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W French Anderson

Publications and source records attributed to W French Anderson.

At least 19 recordsLinked to original sources
PubMed2006

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. We apply the vias to printing nested bioarrays and building autoregulatory devices. A current source is demonstrated, while a diode and a rectifier are derived; all are building blocks for analog circuitry in Newtonian fluids. We also describe microfluidic septa and their applications. Vias lay the foundation for a new generation of microfluidic devices.

Dimethylpolysiloxanes↗
PubMed2006

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. The techniques presented here form the foundation for highly parallel single-cell gene expression studies.

Animals↗
PubMed2006

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. NPC2 protein, in the presence of thrombopoietin (TPO), causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells, a TPO-dependent hematopoietic leukemia cell line, NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly, these results are not observed under the standard 20% O(2) level of the standard incubator, but rather at 7% O(2), the physiological oxygen level of bone marrow. Furthermore, NPC2 protein upregulates hypoxia inducible factor 1-alpha protein level at 7% O(2), but not at 20% O(2). Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O(2).

Animals↗
PubMed2006

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. The device and method presented here may enable highly parallel single cell gene expression analysis.

Humans↗
PubMed2006

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. Next, the relevant scales of both microfluidics and biology are compared, obtaining the promise and limitations of PDMS microfluidics. Finally, the body of work is reclassified in terms of addressed biological applications and compared to the standard methods in cellular and molecular biology, to offer insights for future devices and applications.

Animals↗
PubMed2006

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). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.

Antigens↗
PubMed2005

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. We have used five-color FACS sorting to isolate separate populations of LTR and STR stem cell subsets. The LTR HSC has the phenotype defined as Lin- Sca+ Kit+ 38+ 34-; two subsets of STR HSC were obtained with phenotypes of Lin- Sca+ Kit+ 38+ 34+ and Lin- Sca+ Kit+ 38- 34+. The microarray profiling study reported here was able to identify genes specific for LTR functions. In the interrogated genes (approximately 12,000 probe sets corresponding to 8,000 genes), 210 genes are differentially expressed, and 72 genes are associated with LTR activity, including membrane proteins, signal transduction molecules, and transcription factors. Hierarchical clustering of the 210 differentially expressed genes suggested that they are not bone marrow-specific but rather appear to be stem cell-specific. Transcription factor-binding site analysis suggested that GATA3 might play an important role in the biology of LTR HSC.

Animals↗
PubMed2005

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. MATERIALS AND METHODS: Animals with an implanted TheraCyte immunoisolation device (TID) received lethal-dose radiation and then normal bone marrow Lin- cells were loaded into the device (thereby preventing direct interaction between donor and recipient cells). Animal survival was evaluated and stem cell activity was tested with secondary bone marrow transplantation and flow cytometry analysis. Donor cell gene expression of five antiapoptotic cytokines was examined. RESULTS: Bone marrow Lin- cells rescued lethally irradiated animals via soluble factor(s). Bone marrow cells from the rescued animals can rescue and repopulate secondary lethally irradiated animals. Within the first 6 hours post-lethal-dose radiation, there is no significant change of gene expression of the known radioprotective factors TPO, SCF, IL-3, Flt-3 ligand, and SDF-1. CONCLUSION: Hematopoietic stem cells can be protected in lethally irradiated animals by soluble factors produced by bone marrow Lin- cells.

Animals↗
PubMed2005

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. Handguns shot different caliber bullets at armor that had a 10%-ordnance gelatin backing. The gelatin blocks were tissue simulants. The gunshot data tuned the additive fuzzy function approximator. The fuzzy system's conditional variance V[Y/X = x] described the second-order uncertainty of the function approximation. Handguns with different barrel lengths shot bullets over a fixed distance at armor-clad gelatin blocks that we made with Type 250 A Ordnance Gelatin. The bullet-armor experiments found that a bullet's weight and momentum correlated with the depth of its impact on armor-clad gelatin (R2 = 0.881 and p-value < 0.001 for the null hypothesis that the regression line had zero slope). Related experiments on plumber's putty showed that highspeed baseball impacts compared well to bullet-armor impacts for large-caliber handguns. A baseball's momentum correlated with its impact depth in putty (R2 = 0.93 and p-value < 0.001). A bullet's momentum similarly correlated with its armor-impact in putty (R2 = 0.97 and p-value < 0.001). A Gujarati-Chow test showed that the two putty-impact regression lines had statistically indistinguishable slopes for p-value = 0.396. Baseball impact depths were comparable to bullet-armor impact depths: Getting shot with a .22 caliber bullet when wearing soft body armor resembles getting hit in the chest with a 40-mph baseball. Getting shot with a .45 caliber bullet resembles getting hit with a 90-mph baseball.

Computer Simulation↗
PubMed2004

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. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.

Animals↗
PubMed2004

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. This strategy was devised to provide a systemic, antigen-specific signal to pathogenic T cells in the absence of costimulation and, hence, render them anergic. Cytokine analyses of brain and spinal cord lymphocytes demonstrate that the treatment induces an antiinflammatory Th2 profile, indicating that this antigen-specific therapy acts by a cytokine-induced pathway. This study was designed for translation to the clinic. We envision using allogeneic transduced fibroblasts, encapsulated in a chamber, to deliver the antigen-specific signal. This will enable us to use one therapeutic cell line for all patients and to remove the device should the therapy exacerbate disease.

Animals↗
PubMed2004

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. Here we show that D84K virus infection increased 3,000-fold on cells expressing receptor with an rK234A change and 100,000-fold on cells expressing an rK234D change. The stronger complementation of D84K virus infection by rK234D than by the rK234A receptor suggests that although the major reason for loss of infection of D84K and D84R virus is due to steric hindrance and charge repulsion, the loss of an interaction of D84 with receptor appears to contribute as well. Taken together, these results indicate that D84 is very close to rK234 of mATRC1 in the bound complex and there is likely an interaction between them. The definitive localization of the receptor binding site on SU should facilitate the design of chimeric envelope proteins that target infection to new receptors by replacing the receptor binding site with an exogenous ligand sequence.

Animals↗
PubMed2003

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. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

Animals↗
PubMed2003

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., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 +/- 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.

3T3 Cells↗
PubMed2003

Cytoplasmic tail of Moloney murine leukemia virus envelope protein influences the conformation of the extracellular domain: implications for mechanism of action of the R Peptide.

The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptide-truncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein.

Amino Acid Sequence↗