into the culture media for ease of downstream processing. In addition, cell-free protein synthesis (CFPS) systems using E. coli cell extracts or systems using purified components (the PURE system) have seen significant improvements, and are now competing with E. coli cell-based production systems.46,47