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1.  Aqueous two-phase systems enable multiplexing of homogeneous immunoassays 
Technology  2014;2(2):176-.
Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD).
PMCID: PMC4114222  PMID: 25083509
3.  Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA 
Scientific Reports  2014;4:4878.
Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.
PMCID: PMC4007081  PMID: 24786974
4.  Generation of Embryonic Stem Cell Lines from Immature Rabbit Ovarian Follicles 
Stem Cells and Development  2012;22(6):928-938.
In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.
PMCID: PMC3585479  PMID: 23072728
5.  Rapid Generation of Multiplexed Cell Cocultures Using Acoustic Droplet Ejection Followed by Aqueous Two-Phase Exclusion Patterning 
The development of tools for patterning cocultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection and aqueous two-phase system exclusion patterning to introduce a method for patterning cocultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters, such as pulse, duration, and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol solution containing an additional cell type is then added to the culture dish to produce a two-phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Further, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell cocultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding cocultured cells.
PMCID: PMC3427642  PMID: 22356298
6.  Expression of axonal protein degradation machinery in sympathetic neurons is regulated by nerve growth factor 
Journal of neuroscience research  2012;90(8):1533-1546.
Deficiencies in protein degradation and proteolytic function within neurons are linked to a number of neurodegenerative diseases and developmental disorders. Compartmentalized cultures of peripheral neurons were used to investigate the properties and relative abundance of the proteolytic machinery in the axons and cell bodies of sympathetic and sensory neurons. Immunoblotting of axonal proteins demonstrated that LAMP2, LC3 and PSMA2 were abundant in axons, suggesting that lysosomes, autophagosomes and proteasomes were located in axons. Interestingly, the expression of proteins associated with lysosomes and proteasomes were upregulated selectively in axons by NGF stimulation of the distal axons of sympathetic neurons, suggesting that axonal growth and maintenance requires local protein turnover. The regulation of the abundance of both proteasomes and lysosomes in axons by NGF provides a link between protein degradation and the trophic status of peripheral neurons. Inhibition of proteasomes located in axons resulted in an accumulation of ubiquitinated proteins in these axons. In contrast, lysosome inhibition in axons did not result in an accumulation of ubiquitinated proteins or the transferrin receptor, a transmembrane protein degraded by lysosomes. Interestingly, lysosomes were transported both retrogradely and anterogradely, therefore it is likely that ubiquitinated proteins that are normally destined for degradation by lysosomes in axons can be transported to the cell bodies for degradation. In summary, proteasomal degradation occurs locally, whereas proteins degraded by lysosomes can most likely either be degraded locally in axons, or be transported to cell bodies for degradation.
PMCID: PMC3523670  PMID: 22411744
Proteasome; Lysosome; NGF; Transport; Autophagy
7.  Two Novel Techniques to Screen Abies Seedlings for Resistance to the Balsam Woolly Adelgid, Adelges piceae  
Since its introduction into the Southern Appalachians in the 1950s, the balsam woolly adelgid, Adelges piceae Ratzeburg (Hemiptera: Adelgidae), has devastated native populations of Fraser fir, Abies fraseri (Pursh) Poir. (Pinales: Pinaceae), and has become a major pest in Christmas tree plantations requiring expensive chemical treatments. Adelges piceae—resistant Fraser fir trees would lessen costs for the Christmas tree industry and assist in the restoration of native stands. Resistance screening is an important step in this process. Here, four studies directed toward the development of time— and cost—efficient techniques for screening are reported. In the first study, three methods to artificially infest seedlings of different ages were evaluated in a shade—covered greenhouse. Two—year—old seedlings had much lower infestation levels than 7 year—old seedlings. Placing infested bark at the base of the seedling was less effective than tying infested bark to the seedling or suspending infested bolts above the seedling. Although the two latter techniques resulted in similar densities on the seedlings, they each have positive and negative considerations. Attaching bark to uninfested trees is effective, but very time consuming. The suspended bolt method mimics natural infestation and is more economical than attaching bark, but care must be taken to ensure an even distribution of crawlers falling onto the seedlings. The second study focused on the density and distribution of crawlers falling from suspended bolts onto paper gridded into 7.6 × 7.6 cm cells. Crawler density in a 30 cm band under and to each side of the suspended bolt ranged from 400 to over 3000 crawlers per cell (1 to 55 crawlers per cm2). In the third study, excised branches from 4 year—old A. fraseri and A. vetchii seedlings were artificially infested with A. piceae to determine whether this technique may be useful for early resistance screening. The excised A. fraseri branches supported complete adelgid development (crawler to egg—laying adult), and very little adelgid development occurred on A. vetchii branches. The fourth study compared infestation levels and gouting response on excised versus intact branches of 4 year—old A. fraseri seedlings from three different seed sources, and excised branches from 4 year—old and 25 year—old trees. There were no differences in infestation levels between excised versus intact branches nor in very young versus mature trees; gouting response was observed only on intact branches.
PMCID: PMC3391932  PMID: 22239164
Abies fraseri; artificial infestation; excised branches; Fraser fir; host resistance
8.  Directed cell growth on protein-functionalized hydrogel surfaces 
Journal of neuroscience methods  2007;162(1-2):255-263.
Biochemical surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymerized in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labelled proteins. Soft protein lithography (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biological assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphology on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72 hr in vitro, and remained viable on protein stamped areas for more than 4 weeks. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM 1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.
PMCID: PMC2729282  PMID: 17368788
Hydrogel; microcontact printing; acrylamide; LRM55; neuron; FM1-43FX
9.  Polymerase Chain Reaction Preparation of Template for Massively Parallel Pyrosequencing 
Massively parallel pyrosequencing of DNA fragments immobilized on beads has been applied to genome survey sequencing and transcriptome analysis of a variety of eukaryotic organisms, including laboratory model species, agricultural crops and livestock, and species of interest to population biologists and ecologists. Preparation of sufficient high-quality template for sequencing has been an obstacle to sequence analysis of nucleic acids from tissues or cell types available in limited quantities. We report that the use of a biotinylated primer for polymerase chain reaction amplification allows removal of excess primer and poly(A) tract fragments from the sequencing templates, providing much higher yields of useful sequence information from pyrosequencing of amplified templates. This advance allows deep sequencing analysis of nucleic acids isolated from very small tissue samples. Massively parallel pyrosequencing is particularly useful for preliminary investigations of species that have not yet been the subject of significant genomic research, as genomic survey sequences and catalogs of expressed genes provide a means of linking the biology of less intensively studied species to that of more intensively studied model organisms. We obtained over 220 Mb of transcript DNA sequences from Abies fraseri (Pursh) Poir., a conifer species native to the southern Appalachian Mountains of eastern North America. Comparison of the resulting assembled putative transcripts with similar data obtained by other sequencing methods from other conifers demonstrates the utility of the improved sequencing template preparation.
PMCID: PMC2685602  PMID: 19503624
DNA sequences; polymerase chain reaction; transcriptome analysis
10.  Biomedical Technologies for in vitro Screening and Controlled Delivery of Neuroactive Compounds 
Cell culture models can provide information pertaining to the effective dose, toxiciology, and kinetics, for a variety of neuroactive compounds. However, many in vitro models fail to adequately predict how such compounds will perform in a living organism. At the systems level, interactions between organs can dramatically affect the properties of a compound by alteration of its biological activity or by elimination of it from the body. At the tissue level, interaction between cell types can alter the transport properties of a particular compound, or can buffer its effects on target cells by uptake, processing, or changes in chemical signaling between cells. In any given tissue, cells exist in a three-dimensional environment bounded on all sides by other cells and components of the extracellular matrix, providing kinetics that are dramatically different from the kinetics in traditional two-dimensional cell culture systems. Cell culture analogs are currently being developed to better model the complex transport and processing that occur prior to drug uptake in the CNS, and to predict blood-brain barrier permeability. These approaches utilize microfluidics, hydrogel matrices, and a variety of cell types (including lung epithelial cells, hepatocytes, adipocytes, glial cells, and neurons) to more accurately model drug transport and biological activity. Similar strategies are also being used to control both the spatial and temporal release of therapeutic compounds for targeted treatment of CNS disease.
PMCID: PMC2600660  PMID: 19079777
Blood-brain barrier; Cell Culture Model; TEER; Hydrogel; Microfluidic
11.  Lost Loop 
British Medical Journal  1966;2(5526):1391.
PMCID: PMC1944400
13.  Hospital Confinement 
British Medical Journal  1957;2(5045):644.
PMCID: PMC1962147

Results 1-13 (13)