Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells

Abortion education in UK medical schools: a survey of medical educators

AIM: The 2019 National Institute for Health and Care Excellence (NICE) guidance on abortion care emphasised the importance of teaching the topic at undergraduate and postgraduate level. This study aimed to investigate the current provision of undergraduate abortion education in UK medical schools. METHODS: Relevant medical ethics and clinical leads from the 33 established UK medical schools were invited to complete surveys on the ethico-legal or clinical aspects, respectively, of their institution's abortion teaching. The surveys explored how abortion is currently taught, assessed the respondent's opinion on current barriers to comprehensive teaching, and their desire for further guidance on undergraduate abortion teaching. RESULTS: Some 76% (25/33) of medical schools responded to one or both surveys. The number of hours spent on ethico-legal teaching ranged from under 1 hour to over 8 hours, with most clinical teaching lasting under 2 hours. Barriers to teaching were reported by 68% (21/31) of respondents, the most common being difficulty accessing clinical placements, lack of curriculum time, and the perception of abortion as a sensitive topic. Some 74% (23/31) of respondents would welcome additional guidance on teaching abortion to medical undergraduates. CONCLUSIONS: Education on abortion, particularly clinical education, varies widely among UK medical schools. Most educators experience barriers to providing comprehensive abortion teaching and would welcome up-to-date guidance on teaching both the clinical and ethico-legal aspects of abortion to medical students. It is essential that medical schools address the barriers to teaching, to ensure all medical students have the knowledge, skills and attitudes to provide competent and respectful abortion-related care once qualified

Clonal Strain Persistence of Candida albicans Isolates from Chronic Mucocutaneous Candidiasis Patients

Funding: The study was supported by the MRC for a PhD studentship for AM and the Wellcome Trust for funding (086827, 075470, 097377 & 101873). Acknowledgments We thank Professor Frank Odds for MLST central database curation and useful discussions.Peer reviewedPublisher PD

'I just want a job' : what do we really know about young people in jobs without training?

Over recent years, a central concern of policy has been to drive up post-16 participation rates in full-time education and address the needs of young people not in education, employment or training (NEET). As a result, young people who enter work which is classified as 'without training' at 16/17 have largely been ignored. However, the decision to Raise the Participation Age (RPA) for continuing in learning for all 17-year olds from 2013 and for all 18-year olds from 2015 in England, together with a growing unease about the impact of the current recession on youth unemployment rates, have revived interest in the 'jobs without training' (JWT) group. This paper draws on the findings from two studies: first, a qualitative study in two contrasting local labour markets, of young people in JWT, together with their employers and parents; and second, an evaluation of the Learning Agreement Pilots (LAP), which was the first policy initiative in England targeted at the JWT group. Both studies reveal a dearth of understanding about early labour market entrants and a lack of policy intervention and infrastructure to support the needs of the JWT group throughout the UK. From this, it is concluded that questionable assumptions have been made about the composition and the aspirations of young people in JWT, and their employers, on the basis of little or no evidence. As a consequence, a policy 'quick fix' to satisfy the RPA agenda will not easily be achieved. If the decision to raise the participation age is adopted also by the Welsh and Scottish parliaments, similar challenges may have to be faced

Analysis of ancestry heterozygosity suggests that hybrid incompatibilities in threespine stickleback are environment dependent

Hybrid incompatibilities occur when interactions between opposite ancestry alleles at different loci reduce the fitness of hybrids. Most work on incompatibilities has focused on those that are “intrinsic,” meaning they affect viability and sterility in the laboratory. Theory predicts that ecological selection can also underlie hybrid incompatibilities, but tests of this hypothesis using sequence data are scarce. In this article, we compiled genetic data for F(2) hybrid crosses between divergent populations of threespine stickleback fish (Gasterosteus aculeatus L.) that were born and raised in either the field (seminatural experimental ponds) or the laboratory (aquaria). Because selection against incompatibilities results in elevated ancestry heterozygosity, we tested the prediction that ancestry heterozygosity will be higher in pond-raised fish compared to those raised in aquaria. We found that ancestry heterozygosity was elevated by approximately 3% in crosses raised in ponds compared to those raised in aquaria. Additional analyses support a phenotypic basis for incompatibility and suggest that environment-specific single-locus heterozygote advantage is not the cause of selection on ancestry heterozygosity. Our study provides evidence that, in stickleback, a coarse—albeit indirect—signal of environment-dependent hybrid incompatibility is reliably detectable and suggests that extrinsic incompatibilities can evolve before intrinsic incompatibilities

The Selective Rat Toxicant Norbormide Blocks KATP Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells

Norbormide is a toxicant selective for rats to which it induces a widespread vasoconstriction. In a recent paper, we hypothesized a role of ATP-sensitive potassium (KATP) channels in norbormide-induced vasoconstriction. The current study was undertaken to verify this hypothesis by comparing the effects of norbormide with those of glibenclamide, a known KATP channel blocker. The whole-cell patch-clamp method was used to record KATP currents in myocytes freshly isolated from the rat and mouse caudal artery and from the rat gastric fundus, as well as in insulin-secreting pancreatic beta cells (INS-1 cells). Smooth muscle contractile function was assessed on either rat caudal artery rings or gastric fundus strips. Molecular modeling and docking simulation to KATP channel proteins were investigated in silico. Both norbormide (a racemic mixture of endo and exo isomers) and glibenclamide inhibited KATP currents in rat and mouse caudal artery myocytes, as well as in gastric fundus smooth muscle cells. In rat INS-1 cells, only glibenclamide blocked KATP channels, whereas norbormide was ineffective. The inhibitory effect of norbormide in rat caudal artery myocytes was not stereo-specific as both the endo isomers (active as vasoconstrictor) and the exo isomers (inactive as vasoconstrictor) had similar inhibitory activity. In rat caudal artery rings, norbormide-induced contraction was partially reverted by the KATP channel opener pinacidil. Computational approaches indicated the SUR subunit of KATP channels as the binding site for norbormide. KATP channel inhibition may play a role in norbormide-induced vasoconstriction, but does not explain the species selectivity, tissue selectivity, and stereoselectivity of its constricting activity. The lack of effect in INS-1 cells suggests a potential selectivity of norbormide for smooth muscle KATP channels

Distinct Patterns of Internalization of Different Calcitonin GeneRelated Peptide Receptors

Calcitonin gene-related peptide (CGRP) is a neuropeptide that is involved in the transmission of pain. Drugs targeting CGRP or a CGRP receptor are efficacious in the treatment of migraine. The canonical CGRP receptor is a complex of a G protein-coupled receptor, the calcitonin-like receptor (CLR), with an accessory protein, receptor activity-modifying protein 1 (RAMP1). A second receptor, the AMY1 receptor, a complex of the calcitonin receptor with RAMP1, is a dual high-affinity receptor for CGRP and amylin. Receptor regulatory processes, such as internalization, are crucial for controlling peptide and drug responsiveness. Given the importance of CGRP receptor activity in migraine we compared the internalization profiles of both receptors for CGRP using novel fluorescent probes and a combination of live cell imaging, fixed cell imaging, and ELISA. This revealed stark differences in the regulation of each receptor with the AMY1 receptor unexpectedly showing little internalization.Peer Reviewe

An NBD derivative of the selective rat toxicant norbormide as a new probe for living cell imaging

Norbormide (NRB) is a unique compound that acts directly on rat vascular myocytes to trigger a contractile process, through an as yet unknown mechanism, which results in the selective contraction of rat peripheral arteries. To gain insight into the mechanisms involved in NRB rat-selective activity, we investigated the subcellular distribution of NRB-AF12, a nitrobenzoxadiazole (NBD)-derivative of NRB, in living NRB-sensitive and NRB-insensitive cells. In both cell types, NRB-AF12 localized to the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and endosomes; however, in NRB-sensitive cells, the fluorescence also extended to the plasma membrane. NRB-AF12 was rapidly internalized into the cells, could easily be washed out and then reloaded back into the same cells, all with a high degree of reproducibility. Cells exposed for 24 h to NRB-AF12 did not show apparent signs of toxicity, even at concentrations of the dye (10 μM) much higher than those required for fluorescence labeling (500 ηM). The distribution pattern of NRB-AF12 fluorescence was near identical to that of ER-Tracker® (Er-Tr), a fluorescent derivative of glibenclamide, a known KATP channel blocker. Displacement tests did not demonstrate, but at the same time did not rule out the possibility of a common target for ER-Tr, NRB-AF12, NRB, and glibenclamide. On the basis of these results we hypothesize a common target site for NRB-AF12 and ER-Tr, and a similar target profile for NRB and glibenclamide, and propose NRB-AF12 as an alternative fluorescence probe to ER-Tracker. Furthermore, NRB-based fluorescence derivatives could be designed to selectively label single cellular structures