Outgrowth Endothelial Cells: Characterization and Their Potential for Reversing Ischemic Retinopathy

Purpose. Endothelial progenitor cells (EPCs) have potential for promoting vascular repair and revascularization of ischemic retina. However, the highly heterogeneous nature of these cells causes confusion when assessing their biological functions. The purpose of this study was to provide a comprehensive comparison between the two main EPC subtypes, early EPCs (eEPCs) and outgrowth endothelial cells (OECs), and to establish the potential of OECs as a novel cell therapy for ischemic retinopathy.

Methods. Two types of human blood-derived EPCs were isolated and compared using immunophenotyping and multiple in vitro functional assays to assess interaction with retinal capillary endothelial cells and angiogenic activity. OECs were delivered intravitreally in a mouse model of ischemic retinopathy, and flat mounted retinas were examined using confocal microscopy.

Results. These data indicate that eEPCs are hematopoietic cells with minimal proliferative capacity that lack tube-forming capacity. By contrast, OECs are committed to an endothelial lineage and have significant proliferative and de novo tubulogenic potential. Furthermore, only OECs are able to closely interact with endothelial cells through adherens and tight junctions and to integrate into retinal vascular networks in vitro. The authors subsequently chose OECs to test a novel cell therapy approach for ischemic retinopathy. Using a murine model of retinal ischemia, they demonstrated that OECs directly incorporate into the resident vasculature, significantly decreasing avascular areas, concomitantly increasing normovascular areas, and preventing pathologic preretinal neovascularization.

Conclusions. As a distinct EPC population, OECs have potential as therapeutic cells to vascularize the ischemic retina.

Human Dental Pulp Fibroblasts Express the “Cold-sensing” Transient Receptor Potential Channels TRPA1 and TRPM8

Introduction: Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts.
Methods: Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca2+ microfluorimetry.
Results: Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca2+ microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca2+]i) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists.
Conclusions: Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9(hi), SSEA-1(-) Cells in Embryonic Stem Cell-Derived Embryoid Bodies

Background. Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. Methodology/Principal Findings. We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E-RoSH lines have similar gene expression profiles (r(2) = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi), SSEA-1(-) while ESCs are CD9(lo), SSEA-1(+). Isolation of CD9(hi), SSEA-1(-) cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2) = 0.95) and a propensity to differentiate into endothelial-like cells. Conclusions. By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs.

Preservation methods alter stable isotope values in gelatinous zooplankton: implications for interpreting trophic ecology

Jellyfish are increasingly topical within studies of marine food webs. Stable isotope analysis represents a valuable technique to unravel the complex trophic role of these long-overlooked species. In other taxa, sample preservation has been shown to alter the isotopic values of species under consideration, potentially leading to misinterpretation of trophic ecology. To identify potential preservation effects in jellyfish, we collected Aurelia aurita from Strangford Lough (54(o)22'44.73aEuro(3)N, 5(o)32'53.44aEuro(3)W) during May 2009 and processed them using three different methods prior to isotopic analysis (unpreserved, frozen and preserved in ethanol). A distinct preservation effect was found on delta N-15 values: furthermore, preservation also influenced the positive allometric relationship between individual size and delta N-15 values. Conversely, delta C-13 values remained consistent between the three preservation methods, conflicting with previous findings for other invertebrate, fish and mammalian species. These findings have implications for incorporation of jellyfish into marine food webs and remote sampling regimes where preservation of samples is unavoidable.

Current methods and advances in forecasting of wind power generation

Wind power generation differs from conventional thermal generation due to the stochastic nature of wind. Thus wind power forecasting plays a key role in dealing with the challenges of balancing supply and demand in any electricity system, given the uncertainty associated with the wind farm power output. Accurate wind power forecasting reduces the need for additional balancing energy and reserve power to integrate wind power. Wind power forecasting tools enable better dispatch, scheduling and unit commitment of thermal generators, hydro plant and energy storage plant and more competitive market trading as wind power ramps up and down on the grid. This paper presents an in-depth review of the current methods and advances in wind power forecasting and prediction. Firstly, numerical wind prediction methods from global to local scales, ensemble forecasting, upscaling and downscaling processes are discussed. Next the statistical and machine learning approach methods are detailed. Then the techniques used for benchmarking and uncertainty analysis of forecasts are overviewed, and the performance of various approaches over different forecast time horizons is examined. Finally, current research activities, challenges and potential future developments are appraised. (C) 2011 Elsevier Ltd. All rights reserved.

Standardisation and comparison of methods employed for microbial cell surface hydrophobicity and charge determination

Whilst there are a number of methods available to characterise the cell surface hydrophobicity (CSH) and cell surface charge (CSC) of microorganisms, there is still debate concerning the correlation of results between individual methods. In this study, the techniques of bacterial adherence to hydrocarbons (BATH) and hydrophobic interaction chromatography (HTC) were used to measure CSH. Electrostatic interaction chromatography (ESIC) and zeta potential (ZP) measurements were used to determine CSC. To allow meaningful comparisons between the BATH and HIC tests, between ESIC and ZP and also between CSH and CSC, the buffer systems employed in each test were standardised (phosphate buffered saline, pH 7.3, 0.01 mM). Isolates of Staphylococcus epidermidis derived from microbial biofilm were used as the test organism in this study. The isolates examined exhibited primarily medium to high CSH and a highly negative CSC. Good correlation of CSH measurement was observed between the BATH and HIC tests (r = 0.89). Good correlation was observed between ESIC (anionic exchange column) and ZP measurements. No correlations were observed between isolate CSC and either increased or decreased CSH. It is recommended that whenever comparisons of various methods to determine either CSC or CSH (by partitioning methods), the buffer systems should remain constant throughout to achieve consistency of results.

Pre-hospital body surface potential mapping improves early diagnosis of acute coronary artery occlusion in patients with ventricular fibrillation and cardiac arrest

Aims: To determine whether 80-lead body surface potential mapping (BSPM) improves detection of acute coronary artery occlusion in patients presenting with out-of-hospital cardiac arrest (OHCA) due to ventricular fibrillation (VF) and who survived to reach hospital. Methods and results: Of 645 consecutive patients with OHCA who were attended by the mobile coronary care unit, VF was the initial rhythm in 168 patients. Eighty patients survived initial resuscitation, 59 of these having had BSPM and 12-lead ECG post-return of spontaneous circulation (ROSC) and in 35 patients (age 69±13 yrs; 60% male) coronary angiography performed within 24. h post-ROSC. Of these, 26 (74%) patients had an acutely occluded coronary artery (TIMI flow grade [TFG] 0/1) at angiography. Twelve-lead ECG criteria showed ST-segment elevation (STE) myocardial infarction (STEMI) using Minnesota 9-2 criteria - sensitivity 19%, specificity 100%; ST-segment depression (STD) =0.05. mV in =2 contiguous leads - sensitivity 23%, specificity 89%; and, combination of STEMI or STD criteria - sensitivity 46%, specificity 100%. BSPM STE occurred in 23 (66%) patients. For the diagnosis of TFG 0/1 in a main coronary artery, BSPM STE had sensitivity 88% and specificity 100% (c-statistic 0.94), with STE occurring most commonly in either the posterior, right ventricular or high right anterior territories. Conclusion: Among OHCA patients presenting with VF and who survived resuscitation to reach hospital, post-resuscitation BSPM STE identifies acute coronary occlusion with sensitivity 88% and specificity 100% (c-statistic 0.94). © 2012 Elsevier Ireland Ltd.

Public health education for midwives and midwifery students: a mixed methods study

Background: Current national and international maternity policy supports the importance of addressing public health goals and investing in early years. Health care providers for women during the reproductive and early postnatal period have the opportunity to encourage women to make choices that will impact positively on maternal and fetal health. Midwives are in a unique position, given the emphasis of the philosophy of midwifery care on building relationships and incorporating a holistic approach, to support women to make healthy choices with the aim of promoting health and preventing ill health. However, exploration of the educational preparation of midwives to facilitate public health interventions has been relatively limited. The aim of the study was to identify the scope of current midwifery pre registration educational provision in relation to public health and to explore the perspectives of midwives and midwifery students about the public health role of the midwife.

Methods: This was a mixed methods study incorporating a survey of Higher Educational Institutions providing pre registration midwifery education across the UK and focus groups with midwifery students and registered midwives.

Results: Twenty nine institutions (53% response) participated in the survey and nine focus groups were conducted (59 participants). Public health education was generally integrated into pre registration midwifery curricula as opposed to taught as a discrete subject. There was considerable variation in the provision of public health topics within midwifery curricula and the hours of teaching allocated to them. Focus group data indicated that it was consistently difficult for both midwifery students and midwives to articulate clearly their understanding and definition of public health in relation to midwifery.

Conclusions: There is a unique opportunity to impact on maternal and infant health throughout the reproductive period; however the current approach to public health within midwifery education should be reviewed to capitalise on the role of the midwife in delivering public health interventions. It is clear that better understanding of midwifery public health roles and the visibility of public health within midwifery is required in order to maximise the potential contribution of midwives to achieving short and long term public health population goals.

Mass spectrometric analysis of muscle samples to detect potential antibiotic growth promoter misuse in broiler chickens

Mass spectrometric methods were developed and validated for the analysis in chicken muscle of a range of antibiotic growth promoters: spiramycin, tylosin, virginiamycin and bacitracin, and separately for two marker metabolites of carbadox (quinoxaline-2-carboxylic acid and 1,4-bisdesoxycarbadox), and a marker metabolite of olaquindox (3-methyl-quinoxaline-2-carboxylic acid). The use of these compounds as antibiotic growth promoters has been banned by the European Commission. This study aimed to develop methods to detect their residues in muscle samples as a means of checking for the use of these drugs during the rearing of broiler chickens. When fed growth-promoting doses for 6 days, spiramycin (31.4 mu g kg(-1)), tylosin (1.0 mu g kg(-1)), QCA (6.5 mu g kg(-1)), DCBX (71.2 mu g kg(-1)) and MQCA (0.2 mu g kg(-1)) could be detected in the muscle 0 days after the withdrawal of fortified feed. Only spiramycin could consistently be detected beyond a withdrawal period of 1 day. All analytes showed stability commercial cooking process, therefore raw or cooked muscle could be used for monitoring purposes.

The prediction of intake potential and organic matter digestibility of grass silages by near infrared spectroscopy analysis of undried samples

A study was undertaken to examine a range of sample preparation and near infrared reflectance spectroscopy (NIPS) methodologies, using undried samples, for predicting organic matter digestibility (OMD g kg(-1)) and ad libitum intake (g kg(-1) W-0.75) of grass silages. A total of eight sample preparation/NIRS scanning methods were examined involving three extents of silage comminution, two liquid extracts and scanning via either external probe (1100-2200 nm) or internal cell (1100-2500 nm). The spectral data (log 1/R) for each of the eight methods were examined by three regression techniques each with a range of data transformations. The 136 silages used in the study were obtained from farms across Northern Ireland, over a two year period, and had in vivo OMD (sheep) and ad libitum intake (cattle) determined under uniform conditions. In the comparisons of the eight sample preparation/scanning methods, and the differing mathematical treatments of the spectral data, the sample population was divided into calibration (n = 91) and validation (n = 45) sets. The standard error of performance (SEP) on the validation set was used in comparisons of prediction accuracy. Across all 8 sample preparation/scanning methods, the modified partial least squares (MPLS) technique, generally minimized SEP's for both OMD and intake. The accuracy of prediction also increased with degree of comminution of the forage and with scanning by internal cell rather than external probe. The system providing the lowest SEP used the MPLS regression technique on spectra from the finely milled material scanned through the internal cell. This resulted in SEP and R-2 (variance accounted for in validation set) values of 24 (g/kg OM) and 0.88 (OMD) and 5.37 (g/kg W-0.75) and 0.77 (intake) respectively. These data indicate that with appropriate techniques NIRS scanning of undried samples of grass silage can produce predictions of intake and digestibility with accuracies similar to those achieved previously using NIRS with dried samples. (C) 1998 Elsevier Science B.V.

Improved Detection of Mycobacterium bovis Infection in Bovine Lymph Node Tissue Using Immunomagnetic Separation (IMS)-Based Methods

Immunomagnetic separation (IMS) can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR) or culture (IMS-MGIT). This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL), 74 non-visibly lesioned (NVL)) collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1%) lymph node samples tested positive by culture, 162 (57.8%) by IMS-PCR (targeting IS6110), and 196 (70.0%) by IMS-MGIT culture. Twelve (6.9%) of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 78 M. bovis positive lymph node samples (26 (12.6%) VL and 54 (73.0%) NVL) were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle) decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant) which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 73% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle.. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.

Investigation of the Human Brain Metabolome to Identify Potential Markers for Early Diagnosis and Therapeutic Targets of Alzheimer’s Disease

A study combining high resolution mass spectrometry (liquid chromatography-quadrupole time-of-flight-mass spectrometry, UPLC-QTof-MS) and chemometrics for the analysis of post-mortem brain tissue from subjects with Alzheimer’s disease (AD) (n = 15) and healthy age-matched controls (n = 15) was undertaken. The huge potential of this metabolomics approach for distinguishing AD cases is underlined by the correct prediction of disease status in 94–97% of cases. Predictive power was confirmed in a blind test set of 60 samples, reaching 100% diagnostic accuracy. The approach also indicated compounds significantly altered in concentration following the onset of human AD. Using orthogonal partial least-squares discriminant analysis (OPLS-DA), a multivariate model was created for both modes of acquisition explaining the maximum amount of variation between sample groups (Positive Mode-R2 = 97%; Q2 = 93%; root mean squared error of validation (RMSEV) = 13%; Negative Mode-R2 = 99%; Q2 = 92%; RMSEV = 15%). In brain extracts, 1264 and 1457 ions of interest were detected for the different modes of acquisition (positive and negative, respectively). Incorporation of gender into the model increased predictive accuracy and decreased RMSEV values. High resolution UPLC-QTof-MS has not previously been employed to biochemically profile post-mortem brain tissue, and the novel methods described and validated herein prove its potential for making new discoveries related to the etiology, pathophysiology, and treatment of degenerative brain disorders.

Exchange potential from the common energy denominator approximation for the Kohn-Sham Green's function: Application to (hyper)polarizabilities of molecular chains

An approximate Kohn-Sham (KS) exchange potential v(xsigma)(CEDA) is developed, based on the common energy denominator approximation (CEDA) for the static orbital Green's function, which preserves the essential structure of the density response function. v(xsigma)(CEDA) is an explicit functional of the occupied KS orbitals, which has the Slater v(Ssigma) and response v(respsigma)(CEDA) potentials as its components. The latter exhibits the characteristic step structure with "diagonal" contributions from the orbital densities \psi(isigma)\(2), as well as "off-diagonal" ones from the occupied-occupied orbital products psi(isigma)psi(j(not equal1)sigma). Comparison of the results of atomic and molecular ground-state CEDA calculations with those of the Krieger-Li-Iafrate (KLI), exact exchange (EXX), and Hartree-Fock (HF) methods show, that both KLI and CEDA potentials can be considered as very good analytical "closure approximations" to the exact KS exchange potential. The total CEDA and KLI energies nearly coincide with the EXX ones and the corresponding orbital energies epsilon(isigma) are rather close to each other for the light atoms and small molecules considered. The CEDA, KLI, EXX-epsilon(isigma) values provide the qualitatively correct order of ionizations and they give an estimate of VIPs comparable to that of the HF Koopmans' theorem. However, the additional off-diagonal orbital structure of v(xsigma)(CEDA) appears to be essential for the calculated response properties of molecular chains. KLI already considerably improves the calculated (hyper)polarizabilities of the prototype hydrogen chains H-n over local density approximation (LDA) and standard generalized gradient approximations (GGAs), while the CEDA results are definitely an improvement over the KLI ones. The reasons of this success are the specific orbital structures of the CEDA and KLI response potentials, which produce in an external field an ultranonlocal field-counteracting exchange potential. (C) 2002 American Institute of Physics.

Urban greenways have the potential to increase physical activity levels cost-effectively

BACKGROUND: For many, physical activity has been engineered out of daily life, leading to high levels of sedentariness and obesity. Multi-faceted physical activity interventions, combining individual, community and environmental approaches, have the greatest potential to improve public health, but few have been evaluated. METHODS: Approximately 100 000 people may benefit from improved opportunities for physical activity through an urban regeneration project in Northern Ireland, the Connswater Community Greenway. Using the macro-simulation PREVENT model, we estimated its potential health impacts and cost-effectiveness. To do so, we modelled its potential impact on the burden from cardiovascular disease, namely, ischaemic heart disease, type 2 diabetes mellitus and stroke, and colon and breast cancer, by the year 2050, if feasible increases in physical activity were to be achieved. RESULTS: If 10% of those classified as 'inactive' (perform less than 150 minutes of moderate activity/week) became 'active', 886 incident cases (1.2%) and 75 deaths (0.9%) could be prevented with an incremental cost-effectiveness ratio of £4469/disability-adjusted life year. For effectiveness estimates as low as 2%, the intervention would remain cost-effective (£18 411/disability-adjusted life year). Small gains in average life expectancy and disability-adjusted life expectancy could be achieved, and the Greenway population would benefit from 46 less years lived with disability. CONCLUSION: The Greenway intervention could be cost-effective at improving physical activity levels. Although the direct health gains are predicted to be small for any individual, summed over an entire population, they are substantial. In addition, the Greenway is likely to have much wider benefits beyond health.

Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: Implications for respiratory virus-induced cough reflex sensitivity

ABSTRACT (250 words)
BACKGROUND: The mechanism underlying respiratory virus-induced cough hypersensitivity is unknown. Up-regulation of airway neuronal receptors responsible for sensing physical and chemical stimuli is one possibility and the transient receptor potential (TRP) channel family are potential candidates. We have used an in vitro model of sensory neurones and human rhinovirus (HRV-16) to study the effect of virus infection on TRP expression.
METHODS: IMR32 neuroblastoma cells were differentiated in culture to express three TRP channels, TRPV1, TRPA1 and TRPM8. Flow cytometry and qRT-PCR were used to measure TRP channel protein and mRNA levels following inoculation with live virus, inactivated virus, virus- induced soluble factors or pelleted virus particles. Multiplex bioassay was used to determine nerve growth factor (NGF), interleukin (IL)-1ß, IL-6 and IL-8 levels in response to infection.
RESULTS: Early up-regulation of TRPA1 and TRPV1 expression occurred 2 to4 hours post infection. This was independent of replicating virus as virus induced soluble factors alone were sufficient to increase channel expression 50 and 15 fold, respectively. NGF, IL-6 and IL-8 levels, increased in infected cell supernatants, represent possible candidates. In contrast, TRPM8 expression was maximal at 48 hours (9.6 fold) and required virus replication rather than soluble factors
CONCLUSIONS We show for the first time that rhinovirus can infect neuronal cells. Furthermore, infection causes up-regulation of TRP channels by channel specific mechanisms. Increase in TRPA1 and TRPV1 levels can be mediated by soluble factors induced by infection whereas TRPM8 requires replicating virus. TRP channels may be novel therapeutic targets for controlling virus-induced cough.