The Effect of Wild Blueberry Bioactives on Endothelial Cell Migration and Angiogenesis: an in vitro Mechanistic, Genomic and Proteomic Approach

The goal of this study is to investigate the effects of wild blueberry fractions (Anthocyanins and Phenolic acids) on vascular function and physiology. More specifically the potential effects of the above fractions and their combination in physiological concentrations on endothelial cell migration, angiogenesis, gene expression and proteins synthesis of markers related to the above processes. The objectives are to study whether anthocyanins, phenolic acids and their combinations (ACNs:PAs) affect: a) cell proliferation, b) speed of endothelial cell migration, c) angiogenesis, d) gene expression of genes critical for cell migration and angiogenesis such as RAC1, RHOA, AKT1, eNOS and VEGF and finally e) synthesis of proteins that are critical for cell migration and angiogenesis such as RAC1, RHOA, AKT1, eNOS and VEGF. This project utilized as an experimental model the human umbilical vein endothelial cells (HUV-EC-C [HUVEC] (ATCC® CRL-1730™)). Anthocyanins (ACNs) and phenolic acids (PAs) were extracted from the wild blueberry (WB) powder and a range of concentrations was used (0.002 μg/mL, 8 μg/mL, 15 μg/mL, 60 μg/mL and 120 μg/mL). Cell cytotoxicity experiments were conducted to determine the appropriate concentrations for the following experiments; endothelial cell migration, angiogenesis, gene expression and Western Blot. To determine possible cytotoxicity of the wild blueberry fractions, a broad range of concentrations were used (0.001 μg/mL - 1000 μg/mL for ACNs and 0.001 μg/mL – 500 μg/mL for the PAs) at different time points (30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h). None of the consecrations were cytotoxic to the cells except the 1000 μg/mL of ACNs. Cell migration experiments documented an inhibitory effect on the speed of endothelial cells when ACNs at 60 μg/mL were used. However, PAs had the opposite effect on HUVECs speed. Exposure of the endothelial cells at 0.002 μg/mL, 60 μg/mL and 120 μg/mL significantly increased the speed of endothelial cell migration compared to control. Additionally, combination of both ACNs and PAs (ACNs:PAs) at 8μg/mL:8μg/mL and 60μg/mL:60μg/mL respectively, significantly increased endothelial cell migration rate compared to control. Angiogenesis experiments also revealed similar trends; ACNs inhibited the formation of the endothelial network while PAs and ACNs:PAs promoted a more stable endothelial network. Expression of genes related to the above cellular functions as well as protein analysis support the findings from cell migration and angiogenesis experiments. In conclusion, ACNs, PAs and ACNs:PAs extracted from wild blueberries had a significant effect on endothelial cell function based on type of fraction and concentration not only at the mechanistic but also at the genomic and proteomic level. This may have clinical applications for degenerative diseases such as cancer, diabetic wounds and cardiovascular disease

Synthesis of Aqueous Suspensions of Zero-Valent Iron Nanoparticles (nZVI) from Plant Extracts: Experimental Study and Numerical Modeling

Plant extracts were produced from Camellia sinesis (Green Tea) and Punica granatum (pomegranate), and the total concentration of polyphenols was measured in terms of equivalent concentration of Gallic acid by using the Folin-Ciocalteu method. Zero Valent Iron nanoparticles (nZVIs) were synthesized in a semi-batch reactor by mixing a pre-specified volume of plant extract or Gallic Acid solution with an aqueous solution of iron sulfate heptahydrate (FeSO4·7H2O). To monitor the kinetics of nZVI synthesis, the transient responses of solution pH and redox potential (Eh) were recorded with two probes adequately connected with a data acquisition card. The nanoparticles were characterized by a variety of techniques: Dynamic Light Scattering (DLS), ζ-potential, Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, Transmission Electron Microscopy (TEM). A kinetic parametric model, based on two parallel single electron transfer (SET) and hydrogen atom transfer (HAT) reactions, was suggested to quantify the dynamics of ferrous ions reduction to zero valence, and its parameters were estimated for each experimental system by matching the transient response of pH. The temporal changes of redox potential during nZVI synthesis were indicative of the reaction progress and agreed with the numerical predictions in semi-quantitative basis.  The numerical model enabled us to track the temporal variation of the concentration of iron and polyphenol species, and calculate the yield of ZVI synthesis. The reactivity of nZVIs was assessed by measuring their capacity to reduce hexavalent chromium Cr (VI) in aqueous solutions prepared from potassium dichromate (K2Cr2O7)

The correlation of the steady-state gas/water relative permeabilities of porous media with gas and water capillary numbers

The steady-state gas, k rg, and water, k rw, relative permeabilities are measured with experiments of the simultaneous flow, at varying flow rates, of nitrogen and brine (aqueous solution of NaCl brine) on a homogeneous sand column. Two differential pressure transducers are used to measure the pressure drop across each phase, and six ring electrodes are used to measure the electrical resistance across five segments of the sand column. The electrical resistances are converted to water saturations with the aid of the Archie equation for resistivity index. Both k rw and k rg are regarded as power functions of water, Caw, and gas, Cag, capillary numbers, the exponents of which are estimated with non-linear fitting to the experimental datasets. An analogous power law is used to express water saturation as a function of Caw, and Cag. In agreement to earlier studies, it seems that the two-phase flow regime is dominated by connected pathway flow and disconnected ganglia dynamics for the wetting fluid (brine), and only disconnected ganglia dynamics for the non-wetting fluid (gas). The water saturation is insensitive to changes of water and gas capillary numbers. Each relative permeability is affected by both water and gas capillary numbers, with the water relative permeability being a strong function of water capillary number and gas relative permeability depending strongly on the gas capillary number. The slope of the water relative permeability curve for a gas/water system is much higher than that of an oil/water system, and the slope of the gas relative permeability is lower than that of an oil/water system

HIV persistence during antiretroviral therapy: Characteristics of residual HIV-1 RNA in plasma

Anti-retroviral therapy (ART) is highly effective in suppressing human immunodeficiency virus (HIV) and restoring immunocompetence. However, residual levels of HIV-1 RNA persist in patients with consistent viral suppression (viral load <50 copies/mL) and some immune functions are not reversed. Characterisation of residual viraemia (HIV-1 RNA <10 copies/mL) will facilitate to understand HIV persistence. A systematic review of literature was conducted to identify predictors of low level viraemia (HIV-1 RNA = 10-200 copies/mL) in patients on successful ART. To investigate factors associated with residual viraemia, we recruited a cohort of stably treated patients on first line ART with a non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI) based regimen and no evidence of virological failure. We measured HIV-1 RNA in plasma with our in-house single copy assay (SCA) and we studied total HIV-1 DNA in peripheral blood mononuclear cells (PBMC) and PD1 expression on memory CD4 and CD8 T populations. A subset of our study cohort was sampled twice during consecutive routine visits at the HIV clinic to perform a longitudinal analysis of HIV-1 RNA in plasma, total HIV-1 DNA in PBMC and drug levels. Residual viraemia was identified in over one third (35.9%, n=19 of N=53) of subjects with a median of 3 copies/mL. In the longitudinal cohort (n=32), HIV-1 RNA presence in plasma was reproducible within an interval period of 4 months with 53% (n=17) having undetectable HIV-1 RNA at both time points and 25% (n=8) experiencing consistently detectable HIV-1 RNA. Total HIV-1 DNA in PBMC was strongly associated with residual viraemia (OR: 3.42, 95% CI: 1.32-8.83, p=0.011) in our study population. In the systematic review, low level viraemia was also associated with markers reflecting a bigger cellular reservoir including higher pre-ART viral load, total HIV-1 DNA in PBMC and shorter ART duration. Moreover, low level viraemia was more frequent with reduced adherence and with PI-based ART. The same trends were also seen in our cohort, but the number of patients on PI-based regimens (n=11) and with sub-optimal efavirenz levels (n=6) were small to allow statistical comparisons. Microbial translocation and immune activation were associated with low level viraemia in the review. On the contrary, our patients with undetectable HIV-1 RNA had higher PD1 expression on effector memory CD4 cells (median 43.9% vs. 33.7%, p=0.028), effector memory CD8 cells (median 37.5% vs. 23%, p=0.022) and central memory CD8 cells (median 24.9% vs. 15.7%, p=0.034) compared to those with detectable HIV-1 RNA. Our findings suggest that residual viraemia is probably clonal in our cohort of patients deriving mainly during activation of latently infected cells, which causes bursts of virus production. We noted that common factors were associated with both residual and low level viraemia. Although we consider residual and low level viraemia as two different entities, their sources may overlap. In this context, ongoing viral replication may drive residual viraemia in a small subset of patients. We also made an interesting observation as to PD1 expression on memory CD4 and CD8 subsets which warrants further investigation

The impact of obesity and bariatric surgery on the immune microenvironment of the endometrium

BACKGROUND: The incidence of endometrial cancer is rising in parallel with the obesity epidemic. Obesity increases endometrial cancer risk and weight loss is protective, but the underlying mechanisms are incompletely understood. We hypothesise that the immune microenvironment may influence susceptibility to malignant transformation in the endometrium. The aim of this study was to measure the impact of obesity and weight loss on the immunological landscape of the endometrium. METHODS: We conducted a prospective cohort study of women with class III obesity (body mass index, BMI ≥ 40 kg/m(2)) undergoing bariatric surgery or medically-supervised low-calorie diet. We collected blood and endometrial samples at baseline, and two and 12 months after weight loss intervention. Serum was analysed for inflammatory markers CRP, IL-6 and TNF-α. Multiplex immunofluorescence was used to simultaneously identify cells positive for immune markers CD68, CD56, CD3, CD8, FOXP3 and PD-1 in formalin-fixed paraffin-embedded endometrial tissue sections. Kruskal–Wallis tests were used to determine whether changes in inflammatory and immune biomarkers were associated with weight loss. RESULTS: Forty-three women with matched serum and tissue samples at all three time points were included in the analysis. Their median age and BMI were 44 years and 52 kg/m(2), respectively. Weight loss at 12 months was greater in women who received bariatric surgery (n = 37, median 63.3 kg) than low-calorie diet (n = 6, median 12.8 kg). There were significant reductions in serum CRP (p = 3.62 × 10(−6), r = 0.570) and IL-6 (p = 0.0003, r = 0.459), but not TNF-α levels, with weight loss. Tissue immune cell densities were unchanged except for CD8+ cells, which increased significantly with weight loss (p = 0.0097, r = −0.323). Tissue CD3+ cell density correlated negatively with systemic IL-6 levels (p = 0.0376; r = −0.318). CONCLUSION: Weight loss is associated with reduced systemic inflammation and a recruitment of protective immune cell types to the endometrium, supporting the concept that immune surveillance may play a role in endometrial cancer prevention

Detection of the delayed condensation effect and determination of its impact on the accuracy of gas adsorption pore size distributions

Macroscopic, highly disordered, mesoporous materials present a continuing challenge for accurate pore structure characterization. The typical macroscopic variation in local average pore space descriptors means that methods capable of delivering statistically representative characterizations are required. Gas adsorption is a representative but indirect method, normally requiring assumptions about the correct model for data analysis. In this work we present a novel method to both expand the range, and obtain greater accuracy, for the information obtained from the main boundary adsorption isotherms by using a combination of data obtained for two adsorptives, namely nitrogen and argon, both before and after mercury porosimetry. The method makes use of the fact that nitrogen and argon apparently ‘see’ a different pore geometry following mercury entrapment, with argon, relatively, ‘ignoring’ new metal surfaces produced by mercury porosimetry. The new method permits the study of network and pore–pore co-operative effects during adsorption that substantially affect the accuracy of the characteristic parameters, such as modal pore size, obtained for disordered materials. These effects have been explicitly quantified, for a typical sol-gel silica catalyst support material as a case study. The technique allowed the large discrepancies between modal pore sizes obtained from standard gas adsorption and mercury thermoporometry methods to be attributed to the network-based delayed condensation effect, rather than spinodal adsorption. Once the network-based delayed condensation effect had been accounted for, the simple cylindrical pore model and macroscopic thermodynamic Kelvin-Cohan equation were then found sufficient to accurately describe adsorption in the material studied, rather than needing a more complex microscopic theory. Hence, for disordered mesoporous solids, a proper account of inter-pore interactions is more important than that of intra-pore adsorbate density distribution, to obtain accurate pore size distributions