Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology.     

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics.     

A direct fluorometric assay for tissue transglutaminase

Herein we report the design of a direct and continuous fluorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-(N-carbobenzoxy-l-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of fluorescence (lambda(exc) 330 nm, lambda(em) 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the K(m) of two acceptor substrates, N-acetyl-L-lysine methyl ester and aminoacetonitrile. We also determined the K(m) of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl-L-lysine methyl ester and aminoacetonitrile. We ascertained that the fluorescent substrate was selective toward tissue TGase by testing it with different enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and gamma-glutamyl transpeptidase. 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an efficient fluorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format.     

Automated expression and solubility screening of His-tagged proteins in 96-well format

A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand of postgenomics, and it is now generally accepted that the long-term solution to this problem will be based on automation, on industrial standard-formatted experiments, and on downsizing samples and consumables. In agreement with this consensus, we have set up a fully automated method based on a dot-blot technology and using 96-well format consumables for assessing by immunodetection the amount of total and soluble recombinant histidine (His)-tagged proteins expressed in Escherichia coli. The method starts with the harvest of expression cells and ends with the display of solubility/expression results in milligrams of recombinant protein per liter of culture using a three-color code to assist analysis. The program autonomously processes 160 independent cultures at a time.     

Functional RNA microarrays for high-throughput screening of antiprotein aptamers

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.     

Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium

Here we report the successful implementation of the Plackett-Burman multifactorial design to screen the limiting components for growth and subsequent use of the response surface methodology (RSM) to design a medium that supported exponential growth of the aggregated morphology of the shipworm bacterium, Teredinobacter turnirae. The results obtained with the help of Plackett-Burman design indicated limitations of three components in the growth medium, MnCl2.4H2O, Na2CO3, and K2HPO4. The concentrations of these three components were further optimized using RSM. By increasing the concentrations of the above-mentioned components by 4-fold, 12-fold, and 12-fold, respectively, it became possible to achieve exponential growth of the culture.     

Synthesis of dicarboxylic acylcarnitines

Syntheses of malonyl, methylmalonyl, succinyl, glutaryl, methylglutaryl, dodecanedioyl and hexadecanedioyl carnitines are described. The dicarboxylic acylcarnitines were prepared from eight equivalents of cyclic anhydride or isopropylidene ester of the dicarboxylic acid and carnitine chloride in trifluoroacetic acid solution. Long chain dicarboxylic acylcarnitines were additionally purified by partitioning between water and n-butanol. Stable isotope labeled analogs, containing 3, 6 or 9 deuterium atoms, were also prepared. They are for use as standards in the electrospray ionization tandem mass spectrometric analysis of dicarboxylic acylcarnitines in samples from patients with inherited disorders of fatty acid oxidation.     

A quantitative genetic method for estimating developmental instability

The concept of developmental instability (DI) is frequently used in evolutionary biology, and a range of definitions has been proposed. Moreover, numerous different statistical methods have been used for estimation of DI. The common basis for all methods is that measures need to be obtained from repeated structures within organisms. In the case of fluctuating asymmetry, mirror images could be interpreted as the repeats of each other. All repeats of a trait on one organism should, from a quantitative perspective, have the same genetic foundation. Most previous methods have not accounted for the genetics of the underlying trait. It is here shown how a statistical method from quantitative genetics (the repeated records animal model) can be used for assessment of DI, based on estimation of the variance due to the permanent environment. Moreover, Gibbs sampling is used for inference of the parameters, which provides a Bayesian framework where posterior distributions easily can be calculated from any functions of the variance components. The method is applied to a real dataset from two populations of the plant Scabiosa canescens, and results shows that it works well under realistic situations.     

Estimating the cumulative risk of a false-positive test in a repeated screening program

The goal of screening tests for a chronic disease such as cancer is early detection and treatment with a consequent reduction in mortality from the disease. Screening tests, however, might produce false positive and false-negative results. With an increasing number of screening tests, it is clear that the risk of a false-positive screen, a finding with potentially significant emotional, financial, and health costs, also increases. Elmore et al. (1998, New England Journal of Medicine 338, 1089-1096), Christiansen et al. (2000, Journal of the National Cancer Institute 92, 1657-1666), and Gelfand and Wang (2000, Statistics in Medicine 19, 1865-1879) investigated this problem under the somewhat unrealistic assumption that the choice of making the decision to drop out at the kth screen does not depend upon the results of the earlier k - 1 screens. In this article we obtain sufficient and necessary conditions for their assumption to hold and use one of them to provide a method for testing the validity of the assumption. A new model which does not depend on their assumption is introduced. The maximum likelihood estimator of the cumulative risk of receiving a false-positive screen under the new model is derived and its asymptotic normality is proved. The extension of the new model by incorporating covariate information is also considered. We apply our testing method and the new model to data from the breast cancer screening trial of the Health Insurance Plan of Greater New York.     

Ontogenetic correlates of diet in Malagasy lemurs

There is a well-documented relationship between development and other life-history parameters among anthropoid primates. Smaller-bodied anthropoids tend to mature more rapidly than do larger-bodied species. Among anthropoids of similar body sizes, folivorous species tend to grow and mature more quickly than do frugivorous species, thus attaining adult body size at an earlier age. This pattern conforms to the expectations of Janson and van Schaik's "ecological risk aversion hypothesis," which predicts that rates of growth and maturation should vary in inverse relation to the intensity of intraspecific feeding competition. According to the ecological risk aversion hypothesis (RAH), species experiencing high intraspecific feeding competition will grow and mature slowly to reduce the risk of mortality due to food shortages. Species experiencing low levels of intraspecific feeding competition will shorten the juvenile period to reduce the overall duration of this high-risk portion of the life cycle. This paper focuses on development and maturation in lemurs. We show that folivorous lemurs (such as indriids) grow and mature more slowly than like-sized frugivorous lemurs (e.g., most lemurids), but tend to exhibit faster dental development. Their dental developmental schedules are accelerated on an absolute scale, relative to craniofacial growth, and relative to particular life-history landmarks, such as weaning. Dental development has a strong phylogenetic component: even those lemurids that consume substantial amounts of foliage have slower dental development than those indriids that consume substantial amounts of fruit. Implications of these results for the RAH are discussed, and an explanation for this hypothesis' failure to predict lemur growth schedules is offered. We propose that the differing developmental schedules of folivorous and frugivorous lemurs may reflect different solutions to the ecological problem of environmental instability: some rely on a strategy of low maternal input and slow returns, while others rely on a strategy of high maternal input and fast returns.