A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users

Release of oxidizing fluids in subduction zones recorded by iron isotope zonation in garnet

Subduction zones are key regions of chemical and mass transfer between the Earth’s surface and mantle. During subduction, oxidized material is carried into the mantle and large amounts of water are released due to the breakdown of hydrous minerals such as lawsonite. Dehydration accompanied by the release of oxidizing species may play a key role in controlling redox changes in the subducting slab and overlying mantle wedge. Here we present measurements of oxygen fugacity, using garnet–epidote oxybarometry, together with analyses of the stable iron isotope composition of zoned garnets from Sifnos, Greece. We find that the garnet interiors grew under relatively oxidized conditions whereas garnet rims record more reduced conditions. Garnet δ56Fe increases from core to rim as the system becomes more reduced. Thermodynamic analysis shows that this change from relatively oxidized to more reduced conditions occurred during lawsonite dehydration. We conclude that the garnets maintain a record of progressive dehydration and that the residual mineral assemblages within the slab became more reduced during progressive subduction-zone dehydration. This is consistent with the hypothesis that lawsonite dehydration accompanied by the release of oxidizing species, such as sulfate, plays an important and measurable role in the global redox budget and contributes to sub-arc mantle oxidation in subduction zones

Asynchronous Antarctic and Greenland ice-volume contributions to the last interglacial sea-level highstand

The last interglacial (LIG; ~130 to ~118 thousand years ago, ka) was the last time global sea level rose well above the present level. Greenland Ice Sheet (GrIS) contributions were insufficient to explain the highstand, so that substantial Antarctic Ice Sheet (AIS) reduction is implied. However, the nature and drivers of GrIS and AIS reductions remain enigmatic, even though they may be critical for understanding future sea-level rise. Here we complement existing records with new data, and reveal that the LIG contained an AIS-derived highstand from ~129.5 to ~125 ka, a lowstand centred on 125–124 ka, and joint AIS + GrIS contributions from ~123.5 to ~118 ka. Moreover, a dual substructure within the first highstand suggests temporal variability in the AIS contributions. Implied rates of sea-level rise are high (up to several meters per century; m c−1), and lend credibility to high rates inferred by ice modelling under certain ice-shelf instability parameterisations

A qualitative analysis of environmental policy and children's health in Mexico

<p>Abstract</p> <p>Background</p> <p>Since Mexico's joining the North American Free Trade Agreement (NAFTA) and the Organization for Economic Cooperation and Development (OECD) in 1994, it has witnessed rapid industrialization. A byproduct of this industrialization is increasing population exposure to environmental pollutants, of which some have been associated with childhood disease. We therefore identified and assessed the adequacy of existing international and Mexican governance instruments and policy tools to protect children from environmental hazards.</p> <p>Methods</p> <p>We first systematically reviewed PubMed, the Mexican legal code and the websites of the United Nations, World Health Organization, NAFTA and OECD as of July 2007 to identify the relevant governance instruments, and analyzed the approach these instruments took to preventing childhood diseases of environmental origin. Secondly, we interviewed a purposive sample of high-level government officials, researchers and non-governmental organization representatives, to identify their opinions and attitudes towards children's environmental health and potential barriers to child-specific protective legislation and implementation.</p> <p>Results</p> <p>We identified only one policy tool describing specific measures to reduce developmental neurotoxicity and other children's health effects from lead. Other governance instruments mention children's unique vulnerability to ozone, particulate matter and carbon monoxide, but do not provide further details. Most interviewees were aware of Mexican environmental policy tools addressing children's health needs, but agreed that, with few exceptions, environmental policies do not address the specific health needs of children and pregnant women. Interviewees also cited state centralization of power, communication barriers and political resistance as reasons for the absence of a strong regulatory platform.</p> <p>Conclusions</p> <p>The Mexican government has not sufficiently accounted for children's unique vulnerability to environmental contaminants. If regulation and legislation are not updated and implemented to protect children, increases in preventable exposures to toxic chemicals in the environment may ensue.</p

Beam dynamics corrections to the Run-1 measurement of the muon anomalous magnetic moment at Fermilab

This paper presents the beam dynamics systematic corrections and their uncertainties for the Run-1 dataset of the Fermilab Muon g-2 Experiment. Two corrections to the measured muon precession frequency ωam are associated with well-known effects owing to the use of electrostatic quadrupole (ESQ) vertical focusing in the storage ring. An average vertically oriented motional magnetic field is felt by relativistic muons passing transversely through the radial electric field components created by the ESQ system. The correction depends on the stored momentum distribution and the tunes of the ring, which has relatively weak vertical focusing. Vertical betatron motions imply that the muons do not orbit the ring in a plane exactly orthogonal to the vertical magnetic field direction. A correction is necessary to account for an average pitch angle associated with their trajectories. A third small correction is necessary, because muons that escape the ring during the storage time are slightly biased in initial spin phase compared to the parent distribution. Finally, because two high-voltage resistors in the ESQ network had longer than designed RC time constants, the vertical and horizontal centroids and envelopes of the stored muon beam drifted slightly, but coherently, during each storage ring fill. This led to the discovery of an important phase-acceptance relationship that requires a correction. The sum of the corrections to ω_{a}^{m} is 0.50±0.09 ppm; the uncertainty is small compared to the 0.43 ppm statistical precision of ω_{a}^{m}

Magnetic-field measurement and analysis for the Muon g − 2 Experiment at Fermilab

The Fermi National Accelerator Laboratory (FNAL) Muon g - 2 Experiment has measured the anomalous precession frequency a_{μ}(g_{μ} - 2)/2 of the muon to a combined precision of 0.46 parts per million with data collected during its first physics run in 2018. This paper documents the measurement of the magnetic field in the muon storage ring. The magnetic field is monitored by systems and calibrated in terms of the equivalent proton spin precession frequency in a spherical water sample at 34.7C. The measured field is weighted by the muon distribution resulting in \tilde{ω}'_{p}, the denominator in the ratio \tilde{ω}_{a}/\tilde{ω}'_{p} that together with known fundamental constants yields aμ. The reported uncertainty on \tilde{ω}'_{p} for the Run-1 data set is 114 ppb consisting of uncertainty contributions from frequency extraction, calibration, mapping, tracking, and averaging of 56 ppb, and contributions from fast transient fields of 99 ppb

Measurement of the Positive Muon Anomalous Magnetic Moment to 0.20 ppm

We present a new measurement of the positive muon magnetic anomaly, a_{μ}≡(g_{μ}-2)/2, from the Fermilab Muon g-2 Experiment using data collected in 2019 and 2020. We have analyzed more than 4 times the number of positrons from muon decay than in our previous result from 2018 data. The systematic error is reduced by more than a factor of 2 due to better running conditions, a more stable beam, and improved knowledge of the magnetic field weighted by the muon distribution, ω[over ˜]_{p}^{'}, and of the anomalous precession frequency corrected for beam dynamics effects, ω_{a}. From the ratio ω_{a}/ω[over ˜]_{p}^{'}, together with precisely determined external parameters, we determine a_{μ}=116 592 057(25)×10^{-11} (0.21 ppm). Combining this result with our previous result from the 2018 data, we obtain a_{μ}(FNAL)=116 592 055(24)×10^{-11} (0.20 ppm). The new experimental world average is a_{μ}(exp)=116 592 059(22)×10^{-11} (0.19 ppm), which represents a factor of 2 improvement in precision

Tutorial: Multivariate Classification for Vibrational Spectroscopy in Biological Samples

Vibrational spectroscopy techniques, such as Fourier-transform infrared (FTIR) and Raman spectroscopy, have been successful methods for studying the interaction of light with biological materials and facilitating novel cell biology analysis. Spectrochemical analysis is very attractive in disease screening and diagnosis, microbiological studies and forensic and environmental investigations because of its low cost, minimal sample preparation, non-destructive nature and substantially accurate results. However, there is now an urgent need for multivariate classification protocols allowing one to analyze biologically derived spectrochemical data to obtain accurate and reliable results. Multivariate classification comprises discriminant analysis and class-modeling techniques where multiple spectral variables are analyzed in conjunction to distinguish and assign unknown samples to pre-defined groups. The requirement for such protocols is demonstrated by the fact that applications of deep-learning algorithms of complex datasets are being increasingly recognized as critical for extracting important information and visualizing it in a readily interpretable form. Hereby, we have provided a tutorial for multivariate classification analysis of vibrational spectroscopy data (FTIR, Raman and near-IR) highlighting a series of critical steps, such as preprocessing, data selection, feature extraction, classification and model validation. This is an essential aspect toward the construction of a practical spectrochemical analysis model for biological analysis in real-world applications, where fast, accurate and reliable classification models are fundamental

Transcription analysis of the myometrium of labouring and non-labouring women

An incomplete understanding of the molecular mechanisms that initiate normal human labour at term seriously hampers the development of effective ways to predict, prevent and treat disorders such as preterm labour. Appropriate analysis of large microarray experiments that compare gene expression in non-labouring and labouring gestational tissues is necessary to help bridge these gaps in our knowledge. In this work, gene expression in 48 (22 labouring, 26 non-labouring) lower-segment myometrial samples collected at Caesarean section were analysed using Illumina HT-12 v4.0 BeadChips. Normalised data were compared between labouring and non-labouring groups using traditional statistical methods and a novel network graph approach. We sought technical validation with quantitative real-time PCR, and biological replication through inverse variance-weighted meta-analysis with published microarray data. We have extended the list of genes suggested to be associated with labour: Compared to non-labouring samples, labouring samples showed apparent higher expression at 960 probes (949 genes) and apparent lower expression at 801 probes (789 genes) (absolute fold change ≥1.2, rank product percentage of false positive value (RP-PFP) <0.05). Although half of the women in the labouring group had received pharmaceutical treatment to induce or augment labour, sensitivity analysis suggested that this did not confound our results. In agreement with previous studies, functional analysis suggested that labour was characterised by an increase in the expression of inflammatory genes and network analysis suggested a strong neutrophil signature. Our analysis also suggested that labour is characterised by a decrease in the expression of muscle-specific processes, which has not been explicitly discussed previously. We validated these findings through the first formal meta-analysis of raw data from previous experiments and we hypothesise that this represents a change in the composition of myometrial tissue at labour. Further work will be necessary to reveal whether these results are solely due to leukocyte infiltration into the myometrium as a mechanism initiating labour, or in addition whether they also represent gene changes in the myocytes themselves. We have made all our data available at www.ebi.ac.uk/arrayexpress/ (accession number E-MTAB-3136) to facilitate progression of this work

Volunteer Engagement in Housing Co-Operatives – Civil Society “en miniature”

Housing co‐operatives host miniature versions of civil society. They vitalise a social system that is shaped by formal regulations, economic functions, and a population of private housing units. The study examines factors that influence a person’s willingness to volunteer in civic society using a multilevel analysis based on survey data from 32 co‐operatives and 1263 members. To do so, the social exchange theory is extended to include the member value approach, which connects social engagement with the fulfillment of a range of needs, thus going beyond a narrow economic cost benefit analysis. Study results show that volunteer engagement largely depends on the degree to which members can expect to experience their own achievement. This finding provides an explanation for significant differences in the engagement levels beyond factors that have already been determined (age, level of education). On an organizational level, the study reveals that the age of an organization influences volunteer engagement, but that the size and the degree of professionalization do not have an effect on it