Effect of glycosaminoglycans on growth factor-stimulated trophoblast invasion

Objectives: To determine the effect of glycosaminoglycans and a series of growth factors on the viability and invasion of the extravillous trophoblast cell line SGHPL4. Methods: Cells were cultured in Hams F10 media supplemented with fetal bovine serum and L-glutamine. For viability studies cells were seeded into 96-well culture plates (104 cells/well), maintained in serum free medium for 24h and then incubated with glycosaminoglycans (heparin, heparin sulphate and hyaluronic acid; each 100ng/ml) ± growth factors (VEGF, FGFand HB-EGF). Cell viability was measured in cells using the MTS assay. Cellular invasion was assessed using the FluoroBlok invasion assay. Cells were serum-starved for 24 h, incubated with the fluorescent dye DiIC12(3) (10mg/ml) for 1 hour prior to seeding onto an artificial extracellular matrix coated 8 mm FluoroBlok porous membrane inserts (2.5 x 105 cells per insert). Growth factors ± GAGs were added to the cell suspension and the inserts were lowered into a 96-well plate containing 10% fetal calf serum. Plates were incubated at 37°C for 24h. Invasion was determined by measurement of fluorescence of invaded cells using a fluorescent plate reader (Ex549/Em565 nm). Results: Cell numbers were significantly increased following incubation with VEGF, FGF and HB-EGF. Cell number was also increased after incubation with each of the glycosaminoglycans tested. The largest increase was observed following incubation with heparin sulphate. Cell numbers were further increased when the GFs were incubated with HS and heparin, but not with hyaluronic acid. Invasion was increased following incubation with VEGF, HBEGF and HGF. Heparan sulphate and heparin increased invasiveness in a dose-dependent manner. In contrast, hyaluronic acid had no significant effect. Conclusion: This study demonstrates a role for glycosaminoglycans in key features of trophoblast function

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6–4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA–GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biolog

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02–0.15 and 0.07–0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences

Can quantification of Serum Glycans predict Pre-Eclampsia?

Objectives: To determine if concentrations of placental glycans and glycan components are altered in pre-eclamspia and to determine if serum levels can predict pre-eclampsia. Methods: Serum samples were collected from women in the third trimester of singleton pregnancy but before the onset of pre-eclampsia and also from women during unaffected pregnancies at the samegestational age. Tissues were collected from the basal plate of placentas collected at delivery following uncomplicated singleton pregnancy (term and preterm) and from pregnancies complicated by preeclampsia. Pre-eclampsia was diagnosed according to International Society for the Study of Hypertension in Pregnancy criteria. Glycan components were isolated using a combination of enzyme digestion, molecular weight filtration and ion exchange chromatography, and then derivatised prior to separation using hydrophilic interaction liquid chromatography. Components were detected using electrospray ionisation operated in positive ion mode with single ion monitoring. Results: Specific glycan components (designated glycan 1, 2 and 3) were significantly altered in the serum from women who went on to have preeclampsia compared to those who had an unaffected pregnancy. Interestingly, levels of the same biomarkers were also elevated in nulliparous versus multiparous pregnancy. Biomarkers were also significantly altered in placental tissues from pregnancies complicated by preeclampsia Conclusion: This study suggests that altered glycan levels may contribute to impaired placental development and that the glycome is a potential diagnostic target for pre-eclampsia, and possibly other disorders of pregnancy

When does humoral memory enhance infection?

Antibodies and humoral memory are key components of the adaptive immune system. We consider and computationally model mechanisms by which humoral memory present at baseline might instead increase infection load; we refer to this effect as EI-HM (enhancement of infection by humoral memory). We first consider antibody dependent enhancement (ADE) in which antibody enhances the growth of the pathogen, typically a virus, and typically at intermediate "Goldilocks" levels of antibody. Our ADE model reproduces ADE in vitro and enhancement of infection in vivo from passive antibody transfer. But notably the simplest implementation of our ADE model never results in EI-HM. Adding complexity, by making the cross-reactive antibody much less neutralizing than the de novo generated antibody or by including a sufficiently strong non-antibody immune response, allows for ADE-mediated EI-HM. We next consider the possibility that cross-reactive memory causes EI-HM by crowding out a possibly superior de novo immune response. We show that, even without ADE, EI-HM can occur when the cross-reactive response is both less potent and "directly" (i.e. independently of infection load) suppressive with regard to the de novo response. In this case adding a non-antibody immune response to our computational model greatly reduces or completely eliminates EI-HM, which suggests that "crowding out" is unlikely to cause substantial EI-HM. Hence, our results provide examples in which simple models give qualitatively opposite results compared to models with plausible complexity. Our results may be helpful in interpreting and reconciling disparate experimental findings, especially from dengue, and for vaccination

Does the neutrino magnetic moment have an impact on solar neutrino physics?

Solar neutrino observations coupled with the recent KamLAND data suggest that spin-flavor precession scenario does not play a major role in neutrino propagation in the solar matter. We provide approximate analytical formulas and numerical results to estimate the contribution of the spin-flavor precession, if any, to the electron neutrino survival probability when the magnetic moment and magnetic field combination is small.Comment: 7 pages, 1 figure

Comparison of local frequency shifts between MDI velocity and intensity data

We analyze the frequencies of high–degree p modes using velocity and intensity data from MDI. The study is carried out with the local helioseismic technique of ring diagrams. The resultant power spectra at several different positions on the solar disk are fitted with symmetric and asymmetric profiles to find the center–to–limb variation (CLV). Using symmetric profiles in the anaylsis produces significant differences in frequency between velocity and intensity with the differences increasing from disk center to the limb. The use of asymmetric peak profiles reduces these differences and in the 3 mHz band the frequencies agree reasonably well with each other

Measuring the Solar Radius from Space during the 2003 and 2006 Mercury Transits

The Michelson Doppler Imager (MDI) aboard the Solar and Heliospheric Observatory observed the transits of Mercury on 2003 May 7 and 2006 November 8. Contact times between Mercury and the solar limb have been used since the 17th century to derive the Sun's size but this is the first time that high-quality imagery from space, above the Earth's atmosphere, has been available. Unlike other measurements this technique is largely independent of optical distortion. The true solar radius is still a matter of debate in the literature as measured differences of several tenths of an arcsecond (i.e., about 500 km) are apparent. This is due mainly to systematic errors from different instruments and observers since the claimed uncertainties for a single instrument are typically an order of magnitude smaller. From the MDI transit data we find the solar radius to be 960".12 +/- 0".09 (696,342 +/- 65 km). This value is consistent between the transits and consistent between different MDI focus settings after accounting for systematic effects.Comment: Accepted for publication in The Astrophysical Journal (2012 March 5

Our Sun. IV. The Standard Model and Helioseismology: Consequences of Uncertainties in Input Physics and in Observed Solar Parameters

Helioseismology provides a powerful tool to explore the deep interior of the Sun: for example, the adiabatic sound speed can be inferred with an accuracy of a few parts in 10,000. This has become a serious challenge to theoretical models of the Sun. Therefore, we have undertaken a self-consistent, systematic study of sources of uncertainties in the standard solar model, which must be understood before the helioseismic observations can be used as constraints on theory. We find that the largest uncertainty in the sound speed in the solar interior, namely, 3 parts in 1000, arises from uncertainties in the observed photospheric abundances of the elements; uncertainties of 1 part in 1000 arise from (1) the 4% uncertainty in the OPAL opacities, (2) the 5% uncertainty in the basic pp nuclear reaction rate, (3) the 15% uncertainty in the diffusion constants for the gravitational settling of helium, and (4) the 50% uncertainties in diffusion constants for the heavier elements. (Other investigators have shown that similar uncertainties arise from uncertainties in the interior equation of state and in rotation-induced turbulent mixing.) The predicted pre-main-sequence solar lithium depletion is a factor of order 20 (an order of magnitude larger than that predicted by earlier models that neglected gravitational settling and used older opacities), and is uncertain by a factor of 2. The predicted neutrino capture rate is uncertain by 30% for the Cl-37 experiment and by 3% for the Ga-71 experiments (not including uncertainties in the capture cross sections), while the B-8 neutrino flux is uncertain by 30%.Comment: LaTeX, 38 pages (including 8 figures); ApJ, in press. Added figures/color figurea available at http://www.cita.utoronto.ca/~boothroy/sun4.htm

PENGARUH LOKASI DAN PROMOSI PENJUALAN TERHADAP KEPUTUSAN PEMBELIAN MORNING BREAD BANDUNG (Survei Pada Konsumen Morning Bread Bandung)

ABSTRAK Penelitian ini bertujuan untuk mengetahui pengaruh dari lokasi dan promosi penjualan terhadap keputusan pembelian (survei pada konsumen Morning Bread Bandung) secara simultan maupun parsial. Metode penelitian yang digunakan adalah metode deskriptif dan verifikatif dengan jumlah sampel sebanyak 85 responden. Pengujian instrumen penelitian menggunakan uji validitas dan reliabilitas. Metode analisis data yang digunakan adalah analisis regresi linier berganda, korelasi berganda dan koefisien determinasi. Hasil penelitian menunjukkan bahwa terdapat pengaruh positif dan signifikan antara lokasi dan promosi penjualan terhadap keputusan pembelian. Besarnya pengaruh lokasi dan promosi penjualan terhadap keputusan pembelian secara simultan sebesar 53,6% dan sisanya 46,4% dipengaruhi variabel lain yang tidak diteliti. Secara parsial besarnya pengaruh lokasi terhadap keputusan pembelian sebesar 17,6% dan pengaruh promosi penjualan terhadap keputusan pembelian sebesar 36%, sehingga dapat disimpulkan bahwa promosi penjualan memberikan pengaruh paling besar terhadap keputusan pembelian. Kata kunci : Lokasi, Promosi Penjualan dan Keputusan Pembelia