Chemometric Approaches for Developing Infrared Nanosensors To Image Anthracyclines.

Generation, identification, and validation of optical probes to image molecular targets in a biological milieu remain a challenge. Synthetic molecular recognition approaches leveraging the intrinsic near-infrared fluorescence of single-walled carbon nanotubes are promising for long-term biochemical imaging in tissues. However, generation of nanosensors for selective imaging of molecular targets requires a heuristic approach. Here, we present a chemometric platform for rapidly screening libraries of candidate single-walled carbon nanotube nanosensors against biochemical analytes to quantify the fluorescence response to small molecules, including vitamins, neurotransmitters, and chemotherapeutics. We further show this method can be applied to identify biochemical analytes that selectively modulate the intrinsic near-infrared fluorescence of candidate nanosensors. Chemometric analysis thus enables identification of nanosensor-analyte "hits" and also nanosensor fluorescence signaling modalities such as wavelength shifts that are optimal for translation to biological imaging. Through this approach, we identify and characterize a nanosensor for the chemotherapeutic anthracycline doxorubicin (DOX), which provides a ≤17 nm fluorescence red-shift and exhibits an 8 µM limit of detection, compatible with peak circulatory concentrations of doxorubicin common in therapeutic administration. We demonstrate the selectivity of this nanosensor over dacarbazine, a chemotherapeutic commonly co-injected with doxorubicin. Lastly, we establish nanosensor tissue compatibility for imaging of doxorubicin in muscle tissue by incorporating nanosensors into the mouse hindlimb and measuring the nanosensor response to exogenous DOX administration. Our results motivate chemometric approaches to nanosensor discovery for chronic imaging of drug partitioning into tissues and toward real-time monitoring of drug accumulation.

Variation in transcript abundance during somatic embryogenesis in gymnosperms.

Somatic embryogenesis of Norway spruce (Picea abies L.) is a versatile model system to study molecular mechanisms regulating embryo development because it proceeds through defined developmental stages corresponding to specific culture treatments. Normal embryonic development involves early differentiation of proembryogenic masses (PEMs) into somatic embryos, followed by early and late embryogeny leading to the formation of mature cotyledonary embryos. In some cell lines there is a developmental arrest at the PEM-somatic embryo transition. To learn more about the molecular mechanisms regulating embryogenesis, we compared the transcript profiles of two normal lines and one developmentally arrested line. Ribonucleic acid, extracted from these cell lines at successive developmental stages, was analyzed on DNA microarrays containing 2178 expressed sequence tags (ESTs) (corresponding to 2110 unique cDNAs) from loblolly pine (Pinus taeda L.). Hybridization between spruce and pine species on microarrays has been shown to be effective (van Zyl et al. 2002, Stasolla et al. 2003). In contrast to the developmentally arrested line, the early phases of normal embryo development are characterized by a precise pattern of gene expression, i.e., repression followed by induction. Comparison of transcript levels between successive stages of embryogenesis allowed us to identify several genes that showed unique expression responses during normal development. Several of these genes encode proteins involved in detoxification processes, methionine synthesis and utilization, and carbohydrate metabolism. The potential role of these genes in embryo development is discussed.

Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass.

BACKGROUND: The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. The aim of this study was to investigate C. thermocellum genes required to ferment biomass substrates and to conduct a robust comparison of DNA microarray and RNA sequencing (RNA-seq) analytical platforms. RESULTS: C. thermocellum ATCC 27405 fermentations were conducted with a 5 g/L solid substrate loading of either pretreated switchgrass or Populus. Quantitative saccharification and inductively coupled plasma emission spectroscopy (ICP-ES) for elemental analysis revealed composition differences between biomass substrates, which may have influenced growth and transcriptomic profiles. High quality RNA was prepared for C. thermocellum grown on solid substrates and transcriptome profiles were obtained for two time points during active growth (12 hours and 37 hours postinoculation). A comparison of two transcriptomic analytical techniques, microarray and RNA-seq, was performed and the data analyzed for statistical significance. Large expression differences for cellulosomal genes were not observed. We updated gene predictions for the strain and a small novel gene, Cthe_3383, with a putative AgrD peptide quorum sensing function was among the most highly expressed genes. RNA-seq data also supported different small regulatory RNA predictions over others. The DNA microarray gave a greater number (2,351) of significant genes relative to RNA-seq (280 genes when normalized by the kernel density mean of M component (KDMM) method) in an analysis of variance (ANOVA) testing method with a 5% false discovery rate (FDR). When a 2-fold difference in expression threshold was applied, 73 genes were significantly differentially expressed in common between the two techniques. Sulfate and phosphate uptake/utilization genes, along with genes for a putative efflux pump system were some of the most differentially regulated transcripts when profiles for C. thermocellum grown on either pretreated switchgrass or Populus were compared. CONCLUSIONS: Our results suggest that a high degree of agreement in differential gene expression measurements between transcriptomic platforms is possible, but choosing an appropriate normalization regime is essential.