The Janus face of DNA methylation in aging

Aging is arguably the most familiar yet least-well understood aspect of human biology. The role of epigenetics in aging and age-related diseases has gained interest given recent advances in the understanding of how epigenetic mechanisms mediate the interactions between the environment and the genetic blueprint. While current concepts generally view global deteriorations of epigenetic marks to insidiously impair cellular and molecular functions, an active role for epigenetic changes in aging has so far received little attention. In this regard, we have recently shown that early-life adversity induced specific changes in DNA methylation that were protected from an age-associated erasure and correlated with a phenotype well-known to increase the risk for age-related mental disorders. This finding strengthens the idea that DNA (de-)methylation is controlled by multiple mechanisms that might fulfill different, and partly contrasting, roles in the aging process

Trial of erythropoietin for hypoxic-ischemic encephalopathy in newborns

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy is an important cause of death as well as long-term disability in survivors. Erythropoietin has been hypothesized to have neuroprotective effects in infants with hypoxic-ischemic encephalopathy, but its effects on neurodevelopmental outcomes when given in conjunction with therapeutic hypothermia are unknown. METHODS: In a multicenter, double-blind, randomized, placebo-controlled trial, we assigned 501 infants born at 36 weeks or more of gestation with moderate or severe hypoxic-ischemic encephalopathy to receive erythropoietin or placebo, in conjunction with standard therapeutic hypothermia. Erythropoietin (1000 U per kilogram of body weight) or saline placebo was administered intravenously within 26 hours after birth, as well as at 2, 3, 4, and 7 days of age. The primary outcome was death or neurodevelopmental impairment at 22 to 36 months of age. Neurodevelopmental impairment was defined as cerebral palsy, a Gross Motor Function Classification System level of at least 1 (on a scale of 0 [normal] to 5 [most impaired]), or a cognitive score of less than 90 (which corresponds to 0.67 SD below the mean, with higher scores indicating better performance) on the Bayley Scales of Infant and Toddler Development, third edition. RESULTS: Of 500 infants in the modified intention-to-treat analysis, 257 received erythropoietin and 243 received placebo. The incidence of death or neurodevelopmental impairment was 52.5% in the erythropoietin group and 49.5% in the placebo group (relative risk, 1.03; 95% confidence interval [CI], 0.86 to 1.24; P = 0.74). The mean number of serious adverse events per child was higher in the erythropoietin group than in the placebo group (0.86 vs. 0.67; relative risk, 1.26; 95% CI, 1.01 to 1.57). CONCLUSIONS: The administration of erythropoietin to newborns undergoing therapeutic hypothermia for hypoxic-ischemic encephalopathy did not result in a lower risk of death or neurodevelopmental impairment than placebo and was associated with a higher rate of serious adverse events. (Funded by the National Institute of Neurological Disorders and Stroke; ClinicalTrials.gov number, NCT02811263.)

Efficient influenza vaccine manufacturing: Single MDCK suspension cells in chemically defined medium

Facing the constant global high demand for influenza vaccines, improving production capacity is most important. For influenza vaccine production, cell culture-based processes have advantages regarding flexibility, efficiency, and safety in comparison with the traditional egg-based processes. To avoid problems related to microcarrier-based approaches and serum containing media, growth of suspension cells in chemically-defined media is favoured. In addition, such a process has advantages regarding the improvement of virus titers, the scale-up of the production process, and overall productivity in up- and downstream processing. In this study, a previously developed MDCK suspension cell line [1] was cultivated in an in-house chemically defined medium to evaluate cell growth and virus production. For the purpose of process intensification, virus adaptation and infection strategies were investigated to achieve high cell densities and to maximize virus titers. Therefore, an adapted influenza virus strain (A/PR/8/34 H1N1 RK1) was generated by a series of virus passages with low multiplicity of infection (MOI). Virus infections were carried out by supplementing 100% of fresh medium, infecting cells with a MOI of 10-3, and with trypsin addition at 72 h of cell cultivations in shake flasks and bioreactors. For scale-up, MDCK cells were cultivated in a DASGIP bioreactor system, optimizing stirring speed, time of infection, pH and DO levels both for cell growth and virus infection. Cell count, viability, main extracellular metabolites, and virus titers were measured to compare productivity between shake flasks and bioreactors. In batch culture (shake flasks and bioreactors), single MDCK cells were grown to maximum cell densities of 1.2 x107 cells/ml with cell viabilities exceeding 98% at high cell specific growth rates of 0.036 h-1. Virus adaptation to the MDCK suspension cell line led to a fast infection and stable virus titers over time. Regarding process optimization, optimal pH (cell growth: 7.00, infection: 7.20), DO (40%) and agitation speed (80 rpm) were chosen for influenza A virus production in three parallel bioreactors. Cell densities of 1.0 x107 cells/ml were achieved at time of infection (72 h) before performing a dilution step. Post infection, similar virus infection dynamics were observed in shake flasks and bioreactors. For both cultivation systems maximal HA titers of 3.6 log10(HAU/100µl) were achieved without reduction of cell-specific virus titer (12,000 virions/cell). Overall, a highly efficient and scalable upstream process was realized by cultivation of MDCK suspension cells as single cells in chemically defined medium. This is a strong basis for a promising application in large-scale influenza vaccine manufacturing and potential process intensification towards high cell density virus production. [1] Huang D. et al., PloS One 10 (2015): e0141686. doi: 10.1371/journal.pone.014168

Highly efficient influenza virus production: A MDCK-based high-cell-density process

Seasonal vaccination campaigns for influenza in developed and developing countries create a massive demand for 500 million (2015) vaccine doses every year [1]. Besides egg-based vaccine manufacturing, production platforms based on animal cell culture increasingly contribute to this overall growing market. In order to intensify cell culture-based influenza virus production, high-cell-density (HCD) cultivation of suspension cells can be applied to improve virus titer, process productivity and production costs [2]. For process optimization and evaluation of HCD conditions, cells cultivated using semi-perfusion approaches in small shakers can be used as a scale-down model for bioreactors operating in full perfusion mode [3]. In this study, a previously developed MDCK suspension cell line [4] was adapted to a new serum free medium [5] to facilitate higher growth rate, cell density and virus titer both in batch and in HCD. Therefore, MDCK cells cultivated in Smif-8 medium were slowly adapted to a new cultivation medium (Xeno™) by stepwise increasing the Xeno content. Fully adapted cells were cultivated in shaker flasks to evaluate the performance of influenza A virus production in batch and HCD. Cell densities exceeding 2∙107 cells/mL were achieved in shakers using semi-perfusion, where cell free medium was manually replaced with fresh medium. Volume and time interval of media replacement were chosen to achieve a constant cell-specific perfusion rate of 2.5 pL/(cell h). Cell cultures were infected with influenza virus (A/PR/8/34 H1N1 RKI) with trypsin addition. Cell count, viability, main metabolites and virus titer (HA-assay & TCID50) were monitored pre and post infection. Medium adaptation resulted in a MDCK suspension cell line with morphological, growth, and metabolic characteristics different from parental cells. Cells fully adapted to Xeno medium were growing to higher cell densities (1.4∙107 vs 6∙106 cells/mL) with higher specific growth rate (µmax: 0.036 vs 0.026 1/h), cells were bigger (15-16 vs 13-14 µm) and grew without aggregate formation. Due to higher cell densities at time of infection, virus titers up to 3.6 log10(HAU/100µL) were reached. In semi-perfusion, adapted MDCK cells were grown up to 6∙107 cells/mL, however, maximum virus titer and productivity were observed with 4∙107 cells/mL. In multiple harvests, very high virus titer exceeding 4 log10(HAU/100µL) and up to 9∙109 virions/mL (TCID50) were measured, which corresponded to an accumulated titer of 4.5 log10(HAU/100µL). Cell-specific virus titer was similar or higher compared to the reference batch infections, depending on perfusion and infection strategy. Overall, results in this semi-perfusion scale-down model for influenza A virus production suggest a highly efficient and productive upstream process for influenza virus production, with an up to six-fold improved space time yield compared to batch mode. [1] Palache A. et al., Vaccine 35 (2017): 4681–4686. doi: 10.1016/j.vaccine.2017.07.053 [2] Genzel Y. et al., Vaccine 32 (2014): 2770–2781. doi: 10.1016/j.vaccine.2014.02.016 [3] Vázquez-Ramírez D. et al., Vaccine (2018): article in press. doi: 10.1016/j.vaccine.2017.10.112 [4] Lohr V. et al., Vaccine 28 (2010): 6256–6264. doi: 10.1016/j.vaccine.2010.07.004 [5] Xeno™-S001S MDCK Cell Serum Free Medium (#FG0100402), Bioengine, Shanghai, Chin

Human Tear Fluid Reduces Culturability of Contact Lens-Associated Pseudomonas aeruginosa Biofilms but Induces Expression of the Virulence-Associated Type III Secretion System

Purpose The type III secretion system (T3SS) is a significant virulence determinant for Pseudomonas aeruginosa. Using a rodent model, we found that contact lens(CL)-related corneal infections were associated with lens surface biofilms. Here, we studied the impact of human tear fluid on CL-associated biofilm growth and T3SS expression. Methods P. aeruginosa biofilms were formed on contact lenses for up to 7 days with or without human tear fluid, then exposed to tear fluid for 5 or 24 h. Biofilms were imaged using confocal microscopy. Bacterial culturability was quantified by viable counts, and T3SS gene expression measured by RT-qPCR. Controls included trypticase soy broth, PBS and planktonic bacteria. Results With or without tear fluid, biofilms grew to ∼108 CFU viable bacteria by 24 h. Exposing biofilms to tear fluid after they had formed without it on lenses reduced bacterial culturability ∼180-fold (P\u3c.001). CL growth increased T3SS gene expression versus planktonic bacteria [5.46 ± 0.24-fold for T3SS transcriptional activitor exsA (P=.02), and 3.76 ± 0.36-fold for T3SS effector toxin exoS(P=.01)]. Tear fluid further enhanced exsA and exoS expression in CL-grown biofilms, but not planktonic bacteria, by 2.09 ± 0.38-fold (P=.04) and 1.89 ± 0.26-fold (P\u3c.001), respectively. Conclusions Considering the pivitol role of the T3SS in P. aeruginosa infections, its induction in CL-grown P. aeruginosa biofilms by tear fluid might contribute to the pathogenesis of CL-related P. aeruginosa keratitis

Smog Nitrogen and the Rapid Acidification of Forest Soil, San Bernardino Mountains, Southern California

We report the rapid acidification of forest soils in the San Bernardino Mountains of southern California. After 30 years, soil to a depth of 25 cm has decreased from a pH (measured in 0.01 M CaCl2) of 4.8 to 3.1. At the 50-cm depth, it has changed from a pH of 4.8 to 4.2. We attribute this rapid change in soil reactivity to very high rates of anthropogenic atmospheric nitrogen (N) added to the soil surface (72 kg ha–1 year–1) from wet, dry, and fog deposition under a Mediterranean climate. Our research suggests that a soil textural discontinuity, related to a buried ancient landsurface, contributes to this rapid acidification by controlling the spatial and temporal movement of precipitation into the landsurface. As a result, the depth to which dissolved anthropogenic N as nitrate (NO3) is leached early in the winter wet season is limited to within the top ~130 cm of soil where it accumulates and increases soil acidity

Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V

<p>Abstract</p> <p>Background</p> <p>A unique and essential property of embryonic stem cells is the ability to self-renew and differentiate into multiple cell lineages. However, the possible differences in proliferation and differentiation capabilities among independently-derived human embryonic stem cells (hESCs) are not well known because of insufficient characterization. To address this question, a side-by-side comparison of 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) the responses to directed neural differentiation was made between three NIH registered hES cell lines I3 (TE03), I6 (TE06) and BG01V. Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01.</p> <p>Results</p> <p>Using immunocytochemistry, flow cytometry, qRT-PCR and MPSS, we found that all three cell lines actively proliferated and expressed similar "stemness" markers including transcription factors POU5F1/Oct3/4 and NANOG, glycolipids SSEA4 and TRA-1-81, and alkaline phosphatase activity. All cell lines differentiated into three embryonic germ lineages in embryoid bodies and into neural cell lineages when cultured in neural differentiation medium. However, a profound variation in colony morphology, growth rate, BrdU incorporation, and relative abundance of gene expression in undifferentiated and differentiated states of the cell lines was observed. Undifferentiated I3 cells grew significantly slower but their differentiation potential was greater than I6 and BG01V. Under the same neural differentiation-promoting conditions, the ability of each cell line to differentiate into neural progenitors varied.</p> <p>Conclusion</p> <p>Our comparative analysis provides further evidence for similarities and differences between three hESC lines in self-renewal, and spontaneous and directed differentiation. These differences may be associated with inherited variation in the sex, stage, quality and genetic background of embryos used for hESC line derivation, and/or changes acquired during passaging in culture.</p

Removal of Biofilm from Contact Lens Storage Cases

PURPOSE. Lens case hygiene practices are important in maintaining safe contact lens wear. However, the effectiveness of various lens case cleaning practices have not been evaluated and compared. This in vitro study aimed to evaluate and compare the efficacy of cleaning practices that are most commonly carried out by lens wearers and recommended by practitioners. METHODS. Pseudomonas aeruginosa 122, Serratia marcescens ATCC 13880, and Staphylococcus aureus ATCC 6538 were the challenge bacteria for biofilm formation on unused lens cases from two different manufacturers. After establishment of the biofilm, each lens case was subjected to one of the six cleaning regimens: &quot;rinsed,&quot; &quot;rubbed and rinsed,&quot; &quot;airdried,&quot; &quot;soaked in a multipurpose contact lens solution,&quot; &quot;tissue-wiped,&quot; and &quot;lids recapped.&quot; The level of residual biofilm was quantified at the end of each cleaning regimen. The efficacy of each cleaning regimen was then compared. RESULTS. Mechanical rubbing and wiping of lens cases were the most effective cleaning regimen tested in reducing biofilm. Soaking lenses in disinfecting solution for 6 hours removed the majority of biofilm from lens cases. Rinsing lens cases alone provided only minimal efficacy in reducing biofilm. Air-drying or recapping the cases with the lid without any other additional cleaning methods were the least efficient at removing biofilm. CONCLUSIONS. Based on this study, digital rubbing and rinsing and/or wiping the lens cases with tissue is recommended. Air-drying or recapping the lens case lids after use without any additional cleaning methods should be discouraged with nonantimicrobial lens cases. (Invest Ophthalmol Vis Sci. 2010;51: 6329 -6333