Chlorination of Emerging Contaminants for Application in Potable Wastewater Reuse: Disinfection Byproduct Formation, Estrogen Activity, and Cytotoxicity.

With increasing water scarcity, many utilities are considering the potable reuse of wastewater as a source of drinking water. However, not all chemicals are removed in conventional wastewater treatment, and disinfection byproducts (DBPs) can form from these contaminants when disinfectants are applied during or after reuse treatment, especially if applied upstream of advanced treatment processes to control biofouling. We investigated the chlorination of seven priority emerging contaminants (17ß-estradiol, estrone, 17α-ethinylestradiol, bisphenol A (BPA), diclofenac, p-nonylphenol, and triclosan) in ultrapure water, and we also investigated the impact of chlorination on real samples from different treatment stages of an advanced reuse plant to evaluate the role of chlorination on the associated cytotoxicity and estrogenicity. Many DBPs were tentatively identified via liquid chromatography (LC)- and gas chromatography (GC)-high resolution mass spectrometry, including 28 not previously reported. These encompassed chlorinated, brominated, and oxidized analogs of the parent compounds as well as smaller halogenated molecules. Chlorinated BPA was the least cytotoxic of the DBPs formed but was highly estrogenic, whereas chlorinated hormones were highly cytotoxic. Estrogenicity decreased by ∼4-6 orders of magnitude for 17ß-estradiol and estrone following chlorination but increased 2 orders of magnitude for diclofenac. Estrogenicity of chlorinated BPA and p-nonylphenol were ∼50% of the natural/synthetic hormones. Potential seasonal differences in estrogen activity of unreacted vs reacted advanced wastewater treatment field samples were observed.

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1.

Magnetosomes are biologically-derived magnetic nanoparticles (MNPs) naturally produced by magnetotactic bacteria (MTB). Due to their distinctive characteristics, such as narrow size distribution and high biocompatibility, magnetosomes represent an attractive alternative to existing commercially-available chemically-synthesized MNPs. However, to extract magnetosomes from the bacteria, a cell disruption step is required. In this study, a systematic comparison between three disruption techniques (enzymatic treatment, probe sonication and high-pressure homogenization) was carried out to study their effect on the chain length, integrity and aggregation state of magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 cells. Experimental results revealed that all three methodologies show high cell disruption yields (>89%). Transmission electron microscopy (TEM), dynamic light scattering (DLS) and, for the first time, nano-flow cytometry (nFCM) were employed to characterize magnetosome preparations after purification. TEM and DLS showed that high-pressure homogenization resulted in optimal conservation of chain integrity, whereas enzymatic treatment caused higher chain cleavage. The data obtained suggest that nFCM is best suited to characterize single membrane-wrapped magnetosomes, which can be particularly useful for applications that require the use of individual magnetosomes. Magnetosomes were also successfully labelled (>90%) with the fluorescent CellMask™ Deep Red membrane stain and analysed by nFCM, demonstrating the promising capacity of this technique as a rapid analytical tool for magnetosome quality assurance. The results of this work contribute to the future development of a robust magnetosome production platform.

Core-Shell NaHoF4@TiO2 NPs: A Labeling Method to Trace Engineered Nanomaterials of Ubiquitous Elements in the Environment.

Understanding the fate and behavior of nanoparticles (NPs) in the natural environment is important to assess their potential risk. Single particle inductively coupled plasma mass spectrometry (spICP-MS) allows for the detection of NPs at extremely low concentrations, but the high natural background of the constituents of many of the most widely utilized nanoscale materials makes accurate quantification of engineered particles challenging. Chemical doping, with a less naturally abundant element, is one approach to address this; however, certain materials with high natural abundance, such as TiO2 NPs, are notoriously difficult to label and differentiate from natural NPs. Using the low abundance rare earth element Ho as a marker, Ho-bearing core -TiO2 shell (NaHoF4@TiO2) NPs were designed to enable the quantification of engineered TiO2 NPs in real environmental samples. The NaHoF4@TiO2 NPs were synthesized on a large scale (gram), at relatively low temperatures, using a sacrificial Al(OH)3 template that confines the hydrolysis of TiF4 within the space surrounding the NaHoF4 NPs. The resulting NPs consist of a 60 nm NaHoF4 core and a 5 nm anatase TiO2 shell, as determined by TEM, STEM-EDX mapping, and spICP-MS. The NPs exhibit excellent detectability by spICP-MS at extremely low concentrations (down to 1 × 10-3 ng/L) even in complex natural environments with high Ti background.

Hypoxia-inducible factors 1alpha and 2alpha regulate trophoblast differentiation.

Placental development initially occurs in a low-oxygen (O2) or hypoxic environment. In this report we show that two hypoxia-inducible factors (HIFs), HIF1alpha and HIF2alpha, are essential for determining murine placental cell fates. HIF is a heterodimer composed of HIFalpha and HIFbeta (ARNT) subunits. Placentas from Arnt-/- and Hif1alpha-/- Hif2alpha-/- embryos exhibit defective placental vascularization and aberrant cell fate adoption. HIF regulation of Mash2 promotes spongiotrophoblast differentiation, a prerequisite for trophoblast giant cell differentiation. In the absence of Arnt or Hifalpha, trophoblast stem cells fail to generate these cell types and become labyrinthine trophoblasts instead. Therefore, HIF mediates placental morphogenesis, angiogenesis, and cell fate decisions, demonstrating that O2 tension is a critical regulator of trophoblast lineage determination. This novel genetic approach provides new insights into the role of O2 tension in the development of life-threatening pregnancy-related diseases such as preeclampsia.

Human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates.

Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.

Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration.

The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated Pak1 in vitro. The major site phosphorylated by Akt on Pak1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on Pak1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not Pak1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length Pak1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of Pak1 in vitro with Akt or coexpression of constitutively active Akt with Pak1 reduced Nck binding to Pak1. In HeLa cells, green fluorescent protein-tagged Pak1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of Pak1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.

Effect of socioeconomic conditions on frequent complaints of pain in children: findings from the UK Millennium Cohort Study.

BACKGROUND: Frequent complaints of pain (FCP) are common in high-income countries, affecting about 25% of children, and may have significant adverse consequences including prolonged school absence and disability. Most FCP are unexplained, and the aetiology is poorly understood. This study aimed to identify risk factors for FCP and explore how risk factors explain variation in pain reporting by childhood socioeconomic conditions (SECs). METHODS: Analysis of the UK Millennium Cohort Study, including 8463 singleton children whose parents provided data throughout the study. At 11 years, mothers were asked whether their child frequently complains of pain. Risk ratios (RR) and 95% CIs for FCP were estimated using Poisson regression, according to maternal education. Other risk factors were explored to assess if they attenuated any association between FCP and SECs. RESULTS: 32.3% of children frequently complained of pain. Children of mothers with no educational qualifications were more likely to have FCP than children of mothers with higher degrees (RR 2.06, 95% CI 1.64 to 2.59) and there was a clear gradient across the socioeconomic spectrum. Female sex, fruit consumption, childhood mental health and maternal health measures were associated with childhood FCP in univariable and multivariable analyses. Inclusion of these factors within the model attenuated the RR by 17% to 1.70 (95% CI 1.36 to 2.13). CONCLUSION: In this representative UK cohort, there was a significant excess of FCP reported in less advantaged children that was partially attenuated when accounting for indicators of parental and childhood mental health. Addressing these factors may partially reduce inequalities in childhood FCP.

Activation of p21-activated kinase 1-nuclear factor kappaB signaling by Kaposi's sarcoma-associated herpes virus G protein-coupled receptor during cellular transformation.

Kaposi's sarcoma-associated herpes virus (KSHV) contributes to the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas. KSHV encodes a G protein-coupled receptor (KSHV-GPCR) that signals constitutively and transforms NIH3T3 cells. Here, we show that KSHV-GPCR transformation requires activation of the small G protein Rac1 and its effector, the p21-activated kinase 1 (Pak1). Either transient or sustained expression of KSHV-GPCR activated both Rac1 and Pak1. Furthermore, expression of dominant-negative mutants of Rac (RacN17) or Pak1 (PakR299, Pak-PID) inhibited KSHV-GPCR-induced focus formation and growth in soft agar. We also demonstrate that signaling from Pak1 to nuclear factor-kappaB (NFkappaB) is required for cell transformation induced by KSHV-GPCR. KSHV-GPCR induced transcriptional activation by NFkappaB. This process is inhibited by the PAK-PID, whereas reciprocally, expression of constitutively active Pak1 (PakL107F) activated NFkappaB comparably to KSHV-GPCR. The Pak-PID and RacN17 inhibited the KSHV-GPCR-induced phosphorylation of inhibitor of kappaB kinase-beta and inhibitor of kappaB-alpha, implying that it is Pak1-dependent phosphorylation and subsequent destruction of the inhibitor of kappaB proteins that allows NFkappaB activation. Finally, experiments with the KSHV-GPCR inverse agonist interferon-gamma-inducible protein-10, the Galpha(i) inhibitor pertussis toxin, and an inhibitor of phosphatidylinositol 3'-kinase, wortmannin, indicate that signaling through the Galpha(i) pathway and phosphatidylinositol 3'-kinase contributes to the cell transformation and NFkappaB activation induced by the KSHV-GPCR.

Rho, Rac, Pak and angiogenesis: old roles and newly identified responsibilities in endothelial cells.

Angiogenesis-the develoment of microvasculature-requires, in part, directed endothelial cell motility and responsiveness to external signals. Several of the proteins, which modulate and/or direct endothelial cell motility and morphology in angiogenesis are the Rho GTPases (Rho, Rac, and Cdc42) and Pak (a downstream effector of Rac and Cdc42). Previously, overexpression and activation of Rho GTPases and Pak had been implicated in the development of cancer, through their roles in cancer cell transformation, stimulation of proliferation, inhibition of apoptosis, and migration. Yet regardless of the transformed status of cells within a tumor, without a blood supply most tumors cannot grow larger than 1-2 mm. The blood supply in tumors is provided by capillaries formed of endothelial cells in a process called angiogenesis. Consequently, there is enormous interest in the role of the wild type endothelial cells-and the signaling mechanisms required to support angiogenesis and subsequent growth of metastatic and aggressive cancers. Recent work has begun to uncover the roles of the Rho GTPases and Pak in the regulation of normal endothelial cell function. This review will discuss the current literature regarding the roles of Rho and Rac, and the Rac effector-Pak, in endothelial cells, and we will propose new avenues of research for interaction of the AGC kinase-PKG, with the Rho GTPases and Pak in the cell motility and cell morphology of endothelial cells.

Generating ß-cells in vitro: progress towards a Holy Grail.

PURPOSE OF REVIEW: Diabetes is a debilitating disease characterized by a chronic inability to normalize blood glucose levels. Transplanting cadaveric pancreata or isolated pancreatic islets can restore glucose homeostasis, but organ demand outstrips supply. Consequently, there is significant interest in alternative tissue sources. This review summarizes state-of-the-art efforts to generate scalable, functional ß-cells to treat diabetes. RECENT FINDINGS: Applying knowledge gleaned from developmental biology, human pluripotent stem cells can be treated stepwise with combinations of small molecules, developmentally relevant growth factors, and morphogens to generate pancreatic progenitor cells (PPCs) in vitro. Transplanted PPCs can then further mature in vivo into functional islet-like tissues containing all of the endocrine hormone cells present in adult islets and can reverse hyperglycemia in a diabetic animal model. Recent publications demonstrate that skin, liver, and other cell lineages may also be reprogrammed to functional ß-like cells. SUMMARY: Although generation of fully functional ß-cells in vitro has not yet been achieved, possible intermediate approaches to treat diabetes include using PPCs or reprogramming adult cells to ß-like cells. A cell therapy with either approach will require isolation from the host immune response. Ongoing efforts are addressing this need through the use of immune-isolation devices to avoid immunosuppressive drugs.

Heterozygosity for hypoxia inducible factor 1alpha decreases the incidence of thymic lymphomas in a p53 mutant mouse model.

Hypoxia inducible factors (HIF) are critical mediators of the cellular response to decreased oxygen tension and are overexpressed in a number of tumors. Although HIF1alpha and HIF2alpha share a high degree of sequence homology, recent work has shown that the two alpha subunits can have contrasting and tissue-specific effects on tumor growth. To directly compare the role of each HIFalpha subunit in spontaneous tumorigenesis, we bred a mouse model of expanded HIF2alpha expression and Hif1alpha(+/-) mice to homozygotes for the R270H mutation in p53. Here, we report that p53(R270H/R270H) mice, which have not been previously described, develop a unique tumor spectrum relative to p53(R270H/-) mice, including a high incidence of thymic lymphomas. Heterozygosity for Hif1alpha significantly reduced the incidence of thymic lymphomas observed in this model. Moreover, reduced Hif1alpha levels correlated with decreased stabilization of activated Notch1 and expression of the Notch target genes, Dtx1 and Nrarp. These observations uncover a novel role for HIF1alpha in Notch pathway activation during T-cell lymphomagenesis.

Hypoxia, HIF and the placenta.

Early in mammalian development the placenta, a highly vascularized organ, develops to facilitate exchange of oxygen (O2), nutrients and waste between mother and offspring. This process is intricately regulated by O2 tension and the hypoxic (low O2) uterine environment. Consequently, the placenta provides an excellent model for understanding the relationship between hypoxia (low O2 tension), organogenesis (organ development)and angiogenesis (blood vessel development). Herein we describe recent research on Hypoxia Inducible Factor (HIF), a heterodimeric transcription factor regulated by hypoxia that is crucial for proper placental development. Complete disruption of HIF signaling through loss of the HIFbeta (ARNT) or HIF1alpha and HIF2alpha subunits results in improper placental development, characterized by a diminished spongiotrophoblast layer and insufficient chorio/allantoic fusion. Experiments using placental stem cells (TS cells) derived from Hif1alpha-/- Hif2alpha-/- (Hifalpha-/-) and Arnt-/- mice indicate that there is increased expression of the labyrinthine specific transcription factors GCM and TFEB and a deficiency in the spongiotrophoblast transcription factor Mash2. Furthermore Hifalpha-/- and Arnt-/- TS cells subjected to differentiating conditions tend to adopt a labyrinthine like syncytial fate, and do not form giant cells or spongiotrophoblasts. These observations demonstrate a crucial role for HIF in the formation of the spongiotrophoblast that is probably regulated by Mash2, and suggest a complex interaction between hypoxia, HIF and Mash2 in the formation of the spongiotrophoblast.

cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association.

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.