Search

Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.

Prototype irreversible inhibitors of the cysteinyl protease Der p I were designed, synthesised and evaluated in vitro. Candidates were designed using a modular approach, whereby a peptide sequence was appended with known thiophilic moieties. This hinged on utilizing peptide sequences from substrate specificity data compiled using proprietary RAPiD technology.

The first hyper-variable region (HV1) of the mitochondrial control region (MCR) has been widely used as a molecular tool in population genetics, but inadvertent amplification of nuclear translocated copies of mitochondrial DNA (numts) in gorillas has compromised the use of mitochondrial DNA in population genetic studies. At least three putative classes (I, II, III) of gorilla-specific HV1 MCR numts have been uncovered over the past decade. However, the number, size and location of numt loci in gorillas and other apes are completely unknown. Furthermore, little work to date has assessed the utility of numts as candidate population genetic markers. In the present study, we screened Bacterial Artificial Chromosome (BAC) genomic libraries in the chimpanzee and gorilla to compare patterns of mitochondrial-wide insertion in both taxa. We conducted an intensive BLAST search for numts in the gorilla genome and compared the prevalence of numt loci originating from the MCR with other great ape taxa. Additional gorilla-specific MCR numts were retrieved either through BAC library screens or using an anchored-PCR (A-PCR) amplification using genomic DNA from five unrelated gorillas. Locus-specific primers were designed to identify numt insertional polymorphisms and evaluate their potential as population genetic markers. Mitochondrial-wide surveys of chimpanzee and gorilla BACs showed that the number of numts does not differ between these two taxa. However, MCR numts are more abundant in chimpanzees than in other great apes. We identified and mapped 67 putative gorilla-specific numts, including two that contain the entire HV1 domain, cluster with sequences from two numt classes (I, IIb) and will likely co-amplify with mitochondrial sequences using most published HV1 primers. However, phylogenetic analysis coupled with post-hoc analysis of mitochondrial variation can successfully differentiate nuclear sequences. Insertional polymorphisms were evident in three out of five numts examined, indicating their potential utility as molecular markers. Taken together, these findings demonstrate the potentially powerful insight that numts could make in uncovering population history in gorillas and other mammals.

Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins. © 2013. Published by The Company of Biologists Ltd.

Fibrosis is a common late complication of radiotherapy and/or surgical treatment for head and neck cancers. Fibrosis is difficult to quantify and formal methods of measure are not well recognized. The purpose of this review was to summarize the methods available to quantify neck fibrosis.A PubMed search of articles was carried out using key words "neck" and "fibrosis."Many methods have been used to assess fibrosis, however, there is no preferred methodology. Specific to neck fibrosis, most studies have relied upon hand palpation rating scales. Indentation and suction techniques have been used to mechanically quantify neck fibrosis. There is scope to develop applications of ultrasound, dielectric, bioimpedance, and MRI techniques for use in the neck region.Quantitative assessment of neck fibrosis is sought after in order to compare treatment regimens and improve quality of life outcomes in patients with head and neck cancer.

We disrupted the gene encoding lysophosphatidylinositol-acyltransferase-1 (LPIAT1) in the mouse with the aim of understanding its role in determining cellular phosphoinositide content. LPIAT1(-/-) mice were born at lower than Mendelian ratios and exhibited a severe developmental brain defect. We compared the phospholipid content of livers and brains from LPIAT1(-/-) and LPIAT1(+/+) littermates by LC-ESI/MS. In accord with previous studies, the most abundant molecular species of each phosphoinositide class (PtdIns, PtdInsP, PtdInsP2 and PtdInsP3) possessed a C38∶4 complement of fatty-acyl esters (C18∶0 and C20∶4 are usually assigned to the sn-1 and sn-2 positions, respectively). LPIAT1(-/-) liver and brain contained relatively less of the C38∶4 species of PtdIns, PtdInsP and PtdInsP2 (dropping from 95-97% to 75-85% of the total species measured for each lipid class) and relatively more of the less abundant species (PtdInsP3 less abundant species were below our quantification levels). The increases in the less abundant PtdIns and PtdInsP2 species did not compensate for the loss in C38∶4 species, resulting in a 26-44% reduction in total PtdIns and PtdInsP2 levels in both brain and liver. LPIAT1(-/-) brain and liver also contained increased levels of C18∶0 lyso-PtdIns (300% and 525% respectively) indicating a defect in the reacylation of this molecule. LPIAT1(-/-) brain additionally contained significantly reduced C38∶4 PC and PE levels (by 47% and 55% respectively), possibly contributing to the phenotype in this organ. The levels of all other molecular species of PC, PE, PS and PA measured in the brain and liver were very similar between LPIAT1(-/-) and LPIAT1(+/+) samples. These results suggest LPIAT1 activity plays a non-redundant role in maintaining physiological levels of PtdIns within an active deacylation/reacylation cycle in mouse tissues. They also suggest that this pathway must act in concert with other, as yet unidentified, mechanisms to achieve the enrichment observed in C38∶4 molecular species of phosphoinositides.

Introduction The variety of organisations providing National Health Service (NHS)-funded services in England is growing. Besides NHS hospitals and general practitioners (GPs), they include corporations, social enterprises, voluntary organisations and others. The degree to which these organisational types vary, however, in the ways they manage and provide services and in the outcomes for service quality, patient experience and innovation, remains unclear. This research will help those who commission NHS services select among the different types of organisation for different tasks. Research questions The main research questions are how organisationally diverse NHS-funded service providers vary in their responsiveness to patient choice, NHS commissioning and policy changes; and their patterns of innovation. We aim to assess the implications for NHS commissioning and managerial practice which follow from these differences. Methods and analysis Systematic qualitative comparison across a purposive sample (c.12) of providers selected for maximum variety of organisational type, with qualitative studies of patient experience and choice (in the same sites). We focus is on NHS services heavily used by older people at high risk of hospital admission: community health services; out-of-hours primary care; and secondary care (planned orthopaedics or ophthalmology). The expected outputs will be evidence-based schemas showing how patterns of service development and delivery typically vary between different organisational types of provider. Ethics, benefits and dissemination We will ensure informants' organisational and individual anonymity when dealing with high profile case studies and a competitive health economy. The frail elderly is a key demographic sector with significant policy and financial implications. For NHS commissioners, patients, doctors and other stakeholders, the main outcome will be better knowledge about the relative merits of different kinds of healthcare provider. Dissemination will make use of strategies suggested by patient and public involvement, as well as DH and service-specific outlets.

Haemophagocytic lymphohistiocytosis complicating Epstein-Barr virus positive T-cell lymphoproliferative disease of childhood is a rare and life-threatening entity. We report a child with this condition presenting with a toxic epidermal necrolysis-like eruption.

[1] We present results for multiple Saharan Air Layer (SAL) events and their relationship to tropospheric ozone mixing ratios that were observed in the Eastern Atlantic during the summer of 2010 using ozonesondes. In particular, 5 SAL events are sampled during 2010 in Cape Verde indicting a reduction of ozone mixing ratios throughout much of the SAL layer, except near the base of the SAL. In this layer of enhanced ozone mixing ratio, we find increases of 20–30 ppb in some cases between non-SAL and SAL conditions. In addition, ozone concentrations are enhanced above the SAL layer, with trajectories suggesting enhancements by lightning from the middle/upper troposphere with outflow from Africa. Additional aircraft measurements are required to examine the chemical and aerosol distributions from the Marine Boundary Layer (MBL) through the upper troposphere to determine the heterogeneous chemical processes related to reduced ozone mixing ratio, and further quantify elevated ozone mixing ratios at the base of the SAL and above the SAL.

This chapter reviews and integrates the empirical literature on the influence of organizational factors on hospital financial performance. Five categories of organizational characteristics that research has addressed are identified and examined as part of the review: ownership, governance, integration, management strategy, and quality. With some exceptions, our review reveals a general lack of consistency and conclusiveness across studies in each area. Exceptions were found in the areas of governance (e.g., physician participation and board processes) and integration (e.g., horizontal system centralization). Despite the lack of conclusive findings across studies, our review suggests substantial opportunities for future work, including opportunities for qualitative and exploratory work. Additional implications for theory and management are discussed.

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.

‘Context is king’, announced J. G. A. Pocock in surveying the development of what is generally known as the ‘Cambridge school’ of the history of political thought.1 The contexts for study initially proved to be chiefly early modern ones, and as often contexts set by the recovery of ‘discourses’ or ‘languages’ of politics as by ‘historical and political circumstances’.2 Indeed the reinterpretation of ‘political’ texts by replacing them in contexts which had been later obscured or forgotten was the programmatic insight and lasting achievement of a clearly defined group of scholars, beginning with Peter Laslett in his edition of Filmer in 1949, paradigmatically successful in Laslett’s edition of Locke in 1960,3 extending through John Dunn,4 Pocock5 and Quentin Skinner6 to a cohort of historians in a third generation including Justin Champion, Mark Goldie, John Marshall, Richard Tuck and many others. This work is widely known and acknowledged among academic historians; as appreciations of its contribution are written, it is beginning to be understood by scholars of literature, if a little belatedly, that these insights apply to all texts, not just ‘political’ ones.7 In this essay the same methods are applied to invoke a forgotten context for two neglected texts bearing on the same episode, one text written by Samuel Johnson, the other by Mrs Thrale, in order to illuminate their meanings. Such an exercise entails a substantial reinterpretation of Johnson’s religion and politics in the last decade of his life, with wider implications explored here.

The polyphosphoinositides are key signaling lipids whose levels are tightly regulated within cells. As with other cellular lipids multiple species exist with distinct acyl chain makeups. There are methods which analyze the phosphoinositides as their deacylated derivatives which cannot address these distinct forms. Lipidomic analysis of the polyphosphoinositides has been hampered by difficulties with extraction and problems associated with binding of the lipids to surfaces. This review outlines the available MS methodologies, highlighting the difficulties associated with each. However, at present, no single methodology is available that can successfully and reproducibly quantitate each inositol phospholipid. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.

To review the outcomes of the patients who undergo orbital exenteration for malignancy.A retrospective review of patients who underwent orbital exenteration performed at Royal Prince Alfred and Concord Hospitals between 1990 and 2008.A total of 38 patients were identified. The most common malignancies were squamous cell carcinoma (14 cutaneous and five mucosal origin), basal cell carcinoma (12 cutaneous) and melanoma (five patients). Orbital invasion was the main indication for orbital exenteration. Additional procedures were performed in 29 (76%) patients at the time of exenteration, including craniotomies in eight patients and maxillectomy in 15 patients. Flap reconstructions were required in 35 (92%) patients, including 33 free flap repairs and two local flap repairs. Post-operative complications included cerebrospinal fluid leak (16%) and wound-related complications (13%). The disease-specific survival rate was 97% at 1 year and 92% at 5 years. The local control rate was 83% at 1 year and 55% at 5 years. Fifty per cent of patient mortality and 63% of local recurrence occurred within the first post-operative year.In this series, locoregional control and survival were high, suggesting that aggressive surgical measures such as orbital exenteration are not unreasonable in well-selected patients. Free flap reconstruction is a reliable reconstructive technique which may reduce major complications. The first post-operative year is the most important period when monitoring for recurrence.

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110δ during the activation and differentiation of naive T cells, and p110δ inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110δ activity is required for interferon-γ production. Moreover, acute inhibition of p110δ inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110δ played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor–induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110δ inhibition as naive T cells and show that mouse models accurately predict p110δ function in human T cells. There is therefore a strong rationale for p110δ inhibitors to be considered for therapeutic use in T-cell–mediated autoimmune and inflammatory diseases.

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with “detector” proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2′-O-(N-methylanthraniloyl) guanosine 5′-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.

Discovering the origins of Indiana's mysterious nickname "hoosier" requires a knowledge of when the word fir t came into use. Most standard reference works (including the Oxford English Dictionary) erro neously cite as the first known writing of the word a letter written in Missouri in 1826.1 The error is significant because such an early refer ence, from a site so far west, gives credence to the assumption of Jacob R Dunn (writing roughly a hundred years ago) that the word had been a term of contempt in general use in the South before it became specific to

High-performing boards communicate clearly and often and aren't afraid to challenge the status quo. How does your board compare?

Hypocalcemia after neck exploration for hyperparathyroidism is an important postoperative management issue. With increasing acceptance of less invasive surgical approaches, hypocalcemia is less frequent. This study was conducted to evaluate postoperative hypocalcemia after current surgical exploration techniques in patients with untreated primary hyperparathyroidism. From the University of Louisville parathyroid database, charts of patients undergoing surgery for untreated primary hyperparathyroidism from May 1, 1998 to May 30, 2004 were reviewed. Data was analyzed based on age, sex, preoperative calcium and parathyroid hormone levels, preexisting diseases, and extent of neck exploration. One hundred sixty-nine patients were identified with adequate data for analysis. Transient postoperative hypocalcemia occurred in 21 per cent (36/169) for the total group, in 18 per cent (22/125) after minimally invasive radio-guided parathyroidectomy, and in 32 per cent (14/44) after bilateral neck exploration. Patients with postoperative hypocalcemia had a statistically significant association with older age and pre-existing hypertension. Patients with postoperative hypocalcemia were more likely to have undergone longer surgical procedures and were more likely to have had pre-existing diabetes and mental disorders. These findings were not statistically significant and were considered trends. The frequency of osteoporosis in the hypocalcemia group was increased but was not significant. Transient hypocalcemia occurred in 21 per cent of patients after parathyroid surgery. It was more likely after bilateral neck exploration, a longer duration of surgery, and with hypertension, diabetes, and mental disorders.