Abrupt and dynamic changes in gene expression revealed by live cell arrays.

A description of the noise associated with gene expression is presented, based on a simplified form of the combined multistep processes of transcription and translation. These processes are influenced by numerous factors, including the accessibility of promoter regions to the transcriptional machinery, the kinetics of assembly of the transcription complexes, and the synthesis and degradation of both mRNA and proteins, among others. Ultimately, stochasticity in cellular processes results in variation in protein levels. Here we constructed a rationally designed RNA-based transcriptional activator to reduce these variables and provide a cleaner, more detailed portrayal of cellular noise. Functioning at a level comparable to natural transcription activation, this activator is isolated to a lacZ reporter gene in yeast cells to quantitatively describe the efficiency of the combined processes of transcription and translation. By employing single-cell array techniques to monitor individual cells simultaneously and in real time, a statistical approach to investigate noise inherent in gene expression is possible. Live cell arrays enabled cell populations to be characterized temporally at the individual cell level. The array platform allowed for a relative measure of protein production in real time and could characterize protein bursts with variable size and random timing, such that bursts occurred in a temporally indiscriminate fashion. The inherent variability and randomness of these processes is characterized, with almost half (47%) of cells experiencing bursting behavior at least once over the course of the experiment. We demonstrate that cells identified on the upper periphery of activity exhibit behaviors that are substantially different from the majority of the population, and such variable activities within a population will provide a more accurate characterization of the population.

Rapid identification of synthetic cannabinoids in herbal samples via direct analysis in real time mass spectrometry.

RATIONALE: Dozens of synthetic cannabinoid analogs purposefully meant to circumvent legal restrictions associated with controlled substances continue to be manufactured and promoted as producing 'legal highs'. These designer drugs are difficult to identify in conventional drug screens not only because routine protocols have not been developed for their detection, but also because their association with complex plant matrices during manufacture generally requires labor-intensive extraction and sample preparation for analysis. To address this new and important challenge in forensic chemistry, Direct Analysis in Real Time Mass Spectrometry (DART-MS) is applied to the analysis of these designer drugs. METHODS: DART-MS was employed to sample synthetic cannabinoids directly on botanical matrices. The ambient ionization method associated with DART-MS permitted the analysis of solid herbal samples directly, without the need for extraction or sample preparation. The high mass resolution time-of-flight analyzer allowed identification of these substances despite their presence within a complex matrix and enabled differentiation of closely related analogs. RESULTS: DART-MS was performed to rapidly identify the synthetic cannabinoids AM-251 and JWH-015. For each cannabinoid, three hundred micrograms (300 µg) of material was easily detected within an excess of background matrix by mass. CONCLUSIONS: New variations of herbal blends containing a wide range of base components and laced with synthetic cannabinoids are being produced, making their presence difficult to track by conventional methods. DART-MS permits rapid identification of trace synthetic cannabinoids within complex biological matrices, with excellent sensitivity and specificity compared with standard methods.

Cellular heterogeneity and live cell arrays.

In the past decade, the tendency to move from a global, one-size-fits-all treatment philosophy to personalized medicine is based, in part, on the nuanced differences and sub-classifications of disease states. Our knowledge of these varied states stems from not only the ability to diagnose, classify, and perform experiments on cell populations as a whole, but also from new technologies that allow interrogation of cell populations at the individual cell level. Such departures from conventional thinking are driven by the recognition that clonal cell populations have numerous activities that manifest as significant levels of non-genetic heterogeneity. Clonal populations by definition originate from a single genetic origin so are regarded as having a high level of homogeneity as compared to genetically distinct cell populations. However, analysis at the single cell level has revealed a different phenomenon; cells and organisms require an inherent level of non-genetic heterogeneity to function properly, and in some cases, to survive. The growing understanding of this occurrence has lead to the development of methods to monitor, analyze, and better characterize the heterogeneity in cell populations. Following the trend of DNA- and protein microarrays, platforms capable of simultaneously monitoring each cell in a population have been developed. These cellular microarray platforms and other related formats allow for continuous monitoring of single live cells and simultaneously generate individual cell and average population data that are more descriptive and information-rich than traditional bulk methods. These technological advances have helped develop a better understanding of the intricacies associated with biological processes and afforded greater insight into complex biological systems. The associated instruments, techniques, and reagents now allow for highly multiplexed analyses, which enable multiple cellular activities, processes, or pathways to be monitored simultaneously. This critical review will discuss the paradigm shift associated with cellular heterogeneity, speak to the key developments that have lead to our better understanding of systems biology, and detail the future directions of the discipline (281 references).

Quantum dots for positional registration in live cell-based arrays.

For a better understanding of complex biological processes, it is desirable to simultaneously follow the dynamics of multiple components in living cells or organisms in real time. An encoding scheme was developed that enables the observation of multiple cell populations with single-cell resolution. Specifically, different yeast cell types were labeled with quantum dots and added to an array of microwells, where they randomly self-assemble into the complementary-sized cavities. Quantum dots conjugated to cells externally, internally, or in combination generated unique optical patterns to differentiate various cell types in the array. For the model system described herein, cells were monitored for their lacZ expression levels through the processing of a fluorescent precursor by ss-galactosidase. The encoding schemes employed were independent of the reporter emission and had no affect on the cellular activity. The live cell array platform allowed analysis of hundreds of individual cells simultaneously and continuously in real time. By coupling this platform with quantum dot cell labeling, the utility of this array format is extended to mixed cellular populations.

Quantum dots for live cell and in vivo imaging.

In the past few decades, technology has made immeasurable strides to enable visualization, identification, and quantitation in biological systems. Many of these technological advancements are occurring on the nanometer scale, where multiple scientific disciplines are combining to create new materials with enhanced properties. The integration of inorganic synthetic methods with a size reduction to the nano-scale has lead to the creation of a new class of optical reporters, called quantum dots. These semiconductor quantum dot nanocrystals have emerged as an alternative to organic dyes and fluorescent proteins, and are brighter and more stable against photobleaching than standard fluorescent indicators. Quantum dots have tunable optical properties that have proved useful in a wide range of applications from multiplexed analysis such as DNA detection and cell sorting and tracking, to most recently demonstrating promise for in vivo imaging and diagnostics. This review provides an in-depth discussion of past, present, and future trends in quantum dot use with an emphasis on in vivo imaging and its related applications.