Sumith Kottegoda

Chemical Analysis of Single Cells

Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

Demonstration of low flow push-pull perfusion.

Methods to follow in vivo chemical composition provide information regarding the processes of intercellular communication. There is a need for methods that provide chemical information from small volumes of the central nervous system (CNS) without sacrificing neurochemical recovery. One method that offers potential for providing such information is push-pull perfusion. In this study a low flow push-pull perfusion system is introduced that provides high (70-80%) in vitro recoveries. A concentric probe design is used with a 27-gauge stainless steel outer cannula for saline infusion and an inner fused silica capillary for fluid withdrawal. Flow rates of 10-50 nl/min were reliably generated and were well matched in vitro. Sampling was performed in the striatum of an anesthetized rat generating a 0.5 microl sample every 12 min. Capillary electrophoresis was used to determine glutamate levels in each sample; the basal level was found to be 1.97+/-0.70 microM. The method described was also demonstrated to deliver L-trans-pyrrolidine-2,4-dicarboxylic acid through the perfusion solution while sampling. Post-sampling histological analysis demonstrates little tissue disturbance to the sampled region. These data provide evidence that low flow push-pull method is a viable alternative for studying neurochemical signaling in the CNS.

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The KM of sphingosine kinase for its fluorescently labeled substrate was 38 +/- 18 microM with a Vmax of 0.4 +/- 0.2 microM/min and a kcat of 3900 s-1. Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.

Separations in poly(dimethylsiloxane) microchips coated with supported bilayer membranes

Hybrid microchannels composed of poly(dimethylsiloxane) and glass were coated with supported bilayer membranes (SBMs) by the process of vesicle fusion. The electroosmotic mobility (mu(eo)) of zwitterionic, positively charged, and negatively charged phospholipid membranes was measured over a 4 h time to evaluate the stability of the coatings in an electric field. Coated microchips with a simple cross design were used to separate the fluorescent dyes fluorescein and Oregon Green. Migration time reproducibility was better than 5% RSD over 70 min of continuous separations. Separation of Oregon Green and fluorescein in channels coated with zwitterionic phosphatidylcholine (PC) membranes yielded efficiencies of 611,000 and 499,000 plates/m and a resolution of 2.4 within 2 s. Both zwitterionic and negatively charged membranes were used to separate peptide substrates from their phosphorylated analogues with efficiencies of 200,000-400,000 plates/m. Notably, separations of fluorescently labeled ABL substrate peptide from its phosphorylated counterpart were achieved using a high-salt physiological buffer with near-baseline resolution in 10 s. PC-coated devices were used to successfully separate enhanced green fluorescent protein (eGFP) from a fusion protein (eGFP-Crakl) with an efficiency of 358,000 and 278,000 plates/m respectively in less than 12 s. These SBM-based coatings may enable the separation of a broad range of analytes and may be ideal in biological applications for microfluidics.

An Immature Retroviral RNA Genome Resembles a Kinetically Trapped Intermediate State

ABSTRACT Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. IMPORTANCE The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.