Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

Calibration of a complex activated sludge model for the full-scale wastewater treatment plant

In this study, the results of the calibration of the complex activated sludge model implemented in BioWin software for the full-scale wastewater treatment plant are presented. Within the calibration of the model, sensitivity analysis of its parameters and the fractions of carbonaceous substrate were performed. In the steady-state and dynamic calibrations, a successful agreement between the measured and simulated values of the output variables was achieved. Sensitivity analysis revealed that upon the calculations of normalized sensitivity coefficient (Si,j) 17 (steady-state) or 19 (dynamic conditions) kinetic and stoichiometric parameters are sensitive. Most of them are associated with growth and decay of ordinary heterotrophic organisms and phosphorus accumulating organisms. The rankings of ten most sensitive parameters established on the basis of the calculations of the mean square sensitivity measure (δjmsqr) indicate that irrespective of the fact, whether the steady-state or dynamic calibration was performed, there is an agreement in the sensitivity of parameters

The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C

Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S25E, S45E, T435E, S595E). We also analyzed lamin C (A-type) and its mutant S37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R64H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S45E mutant was insoluble, in contrast to lamin C S37E. Lamin Dm T435E (C-terminal cdc2 site) and R64H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S45E and T435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T435E was cytoplasmic and showed higher mobility in FRAP assay

Discordant Gene Expression Signatures and Related Phenotypic Differences in Lamin A- and A/C-Related Hutchinson-Gilford Progeria Syndrome (HGPS)

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disorder displaying features reminiscent of premature senescence caused by germline mutations in the LMNA gene encoding lamin A and C, essential components of the nuclear lamina. By studying a family with homozygous LMNA mutation (K542N), we showed that HGPS can also be caused by mutations affecting both isoforms, lamin A and C. Here, we aimed to elucidate the molecular mechanisms underlying the pathogenesis in both, lamin A- (sporadic) and lamin A and C-related (hereditary) HGPS. For this, we performed detailed molecular studies on primary fibroblasts of hetero- and homozygous LMNA K542N mutation carriers, accompanied with clinical examinations related to the molecular findings. By assessing global gene expression we found substantial overlap in altered transcription profiles (13.7%; 90/657) in sporadic and hereditary HGPS, with 83.3% (75/90) concordant and 16.7% (15/90) discordant transcriptional changes. Among the concordant ones we observed down-regulation of TWIST2, whose inactivation in mice and humans leads to loss of subcutaneous fat and dermal appendages, and loss of expression in dermal fibroblasts and periadnexial cells from a LMNAK542N/K542N patient further confirming its pivotal role in skin development. Among the discordant transcriptional profiles we identified two key mediators of vascular calcification and bone metabolism, ENPP1 and OPG, which offer a molecular explanation for the major phenotypic differences in vascular and bone disease in sporadic and hereditary HGPS. Finally, this study correlates reduced TWIST2 and OPG expression with increased osteocalcin levels, thereby linking altered bone remodeling to energy homeostasis in hereditary HGPS

Fate of Trichloroethylene in Activated Sludge Systems

Abstract This study was undertaken to investigate the fate of a volatile organic priority pollutant, trichloroethylene (TCE), in activated sludge systems treating municipal wastewater. Batch stripping tests were carried out in a 2 L glass vessel to develop sampling techniques for vapour and liquid phases and to refine analytical protocols. The vapour sampling technique found to be most reliable involved collection of all exhaust vapours in a 40 L Tedlar bag. Equipment which was impervious to the volatile characteristics of TCE was imperative. Continuous flow experiments were carried out in a 15 L gas-tight bench-scale activated sludge system. Experiments were conducted for influent TCE concentrations ranging from 20 to 150 Mg/L and at aeration rates of 0.13 and 0.26 volume of air/volume of mixed liquor-minute. Mass balances for TCE were carried out when the TCE adsorbed by the biomass had reached an equilibrium concentration. A minimum of four hydraulic retention times (HRTs) were required before constant biomass TCE concentrations were observed. Subsequently, grab samples of influent, effluent and mixed liquor were taken within a discrete time interval during which a volume of exhaust gas was collected in a Tedlar bag. The mass balances were based upon TCE analysis of these samples. The first experiments were carried out without mixed liquor to eliminate possible loss of TCE by biodegradation. Approximately 10% of the influent TCE could not be accounted for in these experiments. This loss was attributed to sampling and analytical errors. With mixed liquor present, 60 to 70 % mass balance closures were achieved. Approximately 20 to 30 % of the TCE losses were attributed to biodegradation.</jats:p

Aerobic and Anaerobic Biological Treatment and Characterization of Condensate Wastewaters from the Taciuk Oil Process

Abstract Bench-scale investigations were conducted at Environment Canada's Wastewater Technology Centre to assess the feasibility of using biological activated sludge systems to treat fractionator sour water (PW5) and processor preheat steam vent water (Preheat) from the AOSTRA Taciuk Process when applied to bitumen recovery from oil sands. The PW5 wastewater sample was approximately 10 times more concentrated with regard to COD, TOC and ammonia concentrations than the Preheat sample. Phosphorus was lacking in each wastewater. High levels of cyanide were present in the Preheat sample and high levels of zinc in the PW5 sample. Aerobic treatment of the Preheat wastewater was shown to be feasible with regard to organic carbon removal. At a sludge retention time (SRT) of 20 days and hydraulic retention times (HRTs) ranging from 0.9 to 2.4 days, organic carbon removal of the Preheat wastewater in terms of BOD5 was greater than 95%. Nitrification was also achieved with ammonia removals of greater than 95%. The PW5 treatment system, operated at a 20-day SRT and HRTs ranging from 5 to 10 days, achieved BOD5 removals of approximately 90%. However, residual carbon levels of approximately 200-400 mg/L remained in the effluent. The addition of powdered activated carbon at the 5-day HRT level did not significantly improve system performance. Excessive foaming and effluent suspended solids losses contributed to operating problems. Nitrification was not established, possibly due to high influent ammonia values. Anaerobic toxicity testing showed that the Preheat wastewater was a relatively weak substrate which did not inhibit anaerobic microorganisms. The PW5 wastewater was toxic to these organisms and this was a substrate concentration-related phenomenon.</jats:p

The Application of Predenitrification Nitrification Technology for Trace Contaminant Control

Predenitrification nitrification (PDN) technology was applied in two stage anoxic-aerobic systems treating coke plant wastewaters and coal liquefaction process condensates. Both activated sludge and biological fluidized bed modes of operation were investigated for their potential to remove ammonia, phenol, thiocyanate, cyanide and a wide range of trace organic contaminants. It was demonstrated that during nitrification of these complex wastewaters, simultaneous removal of most of the conventional and trace organic contaminants was achieved. Specifically, of the base/neutral extractable organic compounds, only di-n-butyl phthalate was consistently found in treated effluent samples at greater than trace levels (0.01 mg/l). It appears that trace organic contaminant control in complex industrial wastewaters can be achieved by the application of PDN technology.</jats:p

Activated Sludge Process Response to Variable Inputs of Volatile Organic Contaminants

Laboratory-scale activated sludge treatment systems were operated under dynamic loading conditions to investigate the non-steady state behaviour of volatile organic contaminants (VOCs) under controlled conditions. Four step tests were conducted in which an incremental increase in the concentrations of selected contaminants was applied to the reactor feed from background levels of about 15 µg/L to levels of about 100 µg/L for a duration of approximately three hydraulic retention times. Although it was not possible to define the relative contributions of stripping and biodegradation to the removal of the test VOCs, different mechanisms are responsible for the removal of short-term, non-steady state inputs of chlorinated and non-chlorinated VOCs. No apparent effect of SRT or HRT on VOC removal was observed at the conditions tested.</jats:p

Activated Sludge Process Response to Variable Inputs of Heavy Metals

Laboratory-scale activated sludge treatment systems were operated under dynamic loading conditions to investigate the non-steady state behaviour of heavy metal contaminants under controlled conditions. Four step tests were conducted in which an incremental increase in the concentrations of selected contaminants was applied to the reactor feed from background levels of about 100 µg/L to levels of about 1000 µg/L for each metal over a period of approximately three hydraulic retention times. Effluent metal concentrations rose significantly to levels of approximately 500 µg/L. They remained elevated for long periods after termination of the metal perturbations. Solids retention time (SRI) did not appear to influence metal removal efficiency over the range tested. Hydraulic retention time (HRT) effects were difficult to discern from the confounding effect of influent metal concentration.</jats:p