Frances L. Shaw

A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity

Since the discovery that neural tissue contains a population of stem cells that form neurospheres in vitro, sphere-forming assays have been adapted for use with a number of different tissue types for the quantification of stem cell activity and self-renewal. One tissue type widely used for stem cell investigations is mammary tissue, and the mammosphere assay has been used in both normal tissue and cancer. Although it is a relatively simple assay to learn, it can be difficult to master. There are methodological and analytical aspects to the assay which require careful consideration when interpreting the results. We describe here a detailed mammosphere assay protocol for the assessment of stem cell activity and self-renewal, and discuss how data generated by the assay can be analysed and interpreted.

No impairment of monocyte-derived Langerhans cell phenotype or function in early-onset psoriasis.

Background.  Migration of epidermal Langerhans cells (LCs) in response to the cytokines interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α is impaired in uninvolved skin of patients with early‐onset psoriasis.

Hyperbaric oxygen treatment induces platelet aggregation and protein release, without altering expression of activation molecules

OBJECTIVES To investigate the effect of hyperbaric oxygen (HBO) on platelet physiology. DESIGN AND METHODS Human platelets were exposed to HBO (97.7% O(2), balance CO(2) at 2.2 ata) or control (CON; 5% CO(2), balance air at 1 ata) for 90 min, and analyzed for aggregation, protein release, ()NO production, and activation. RESULTS HBO induced 29.8+/-3.0% of platelets to aggregate compared with CON (5.5+/-0.9%). Proteins observed to be released in greater abundance from HBO- compared with CON-treated platelets included 14-3-3 zeta and alpha-2-macroglobulin. Release of ()NO by platelets was unaffected following exposure to HBO, as was platelet activation as measured by surface expression of PECAM-1, CD62P and the activated form of alpha(IIB)beta(IIIa). CONCLUSIONS Exposure to HBO induces both platelet aggregation and protein release. Further study will better define the precise mechanisms and effects of HBO on platelet activation.

Determination of S-Nitrosothiols in Biological and Clinical Samples Using Electron Paramagnetic Resonance Spectrometry with Spin Trapping

S-Nitroso moieties, such as the S-nitroso group within S-nitrosated albumin, constitute a potential endogenous reservoir of nitric oxide (NO.) in human tissues and other biological systems. Moreover, S-nitroso compounds are under investigation as therapeutic agents in humans. Therefore, it is important to be able to detect S-nitrosothiols (RSNOs) in human extracellular fluids, such as plasma and synovial fluid, as well as other biological samples. This chapter describes a method for the determination of S-nitrosothiols in biofluids. The method is based on electron paramagnetic resonance (EPR) spectrometry, in combination with spin trapping using a ferrous ion complex of the iron chelator N-methyl-d-glucamine dithiocarbamate under alkaline conditions. This iron complex mediates the decomposition of RSNO to NO., as well as spin trapping the generated NO.. The resulting spin adduct has a unique EPR signal that can be quantified.

The intelligent anatomy spotter: A new approach to incorporate higher levels of Bloom's taxonomy.

The spotter test is an assessment that has been used widely to test practical knowledge of anatomy. Traditional spotter formats often focus solely on knowledge recall, in addition to being an onerous marking burden on staff where consistency in marking free text responses can be questioned. First‐year optometry students at the University of Manchester study the functional anatomy of the eye in the first semester of their first year. Included in the assessment of this unit is a spotter examination worth 45% of the total unit mark. Due to the factors listed above, a new spotter format was designed. Students had to answer three questions per specimen where the answers to the questions were the labeled structures themselves (A, B, C, or D). They had to work out the answer to the question and then work out which of the labeled structures was the correct structure, negating the “cueing effect” of standard multiple choice questions. Examination results were analyzed over a six‐year period (control groups 2008/2009, 2009/2010, 2010/2011; treatment groups 2011/2012, 2012/2013, 2013/2014). There were no significant differences between marks obtained for the new spotter format when compared with the traditional format. The new format spotter tested comprehension rather than just knowledge, and facilitated marking because subjectiveness was erased, and less time was spent determining whether an answer was correct or not. Anat Sci Educ 9: 440–445.