Palladium-Catalyzed Enantioselective C-H Activation of Aliphatic Amines Using Chiral Anionic BINOL-Phosphoric Acid Ligands

The design of an enantioselective Pd(II)-catalyzed C-H amination reaction is described. The use of a chiral BINOL phosphoric acid ligand enables the conversion of readily available amines into synthetically valuable aziridines in high enantiomeric ratios. The aziridines can be derivatized to afford a range of chiral amine building blocks incorporating motifs readily encountered in pharmaceutically relevant molecules

Continuous-Flow Synthesis and Derivatization of Aziridines through Palladium-Catalyzed C(sp 3 )−H Activation

A continuous‐flow synthesis of aziridines by palladium‐catalyzed C(sp3)−H activation is described. The new flow reaction could be combined with an aziridine‐ring‐opening reaction to give highly functionalized aliphatic amines through a consecutive process. A predictive mechanistic model was developed and used to design the C−H activation flow process and illustrates an approach towards first‐principles design based on novel catalytic reactions.We are grateful to the Department of Chemical Engineering and Biotechnology (J.Z.) and the EPSRC (A.P.S.) for studentships, and to the ERC and EPSRC (EP/100548X/1) for fellowships (M.J.G.). Mass spectroscopy data were acquired at the EPSRC UK National Mass Spectroscopy Facility at Swansea Universit

CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells

INTRODUCTION: Breast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells. METHODS: Mouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation. RESULTS: Three populations of CD24 expressing cells were identified: CD24(Negative), CD24(Low )and CD24(High). Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24(Low )cells, whilst CD24(High )cells have limited repopulation capacity. CONCLUSION: Differential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24(Low )population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis

A general catalytic β-C-H carbonylation of aliphatic amines to β-lactams

Methods for the synthesis and functionalization of amines are intrinsically important to a variety of chemical applications. We present a general carbon-hydrogen bond activation process that combines readily available aliphatic amines and the feedstock gas carbon monoxide to form synthetically versatile value-added amide products. The operationally straightforward palladium-catalyzed process exploits a distinct reaction pathway, wherein a sterically hindered carboxylate ligand orchestrates an amine attack on a palladium anhydride to transform aliphatic amines into β-lactams. The reaction is successful with a wide range of secondary amines and can be used as a late-stage functionalization tactic to deliver advanced, highly functionalized amine products of utility for pharmaceutical research and other areas.We gratefully acknowledge funding from the European Research Council and the UK Engineering and Physical Sciences Research Council (EPSRC) (to D.W., K.F.H., A.P.S., and M.J.G.), the Herchel Smith Trust (to B.G.N.C.), the Marie Curie Foundation (to J.C.), and the Royal Society (Wolfson Merit Award to M.J.G.). Mass spectrometry data were acquired at the EPSRC UK National Mass Spectrometry Facility at Swansea University. Computational work was performed with the Darwin Supercomputer of the University of Cambridge High Performance Computing Service (http://www.hpc.cam.ac.uk/), provided by Dell, using Strategic Research Infrastructure Funding from the Higher Education Funding Council for England

A spectroscopic study of the open cluster NGC 6250

We present the chemical abundance analysis of 19 upper main-sequence stars of the young open cluster NGC 6250 (log t∼7.42 yr). Thiswork is part of a project aimed at setting observational constraints on the theory of atomic diffusion in stellar photospheres, by means of a systematic study of the abundances of the chemical elements of early F-, A- and late B-type stars of well-determined age. Our data set consists of low-, medium- and high-resolution spectra obtained with the Fibre Large Array Multi Element Spectrograph (FLAMES) instrument of the ESO Very Large Telescope (VLT). To perform our analysis, we have developed a new suite of software tools for the chemical abundance analysis of stellar photospheres in local thermodynamical equilibrium. Together with the chemical composition of the stellar photospheres, we have provided new estimates of the cluster mean radial velocity, proper motion, refined the cluster membership, and we have given the stellar parameters including masses and fractional age. We find no evidence of statistically significant correlation between any of the parameters, including abundance and cluster age, except perhaps for an increase in Ba abundance with cluster age.We have proven that our new software tool may be successfully used for the chemical abundance analysis of large data sets of stellar spectra

Functional and molecular characterisation of mammary side population cells

BACKGROUND: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. METHODS: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. RESULTS: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2–0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures. CONCLUSION: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology

Mouse mammary stem cells express prognostic markers for triple-negative breast cancer

Introduction Triple negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis. Methods Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power. Results A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis. Conclusions Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC

From normal cell types to malignant phenotypes

The phenotypic diversity of breast cancer has been proposed to result from different target cell types undergoing oncogenic transformation and giving rise to cancer stem cells. Global gene expression profiling revealed distinct molecular phenotypes and some of these signatures were held to reflect the cell of origin, with the basal carcinomas arising from basal/progenitor cells. Recent work challenges this view by providing evidence that luminal precursor cells are involved in the pathogenesis of basal breast cancers and has made new links between normal cell populations and molecular tumor phenotypes

Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells

Introduction In humans, an early full-term pregnancy reduces lifetime breast cancer risk by up to 50% whereas a later pregnancy (>35 years old) can increase lifetime risk. Several mechanisms have been suggested, including changes in levels of circulating hormones, changes in the way the breast responds to these hormones, changes in gene expression programmes which may alter susceptibility to transformation and changes to mammary stem cell numbers or behaviour. Previous studies have shown that the mammary tissue isolated from both virgin and parous mice has the ability to repopulate a cleared mammary fat pad in transplant experiments. Limited dilution transplant assays have demonstrated that early pregnancy (at 5 weeks of age) reduces stem/progenitor cell numbers in the mouse mammary epithelium by twofold. However, the effects on stem/progenitor cell numbers in the mammary epithelium of a pregnancy in older animals have not yet been tested. Methods Mice were put through a full-term pregnancy at 9 weeks of age, when the mammary epithelium is mature. The total mammary epithelium was purified from parous 7-week post-lactation and age-matched virgin mice and analysed by flow cytometry and limiting dilution cleared fat pad transplants. Results There were no significant differences in the proportions of different mammary epithelial cell populations or numbers of CD24+/Low Sca-1- CD49fHigh cells (stem cell enriched basal mammary epithelial compartment). There was no significant difference in stem/progenitor cell frequency based on limiting dilution transplants between the parous and age-matched virgin epithelium. Conclusions Although differences between parous and virgin mammary epithelium at later time points post lactation or following multiple pregnancies cannot be ruled out, there are no differences in stem/progenitor cell numbers between mammary epithelium isolated from parous animals which were mated at 9 weeks old and virgin animals. However, a recent report has suggested that animals that were mated at 5 weeks old have a twofold reduction in stem/progenitor cell numbers. This is of interest given the association between early, but not late, pregnancy and breast cancer risk reduction in humans. However, a mechanistic connection between stem cell numbers and breast cancer risk remains to be established

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites