Mass spectrometry of murine leukemia virus core proteins.

A mass spectrometry (MS) approach was used to analyze viral core proteins of the murine leukemia virus (MuLV)-based gene delivery vector. The retroviral particles produced by traditional methods were concentrated and purified by ultracentrifugation and spin column for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS. MALDI application detected all core MuLV proteins, partial degradation of p10, phosphorylation of p12, as well as the previously unknown formation of a polymeric supramolecular complex between p15 and p30 core proteins.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

Background: Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time.

Internally calibrated quantification of VEGF in human plasma by fluorescence immunoassays in disposable elastomeric microfluidic devices.

Herein we report on a proof of principle for the reproducible quantification of Vascular Endothelial Growth Factor (VEGF) in human plasma by fluorescence sandwich immunoassays using disposable polydimethylsiloxane (PDMS) microfluidic chips. The system requires 100 times less sample than typical clinical blood tests, while its current quantification limit is established at 4pM. The in-built calibration method of spiking the plasma with known concentrations of commercially available antigen avoids common sources of error and improves the reliability of the test results.

Membrane-proximal cytoplasmic domain of Moloney murine leukemia virus envelope tail facilitates fusion.

Removal of the R peptide (residues 617-632) from the Moloney murine leukemia virus (MoMuLV) envelope protein (Env) cytoplasmic tail potentiates fusion. We examined the role of the membrane-proximal cytoplasmic domain (598-616) of the MoMuLV Env in the Env-mediated membrane fusion and incorporation. The Env truncated at 616 exhibits maximum fusogenicity in cell-to-cell fusion assay.

Membrane activity of an amphiphilic alpha-helical membrane-proximal cytoplasmic domain of the MoMuLV envelope glycoprotein.

In the Moloney murine leukemia virus (MoMuLV) envelope glycoprotein (Env) we identified a membrane-proximal cytoplasmic domain (residues 598-616) that facilitates the Env incorporation into virions and Env-mediated fusion [Rozenberg, Y., Conner, J., Aguilar-Carreno, H.

Downregulation of NPM-ALK by siRNA causes anaplastic large cell lymphoma cell growth inhibition and augments the anti cancer effects of chemotherapy in vitro.

The fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), results from the chromosome translocation t(2;5)(p23;q25) and is present in 50-70 percent of anaplastic large-cell lymphomas (ALCLs). NPM-ALK is a constitutively activated kinase that transforms cells through stimulating several mitogenic signaling pathways. To examine if the NPM-ALK is a potential therapeutic target in ALCL, we used siRNA to specifically downregulate the expression of the NPM-ALK in ALCL cell lines.

Electrical microfluidic pressure gauge for elastomer microelectromechanical systems.

We report on an electrical microfluidic pressure gauge. A polydimethylsiloxane microvalve closes at a characteristic applied pressure determined by the material's properties and the valve's dimensions. Hence, when the same pressure is applied to all valves of a heterogeneous valve array, some valves close while others remain open.

Experimentally validated quantitative linear model for the device physics of elastomeric microfluidic valves.

A systematic experimental study and theoretical modeling of the device physics of polydimethylsiloxane "pushdown" microfluidic valves are presented. The phase space is charted by 1587 dimension combinations and encompasses 45-295 μm lateral dimensions, 16-39 μm membrane thickness, and 1-28 psi closing pressure. Three linear models are developed and tested against the empirical data, and then combined into a fourth-power-polynomial superposition.

The analytical approach to polydimethylsiloxane microfluidic technology and its biological applications.

This review article discusses PDMS (polydimethylsiloxane) microfluidic devices and their biological applications. First, the already developed devices are classified from the viewpoints of underlying technology within a common logical framework comprising single-layer, multilayer, and integrated devices, as well as surface chemistry modifications of PDMS. Combinatorial techniques are applied to re-derive existing devices within this framework.

Microfluidic vias enable nested bioarrays and autoregulatory devices in Newtonian fluids.

We report on a fundamental technological advance for multilayer polydimethylsiloxane (PDMS) microfluidics. Vertical passages (vias), connecting channels located in different layers, are fabricated monolithically, in parallel, by simple and easy means. The resulting 3D connectivity greatly expands the potential complexity of microfluidic architecture.

Microfluidic single-cell mRNA isolation and analysis.

Single-cell gene expression analysis holds great promise for studying diverse biological systems, but methodology to process these precious samples in a reproducible, quantitative, and parallel fashion remains challenging. Here, we utilize microfluidics to isolate picogram and subpicogram mRNA templates, as well as to synthesize cDNA from these templates. We demonstrate single-cell mRNA isolation and cDNA synthesis, provide quantitative calibrations for each step in the process, and measure gene expression in individual cells.

Involvement of Niemann-Pick type C2 protein in hematopoiesis regulation.

Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease, an inherited neurodegenerative disorder. When analyzing gene expression profile, we noticed high expression of both NPC2 and its receptor, mannose 6-phosphate receptor (MPR), in murine hematopoietic stem cells.

High-throughput multi-antigen microfluidic fluorescence immunoassays.

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF).

Parallel picoliter rt-PCR assays using microfluidics.

The development of microfluidic tools for high-throughput nucleic acid analysis has become a burgeoning area of research in the post-genome era. Here, we have developed a microfluidic chip to perform 72 parallel 450-pL RT-PCRs. We took advantage of Taqman hydrolysis probe chemistry to detect RNA templates as low as 34 copies.

Modeling gunshot bruises in soft body armor with an adaptive fuzzy system.

Gunshots produce bruise patterns on persons who wear soft body armor when shot even though the armor stops the bullets. An adaptive fuzzy system modeled these bruise patterns based on the depth and width of the deformed armor given a projectile's mass and momentum. The fuzzy system used rules with sinc-shaped if-part fuzzy sets and was robust against random rule pruning: Median and mean test errors remained low even after removing up to one fifth of the rules.

Soluble factor(s) from bone marrow cells can rescue lethally irradiated mice by protecting endogenous hematopoietic stem cells.

Objective: Ionizing radiation-induced myeloablation can be rescued via bone marrow transplantation (BMT) or administration of cytokines if given within 2 hours after radiation exposure. There is no evidence for the existence of soluble factors that can rescue an animal after a lethal dose of radiation when administered several hours postradiation. We established a system that could test the possibility for the existence of soluble factors that could be used more than 2 hours postirradiation to rescue animals.

Gene expression profile of murine long-term reconstituting vs. short-term reconstituting hematopoietic stem cells.

The hematopoietic stem cell (HSC) compartment is composed of long-term reconstituting (LTR) and short-term reconstituting (STR) stem cells. LTR HSC can reconstitute the hematopoietic system for life, whereas STR HSC can sustain hematopoiesis for only a few weeks in the mouse. Several excellent gene expression profiles have been obtained of the total hematopoietic stem cell population.

Complementation of a binding-defective retrovirus by a host cell receptor mutant.

The entry of ecotropic murine leukemia virus (MLV) into cells requires the interaction of the envelope protein (Env) with its receptor, mouse cationic amino acid transporter 1 (mATRC1). An aspartic acid-to-lysine change at position 84 (D84K) of ecotropic Moloney MLV Env abolishes virus binding and infection. We recently identified lysine 234 (rK234) in mATRC1 as a residue that influences virus binding and infection.

A nanoliter-scale nucleic acid processor with parallel architecture.

The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment.

Gene therapy in a murine model for clinical application to multiple sclerosis.

Female SJL/J mice, suffering from experimental autoimmune encephalomyelitis (EAE), were injected with 1 x 10(7) cells from a syngeneic fibroblast line transduced with a retroviral vector designed to encode proteolipid protein (101-157) targeted for secretion. A striking abrogation of both clinical and histological signs of disease resulted. The treatment was efficacious when given after the first or the third relapses, protected naive mice from challenge with spinal cord homogenate, and was dose dependent.

Antibody-mediated targeting of replication-competent retroviral vectors.

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells.

Determination of infectious retrovirus concentration from colony-forming assay with quantitative analysis.

The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g.