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Whole cell proteomics of NDUFAF8 knock-out cell line and NDUFAF5 knock-out cell line

ABSTRACT: Comparison of whole cell proteome of NDUFAF8 KO and NDUFAF5 KO to WT cell line

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Prerana Wagle

LAB HEAD: Jan Riemer

PROVIDER: PXD040441 | Pride | 2023-05-08

REPOSITORIES: Pride

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			Action	DRS
	20210310_Q2_ColID_193_2483_01.raw	Raw
	20210310_Q2_ColID_193_2483_02.raw	Raw
	20210310_Q2_ColID_193_2483_03.raw	Raw
	20210310_Q2_ColID_193_2483_04.raw	Raw
	20210310_Q2_ColID_193_2483_09.raw	Raw

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The choice of feed composition exerts a considerable influence on phenotypic, metabolic and proteomic outcome in BL/6 male mice

Project description:The multitude of obesogenic diets used in rodent studies is enormous and thus hardly manageable. Since standardization is missing and it is presumed that individual compositions provoke individual effects, the choice of quality, quantity and combination of diet ingredients seems to be crucial for the outcome and interpretation of obesity studies. Therefore, the present study was conducted to compare the effects of three commonly used obesogenic diets on selected parameters. Besides basic phenotypic and metabolic characterization, one main aspect was a comparative liver proteome analysis. As expected, the obtained results picture differentiated consequences mainly depending on fat source and/or fat- and sugar quantity. By confirming the general presumption that the choice of nutritional composition is a pivotal factor, the present findings demonstrate that a conscious selection is indispensable for obtaining reliable and sound results in obesity research.In conclusion, we strongly recommend a thorough selection of the appropriate obesogenic diet prior to an experiment and in consideration of the individual research question.

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Interactome of NDUFAF8-HA overexpressing cell line compared to mock

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A compound-based therapy approach for epidermolysis bullosa simplex (EBS)

Project description:Keratin cytoskeletal proteins are crucial for the maintenance of skin integrity. Mutations in genes coding for K5 and K14 cause the human skin disorder epidermolysis bullosa simplex (EBS) leading to substantial alterations in keratin assembly and collapse of keratin filaments into cytoplasmic protein aggregates. The phenotypic consequences of K5 and K14 mutations comprise fragility of basal keratinocytes and skin blistering upon mild mechanical trauma. Treatment of EBS is only supportive and consists primarily of wound care and avoidance of mechanical stress. Besides symptomatic care, no efficient therapeutic treatment is available for EBS. In the present study, we used patient-derived keratinocytes carrying the most frequent K14.R125C mutation as a reproducible EBS model to understand EBS pathomechanisms and to develop a therapy approach aimed to restore a functional keratin network. Numerous post-translational modifications (PTMs) such as phosphorylation have been reported to occur on keratins, which affect the organization of keratin networks. Whether keratin mutations affect the occurrence of PTMs and thereby keratin aggregation in EBS is yet unknown. We find that the K14.R125C mutation alters keratin and keratin-associated protein PTMs in distinct ways and suggest that disease mutations and altered PTMs aggravate keratin aggregation. We reason that chemical compounds affecting the interplay of mutations and PTMs enable the reformation of a keratin cytoskeleton from aggregates are potential candidates for combating EBS.

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Global proteome of Atg5flox:CAG-CreTmx WT and KO neurons

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HEK293 WT and TOM70 KD cell lines expressing HA-tagged Orf9b grown on galactose media

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Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system.

Project description:The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.

| S-EPMC6269370 | biostudies-literature

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