Genetic Silencing of AKT Induces Melanoma Cell Death via mTOR Suppression.

Aberrant activation of the PI3K-AKT pathway is common in many cancers, including melanoma, and AKT1, 2 and 3 (AKT1-3) are bona fide oncoprotein kinases with well-validated downstream effectors. However, efforts to pharmacologically inhibit AKT have proven to be largely ineffective. In this study, we observed paradoxical effects following either pharmacologic or genetic inhibition of AKT1-3 in melanoma cells. Although pharmacological inhibition was without effect, genetic silencing of all three AKT paralogs significantly induced melanoma cell death through effects on mTOR. This phenotype was rescued by exogenous AKT1 expression in a kinase-dependent manner. Pharmacological inhibition of PI3K and mTOR with a novel dual inhibitor effectively suppressed melanoma cell proliferation in vitro and inhibited tumor growth in vivo. Furthermore, this single-agent-targeted therapy was well-tolerated in vivo and was effective against MAPK inhibitor-resistant patient-derived melanoma xenografts. These results suggest that inhibition of PI3K and mTOR with this novel dual inhibitor may represent a promising therapeutic strategy in this disease in both the first-line and MAPK inhibitor-resistant setting.

A High Voltage Power Supply That Mitigates Current Reversals in Capillary Zone Electrophoresis-Electrospray Mass Spectrometry.

Capillary electrophoresis coupled with electrospray ionization typically employs two power supplies, one at each end of the capillary. One power supply is located at the proximal (injection) end of the capillary. The power supply located at the distal (detector) end of the capillary drives the electrospray. Electrophoresis is driven by the difference in potential between these power supplies. Separations that employ large capillary inner diameter, high conductivity background electrolyte, and high separation potentials generate higher current than that produced by the electrospray. Excess current flows through the electrospray power supply. Most power supplies are not designed to sink current, and the excess current will cause the electrospray voltage to deviate from its set point. We report a simple circuit to handle this excess current, allowing separations under a wide range of electrophoretic conditions. Graphical Abstract ᅟ.

Capillary electrophoresis coupled with automated fraction collection.

A fraction collector based on a drop-on-demand ink-jet printer was developed to interface capillary zone electrophoresis with a 96 well microtiter plate. We first evaluated the performance of the collector by using capillary zone electrophoresis to analyze a 1mM solution of tetramethylrhodamine; a fluorescent microtiter plate reader was then used to detect the analyte and characterize fraction carryover between wells. Relative standard deviation in peak height was 20% and the relative standard deviation in migration time was 1%. The mean and standard deviation of the tetramethylrhodamine peak width was 5 ± 1 s and likely limited by the 4-s period between droplet deposition. We next injected a complex mixture of DNA fragments and used real-time PCR to quantify the product in a CE-SELEX experiment. The reconstructed electrophoretic peak was 27 s in duration. Finally, we repeated the experiment in the presence of a 30-µM thrombin solution under CE-SELEX conditions; fractions were collected and next-generation sequencing was used to characterize the DNA binders. Over 25,000 sequences were identified with close matches to known thrombin binding aptamers.

Ultrasensitive online SERS detection of structural isomers separated by capillary zone electrophoresis.

A mixture of structural isomers was separated and identified at nanomolar concentrations (∼100,000 molecules) by incorporating capillary zone electrophoresis (CZE) with a sheath flow surface-enhanced Raman scattering (SERS) detector. Baseline resolution was obtained from three structural isomers of rhodamine using a planar silver SERS substrate, demonstrating the utility of this approach for trace chemical analysis.

Nicked-sleeve interface for two-dimensional capillary electrophoresis.

We report an improved interface for two-dimensional capillary electrophoresis. This interface is based on capillary tubing and a Plexiglas chip, both of which were milled using a micro-dicing saw. The interface was evaluated and compared to a traditional interface design for both pseudo one-dimensional and two-dimensional capillary electrophoresis. We observe less than 70% transfer efficiency for the traditional design and greater than 90% transfer efficiency with this new interface.

Cyclodextrins as complexation and extraction agents for pesticides from contaminated soil.

The binding constants of seven commonly used pesticides (2,4-D, acetochlor, alachlor, dicamba, dimethenamid, metolachlor, and propanil) with native and derivatized cyclodextrins (α-CD, ß-CD, γ-CD, hydroxypropyl-ß-CD, methyl-ß-CD, sulfated-ß-CD, and carboxymethyl-ß-CD) were measured using affinity capillary electrophoresis. All cyclodextrins showed significant binding interactions with each of the seven pesticides investigated, with the exception of sulfated-ß-CD which exhibited negligible binding to acetochlor, alachlor, and metolachlor. Propanil was found to bind most strongly to the cyclodextrins in this study. The ability of cyclodextrins to extract these pesticides from contaminated soil was also assessed. A general correlation between the pesticide-cyclodextrin binding constants and the percent extraction enhancements was found. In most cases, aqueous cyclodextrin extraction of pesticides from soil produced soluble pesticide-cyclodextrin complexes with a Type AL solubility diagram. Hydroxypropyl-ß-CD and methyl-ß-CD generally displayed the greatest levels of extraction enhancement. However, most pesticides with γ-CD (and a few cases with α-CD and ß-CD) produced relatively insoluble pesticide-cyclodextrin complexes in these soil extraction studies, resulting in Type BS solubility diagrams. Therefore, the measured aqueous extraction level for these pesticide-cyclodextrin combinations was lower relative to the control (1.0mM phosphate at pH=7.0). The results of this study may be used for future novel methods of contaminated soil remediation, which overcome the disadvantages of organic solvent and surfactant use. In addition, such binding studies may be applicable toward the development of pesticide-cyclodextrin formulations.