Effects Of Selective Agents Research Articles

Abstract 605: Building better cell models for screening oncology compounds

Abstract The NCI60 screen is a valuable compound screening service offered to researchers for 20+ years. The screen allows the submission of small molecules, biologics and natural products for testing in the NCI60 cell line panel (60 cell lines covering 9 major tumor types). Sophisticated tools such as COMPARE allow stratification of compounds based on pattern of cell line response and these tools elucidated several compounds with differential specificity for a disease type (e.g. breast cancer over melanoma). Since inception the NCI60 screen has been performed in 96-well plates on monolayer cultures of cells. SulfoRhodamine B was used to measure cell viability after a 48h exposure to the compounds at either a single concentration or 5 point concentration response assay. However, recent work has suggested that cells in 3D culture may respond differently to compounds than in 2D. 3D culture systems produce tumor cell spheroids that exhibit features seen in vivo including greater cell-cell contact, and gradients of nutrients and oxygen between the periphery and the center of the spheroid. We sought to optimize the NCI60 cells for use in a 3D spheroid model that would be suitable for screening compounds. By varying NCI60 cell plating density and incubation times, we optimized the formation of spheroids of a given diameter (500um) in Ultra Low Attachment (ULA) plates. Spheroids were imaged using a high content imaging plate reader and classified into 4 categories based on their apparent morphologies. Comparisons were made in drug sensitivity between 2D and 3D model systems using the NCI60 and cells derived from PDX samples. Data on the optimal cell densities of the NCI60 lines used for spheroid formation as well as concentration response effects of select agents compared in 2D and 3D models are presented. These data will allow others to rapidly utilize these 3D models to determine the differential drug sensitivity of select cell types. Funded by NCI Contract No. HHSN261200800001E. Citation Format: David M. Evans, Michael Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Thomas Silvers, Ralph E. Parchment, Beverly Teicher. Building better cell models for screening oncology compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 605.

In Vitro Selection for Improved Plant Resistance to Toxin‐Producing Pathogens

AbstractSimultaneously with the progress in plant biotechnology since the 1980s, new methods in plant pathology have been developed. This review summarizes papers that cover basic research on the effects of selective agents on in vitro cultures of host plants, as well as applications of agents in regeneration systems that result in lines with increased variability in resistance or susceptibility. The first part of the study deals with theoretical aspects of the interactions between plants and toxin‐producing pathogens, mode of phytotoxic action, and host‐ and non‐host‐selective toxins. The second part lists and describes various agents used for selections in vitro. In the last two decades more than 100 publications focused on these selections for the improvement of resistance to plant pathogens. Over 30 plant species were examined to utilise various selection agents extracted from about 40 plant pathogens. The review covers basic research studies and methods that elucidate the relationships between in vitro and in vivo mechanisms of resistance, but also try to develop practical applications to obtain resistant breeding lines. Such methods often utilise some type of explant cultures of the host plants that are treated with various selective agents (culture filtrates, toxins, elicitors), which then elicit typical reactions that parallel those by the pathogens. Their application successfully resulted in resistant lines in banana, carnation, grapevine, strawberry and wheat. Nowadays, these techniques are an important complement to classical breeding methods.

Persistence of unselected transgenic DNA during a plastid transformation and segregation approach to herbicide resistance.

The use of a nonlethal selection scheme, most often using the aadA gene that confers resistance to spectinomycin and streptomycin, has been considered critical for recovery of plastid transformation events. In this study, the plastid-lethal markers, glyphosate or phosphinothricin herbicides, were used to develop a selection scheme for plastids that circumvents the need for integration of an antibiotic resistance marker. The effect of selective agents on tobacco (Nicotiana tabacum) mesophyll chloroplasts was first examined by transmission electron microscopy. We found that at concentrations typically used for selection of nuclear transformants, herbicides caused rapid disintegration of plastid membranes, whereas antibiotics had no apparent effect. To overcome this apparent herbicide lethality to plastids, a "transformation segregation" scheme was developed that used two independent transformation vectors for a cotransformation approach and two different selective agents in a phased selection scheme. One transformation vector carried an antibiotic resistance (aadA) marker used for early nonlethal selection, and the other transformation vector carried the herbicide (CP4 or bar) resistance marker for use in a subsequent lethal selection phase. Because the two markers were carried on separate plasmids and were targeted to different locations on the plastid genome, we reasoned that segregation of the two markers in some transplastomic lines could occur. We report here a plastid cotransformation frequency of 50% to 64%, with a high frequency (20%) of these giving rise to transformation segregants containing exclusively the initially nonselected herbicide resistance marker. Our studies indicate a high degree of persistence of unselected transforming DNA, providing useful insights into plastid chromosome dynamics.

Embryogenic callus induction from leaf explants of the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton : Histological study and response to selective agents

Highly embryogenic callus cultures were established from leaf explants in the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton, as the first step toward the development of an efficient transformation system. Embryogenic calli were induced and then maintained by monthly subculturing onto a medium containing 1 mg l −1 picloram. Upon transfer to a plant growth regulator-free medium, the calli produced numerous somatic embryos, most of which could develop into plantlets. Histological observations revealed that, following the transfer of the embryogenic calli to a plant growth regulator-free medium, 2- to 6-cell proembryos, probably of unicellular origin, were produced, which passed through the globular and oval stages, and developed into club-shaped embryos with cotyledon, shoot apex and radicle. For establishing an efficient selection system in future transformation, the effects of selective agents (kanamycin, G418, hygromycin and bialaphos) as well as antibiotics for eliminating Agrobacterium (carbenicillin and cefotaxime) were examined on the growth and development of the embryogenic calli. Callus growth was completely inhibited by 50 mg l −1 hygromycin or 4 mg l −1 bialaphos, and somatic embryo formation was completely inhibited by 50 mg l −1 hygromycin, 75 mg l −1 G418 or 3 mg l −1 bialaphos. On the other hand, carbenicillin and cefotaxime rather promoted both growth and development of the embryogenic calli.

The clinical pharmacologic profile of reboxetine: does it involve the putative neurobiological substrates of wellbeing?

Following a review of the clinical trials of reboxetine, a new nonadrenegic reuptake inhibitor antidepressant, this paper presents a heuristic theoretical framework to better understand selective antidepressant action. For over three decades, the dominant views of antidepressant action have seen these agents active across all constitutional types and regardless of social setting. An increasing number of studies using quality of life methods are at odds with this view. This paper summarizes several of these studies, along with two studies of the effects of reboxetine on the quality of life, which reveal differential effects of selective agents that demand alternative explanations to the conventional monoamine theories. The authors submit that any revisions in our understanding of what is happening will have to pay attention to temperamental inputs that antedate affective episodes and to the sense of wellbeing and level of residual symptoms patients have on treatment after the acute phase of their illness has remitted. Obviously much more research needs to be done in this area. This invited paper sketches out, in very general terms, some provocative possibilities of how future understanding of antidepressants, temperament and their neurobiologic substrates could lead to better matching of specific antidepressants to specific temperament types.

The use of selective and non-selective enrichment broths for the isolation of Listeria species from meat

The effects of selective agents in commonly used Listeria selective broths on the growth kinetics of Listeria were investigated. The data obtained were subjected to linear regression analysis and the lag phases and growth rates calculated. The selective agents significantly lengthened the lag phase (P < 0·01) but resulted in a faster growth rate (P < 0·001). The growth kinetics of the meat microflora were unaffected by the selective agents in the broths investigated. The use of buffered peptone water as a non-selective enrichment medium was found to offer advantages in the recovery of Listeria species from retail meat samples (n = 120). Buffered peptone water gave higher recovery rates at shorter incubation times than selective enrichment media and also gave improved recovery of Listeria monocytogenes.