High-throughput high-content imaging assays for identification and characterization of selective AXL pathway inhibitors.

Receptor tyrosine kinases (RTKs) regulate a wide range of important biological activities, including cell proliferation, differentiation, migration, and apoptosis. Abnormalities in RTKs are involved in numerous diseases, including cancer and other proliferative disorders. AXL belongs to the TAM (Tyso3, AXL, and Mer) family of RTKs. The AXL signaling pathway represents an attractive target for the treatment of diseases, such as cancer. Using phospho-AKT as readout, a high-throughput 384-well cell-based assay was established in the NCI-H1299 human non-small cell lung carcinoma cell line to evaluate compound potency in inhibiting AXL pathway activation. In addition, a counter screen assay was established in the same cellular background to differentiate AXL kinase inhibitors from AXL receptor antagonists, which block the interaction of AXL and its natural ligand GAS6. These cell-based functional assays are useful tools in the identification and optimization of small molecules and biological reagents for potential therapeutics for the treatment of GAS6/AXL-related diseases.

Contemporary psychological approaches to life at the end of life.

When people have a serious life-limiting illness, physical symptoms are often prominent, both in the experience of the illness and in its treatment. No less important, however, are psychological symptoms. A holistic, bio-psycho-social-spiritual approach to quality of life near the end of life must address psychological distress of all types, including frank psychopathology, more moderate problems with living, and existential distress. Responding to mental health issues at the end of life requires (1) systematic and careful assessment, and (2) deployment of evidence-based treatments. In recent years, standardized assessment tools have been adapted or developed for use with people who have serious illness, and the same has happened with psychological treatments. Practitioners have several resources available to them. Given their practice orientation centered on meaningful engagement, occupational therapists can play an important role in responding to mental distress in patients with serious illness whose lives are becoming more circumscribed because of their medical condition or because of the mental distress itself. High-quality end-of-life care depends on scrupulous attention to the full spectrum of thoughts, feelings, and behaviors that unfold as death draws near.

In vivo release of lectins from the green alga Ulva fasciata.

The green alga Ulva fasciata Delile (Ulvaceae), after thawing from storage at -20 degrees C, has been used to study the in vivo biosynthesis and release of lectins. The alga was made to resume viable growth by immersion in a PBS buffer, pH 7.4, containing 0.01% w/v sodium azide and irradiating with a halophosphate lamp. The growing alga readily took up 14C leucine, when this was added to the buffer, as seen by a decrease in a sample count rate of approximately 8000 cpm over a period of twenty minutes. The transfer of the radioactivity fed algae into fresh PBS buffer resulted in 14C labeled proteins being subsequently released into solution. As well as observing changes in levels of radioactivity, the release of proteins was also monitored by UV absorption at 280 nm. Both techniques indicated an initial steady release over the first twelve hours, followed by a slower approach to a plateau value. Transfer of the algae that had undergone an initial period of protein release into a subsequent second and third volume of fresh PBS buffer produced similar UV absorption profiles, but the total quantities of material released were reduced. Identification of the released proteins was obtained from their ability to agglutinate red blood cells, which was inhibited by L-fucose, and their electrophoretic mobilities when compared with earlier isolated samples of the U. fasciata lectin. The reference lectin was obtained by affinity chromatography, following the selective precipitation of the water soluble algal proteins with ammonium sulfate. We postulate that the observed release profiles support the previously suggested concept that lectins have the ability to function as protection agents for living marine algae.

Protein expression plasmids produced rapidly: streamlining cloning protocols and robotic handling.

As many processes in the preclinical drug discovery process become highly parallel, the need to also produce a large number of different proteins in parallel has become acute, such as for protein crystallization and activity screening. In turn, the requisite DNA constructions to produce these proteins must now be done at a rate that requires automated cloning procedures, each with an intrinsic low failure probability per sample. The high-throughput cloning solutions presented here achieve production of 192 different expression plasmids at a success rate of greater than 95% of the targeted open reading frames. Time for completion of the set by one person is reduced to approximately 11 working days, starting with polymerase chain reactions for a number of source clones and ending with purified expression plasmids. Achievement of this throughput utilizes the following: (1) the Beckman Coulter (Fullerton, CA) Biomek FX liquid handler for most manipulations, (2) Gateway cloning technology (Invitrogen Corp., Carlsbad, CA), and (3) computer programs designed for parallel processing of all sample information, including primer design and the resulting DNA and protein sequence assembly. Exemplary data are presented for discovery of a form of the Rho-kinase that crystallizes (ROCK2).