A simple and high -performance immobilization technique of membrane protein from crude cell lysate sample for a membrane-based immunoassay application.

Membrane proteins are difficult to be extracted and to be coated on the substrate of the immunoassay reaction chamber because of their hydrophobicity. Traditional method to prepare membrane protein sample requires many steps of protein extraction and purification that may lead to protein structure deformation and protein dysfunction. This work proposes a simple technique to prepare and immobilize the membrane protein suspended in an unprocessed crude cell lysate sample. Membrane fractions in crude cell lysate were incorporated with the large unilamellar vesicle (LUV) that was mainly composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) before coating in the polystyrene plate by passive adsorption technique. Immunofluorescence staining and the Enzyme-Linked Immunosorbent Assay (ELISA) examination of a strictly conformation-dependent integral membrane protein, Myelin Oligodendrocyte Glycoprotein (MOG), demonstrate that LUV incorporated cell lysate sample obviously promotes MOG protein immobilization in the microplate well. With LUV incorporation, the dose-response curve of the MOG transfected cell lysate coating plate can be 2-9 times differentiated from that of the untransfected cell lysate coating plate. The LUV incorporated MOG transfected cell lysate can be efficiently coated in the microplate without carbonate/bicarbonate coating buffer assistance.

Extracellular Vesicle-Mediated IL-1 Signaling in Response to Doxorubicin Activates PD-L1 Expression in Osteosarcoma Models.

The expression of programmed cell death ligand 1 (PD-L1) in tumors is associated with tumor cell escape from T-cell cytotoxicity, and is considered a crucial effector in chemoresistance and tumor relapse. Although PD-L1 induction has been observed in patients after chemotherapy treatment, the mechanism by which the drug activates PD-L1 expression remains elusive. Here, we identified the extracellular vesicles (EVs) as a molecular mediator that determines the effect of doxorubicin on PD-L1 expression in osteosarcoma models. Mechanistically, doxorubicin dependently stimulates the release of extracellular vesicles, which mediate autocrine/paracrine signals in osteosarcoma cells. The recipient cells were stimulated by these EVs and acquired the ability to promote the expression of inflammatory cytokines interleukin (IL)-1ß and IL-6. In response to doxorubicin, IL-1ß, but not IL-6, allowed- osteosarcoma cells to promote the expression of PD-L1, and the elimination of IL-1ß/IL-1 receptor signaling with IL-1 receptor antagonist reduced PD-L1 expression. Together, these findings provided insights into the role of EV release in response to chemotherapy that mediates PD-L1 expression via the IL-1 signaling pathway, and suggested that the combination of a drug targeting IL-1 or PD-L1 with chemotherapy could be an effective treatment option for osteosarcoma patients.

Formation of double emulsion micro-droplets in a microfluidic device using a partially hydrophilic-hydrophobic surface.

The objective of this paper is to propose a surface modification method for preparing PDMS microfluidic devices with partially hydrophilic-hydrophobic surfaces for generating double emulsion droplets. The device is designed to be easy to use without any complicated preparation process and also to achieve high droplet encapsulation efficiency compared to conventional devices. The key component of this preparation process is the permanent chemical coating for which the Pluronic surfactant is added into the bulk PDMS. The addition of Pluronic surfactant can modify the surface property of PDMS from a fully hydrophobic surface to a partially hydrophilic-hydrophobic surface whose property can be either hydrophilic or hydrophobic depending on the air- or water-treatment condition. In order to control the surface wettability, this microfluidic device with the partially hydrophilic-hydrophobic surface undergoes water treatment by injecting deionized water into the specific microchannels where their surface property changes to hydrophilic. This microfluidic device is tested by generating monodisperse water-in-oil-in-water (w/o/w) double emulsion micro-droplets for which the maximum droplet encapsulation efficiency of 92.4% is achieved with the average outer and inner diameters of 75.0 and 57.7 µm, respectively.

Cell proliferation and migration inside single cell arrays.

Cell proliferation and migration are fundamental processes in determining cell and tissue behaviour. In this study we show the design and fabrication of a new single cell microfluidic structure, called a "vertically integrated array" or "VIA" trap to explore quantitative functional assays including single cell attachment, proliferation and migration studies. The chip can be used in a continuous (flow-through) manner, with a continuous supply of new media, as well as in a quiescent mode. We show the fabrication of the device, together with the flow characteristics inside the network of channels and the single cell traps. The flow patterns inside the device not only facilitate cell trapping, but also protect the cells from mechanical flow-induced stress. MDA-MB-231 human breast cancer cells were used to study attachment and detachment during the cell cycle as well as explore the influences of the chemokine SDF-1 (enabling the quantification of the role of chemokine gradients both on pseudopod formation and directional cell migration).

Spatially selecting a single cell for lysis using light-induced electric fields.

An optoelectronic tweezing (OET) device, within an integrated microfluidic channel, is used to precisely select single cells for lysis among dense populations. Cells to be lysed are exposed to higher electrical fields than their neighbours by illuminating a photoconductive film underneath them. Using beam spot sizes as low as 2.5 µm, 100% lysis efficiency is reached in <1 min allowing the targeted lysis of cells.

Expression of membrane-associated proteins within single emulsion cell facsimiles.

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

Intracellular protein determination using droplet-based immunoassays.

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 µM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

Inhibition of Taura syndrome virus replication in Litopenaeus vannamei through silencing the LvRab7 gene using double-stranded RNA.

Taura syndrome virus (TSV) is a major cause of high mortality in Pacific white shrimp (Litopenaeus vannamei, Lv). Previously, silencing of Penaeus monodon Rab7 (PmRab7) by injecting double-stranded RNA corresponding to PmRab7 (dsRNA-PmRab7) prevented white spot syndrome virus or yellow head virus infection. Rab7 is proposed to be involved in intracellular trafficking of the viruses. This study aimed to investigate whether knockdown of Rab7 in L. vannamei by dsRNA-PmRab7 could inhibit replication of TSV. RNA interference (RNAi) technology using dsRNA targeting the LvRab7 gene was used to silence the mRNA expression of LvRab7. The silencing of the LvRab7 gene inhibited TSV replication dramatically when compared to groups receiving dsRNA-GFP or NaCl. This is the first demonstration that dsRNA targeting the endogenous shrimp gene LvRab7 strongly reduces TSV replication. It provides further evidence that LvRab7 is involved in the endosomal trafficking pathway of viruses infecting penaeid shrimp.

Suppression of PmRab7 by dsRNA inhibits WSSV or YHV infection in shrimp.

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.