Synthesis of the oxysterol, 24(S), 25-epoxycholesterol, parallels cholesterol production and may protect against cellular accumulation of newly-synthesized cholesterol

AIM: The effects of 24(S),25-epoxycholesterol (24,25EC) on aspects of cholesterol homeostasis is well-documented. When added to cells, 24,25EC decreases cholesterol synthesis and up-regulates cholesterol efflux genes, including ABCA1. Synthesis of 24,25EC occurs in a shunt of the mevalonate pathway which also produces cholesterol. Therefore, 24,25EC synthesis should be subject to the same negative feedback regulation as cholesterol synthesis. To date, no role has been ascribed to 24,25EC in light of the fact that increased accumulation of cholesterol should decrease formation of this oxysterol through feedback inhibition. This leads to the intriguing paradox: why inhibit production of an apparently important regulator of cholesterol homeostasis when it is needed most? METHODS: We used a combination of pharmacological and genetic approaches in Chinese Hamster Ovary cell-lines to investigate this paradox. Endogenous synthesis of 24,25EC was manipulated using partial inhibition of the enzyme, Oxidosqualene Cyclase. Changes in cholesterol and 24,25EC synthesis were determined using metabolic labelling with [1-(14)C]-acetate, thin-layer chromatography and phosphorimaging. Transcriptional effects mediated via SREBP and LXR were analysed by luciferase reporter assays. RESULTS: We showed that cholesterol addition to cells lead to a rapid and preferential inhibition of 24,25EC synthesis. Addition of 24,25EC resulted in parallel inhibition of 24,25EC and cholesterol synthesis. Furthermore, we used a variety of approaches to examine the relationship between cholesterol and 24,25EC synthesis, including cell-lines with different rates of cholesterol synthesis, varying cholesterol synthetic rates by pre-treatment with a statin, or lipoprotein cholesterol loading of macrophages. In all cases, we showed that 24,25EC synthesis faithfully tracked cholesterol synthesis. Moreover, changes in 24,25EC synthesis exerted downstream effects, reducing SREBP transcriptional activity whilst increasing ABCA1 and LXR transcriptional activity. CONCLUSION: Our results show that 24,25EC synthesis parallels cholesterol synthesis, consistent with this oxysterol functioning as a safety valve to protect against the accumulation of newly-synthesised cholesterol (as opposed to exogenously-derived cholesterol). Considering that 24,25EC is capable of being produced in all cholesterogenic cells, we propose that production of 24,25EC may represent a ubiquitous defence mechanism

Revision workshops in elementary mathematics enhance student performance in routine laboratory calculations

The ability to understand and implement calculations required for molarity and dilution computations that are routinely undertaken in the laboratory are essential skills that should be possessed by all students entering an undergraduate Life Sciences degree. However, it is increasingly recognized that the majority of these students are ill equipped to reliably carry out such calculations. There are several factors that conspire against students' understanding of this topic, with the alien concept of the mole in relation to the mass of compounds and the engineering notation required when expressing the relatively small quantities typically involved being two key examples. In this report, we highlight teaching methods delivered via revision workshops to undergraduate Life Sciences students at the University of Nottingham. Workshops were designed to 1) expose student deficiencies in basic numeracy skills and remedy these deficiencies, 2) introduce molarity and dilution calculations and illustrate their workings in a step-by-step manner, and 3) allow students to appreciate the magnitude of numbers. Preworkshop to postworkshop comparisons demonstrated a considerable improvement in students' performance, which attenuated with time. The findings of our study suggest that an ability to carry out laboratory calculations cannot be assumed in students entering Life Sciences degrees in the United Kingdom but that explicit instruction in the form of workshops improves proficiency to a level of competence that allows students to prosper in the laboratory environment

Is Seladin-1 really a selective Alzheimer\u27s disease indicator?

Selective Alzheimer\u27s Disease Indicator-1 (Seladin-1) was originally identified by its down-regulation in the brains of Alzheimer\u27s disease (AD) patients. Here, we re-examine existing data and present new gene expression data that refutes its role as a selective AD indicator. Furthermore, we caution against the use of the name “Seladin-1” and instead recommend adoption of the approved nomenclature, 3β-hydroxysterol Δ24-reductase (or DHCR24), which describes its catalytic function in cholesterol synthesis. Further work is required to determine what link, if any, exists between DHCR24 and AD

Angular Dependence in Proton-Proton Correlation Functions in Central 40Ca+40Ca^{40}Ca+^{40}Ca and 48Ca+48Ca^{48}Ca+^{48}Ca Reactions

The angular dependence of proton-proton correlation functions is studied in central $^{40}Ca+^{40}Ca$ and $^{48}Ca+^{48}Ca$ nuclear reactions at E=80 MeV/A. Measurements were performed with the HiRA detector complemented by the 4$\pi$ Array at NSCL. A striking angular dependence in the laboratory frame is found within p-p correlation functions for both systems that greatly exceeds the measured and expected isospin dependent difference between the neutron-rich and neutron-deficient systems. Sources measured at backward angles reflect the participant zone of the reaction, while much larger sources observed at forward angles reflect the expanding, fragmenting and evaporating projectile remnants. The decrease of the size of the source with increasing momentum is observed at backward angles while a weaker trend in the opposite direction is observed at forward angles. The results are compared to the theoretical calculations using the BUU transport model.Comment: 8 pages, 3 figures, submitted to PR

Analysis of phosphatases in ER-negative breast cancers identifies DUSP4 as a critical regulator of growth and invasion.

Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple-negative" breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease- Associated ‘‘Toggle’’ Calcium-Sensing Receptor Mutations

The Ca2+-sensing receptor (CaSR) regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+ ([Ca2+]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-offunction CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding

Identification of an L-Phenylalanine Binding Site Enhancing The Cooperative Responses of The Calcium Sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca2+]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors (GPCRs), by [Ca2+]o or amino acids elicits intracellular Ca2+ ([Ca2+]i) oscillations. Here, we report the central role of predicted Ca2+-binding Site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+]o. Next, we identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+]o and L- Phe in eliciting CaSR-mediated [Ca2+]i oscillations. The hetero-communication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+]o signaling by positively impacting multiple [Ca2+]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+]o and amino acids into intracellular signaling events

Investigations of three, four, and five-particle exit channels of levels in light nuclei created using a 9C beam

The interactions of a E/A=70-MeV 9C beam with a Be target was used to populate levels in Be, B, and C isotopes which undergo decay into many-particle exit channels. The decay products were detected in the HiRA array and the level energies were identified from their invariant mass. Correlations between the decay products were examined to deduce the nature of the decays, specifically to what extent all the fragments were created in one prompt step or whether the disintegration proceeded in a sequential fashion through long-lived intermediate states. In the latter case, information on the spin of the level was also obtained. Of particular interest is the 5-body decay of the 8C ground state which was found to disintegrate in two steps of two-proton decay passing through the 6Beg.s. intermediate state. The isobaric analog of 8Cg.s. in 8B was also found to undergo two-proton decay to the isobaric analog of 6Beg.s. in 6Li. A 9.69-MeV state in 10C was found to undergo prompt 4-body decay to the 2p+2alpha exit channel. The two protons were found to have a strong enhancementin the diproton region and the relative energies of all four p-alpha pairs were consistent with the 5Lig.s. resonance

Uncertainty in estuarine extreme water level predictions due to surge-tide interaction

Storm surge is often the greatest threat to life and critical infrastructures during hurricanes and violent storms. Millions of people living in low-lying coastal zones and critical infrastructure within this zone rely on accurate storm surge forecast for disaster prevention and flood hazard mitigation. However, variability in residual sea level up-estuary, defined here as observed sea level minus predicted tide, can enhance total water levels; variability in the surge thus needs to be captured accurately to reduce uncertainty in site specific hazard assessment. Delft3D-FLOW is used to investigate surge variability, and the influence of storm surge timing on barotropic tide-surge propagation in a tide-dominant estuary using the Severn Estuary, south-west England, as an example. Model results show maximum surge elevation increases exponentially up-estuary and, for a range of surge timings consistently occurs on the flood tide. In the Severn Estuary, over a distance of 40 km from the most upstream tide gauge at Oldbury, the maximum surge elevation increases by 255%. Up-estuary locations experience short duration, high magnitude surge elevations and greater variability due to shallow-water effects and channel convergence. The results show that surge predictions from forecasting systems at tide gauge locations could under-predict the magnitude and duration of surge contribution to up-estuary water levels. Due to the large tidal range and dynamic nature of hyper-tidal estuaries, local forecasting systems should consider changes in surge elevation and shape with distance up-estuary from nearby tide gauge sites to minimize uncertainties in flood hazard assessment

Tracheostomy Following Surgery for Congenital Heart Disease: 14-year Institutional Experience

Background: Tracheostomy following congenital heart disease (CHD) surgery is a rare event and associated with significant mortality. Hospital survival has been reported from 20% to 40%. Late mortality for these patients is not well characterized. Methods: We performed a retrospective observational study of patients who had a tracheostomy following CHD surgery (excluding isolated patent ductus arteriosus ligation) between January 2000 and December 2013. Patients were categorized into single-ventricle or biventricular physiology groups. Demographics, genetic syndromes, pulmonary disease, and comorbidities were collected. Outcomes including hospital survival, long-term survival, and weaning from positive pressure ventilation are reported. Bivariate and time-to-event models were used. Results: Over a 14-year period, 61 children (0.9% incidence) had a tracheostomy placed following CHD surgery. There were 12 single-ventricle patients and 49 biventricular patients. Prematurity, genetic syndromes, lung/airway disease, and other comorbidities were common in both CHD groups. Gastrostomy tubes were used more frequently in biventricular physiology patients (91.8%) versus single-ventricle patients (66.7%, P = .04). Survival to hospital discharge was 50% in the single-ventricle group compared to 86% in biventricular patients (P = .01). Long-term survival continued to be poor in the single-ventricle group comparatively (three years, 27.8% vs 64.8%, P = .01). Gastrostomy tube placement was independently associated with survival in both groups (P = .002). Conclusion: Tracheostomy is performed following many types of surgery for CHD and is commonly associated with other comorbidities. Both hospital and long-term survival are substantially lower in children with single-ventricle physiology as compared to patients with biventricular physiology