RESUMO
The accurate determination of the molar concentration or the number concentration of particles in a defined volume is important but challenging. Since particle diversity and heterogeneity cannot be ignored in particle quantification, single particle counting has become quite important. However, most methods require standard samples (calibrators) which are usually difficult to obtain. The authors describe a method for single particle counting that is based on the combination of digital counting and formation of microdroplets in a microchip. By compartmentalizing particles into picoliter droplets, positive droplets encapsulating particles were counted and particle concentrations were calculated by Poisson statistics. The concentration of particles over a wide range (from 5.0 × 103 to 1.8 × 107 particles per mL) were accurately determined without the need for using a calibrator. A microdroplet chip including a T-junction channel achieved a 9-fold increase of signal-to-background ratio compared to the traditional flow-focusing chip. This makes the digital counting system a widely applicable tool for quantification of fluorescent particles. Various particles including differently sized fluorescent microspheres and bacteria with large heterogeneity in shape such as Escherichia coli DH5α-pDsRed were accurately quantified by this method. Graphical abstract Schematic representation of the digital single particle counting system for absolute quantification of particles. Particles compartmentalized in picoliter droplets were counted and the number concentration of particles was determined using digital analysis.
RESUMO
Background: OTUB1, an essential deubiquitinating enzyme, is upregulated in various types of cancer. Previous studies have shown that OTUB1 may be an oncogene in glioblastoma multiforme (GBM), but its specific regulatory mechanism remains unclear. This study aimed to investigate the mechanism by which OTUB1 and the JAK2/STAT1 signaling pathway co-regulate the growth of GBM. Methods: Using bioinformatics, GBM tissues, and cells, we evaluated the expression and clinical significance of OTUB1 in GBM. Subsequently, we explored the regulatory mechanisms of OTUB1 on malignant behaviors in GBM in vitro and in vivo. In addition, we added the JAK2 inhibitor AZD1480 to explore the regulation of OTUB1 for JAK2/STAT1 pathway in GBM. Results: We found that OTUB1 expression was upregulated in GBM. Silencing OTUB1 promotes apoptosis and cell cycle arrest at G1 phase, inhibiting cell proliferation. Moreover, OTUB1 knockdown effectively inhibited the invasion and migration of GBM cells, and the opposite phenomenon occurred with overexpression. In vivo experiments revealed that OTUB1 knockdown inhibited tumor growth, further emphasizing its crucial role in GBM progression. Mechanistically, we found that OTUB1 was negatively correlated with the JAK2/STAT1 pathway in GBM. The addition of the JAK2 inhibitor AZD1480 significantly reversed the effects of silencing OTUB1 on GBM. Conclusion: Our study reveals a novel mechanism by which OTUB1 inhibits the JAK2/STAT1 signaling pathway. This contributes to a better understanding of OTUB1's role in GBM and provides a potential avenue for targeted therapeutic intervention.