Functional study of popx2 phosphatase in breast cancer metastasis

POPX2, a serine/threonine phosphatase belonging to the PP2C family, is involved in the regulation of actin cytoskeleton, mDia-SRF (serum response factor) mediated transcription and breast cancer cell motility and invasiveness. In this project, we propose to use three different approaches to explore the biological functions of POPX2 in breast cancer. Firstly, we used an integrative transcriptome and proteome approach to decipher the roles of POPX2 in cancer motility and related signaling. Our study suggests that POPX2 is involved in cancer cell migration through modulating MAPK1/3-stathmin-mediated microtubule dynamics, in collaboration with other possible signaling molecules to modulate actin cytoskeleton and focal adhesions. POPX2 is also implicated in transcriptome regulation possibly through modulating MAPK1/3 and GSK3β as well as the downstream transcription factors. To further explore the role of POPX2 in angiogenesis, we also initiated a combined phosphoproteome and secretome study. Our phosphoproteome data implicate the participation of POPX2 in CDK1-mediated cell cycle progression. Secretome analysis combined with extensive biological validation verified that there is exosome enrichment and accelerated EMT progression in response to POPX2 silencing, which could further contribute to angiogenesis. Apart from the –omics approaches to understand the signaling network regulated by POPX2, we have also taken a candidate approach to specifically study candidate proteins which might be regulated by POPX2. RhoGDIs, the candidate proteins related to POPX2-regulated signaling, are also examined as we have earlier found that the levels of RhoGDI are affected by POPX2 knockdown1. The effects of RhoGDIs on actin cytoskeleton are studied. Overexpression of RhoGDI1 and RhoGDI2 result in cell rounding with concomitant loss of stress fiber and focal adhesions to different extent. RhoGDI1 appears to exert a more prominent effect compared to that of RhoGDI2. This might be due to their differential binding affinities to RhoGTPases, one of the major regulators of the actin cytoskeleton. Further characterization of RhoGDIs’ interaction with the different RhoGTPases reveals two conserved leucine residues, which are critical to RhoGTPases binding. We also verify other mutants, which affect RhoGDI binding affinity to various RhoGTPases. These mutants help to elucidate that RhoGDIs affect the actin cytoskeleton via RhoGTPases.DOCTOR OF PHILOSOPHY (SBS

Low-cost and facile implementation of microfluidic colour-changing devices using dry film photoresist-based moulds

In this work, different microfluidic colour-changing devices are implemented by using dry film photoresist-based moulds instead of standard photolithography moulds. EtertecHT-115T negative dry film photoresist is employed to realise the rapid fabrication of the moulds for colour-changing layers. The major factors that may affect the fidelity of the dry film moulds during fabrication are summarised and analysed, including the optimum exposure times and the appropriate developing times. Especially, the impacts of different concentrations of sodium carbonate (Na2CO3) solution on developing rate are investigated for 1–5 layers (50–250 μm thick) of EtertecHT-115T dry film photoresists by experiments. The created dry film moulds show the advantages of low cost, high manufacturing efficiency and requiring no professional training. Each application of the microfluidic colour-changing devices presents high transparency and good colour-changing effect. The microfluidic colour-changing layers based on dry film moulds can be used in different wearable devices of human, and also can be applied for realising surface camouflage and display functions of soft machines/robotics

Damping Force Modeling and Suppression of Self-Excited Vibration due to Magnetic Fluids Applied in the Torque Motor of a Hydraulic Servovalve

As a key component of hydraulic control systems, hydraulic servovalves influence their performance significantly. Unpredictable self-excited noise inside hydraulic servovalves may cause instability and even failure. Being functional, with higher saturation magnetization and increased viscosity when exposed to a magnetic field, magnetic fluids (MFs) have been widely used in dampers, sealing, and biomedical treatment. In this paper, magnetic fluids are applied in the torque motor of a hydraulic servovalve to exert damping and resistance for vibration and noise suppression. Construction of the torque motor armature with magnetic fluids is introduced and the forces due to magnetic fluids on the torque motor armature are studied. Based on a bi-viscosity-constituted relationship, a mathematical model of the damping force from magnetic fluids is built when magnetic fluids are filled in the working gaps of the torque motor. Measurements of the properties of an Fe3O4 composite magnetic fluid are carried out to calculate the parameters of this mathematical model and to investigate the influence of magnetic fluids on the vibration characteristics of the armature assembly. The simulated and tested harmonic responses of the armature with and without magnetic fluids show the good suppression effects of magnetic fluids on the self-excited noise inside the servovalve

POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

The Hippo pathway regulates cell proliferation, survival, apoptosis and differentiation. During carcinogenesis, members of the Hippo pathway are mutated to avoid anoikis and promote anchorage independent growth. Although many regulators of the Hippo pathway have been reported, negative regulators of the hippo kinases are not well studied. Through an interactome screen, we found that POPX2 phosphatase interacts with several of the Hippo pathway core kinases, including LATS1 which is the direct kinase regulating the transcription co-activators, YAP and TAZ. Phosphorylated YAP/TAZ are retained in the cytoplasm and prevented from translocation into the nucleus to activate transcription of target genes. We found that POPX2 could dephosphorylate LATS1 on Threonine-1079, leading to inactivation of LATS1 kinase. As a result, YAP/TAZ are not phosphorylated and are able to translocate into the nucleus to activate target genes involved in cell proliferation. Furthermore, POPX2 knock-out using CRISPR in the highly metastatic MDA-MB-231 breast cancer cells results in decreased cell proliferation and impairment of anchorage independent growth. We propose that POPX2 act as a suppressor of the Hippo pathway through LATS1 dephosphorylation and inactivation.MOE (Min. of Education, S’pore)Published versio

Identification Algorithm and Improvement of Modal Damping Ratios for Armature Assembly in a Hydraulic Servo-Valve with Magnetic Fluid

The high-frequency vibration and resonance of armature assembly in the hydraulic servo valve are the main reasons for instability and failure. Magnetic fluid (MF) operating in the squeeze mode can be taken as an effective damper for resonance suppression in the servo valve. Due to excitation difficulty and the low signal-to-noise ratio of high-frequency vibration signals, the capability of MF to modify multiple-order modal damping ratios in a multi-degree-of-freedom system is still unclear. To reveal the mechanism of magnetic fluid for improving modal damping ratios, an algorithm for modal damping ratio identification is proposed. The modal damping ratios of the armature assembly with and without magnetic fluid are identified based on the tested resonance free decay responses. Four resonance frequencies of armature assembly are observed, and the corresponding damping ratios are identified. The equivalent modal damping ratios due to squeeze flow of MF are obtained. The results show that the proposed algorithm can identify damping ratios with an accuracy of up to 98.79%. The damping ratios are improved by double or more due to the magnetic fluid, and the maximum resonance amplitudes are significantly reduced by 65.2% (from 916.5 μm to 318.6 μm)

Enhanced Delta-Notch lateral inhibition model incorporating intracellular notch heterogeneity and tension-dependent rate of Delta-Notch binding that reproduces sprouting angiogenesis patterns

Endothelial cells adopt unique cell fates during sprouting angiogenesis, differentiating into tip or stalk cells. The fate selection process is directed by Delta-Notch lateral inhibition pathway. Classical Delta-Notch models produce a spatial pattern of tip cells separated by a single stalk cell, or the salt-and-pepper pattern. However, classical models cannot explain alternative tip-stalk patterning, such as tip cells that are separated by two or more stalk cells. We show that lateral inhibition models involving only Delta and Notch proteins can also recapitulate experimental tip-stalk patterns by invoking two mechanisms, specifically, intracellular Notch heterogeneity and tension-dependent rate of Delta-Notch binding. We introduce our computational model and analysis where we establish that our enhanced Delta-Notch lateral inhibition model can recapitulate a greater variety of tip-stalk patterning which is previously not possible using classical lateral inhibition models. In our enhanced Delta-Notch lateral inhibition model, we observe the existence of a hybrid cell type displaying intermediate tip and stalk cells’ characteristics. We validate the existence of such hybrid cells by immuno-staining of endothelial cells with tip cell markers, Delta and CD34, which substantiates our enhanced model.MOE (Min. of Education, S’pore)Published versio

USP5 promotes lipopolysaccharide-induced apoptosis and inflammatory response by stabilizing the TXNIP protein

Background:. The role of thioredoxin-interacting protein (TXNIP) in lipopolysaccharide-induced liver injury in mice has been reported, but the underlying mechanisms are poorly understood. Methods:. We overexpressed deubiquitinase in cells overexpressing TXNIP and then detected the level of TXNIP to screen out the deubiquitinase regulating TXNIP; the interaction between TXNIP and deubiquitinase was verified by coimmunoprecipitation. After knockdown of a deubiquitinase and overexpression of TXNIP in Huh7 and HepG2 cells, lipopolysaccharide was used to establish a cellular inflammatory model to explore the role of deubiquitinase and TXNIP in hepatocyte inflammation. Results:. In this study, we discovered that ubiquitin-specific protease 5 (USP5) interacts with TXNIP and stabilizes it through deubiquitylation in Huh-7 and HepG2 cells after treatment with lipopolysaccharide. In lipopolysaccharide-treated Huh-7 and HepG2 cells, USP5 knockdown increased cell viability, reduced apoptosis, and decreased the expression of inflammatory factors, including NLRP3, IL-1β, IL-18, ASC, and procaspase-1. Overexpression of TXNIP reversed the phenotype induced by knockdown USP5. Conclusions:. In summary, USP5 promotes lipopolysaccharide-induced apoptosis and inflammatory response by stabilizing the TXNIP protein