RESUMO
The proteasome-associated deubiquitinase USP14 is a potential drug target. Using an inducible USP14 knockout system in colon cancer cells, we found that USP14 depletion impedes cellular proliferation, induces cell cycle arrest, and leads to a senescence-like phenotype. Transcriptomic analysis revealed altered gene expression related to cell division and cellular differentiation. USP14 knockout cells also exhibited changes in morphology, actin distribution, and expression of actin cytoskeletal components. Increased ubiquitin turnover was observed, offset by upregulation of polyubiquitin genes UBB and UBC. Pharmacological inhibition of USP14 with IU1 increased ubiquitin turnover but did not affect cellular growth or morphology. BioGRID data identified USP14 interactors linked to actin cytoskeleton remodeling, DNA damage repair, mRNA splicing, and translation. In conclusion, USP14 loss in colon cancer cells induces a transient quiescent cancer phenotype not replicated by pharmacologic inhibition of its deubiquitinating activity.
Assuntos
Proliferação de Células , Senescência Celular , Neoplasias Colorretais , Ubiquitina Tiolesterase , Humanos , Senescência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Linhagem Celular Tumoral , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Ubiquitina/metabolismoRESUMO
In clinical practice, a variety of diagnostic applications require the identification of target cells. Density has been used as a physical marker to distinguish cell populations since metabolic activities could alter the cell densities. Magnetic levitation offers great promise for separating cells at the single cell level within heterogeneous populations with respect to cell densities. Traditional magnetic levitation platforms need bulky and precise optical microscopes to visualize levitated cells. Moreover, the evaluation process of cell densities is cumbersome, which also requires trained personnel for operation. In this work, we introduce a device (HologLev) as a fusion of the magnetic levitation principle and lensless digital inline holographic microscopy (LDIHM). LDIHM provides ease of use by getting rid of bulky and expensive optics. By placing an imaging sensor just beneath the microcapillary channel without any lenses, recorded holograms are processed for determining cell densities through a fully automated digital image processing scheme. The device costs less than $100 and has a compact design that can fit into a pocket. We perform viability tests on the device by levitating three different cell lines (MDA-MB-231, U937, D1 ORL UVA) and comparing them against their dead correspondents. We also tested the differentiation of mouse osteoblastic (7F2) cells by monitoring characteristic variations in their density. Last, the response of MDA-MB-231 cancer cells to a chemotherapy drug was demonstrated in our platform. HologLev provides cost-effective, label-free, fully automated cell analysis in a compact design that could be highly desirable for laboratory and point-of-care testing applications.
Assuntos
Holografia , Microscopia , Animais , Processamento de Imagem Assistida por Computador , Fenômenos Magnéticos , Magnetismo , CamundongosRESUMO
The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15-35 % hydrophilic ILs, even at 75-90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15-25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower V max. However, only minor changes in kinetic parameter K m showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential.
