Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

Background

Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

Methods

CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

Results

Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2^′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O₂.

Conclusion

These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.     

Inhibition by levamisole of metastases by cells transformed by herpes simplex virus type I.

Levamisole was tested to determine whether the drug could reduce metastases by HSV-1-transformed cells in a model hamster system. The results presented reveal an inhibition of metastases to the lungs even when the drug is inoculated after development of subcutaneous tumors at the site of inoculation of the cells.     

Cellulose degradation and monitoring of viscosity decrease in cultures of Cellulomonas uda grown on printed newspaper

The rheological behavior of cultures of Cellulomonas uda with shredded printed newspaper as the carbon source was studied. The initial substrate concentrations ranged from 23 to 60 g/L. The changes in apparent viscosity were followed on-line by applying a commercially available process viscometer and discretely using a rotational viscometer with an anchor impeller. During the time of highest cellulose degradation, the broths exhibited a pseudoplastic behavior which could be explained satisfactorily by the power-law model. At the end of cultivation when cellulose degradation slowed down, the broths became Newtonian in behavior. Endo-1,4-beta-glucanase, 1,4-beta-xylanase, beta-glucosidase, and beta-xylosidase activities were also determined during cultivation as well as cellulose degradation and cell mass production. The beginning of endoglucanase formation and the start of the final viscosity decrease of the bacterial paper pulp suspensions could be correlated.     

Social predation in electric eels

Social predation—when groups of predators coordinate actions to find and capture prey—is a common tactic among mammals but comparatively rare in fishes. We report the unexpected social predation by electric eels, an otherwise solitary predator in the Amazon rainforest. Observations made in different years and recorded on video show electric eels herding, encircling shoals of small nektonic fishes, and launching joint predatory high‐voltage strikes on the prey ball. These findings challenge the hypothesis that electric eels may have a single foraging strategy and extend our knowledge on social predation to an organism that employs high‐voltage discharge for hunting. Thereby offering a novel perspective for studies on the evolutionary interplay between predatory and escape tactics.     

More diversity,less tolerance? The effect of type of cultural diversity on the erosion of tolerance in Swiss municipalities

Immigration and the ways in which host societies receive newcomers pose challenges for modern civil societies. This article contributes to the ongoing discussion about how ethnic diversity influences tolerance towards immigrants. Compared to previous studies, we analyse tolerance as a sequential concept in order to uncover the effects of contextual diversity on attitudes towards immigrants and the granting of certain rights to this group. Moreover, we distinguish different shares of ethnic groups based on their ethnic and cultural origins both on the independent and dependent variable. The analysis relies on a subnational survey of sixty municipalities in Switzerland, revealing that only certain ethnic groups are seen as an economic and cultural threat.     

Properties of Hamster Embryo Fibroblasts Transformed In Vitro After Exposure to Ultraviolet-Irradiated Herpes Simplex Virus Type 2

An in vitro method which led to the transformation of hamster embryo fibroblasts after exposure to herpes simplex virus type 2 (HSV-2) inactivated with ultraviolet irradiation is described. The transformed cells (333-8-9) produced tumors when inoculated into newborn Syrian hamsters but not when injected into weanling Syrian hamsters of the same LSH inbred strain. However, after one in vivo passage, the 333-8-9 cells became highly oncogenic in weanling hamsters. No infectious virus was recovered from these cells. Herpes simplex virus antigens were detected in the transformed cells by the indirect immunofluorescence technique. Sera from tumor-bearing hamsters contained antibody with highly specific neutralizing activity against HSV-2. These studies indicate the continued involvement of the HSV-2 genome in an oncogenic cell line.     

High-density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.     

Risk Perception and Risk-Taking Behaviour during Adolescence: The Influence of Personality and Gender

This study investigated the influence of personality characteristics and gender on adolescents’ perception of risk and their risk-taking behaviour. Male and female participants (157 females: 116 males, aged 13–20) completed self-report measures on risk perception, risk-taking and personality. Male participants perceived behaviours as less risky, reportedly took more risks, were less sensitive to negative outcomes and less socially anxious than female participants. Path analysis identified a model in which age, behavioural inhibition and impulsiveness directly influenced risk perception, while age, social anxiety, impulsiveness, sensitivity to reward, behavioural inhibition and risk perception itself were directly or indirectly associated with risk-taking behaviour. Age and behavioural inhibition had direct relationships with social anxiety, and reward sensitivity was associated with impulsiveness. The model was representative for the whole sample and male and female groups separately. The observed relationship between age and social anxiety and the influence this may have on risk-taking behaviour could be key for reducing adolescent risk-taking behaviour. Even though adolescents may understand the riskiness of their behaviour and estimate their vulnerability to risk at a similar level to adults, factors such as anxiety regarding social situations, sensitivity to reward and impulsiveness may exert their influence and make these individuals prone to taking risks. If these associations are proven causal, these factors are, and will continue to be, important targets in prevention and intervention efforts.     

Kinetics of expression of infectious ecotropic, xenotropic, and mink cell focus-forming murine leukemia virus after 5-iododeoxyuridine induction of cells from high- and low-leukemia mouse strains

Endogenous murine leukemia virus (MuLV) was induced with 5-iododeoxyuridine (IdUrd) from the high-leukemia mouse strain AKR and from two low-leukemia strains, C3H/He and BALB/c. A virus-free cell line from strain AKR readily gave rise to infectious, XC-positive MuLV upon treatment with IdUrd, whereas cells from strains C3H/He and BALB/c produced replication-deficient, XC-negative MuLV. IdUrd-induced cells also produced xenotropic and mink cell focus-forming MuLV. Xenotropic virus emerged at a higher titer from both AKR and BALB/c cells during two discrete time intervals, first at day 3 after induction and a second time during spread of the induced ecotropic MuLV. Xenotropic and mink cell focus-forming MuLVs were also produced by IdUrd-induced C3H/He cells but required another round of infection in Sc-1 cells for detection. The in vitro infectivity of endogenous ecotropic MuLV isolated by IdUrd induction from C3H/He cells correlated with pathogenicity in the Fv-1-compatible, leukemia-negative mouse strain NFS/N. Thus, the virulence of endogenous ecotropic MuLV may be an important factor in determining the leukemia incidence in these inbred strains of mice.