alzheimer\’s disease DNA fragmentation is an essential component of PCD Of several

DNA fragmentation is an essential component of PCD. Of several nucleases that were tested for their involvement in charontosis, EndoG emerged as a likely candidate, although a role for CAD cannot be discounted due to the degradation of ICAD observed 44 h after ETO treatment. EndoG is activated in alzheimer\’s disease undergoing oxidative stress and translocates to the nucleus to fragment DNA (Higgins et al., 2009; Ishihara and Shimamoto, 2006). ETO induces oxidative stress and reactive oxygen species (ROS) in a variety of cell types (England et al., 2004; Hirano et al., 2004; Rojas et al., 2009), an observation we have observed in mESCs after ETO treatment (Supplemental Fig. 10), adding credence to the involvement of EndoG in this process. Finally, since pifithrin μ inhibits p53 association with mitochondria and results in decreased PCD in ETO-treated mESCs, and since EndoG is released from mitochondria to promote DNA fragmentation in response to ETO treatment, p53 may contribute to EndoG release.
In summary, we have shown that ETO induces massive DNA DSBs, an accumulation of cells in the G2 phase of the cell cycle, and extensive PCD in mESCs (Fig. 1; Supplemental Fig. 11). This PCD appears to be independent of caspase activity and of RIP kinases, which are active in necroptosis. Knockdown of proteins that are integral to autophagy did not reduce PCD, whereas chemical inhibitors of autophagy did significantly decrease the high levels of cell death after treatment with ETO. The possibility that autophagy itself promotes PCD was excluded, since the activation of autophagy during ETO treatment promoted cell survival and not cell death. When mESCs were exposed to a broad spectrum inhibitor of cathepsins, the level of cell death was significantly reduced suggesting that these lysosomal proteases were involved in PCD. The involvement of p53 was also queried by the use of inhibitors that prevent p53 transactivation or mitochondrial translocation. Inhibitors of both p53 activities significantly protected ESCs from ETO-induced PCD, albeit at varying degrees. Lastly, we have identified a novel role for EndoG in the ETO-induced PCD pathway alzheimer\ in mESCs, a finding that has not been described in ETO-induced PCD in any other cell type. We have coined the term charontosis, after Charon, the ferryman on the river Styx in Greek mythology, to describe this PCD pathway, which is summarized in Fig. 6.
The following are the supplementary data related to this article.

Conflict of interest

We thank P Hexley and G Babcock for assistance with flow cytometry, performed at Shriners Hospitals for Children — Cincinnati, supported by a grant from the Shriners of North AmericaSSF 84070. We would also like to thank N White and the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children\’s Hospital Medical Center, supported in part by NIHAR-47363, NIH DK78392 and NIH DK90971, for assistance with Imagestream flow cytometry data acquisition and analysis. We also thank S. Mylavarapu and H. Ma for assistance with data acquisition. This work was supported in part by grants R01 ES012695 and R01 ES12695-4S1 to PJS from the National Institutes of Health and the Center for Environmental Genetics and grant P30 ES006096 from NIEHS. EDT was supported by a NIH training grant T32 ES007250.

Definitive erythropoiesis occurs in the bone marrow, where a set of complex interactions encourages red blood cell (RBC) production. The central macrophage in the erythropoietic niches called erythroblastic islands plays a critical role in controlling the maturation, differentiation, and enucleation of erythroid cells (Bessis, 1958; Chasis and Mohandas, 2008). It is known that the main route of signal transport between macrophages and erythroid cells is physical contact via several ligands and receptors (Rhodes et al., 2008; Soni et al., 2006; Spring and Parsons, 2000; Telen, 2000). Various adhesion molecules responsible for maintaining this cell-to-cell contact within the erythroid islands have been identified, mainly through the relationship between macrophages and erythroid cells. However, not every erythroid cell is attached to a macrophage (Rhodes et al., 2008), and some erythroid cells contact only other erythroid cells. Erythroid cells seem to be able to autonomously regulate erythropoiesis, albeit with reduced efficiency (Chasis and Mohandas, 2008). Based on these observations, we speculated that there must be factors regulating erythroid cells that are not based on direct contact with macrophages.

Introduction The stem cell niche is a

The stem cell niche is a unique microenvironment that maintains long-term repopulation of a specific tissue by undergoing self-renewal of stem GDC-0941 and by producing progenies of increasingly differentiated cells. A large amount of knowledge concerning the regulation of stem cell self-renewal and differentiation by niche cells is described in a subpopulation of adult osteogenic and adult hematopoietic stem cells (HSCs) (Bianco, 2011; Calvi et al., 2003; Zhang et al., 2003). In particular, recent studies showed that specialized spindle-shaped N-cadherin (N-cad) expressing osteoblasts (SNO) are a key component of the bone marrow stem cell niche, where HSCs directly interact with SNO via N-cad interaction (Nilsson et al., 2001; Whetton and Graham, 1999; Zhang et al., 2003; Zhu and Emerson, 2004).
The corneal epithelial stem cells are located in the cornea limbus, a ring of tissue surrounding the peripheral clear cornea (Cotsarelis et al., 1989; Schermer et al., 1986), and continue to supply corneal epithelial cells to the central cornea. Hayashi, et al. recently reported that limbal basal epithelial cells in the peripheral cornea express N-cad as a possible marker of putative epithelial stem cells, and that melanocytes may be associated with these cells through homotypic adhesion by N-cad in the human limbal epithelial stem cell niche (Hayashi et al., 2007). We recently found that 3T3 feeder cells lacking N-cad expression significantly lost the ability to support clonal growth and maintain basal Keratin 15 positive (K15+) limbal phenotype in epithelial sheets using colony forming assays and the duplex feeder system, an in vitro model of the limbal epithelium (Higa et al., 2009; Miyashita et al., 2008). Recently, Chen et al. demonstrated that isolation of human limbal progenitor cells using collagenase digestion dramatically maintained close association with their niche cells compared to dispase digestion (S.Y. Chen et al., 2011). These findings strongly suggest that a unique niche cell exists in the corneal limbus that regulates limbal epithelial progenitors/stem cells. Although there are several reports concerning the corneal limbal progenitor/stem cells, studies describing the limbal niche are few. In addition, whether corneal epithelial stem cells directly interact with subepithelial niche cells via N-cad interaction remains to be shown. In this paper, by using collagenase digestion we demonstrate that aquaporin1 (AQP1) positive niche-like cells exist immediately beneath the limbal epithelial basement membrane, and directly interact with N-cad positive limbal basal epithelial cells in a calcium-dependent manner.

Material and methods


In this study, we showed that large dendritic AQP1+ stromal cells exist beneath N-cad+ limbal basal progenitor/stem clusters by using collagenase digestion. Chen et al. recently reported that collagenase enables the isolation of the corneal limbal progenitors/stem cell microenvironment, including the basement membrane, compared with dispase isolation (S.Y. Chen et al., 2011). In addition, they also isolated cytokeratin and p63 negative, vimentin positive 5–10μm small diameter cells by collagenase isolation (S.Y. Chen et al., 2011). However, the vimentin+, AQP1+ dendritic cells that we isolated were approximately 30–50μm in diameter, which is several fold larger than the cells that they have reported (Fig. 4C, E). They also reported that collagenase isolation maintained basement membrane components such as laminin 5 (S.Y. Chen et al., 2011). Preservation of basement membrane components during isolation is important for rapid re-synthesis and deposition of it components in association with limbal epithelial progenitor cells and corneal endothelial cells (S.Y. Chen et al., 2011; Li et al., 2007). Although we also isolated limbal epithelial cells with stromal cells using different collagenase conditions, basement membrane components were still maintained after both collagenase and accutase digestions (Figs. 5 and 7). We used accutase for adhesion assays since it is milder than trypsin for preserving cadherin, and because we failed to isolate the large AQP1+ cells using trypsin (data not shown). Due to the slight difference in isolation protocol, there is the possibility that the cells we isolated were a completely different cell type that support limbal basal progenitor cells.

Introduction The vascular endothelium the single cell

The vascular endothelium, the single-cell layer lining blood vessels, is a multifunctional interface that displays a striking phenotypic plasticity necessary for maintaining vascular homeostasis. In this context, the vascular endothelium is critical to initiate an inflammatory response, trigger thrombosis, regulate vasomotor tone, and control vascular permeability. Dysfunction of the endothelium plays a significant pathogenic role in cardiovascular diseases, namely, atherosclerosis and its consequences: heart attacks and strokes (Gimbrone et al., 2000; Hansson, 2005). Notably, studies at the genetic and molecular level of human endothelium have been limited by the availability of relevant tissue derived from cadaveric, discarded surgical, or umbilical vasculature sources.
Recent developments in stem cell biology promise new resources for modeling genetic diseases. In particular, induced pluripotent stem oxycodone hydrochloride (iPSCs) offer the ability to study the effects of genetic alterations and mechanisms of genetic diseases in currently inaccessible cell types (Takahashi and Yamanaka, 2006). Although iPSCs have been differentiated into many cell types including endothelium (Choi et al., 2009; Homma et al., 2010; Li et al., 2011; Park et al., 2010; Rufaihah et al., 2011, 2013; Taura et al., 2009; White et al., 2013), the fidelity and functional mimicry of stem cell-derived tissues and their relevance to human disease remain poorly characterized. This functionality must be carefully assessed before their scientific and therapeutic potential can be realized (Soldner and Jaenisch, 2012).


In this study, we have generated vascular ECs from human iPSCs and characterized their humoral-, pharmacological-, and biomechanical-induced functional phenotypes, advancing iPSC-ECs as an experimental platform to study endothelial biology. The methodology to differentiate iPSCs into vascular endothelium was reproducible and robust across several iPSC lines created with different technologies. Previous studies have differentiated iPSCs to isolate ECs based on expression of CD31 or KDR (Choi et al., 2009; Homma et al., 2010; Li et al., 2011; Park et al., 2010; Rufaihah et al., 2011, 2013; Taura et al., 2009; White et al., 2013). In contrast, we isolated populations of ECs from the EBs based on VE-cadherin expression, a strong marker of endothelial identity, as we observed CD31+, and KDR+ cells subpopulations lacking VE-cadherin, and a CD31+ subpopulation expressing CD45.
After isolation and culture, iPSC-ECs displayed molecular markers of vascular endothelium and contained vWF-positive WPBs that can be rapidly exocytosed, a critical function for hemostasis. It is possible that the perinuclear localization observed in many of the WPBs may suggest an immature stage in WPB biogenesis (Valentijn et al., 2011; Zenner et al., 2007). iPSC-ECs are competent for the functional repertoire displayed by vascular endothelium critical in pathophysiological settings. Specifically, we demonstrated that proinflammatory stimuli induce an activated proinflammatory phenotype that included expression of leukocyte adhesion molecules, secretion of proinflammatory cytokines, and support for human leukocyte transmigration. We also observed that iPSC-EC monolayers display a dynamic permeability in response to a panel of physiological stimuli.
Human iPSC-ECs are a promising platform for studying endothelial contributions to cardiovascular diseases in the context of specific patients’ genetic backgrounds. We demonstrated that iPSC-ECs have the plasticity to acquire distinct flow-dependent phenotypes, namely atheroprotective and atheroprone phenotypes critical for atherosclerosis resistance and susceptibility. We observed that iPSC-ECs respond to atheroprotective shear stress by activating an atheroprotective gene expression program transcriptionally mediated by KLF2 and KLF4 expression, shown to integrate the flow-mediated endothelial atheroprotective functional phenotype by regulating leukocyte adhesion, redox state, and thrombotic function in cultured human ECs (Dekker et al., 2006; Lin et al., 2005; Parmar et al., 2006; SenBanerjee et al., 2004; Villarreal et al., 2010). The ability to direct the acquisition of flow-dependent functional and dysfunctional phenotypes using patient-specific endothelium should allow the study of specific genetic contributions to endothelial function and dysfunction in the context of human cardiovascular disease. Furthermore, we demonstrated that simvastatin activated an atheroprotective gene expression program and downregulated genes associated with an atheroprone phenotype in iPSC-ECs. This suggests that iPSC-ECs may be a suitable surrogate to assess the effects of drugs on the endothelia of specific patients.

Introduction The p functions are ubiquitously altered in cancer cells

The p53 functions are ubiquitously altered in cancer cells by mutations/perturbation of its signaling pathways, and loss of p53 activity is a prerequisite for cancer development. Mutant p53 is thought to play a pivotal role in promoting invasion, favoring cancer cell exit from the primary tumor site and dissemination, ultimately leading to metastasis formation (Gadea et al., 2007; Muller et al., 2009; Roger et al., 2010; Vinot et al., 2008).
Recent reports have documented a p53 role in stem cell homeostasis and pluripotency. Wild-type (WT) p53 counteracts somatic cell reprogramming (Hong et al., 2009; Kawamura et al., 2009; Liu et al., 2009; Utikal et al., 2009), whereas mutant p53 stimulates induced pluripotent stem (iPS) cell formation (Sarig et al., 2010). Depletion of p53 significantly increases cell reprogramming efficacy and facilitates iPS cell generation (Kawamura et al., 2009). Consequently, p53 might be considered as the guardian of the Cyclo and also of reprogramming.
All these functions are associated with full-length p53 (i.e., the TAp53α isoform). However, the TP53 gene encodes at least 12 different physiological isoforms (TAp53 [α, β, and γ], Δ40p53 [α, β, and γ], Δ133p53 [α, β, and γ], and Δ160p53 [α, β, and γ]) (Bourdon, 2007) via several mechanisms: alternative promoters (the TA and Δ133 isoforms), alternative intron splicing (intron 2: Δ40 isoforms and intron 9: α, β, and γ isoforms), and alternative translational initiation sites (Δ40 and Δ160 isoforms). The TAp53α isoform is the best described and classically mentioned in the literature as p53. Basically, p53 isoforms can be divided into two groups as follows: (1) long isoforms that contain the transactivation domain (TA and Δ40), and (2) short isoforms without the transactivation domain (Δ133 and Δ160). Furthermore, the β and γ isoforms do not contain the canonical C-terminal oligomerization domain, but an additional domain with unknown function(s) (Khoury and Bourdon, 2011).
The p53 isoforms modify p53 transcriptional activity in many processes, such as cell-cycle progression, programmed cell death, replicative senescence, cell differentiation, viral replication, and angiogenesis (Aoubala et al., 2011; Bernard et al., 2013; Bourdon et al., 2005; Marcel et al., 2012; Terrier et al., 2011, 2012). Importantly, p53 isoforms are specifically deregulated in human tumors (Machado-Silva et al., 2010). However, the functions of p53 isoforms in cancer stem cell (CSC) homeostasis have never been explored.
Here, we show that the Δ133p53β isoform is specifically involved in promoting cancer cell stemness. Overexpression of Δ133p53β in human breast cancer cell lines stimulated mammosphere formation and the expression of key pluripotency and stemness regulators (SOX2, OCT3/4, and NANOG and CD24/CD44), but not C-MYC. Furthermore, using MDA-MB-231-based cell lines, we show that increased expression of Δ133p53 isoforms correlates with the increased metastatic potential and with mammosphere formation. Finally, incubation of MCF-7 and MDA-MB-231 cells with the anti-cancer drug etoposide also promoted cell stemness in a Δ133p53-dependent manner. Our results demonstrate that short p53 isoforms positively regulate CSC potential regardless of any p53 mutation. Consequently, WT TP53, which is considered a tumor suppressor gene, also can act as an oncogene through Δ133p53β expression.


In this work, by modulating p53 isoform expression in breast cancer cell lines, we show that Δ133p53 isoforms have a role in regulating their stemness potential. Surprisingly, depletion of all p53 isoforms in MCF-7 cells significantly reduced mammosphere formation (a hallmark of CSC potential), although previous reports indicate that TAp53α hinders cell reprogramming. Conversely, selective depletion of TAp53 and Δ40p53 isoforms with the Sh2 shRNA did not affect mammosphere formation, suggesting that Δ133p53 isoforms are responsible for this activity. We then confirmed this hypothesis by showing that mammosphere formation was strongly reduced upon knockdown of these small isoforms (Figure 1). Similarly, depletion of the β isoforms had a deleterious effect on the capacity of MCF-7 cell to form mammospheres, while depletion of the α isoforms did not have any effect. Moreover, all changes in p53 isoform expression, particularly Δ133p53, were associated with variations in the expression of SOX2, OCT3/4, and NANOG, key cell pluripotency/reprogramming genes, but not of C-MYC (Figures 1 and 2). Furthermore, the finding that Δ133p53β isoform specifically promoted mammosphere formation and increased the proportion of CD44+/CD24− cells indicates that this isoform positively regulates CSC potential in MCF-7 breast cancer cells. Indeed, our data show that Δ133p53β expression positively correlates with SOX2, OCT3/4, and NANOG expression, genes responsible for cell pluripotency induction and maintenance (Figure 2). Finally, using a breast cell model of tumor aggressiveness, we show that higher metastatic potential and chemoresistance are coupled with increased expression of the Δ133p53 isoforms, CSC stemness, and increased expression of key pluripotency/reprogramming genes (Figures 3 and 4).

br Results br Discussion The transplant created chimera enables us


The transplant-created chimera enables us to investigate the effects of human neural order THZ1 Hydrochloride in the adult mouse spinal cord (Chen et al., 2015). Using this model, we have shown that neural cells from sALS patient iPSCs, especially astrocytes, integrate into the mouse spinal cord to a similar degree as healthy cells. It is the sALS cells that induce degenerative changes in both MNs and non-MNs of the host, which corresponds to the mouse motor behavioral deficits. By taking advantage of the time-dependent integration of human cells in this model, we have discovered that non-MNs are lost earlier than MNs, with a corresponding reduction of inhibitory nerve terminals in MNs. Thus, the effect of sALS astrocytes on neural degeneration is not specific to MNs, and non-MNs may mediate MN degeneration.
Involvement of glial cells in the pathogenesis of ALS has been suggested from a series of studies where disease-causing proteins are specifically expressed in glial cells in transgenic animals (Yamanaka et al., 2008) and when MNs are cultured with glial cells that express mutant ALS proteins (Di Giorgio et al., 2007, 2008; Nagai et al., 2007). The involvement of glia in sALS is suggested by recent observations that astrocytes, derived from neural progenitor cells that were reprogrammed from sALS patient fibroblasts (i-astrocytes), impaired the survival of MNs (Meyer et al., 2014). Nevertheless, astrocytes generated in a similar manner (via iPSCs) had no obvious effects (Re et al., 2014; Serio et al., 2013), casting a possibility that the in vitro toxic effects of astrocytes may be influenced by culture conditions. Our present study offers in vivo evidence that non-MN cells, especially astrocytes, may participate in the neural degeneration in sALS. This is demonstrated by the fact that neural progenitors differentiated from sALS patient but not healthy PSCs, generate non-MN cells following transplantation into the spinal cord of SCID mice, and cause neuronal degeneration and corresponding motor deficits in mice. It should be noted that one of the control transplants was performed at a different time yet the result was very similar, highlighting the consistency of the effect of the astrocytes and the reproducibility of the experiments. Furthermore, we show here that cells from both sALS patients have similar effects. ALS is heterogeneous. Although we excluded the possibility of C9orf72 mutations, the main contributor of genetic components to sALS, we cannot rule out other albeit extremely rare mutations and those that have not yet been discovered. Since the majority of the differentiated cells are astrocytes, astrocytes may play important roles in neuronal degeneration. In addition, the neuronal degeneration occurs mostly in the transplant center or near the center but not the distal area. Astrocytes in and around the transplant center are more mature than those in the distal area. This suggests that mature but not immature astrocytes exert toxic effects. This may in part explain why astrocytes generated from ALS patient iPSCs have limited effects (Re et al., 2014; Serio et al., 2013). Astrocytes generated from human ESCs or iPSCs usually exhibit immature phenotypes (Krencik et al., 2011).
Co-culture studies have suggested a specific effect on MNs by ALS astrocytes (Di Giorgio et al., 2007, 2008; Nagai et al., 2007). The reason for the specific effect of ALS astrocytes on MNs remains a mystery. One possibility is that MNs are particularly vulnerable to toxicity, especially in the cell culture environment. In some cases, such a phenomenon may be misinterpreted due to the fact that changes in MN but not non-MN numbers are readily discerned because MNs are usually the minority in the culture system. Because of this mystery, we have paid a particular attention to the effect on MNs and non-MNs. Contrary to the in vitro observations, we found that both MNs and non-MNs degenerate in the spinal cord transplanted with sALS cells. This result suggests that the effect of astrocytes is not specific to MNs. An attempt to identify MN-specific astrocytic factors may be elusive.

Overexpression of the sigma receptor also leads to its

Overexpression of the sigma-1 receptor also leads to its increased translocation to the plasma membrane [17]. It was demonstrated in a mouse model [18] that chronic alcohol consumption causes increased expression (and therefore, possibly, translocation to the plasma membrane) of the sigma-1 receptor in the brain. At the same time, a recent study by Yao et al. [19] has revealed that cocaine causes the sigma-1 receptor to translocate from the ER to the lipid rafts of the plasma membrane, where chemokines CCL2 are induced in microglia via Src-kinase activation. It casein kinase is also shown that overexpression of the sigma-1 receptor in cortical neurons increases the binding of the tyrosine kinase receptor B and phospholipase C [20].
After translocating to the plasma membrane, the sigma-1 receptor interacts with various ion channels, receptors and kinases [21]. In fact, it was demonstrated using the patch-clamp on pituitary gland casein kinase that pentazocine, which is a sigma-1 receptor agonist, inhibits the outward current of potassium ions (K+), and this phenomenon can be reversed by the sigma-1 receptor antagonist NE-100 [22]. In addition to direct physical interaction and regulation of the activity of voltage-gated K+ channels in mouse nerve terminals of the posterior lobe of the pituitary gland [23], the sigma-1 receptor regulates the activity of the K+ channel in rat hippocampal slices, intracardiac neurons and cancer cells [24]. The sigma-1 receptor ligands modulate several types of presynaptic Са2+ channels in rat sympathetic and parasympathetic neurons [25]. The sigma-1 receptor also modulates the NMDA receptor activity [26] and affects synaptic plasticity through the small-conductance Са2+-activated K+ channels [27]. The sigma-1 receptor has been shown to modulate cardiac voltage-gated Na+ channels in HEK293 and COS- cells, as well as in neonatal mouse cardiomyocytes [28]. SKF-10047, a sigma-1 receptor agonist, inhibits calcium ion currents in cultured retinal ganglion cells. Direct association between the sigma-1 receptor and the L-type Ca2+-channel was performed using immunoprecipitation [29].
There is also data indicating that the sigma-1 receptor regulates neurotransmitter release in dopaminergic, serotonergic and cholinergic transmission, and is involved in cell differentiation, cellular responses to inflammation, and in pathogenesis of extrapyramidal disorders [21].
Interestingly, the sigma-1 receptor was detected in the extracellular space of NG-108 cells exposed to cocaine, indicating that the receptor possibly acts as a chaperone in the extracellular space [7].

The structure of the sigma-1 receptor
The sigma-1 receptor is an integral membrane receptor, and it is predominantly localized in the ER membranes associated with mitochondria [16].
Even though the detailed atomic structure of the receptor has not yet been determined, a number of studies have been focused on establishing the protein topology and on mapping its active site. Initially, the sigma-1 receptor has been characterized as a type I transmembrane protein with a single transmembrane domain [30]. Currently, a fairly large number of experimental data indicates that there are two alpha-helical transmembrane domains. This data was obtained via bioinformatic analysis, molecular simulation, epitope mapping techniques, limited proteolysis, and NMR spectroscopy.
The number and localization of transmembrane domains are different and depend on the specific algorithm chosen for predicting hydrophobic domains based on the amino acid sequence (Fig. 1) [31–37]. Most algorithms indicate the presence of two or three hydrophobic regions (see. Fig. 1). The first two domains have been identified as highly ordered transmembrane alpha-helices (TM1, TM2). The second transmembrane helix has amphipathic properties [38]. Amino acids 91–109 and 176–194 contain highly conserved sequences homologous to the yeast and fungal sterol C8–C7 isomerase. Due to their homology, these sequences were named steroid-binding domain-like I and II (SBDLI and SBDLII) (Fig. 2a) [39].

The gene of the sigma receptor is

The gene of the sigma-1 receptor is located on chromosome 9p13, known due to its being associated with psychotic disorders [6]. The sigma-1 receptor, with its small size (223 amino-acid residue), binds with medium or high affinity to a wide range of chemical compounds of very different structural classes with various pharmacological and therapeutic properties. Among its ligands there are such compounds as benzomorphans (SKF-10047, pentazocine, dextromethorphan), antipsychotics (haloperidol), antidepressants (fluvoxamine), steroids (progesterone), antihistamines (chlorpheniramine), nuclear hormone receptor ligands (tamoxifen), Са2+ channel antagonists (verapamil, emopamil), antifungals (fenpropimorph, tridemorph) and drugs of abuse (methamphetamine, cocaine, and N,N-dimethyltryptamine) [7]. However, the sigma-1 receptor knockout mice are viable as well as fertile, and do not exhibit any apparent changes in the phenotype except for a reduced hypermotor activity in response to SKF-10047 stimulation compared to wild-type mice. This fact supports the idea that the sigma-1 receptor is involved in response to psychostimulatory actions [8].
Sigma-1 receptors are widely spread in the central nervous system, liver, kidneys, and lungs, in the endocrine, immune and reproductive tissues [9]. This receptor is a transmembrane protein specifically located in ceramide- and cholesterol-rich lipid microdomains associated with the mitochondria of the ER membrane. It regulates the function of the inositol-3-phosphate receptor, stabilizing calcium signaling between the ER and the mitochondrion. It has been shown that the sigma-1 receptor forms Са2+-regulating trimeric complex with ankyrin-B and the inositol-3-phosphate receptor in NG-108 neuroblastoma cells [10].
Additionally, by adjusting the levels of reactive oxygen species, the sigma-1 receptor controls the levels of Rac GTPase in the plasma membrane, thus responsible for the formation of spines in the hippocampus, which is a central ck1 inhibitor region responsible for memory formation [11]. Through regulating the level of reactive oxygen species the sigma-1 receptor also activates NF-κB transcription factor, which controls the expression of the Bcl-2 anti-apoptotic protein [7], and is therefore involved in supporting neuronal life. It was demonstrated on cortical cell cultures that SA4503, a sigma-1 receptor agonist, increased the number of surviving cells following oxidative stress through the suppression of the MAP kinase pathway and the expression of glutamate receptors [12].
Even though the sigma-1 receptor does not directly interact with the G-protein [13], physical and functional connections have been revealed between the sigma-1 receptor and the cloned opioid μ-receptor (which binds the G-protein) [14]. These interactions, which occur only in a form of the sigma-1 receptor related to the antagonist, manifested as significantly facilitated activation of the G-protein by the agonist of μ-receptor DAMGO, which is confirmed by the enhancement (observed in vivo) of morphine-induced analgesia by ck1 inhibitor the antagonists of the sigma-1 receptor.
Functional activity and location of the sigma-1 receptor in a cell depend on the state of the cell, on the stimulation of the receptor by ligands and the level of calcium concentration in the ER. The sigma-1 receptor may be both active and inactive. The most recent studies indicate that this receptor interacts with chaperones, and is itself an ER chaperone [3]. When inactive or stimulated by antagonists (for example, NE-100 or haloperidol), the sigma-1 receptor is linked to another ER protein, a BiP chaperone [10]. When stimulated by agonists (for example, cocaine or pentazocine) at a saturation concentration, or if subjected to prolonged cellular stress caused by, for example, hypoglycemia or depletion of calcium reserves in the ER under the action of thapsigargin, the sigma-1 receptor translocates either to ER regions near to the plasma membrane, or directly into the plasma membrane [15, 16].

br Introduction The pattern of maternal

The pattern of maternal alcohol consumption over the reproductive life course is greatly understudied. Research involving women׳s use of alcohol has tended to focus on short periods of their lives, generally during pregnancy (Anderson et al., 2013; Maloney et al., 2011; Liu, Mumford & Petras, 2014) or old age (Brennan et al., 2011; Molander, Yonker & Krahn, 2010), with little interest in the maternal reproductive life course. The reproductive life course stage for women involves a period of relatively good health but with numerous competing commitments. Childrearing may be the central activity during this period but the formation and maintenance of a dyadic partnership, the buy LY2874455 and development of a career, the maintenance and enhancement of social networks, and major transitions involving the death of parents, marital breakdown, and the growing independence of children are all characteristic exposures during the reproductive life course (Mishra, Cooper & Kuh, 2010; Evans, 1985; Neve, Lemmens & Drop, 2000). There is a need to know more about how women transition through this life stage.

Materials and methods


The maternal cohort was recruited early in pregnancy and followed up over a 21 year period. We found four distinct trajectories of alcohol consumption by women over their reproductive life course. The largest trajectory group comprised low-stable drinkers (58.0%), followed by moderate-escalating drinkers (25.3%), abstainers (11.9%), and heavy-escalating drinkers (4.8%). Our study did not identify the decreasing or curvilinear pattern found in other research (Brennan et al., 2011; Powers & Young, 2008).
The four trajectories found in this study exhibit evidence of a high level of stability for abstainers and low-stable drinkers, but changeability for moderate and heavy-escalating drinkers over their reproductive life course. Members of the abstainers and the low-stable group consistently did not consume alcohol, or consumed at a very low level, over 21 years. The biggest reduction is observed during the period of pregnancy and delivery among moderate and heavy drinking mothers, consistent with the findings from previous research (Maloney et al., 2011; Bachman et al., 2013; Tran et al., 2014). However, six months after the birth of the baby, alcohol consumption levels among the moderate-escalating groups started to increase but remained at a moderate level at the 5, 14 and 21 year follow-ups; while alcohol consumption in the heavy-escalating group sharply increased at Year 5 from moderate to heavy (17.1 glasses per week) and remained at this drinking level at the 21-year follow-up (Fig. 2). The pattern of alcohol consumption in the heavy-escalating group suggests the need to focus on prevention and intervention with these women after they have given birth as they breastfeed their baby while apparently consuming moderate levels of alcohol (Tran et al., 2014; Giglia & Binns, 2007). The result also suggests that interventions may be needed during the child-adolescent motherhood period where children may be influenced by their mother׳s drinking behaviour (Van Der Vorst et al., 2009; Cleveland et al., 2014).
Examinations of baseline predictors associated with four drinking trajectory groups indicate the differences among profiles of abstainers versus moderate and heavy-escalating drinkers. Predictors associated with abstainers are those who had low family income and being married. The results appear inconsistent with some studies (Huckle, You & Casswell, 2010; Karlamangla et al., 2006) but in line with the work from Cerdá and colleagues (Cerdá, Johnson-Lawrence & Galea, 2011). Frequency of religious participation is a protective factor against alcohol consumption (Krause, 2003). This finding may be limited to Australian society as Australians have a low rate of church attendance (Australian Bureau of Statistics, 2013). Our study shows that women with increasing consumption over time tend to be affluent (except for the heavy-escalating group), are more likely to be unmarried, and less religious—consistent with previous studies (Platt et al., 2010; Karlamangla et al., 2006).

br For an isolated particle n where

For an isolated particle, 0 = 0 = n0, where n0 is the concentration of charged particles in unperturbed plasma; besides, the thickness r0 of the perturbed region for that particle is not known in advance.
The charge (the potential) of the particle in the steady-state case is determined by the charge balance equation [1]:
where is the total current density of the emitted electrons.
The ion current density on the surface , as well as the surface potential are determined through solving a system of differential equations in the perturbed region. The required value r0 is found by the optimization method (described in Ref. [13]) from the minimal value of the objective function

This approach allows to determine the size of the perturbed region and obtain the distribution of the plasma parameters in this region, as well as the expression for the density of the ion current onto the particle surface
where and are the normalized concentration and the ion velocity on the particle surface.
In a plasma crystal, the thickness r0 may exceed the radius of the Wigner–Seitz cell
where is the concentration of dust particles.
In this case, the particle cannot be regarded as isolated, so the following ion concentration should be chosen as the optimized parameter at the boundary of the Wigner–Seitz cell:
for the negative charge of the dust particles and 0 < 0 for their positive charge. The quasi-neutrality of the Wigner–Seitz cell is thus maintained.
The balance equations of particle charge and energy
The balance equations of charge and energy on the particle surface [14] were solved simultaneously to determine the dust particle charge taking into account the secondary, ion-electron, photoelectron and thermal field emission processes:
where and are the densities of ion, electron, resonant photon, thermal-field electron and regadenoson currents onto the particle surface (the wall factor W is omitted to simplify the expression); is the temperature of the dust particle surface; and are the secondary-electron emission coefficients for the elastically backscattered and the true secondary electrons, averaged over the electron energy distribution function (EEDF) [15]; γ is the coefficient of the potential ion-electron emission; Y is the quantum yield for the photoelectric effect; is the probability of an electron escaping from the rough surface of the dust particle without a repeated collision [16]; α and α are the accommodation coefficients of atoms and ions [17]; σ0 is the Stefan–Boltzmann constant; a0 is the integrated absorptivity of a dust particle (the emissivity coefficient); ε, εγ, εδ and ε are the kinetic energy quantities of ions and secondary electrons during ion-electron emission, and of true secondary electrons and of photoelectrons; is the excitation energy of the resonant levels.
The factor κ is equal to unity if the charge of the dust particle is negative and to if it is positive, as the emitted electrons experience additional deceleration. The temperature T takes the values and [14] for the respective processes and for the thermal field emission electrons.
Table 1 lists the particle current densities and particle energies carried onto or from the surface of the dust particle that were used in the calculations.

Calculation results
Fig. 1 shows the calculated curves of the normalized potential of a smooth dust particle surface versus τ=, with the above-listed emission processes factored in separately and together. The calculations were performed based on the orbital-motion-limited (OML) theory and on the proposed technique under conditions corresponding to the OML approximation.
We examined aluminum oxide (Al2O3) particles in a neon (Ne) discharge under the following parameters:

These parameters were used to calculate the potentials excluding emission and including all types of emission (see Fig. 1).
A comparison of the obtained data shows that the results of the calculation by the described technique are close to those described by the OML theory. It has been shown that the secondary, ion-electron emission and photoemission from the surface of the smooth particle reduce the absolute value of the potential of the dust particle surface in the whole considered electron temperature range. The effect of thermal field emission on the potential begins at / ≈ 300 and increases with an increase in the electron temperature.

A-1210477 The cylindrical container in which the melt

The cylindrical container in which the melt was placed was made of Plexiglas. The container was surrounded by the inductor uniformly distributed over the height of the melt (Fig. 1).
Fig. 1 schematically shows the characteristic distribution of the Lorentz force (5), induced in the melt in an alternating magnetic field, and the flow lines indicating the main directions in which the fluid moves under the influence of the EM field force. As previously mentioned, the structure of the melt flow in the cylindrical volume at the above-noted density distribution of the Lorentz force is the circulation of two toroidal eddies. The density of the force applied to the melt oscillates at a frequency which is double the frequency of the EM field. At the same time, the change in the Lorentz force density can be neglected, as liquid metals are slow to respond (due to their inertness) to impacts with a frequency higher than 4Hz [10]. Varying the frequency of the EM field leads to a change in the depth to which the field can penetrate the conducting liquid (the skin depth) and in the area of the field\’s action. The density value of the EM field force decreases exponentially within the skin depth whose thickness also depends on the characteristics of the medium acted upon. The thickness of the skin depth can be described by the relation
where is the frequency with which the direction of the EM field changes, μ=1.256·10N/A2 is the magnetic constant, σ is the electric conductivity of the medium.
The δ value for Galinstan was 22mm; for the inside diameter D of the container equal to 62mm, this made up a substantial portion of the melt volume which the field affects by setting the fluid in motion.
The flow parameters were determined experimentally using the Doppler velocity meter. As the name implies, its operating principles are based on the Doppler effect, i.e., on changing the frequency of the ultrasonic wave emitted and received by the A-1210477 as the wave is reflected by the microparticles distributed in the fluid. The particle drift velocity within the ultrasonic beam is determined by the difference between the frequencies of the emitted and the reflected waves. This method is widely used for finding the flow parameters in low-melting liquid metals and model fluids [5,12,13].
In the experiment described, a DOP3010 ultrasonic Doppler velocity meter (Signal Processing SA, Switzerland) was used. During the measurements, the sensor (8mm in diameter) was placed directly into the fluid through the free surface to a depth of 1–2mm and fixed in that position throughout the whole experiment. The frequency of the signal transmitted by the sensor was 1428Hz at the speed of sound in the melt equal to 2730m/s (see Table 1). The sensor was secured on the cylindrical volume axis (point P (r = 0mm) in Fig. 1) for measuring the axial component of the velocity. This velocity component was predominant in the flow structure under investigation. At the same time, the velocity had a negative value if the flow parallel to the signal beam was directed toward the sensor and a positive one if the particles in the fluid were moving away from the sensor.
The experiment used two modes of applying the EM field force to the flow. In the first case, we measured the melt flow velocities under a continuous current in the inductor for the values ranging from 50 to 250Hz. Given that >> 4Hz, the effect on the melt was considered steady (from now on, the EM field parameters are denoted by the index st).
In the second case, we considered the motion of fluid under the action of the pulsed Lorentz force (parameters with the index p). As the force was modulated, the current in the inductor was interrupted to generate pulses with the frequency . If we denote the period of time during which the EM field force acts on the melt as , and the duration of the pause in which the Lorentz force is absent and the fluid moves under the influence of inertia as T0, the expression for determining the frequency and the period of the pulses can be written as follows: