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.
Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life, and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, sym-homospermidine. We demonstrate that homospermidine synthase (HSS) has evolved vertically, primarily in the ␣-Proteobacteria, but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes, and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of co-products diaminopropane and N 1 -aminobutylcadaverine, in addition to sym-homospermidine. We show that sym-homospermidine is required for normal growth of the ␣-proteobacterium Rhizobium leguminosarum. However, sym-homospermidine can be replaced, for growth restoration, by the structural analogues spermidine and sym-norspermidine, suggesting that the symmetrical or unsymmetrical form and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some ␣-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine.Polyamines are primordial, small flexible organic polycations found in almost all cells of bacteria, archaea, and eukaryotes (1). In bacteria and archaea, the key polyamines (see Fig. 1A) are the triamines spermidine, sym-norspermidine, and sym-homospermidine (referred to herein as norspermidine and homospermidine), and occasionally more than one triamine can be found in the same cell. In eukaryotes, which contain spermidine (and in some plants, yeasts, and animals, the tetraamine spermine), polyamines are required for growth, cell proliferation, and normal cellular physiology. Polyamine biosynthesis is essential in the fungi Saccharomyces cerevisiae (2), Schizosaccharomyces pombe (3), Aspergillus nidulans (4), and Ustilago maydis (5), the kinetoplastid parasites Trypanosoma brucei (6) and Leishmania donovani (7), and the diplomonad parasite Giardia lamblia (8). In mouse, polyamines are essential for early embryo development (9, 10), and they are also essential for seed development in the flowering plant Arabidopsis thaliana (11).The universal distribution of polyamines suggests that...
Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s10911-016-9361-8) contains supplementary material, which is available to authorized users.
The food-borne bacterial pathogen Campylobacter jejuni efficiently utilizes organic acids such as lactate and formate for energy production. Formate is rapidly metabolized via the activity of the multisubunit formate dehydrogenase (FDH) enzyme, of which the FdhA subunit is predicted to contain a selenocysteine (SeC) amino acid. In this study we investigated the function of the cj1500 and cj1501 genes of C. jejuni, demonstrate that they are involved in selenium-controlled production of FDH, and propose the names fdhT and fdhU, respectively. Insertional inactivation of fdhT or fdhU in C. jejuni resulted in the absence of FdhA and FdhB protein expression, reduced fdhABC RNA levels, the absence of FDH enzyme activity, and the lack of formate utilization, as assessed by 1 H nuclear magnetic resonance. The fdhABC genes are transcribed from a single promoter located two genes upstream of fdhA, and the decrease in fdhABC RNA levels in the fdhU mutant is mediated at the posttranscriptional level. FDH activity and the ability to utilize formate were restored by genetic complementation with fdhU and by supplementation of the growth media with selenium dioxide. Disruption of SeC synthesis by inactivation of the selA and selB genes also resulted in the absence of FDH activity, which could not be restored by selenium supplementation. Comparative genomic analysis suggests a link between the presence of selA and fdhTU orthologs and the predicted presence of SeC in FdhA. The fdhTU genes encode accessory proteins required for FDH expression and activity in C. jejuni, possibly by contributing to acquisition or utilization of selenium.
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. © 2015 American Association of Anatomists.
Aasen T, Raya A, Barrero MJ et al. (2008) Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26:1276-84 Darling TN, Yee C, Bauer JW et al. (1999) Revertant mosaicism: partial correction of a germ-line mutation in COL17A1 by a framerestoring mutation. J Clin Invest 103:1371-7 Fine JD, Eady RA, Bauer EA et al. (2008) The classification of inherited epidermolysis bullosa (EB): Report of the third international consensus meeting on diagnosis and classification of EB. J Am Acad Dermatol 58:931-50 Frank J, Happle R (2007) Cutaneous mosaicism: right before our eyes. J Clin Invest 117:1216-9 Gostynski A, Deviaene FC, Pasmooij AM et al. (2009) Adhesive stripping to remove epidermis in junctional epidermolysis bullosa for revertant cell therapy. Br J Dermatol 161:444-7 Hall JG (1988) Review and hypotheses: somatic mosaicism: observations related to clinical genetics. Am J Hum Genet 43:355-63 Hirschhorn R (2003) In vivo reversion to normal of inherited mutations in humans. J Med Genet 40:721-8 Jonkman MF, Pasmooij AM (2009) Revertant mosaicism-patchwork in the skin. N Engl J Med 360:1680-2 Jonkman MF, Scheffer H, Stulp R et al. (1997) Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion. Cell 88:543-51 McGrath JA, Ishida-Yamamoto A, O'Grady A et al. (1993) Structural variations in anchoring fibrils in dystrophic epidermolysis bullosa: correlation with type VII collagen expression. J Invest Dermatol 100:366-72 Mellerio JE, Dunnill MG, Allison W et al. (1997) Recurrent mutations in the type VII collagen gene (COL7A1) in patients with recessive dystrophic epidermolysis bullosa. J Invest Dermatol 109:246-9 Pasmooij AM, Pas HH, Deviaene FC et al. (2005) Multiple correcting COL17A1 mutations in patients with revertant mosaicism of epidermolysis bullosa. Am J Hum Genet 77:727-40 Pasmooij AM, Pas HH, Bolling MC et al. (2007) Revertant mosaicism in junctional epidermolysis bullosa due to multiple correcting second-site mutations in LAMB3. J Clin Invest 117:1240-8 Schuilenga-Hut PH, Scheffer H, Pas HH et al. (2002) Partial revertant mosaicism of keratin 14 in a patient with recessive epidermolysis bullosa simplex. J Invest Dermatol 118:626-30 Smith FJ, Morley SM, McLean WH (2004) Novel mechanism of revertant mosaicism in Dowling-Meara epidermolysis bullosa simplex. J Invest Dermatol 122:73-7 Wong T, Gammon L, Liu L et al. (2008) Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa. J Invest Dermatol 128:2179-89 Langerhans Cell Mobilization Distinguishes between Early-Onset and Late-Onset Psoriasis
BackgroundMigration 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.AimTo investigate whether this impairment is a reflection of a systemic defect in dendritic cells (DCs), using an established model of monocyte-derived LC-like cells (mLCs).MethodsCD14+ monocytes isolated from both patients with psoriasis and healthy control volunteers were cultured in a cytokine cocktail for 5 days to promote their differentiation into mLCs, then stimulated for 24 h with TNF-α, IL-1β (both 100 ng/mL) or medium alone. Cellular surface protein expression was quantified by flow cytometry, and the ability of cells to migrate to media supplemented with C-C motif ligand (CCL)19 was assessed using a Transwell migration assay. The cytokine and chemokine content of supernatants was analysed by cytokine array.ResultsCD14+ cells acquired an LC-like phenotype with high expression of CD1a and major histocompatibility complex (MHC) class II. There were no differences in the expression of activation markers or in the secretion of cytokines by mLCs isolated from patients with psoriasis and those isolated from healthy controls. Moreover, mLCs isolated from both groups displayed comparable ability to migrate in vitro.ConclusionsThese data suggest that the failure of LCs to migrate in response to stimulation in patients with psoriasis is not attributable to a systemic defect in DC function, but is rather a reflection of local changes in the epidermal microenvironment.
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