Expression of estrogen receptor (ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation

The aim of the current study is to demonstrate normal and malignant prostatic epithelial cells (PrECs) as targets for receptor-mediated estrogenic and antiestrogenic action. Using an improved protocol, we have successfully isolated and maintained highly enriched populations of normal PrECs from ultrasound-guided peripheral zone biopsies, individually determined to be morphologically normal. Semiquantitative reverse transcription-PCR analyses were used to determine whether transcripts of estrogen receptor (ER)-alpha and those of ER-beta were expressed in our normal PrEC primary cultures, in a commercially available PrEC preparation (PrEC; Clontech), in an immortalized PrEC line established from a benign prostatic hyperplasia specimen (BPH-1), and in three prostatic cancer cell lines (LNCaP, PC-3, and DU145). Expression levels of ER-alpha and ER-beta transcripts were related to those of two estrogen-responsive genes [progesterone receptor (PR) and pS2], at the message levels, to gain insights into the functionality of the ER subtypes in PrECs. Interestingly, only transcripts of ER-beta, but not those of ER-alpha, were found in our primary cultures of normal PrECs, along with both PR and pS2 mRNA. These data strongly suggest that estrogen action was signaled exclusively via ER-beta in normal human PrECs. In contrast, PrEC (Clontech) and BPH-1 cells expressed both ER-alpha and ER-beta transcripts and no PR nor pS2 mRNA in PrEC and only a minimal level of PR mRNA in BPH-1. Among the three prostate cancer cell lines, LNCaP expressed ER-beta mRNA along with transcripts of PR and pS2, DU145 expressed messages of ER-beta and PR, and PC-3 cells exhibited ER-alpha, ER-beta, and pS2 mRNA. Thus, unlike normal PrECs, expression patterns of these genes in malignant PrECs are more variable. Treatment of prostate cancer cells with demethylation agents effectively reactivated the expression of ER-alpha mRNA in LNCaP and DU145 and that of pS2 message in DU145. These findings provide experimental evidence that ER-alpha gene silencing in prostate cancer cells, and perhaps also in normal PrECs, are caused by DNA hypermethylation. To evaluate the potential of using antiestrogens as prostate cancer therapies, we have assessed the growth-inhibitory action of estrogens (estradiol and diethylstilbestrol) and antiestrogens (4-hydroxy-tamoxifen and ICI-182,780) on PC-3 and DU-145 cells. In PC-3 cells, which express both ER subtypes, estrogens as well as antiestrogens are effective inhibitors. In contrast, in DU145 cells, which express only ER-beta, antiestrogens, but not estrogens, are growth inhibitors. By comparison, ICI 182,780 is the more effective cell growth inhibitor. Importantly, the ICI 182,780-induced antiproliferative effects were reversed by cotreatment of DU145 cells with an ER-beta antisense oligonucleotide, hence lending additional support to a central role played by ER-beta in mediating growth-inhibitory action of antiestrogens

Structural and Electrical Characterization of a Novel Mixed Conductor: CeO\u3csub\u3e2\u3c/sub\u3e - Sm\u3csub\u3e2\u3c/sub\u3eO\u3csub\u3e3\u3c/sub\u3e - ZrO\u3csub\u3e2\u3c/sub\u3e Solid Solution

solid solutions were synthesized for the first time by the hydrothermal method. The electrical properties of the solid solutions have been studied in air and under reducing conditions. Solid solutions with the fluorite structure were formed in all of the studied range of substitution after calcination at 1500°C. With increasing substitution up to 30 mol %, the electronic conductivity increases under a reducing atmosphere. The solid solution has good mixed electronic and ionic conductivity; the total conductivity is 0.42 S/cm at and 700°C with an estimated ionic conductivity of ca.

Cell Density Plays a Critical Role in Ex Vivo Expansion of T Cells for Adoptive Immunotherapy

The successful ex vivo expansion of a large numbers of T cells is a prerequisite for adoptive immunotherapy. In this study, we found that cell density had important effects on the process of expansion of T cells in vitro. Resting T cells were activated to expand at high cell density but failed to be activated at low cell density. Activated T cells (ATCs) expanded rapidly at high cell density but underwent apoptosis at low cell density. Our studies indicated that low-cell-density related ATC death is mediated by oxidative stress. Antioxidants N-acetylcysteine, catalase, and albumin suppressed elevated reactive oxygen species (ROS) levels in low-density cultures and protected ATCs from apoptosis. The viability of ATCs at low density was preserved by conditioned medium from high-density cultures of ATCs in which the autocrine survival factor was identified as catalase. We also found that costimulatory signal CD28 increases T cell activation at lower cell density, paralleled by an increase in catalase secretion. Our findings highlight the importance of cell density in T cell activation, proliferation, survival and apoptosis and support the importance of maintaining T cells at high density for their successful expansion in vitro

Stage-Specific Generation of Human Pluripotent Stem Cell Derived Lung Models to Measure CFTR Function

Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic-activated cell sorting system, and the generation of adult-like airway epithelia through air-liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air-liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture

Ageing memory and glassiness of a driven vortex system

Many systems in nature, glasses, interfaces and fractures being some examples, cannot equilibrate with their environment, which gives rise to novel and surprising behaviour such as memory effects, ageing and nonlinear dynamics. Unlike their equilibrated counterparts, the dynamics of out-of- equilibrium systems is generally too complex to be captured by simple macroscopic laws. Here we investigate a system that straddles the boundary between glass and crystal: a Bragg glass formed by vortices in a superconductor. We find that the response to an applied force evolves according to a stretched exponential, with the exponent reflecting the deviation from equilibrium. After the force is removed, the system ages with time and its subsequent response time scales linearly with its age (simple ageing), meaning that older systems are slower than younger ones. We show that simple ageing can occur naturally in the presence of sufficient quenched disorder. Moreover, the hierarchical distribution of timescales, arising when chunks of loose vortices cannot move before trapped ones become dislodged, leads to a stretched-exponential response.Comment: 16 pages, 5 figure

Equilibration and Dynamic Phase Transitions of a Driven Vortex Lattice

We report on the observation of two types of current driven transitions in metastable vortex lattices. The metastable states, which are missed in usual slow transport measurements, are detected with a fast transport technique in the vortex lattice of undoped 2H-NbSe$_2$. The transitions are seen by following the evolution of these states when driven by a current. At low currents we observe an equilibration transition from a metastable to a stable state, followed by a dynamic crystallization transition at high currents.Comment: 5 pages, 4 figure

Edge and Bulk Transport in the Mixed State of a Type-II Superconductor

By comparing the voltage-current (V-I) curves obtained before and after cutting a sample of 2H-NbSe2, we separate the bulk and edge contributions to the transport current at various dissipation levels and derive their respective V- I curves and critical currents. We find that the edge contribution is thermally activated across a current dependent surface barrier. By contrast the bulk V-I curves are linear, as expected from the free flux flow model. The relative importance of bulk and edge contributions is found to depend on dissipation level and sample dimensions. We further show that the peak effect is a sharp bulk phenomenon and that it is broadened by the edge contribution

Epigenetic Changes with Dietary Soy in Cynomolgus Monkeys

Nutritional interventions are important alternatives for reducing the prevalence of many chronic diseases. Soy is a good source of protein that contains isoflavones, including genistein and daidzein, and may alter the risk of obesity, Type 2 diabetes, osteoporosis, cardiovascular disease, and reproductive cancers. We have shown previously in nonhuman primates that soy protein containing isoflavones leads to improved body weight, insulin sensitivity, lipid profiles, and atherosclerosis compared to protein without soy isoflavones (casein), and does not increase the risk of cancer. Since genistein has been shown to alter DNA methylation, we compared the methylation profiles of cynomolgus monkeys, from multiple tissues, eating two high-fat, typical American diets (TAD) with similar macronutrient contents, with or without soy protein. DNA methylation status was successfully determined for 80.6% of the probes in at least one tissue using Illumina's HumanMethylation27 BeadChip. Overall methylation increased in liver and muscle tissue when monkeys switched from the TAD-soy to the TAD-casein diets. Genes involved in epigenetic processes, specifically homeobox genes (HOXA5, HOXA11, and HOXB1), and ABCG5 were among those that changed between diets. These data support the use of the HumanMethylation27 BeadChip in cynomolgus monkeys and identify epigenetic changes associated with dietary interventions with soy protein that may potentially affect the etiology of complex diseases

Quantification of Beat-To-Beat Variability of Action Potential Durations in Langendorff-Perfused Mouse Hearts

Background: Beat-to-beat variability in action potential duration (APD) is an intrinsic property of cardiac tissue and is altered in pro-arrhythmic states. However, it has never been examined in mice.Methods: Left atrial or ventricular monophasic action potentials (MAPs) were recorded from Langendorff-perfused mouse hearts during regular 8 Hz pacing. Time-domain, frequency-domain and non-linear analyses were used to quantify APD variability.Results: Mean atrial APD (90% repolarization) was 23.5 ± 6.3 ms and standard deviation (SD) was 0.9 ± 0.5 ms (n = 6 hearts). Coefficient of variation (CoV) was 4.0 ± 1.9% and root mean square (RMS) of successive differences in APDs was 0.3 ± 0.2 ms. The peaks for low- and high-frequency were 0.7 ± 0.5 and 2.7 ± 0.9 Hz, respectively, with percentage powers of 39.0 ± 20.5 and 59.3 ± 22.9%. Poincaré plots of APDn+1 against APDn revealed ellipsoid shapes. The ratio of the SD along the line-of-identity (SD2) to the SD perpendicular to the line-of-identity (SD1) was 8.28 ± 4.78. Approximate and sample entropy were 0.57 ± 0.12 and 0.57 ± 0.15, respectively. Detrended fluctuation analysis revealed short- and long-term fluctuation slopes of 1.80 ± 0.15 and 0.85 ± 0.29, respectively. When compared to atrial APDs, ventricular APDs were longer (ANOVA, P < 0.05), showed lower mean SD and CoV but similar RMS of successive differences in APDs and showed lower SD2 (P < 0.05). No difference in the remaining parameters was observed.Conclusion: Beat-to-beat variability in APD is observed in mouse hearts during regular pacing. Atrial MAPs showed greater degree of variability than ventricular MAPs. Non-linear techniques offer further insights on short-term and long-term variability and signal complexity