Indoleamine 2,3-dioxygenase-1 (IDO1) mediates the degradation of L-tryptophan (L-Trp) and is constitutively expressed in the chorionic vascular endothelium of the human placenta with highest levels in the microvasculature. Given that endothelial expression of IDO1 has been shown to regulate vascular tone and blood pressure in mice under the condition of systemic inflammation, we asked whether IDO1 is also involved in the regulation of placental blood flow and if yes, whether this function is potentially impaired in intrauterine growth restriction (IUGR) and pre-eclampsia (PE). In the large arteries of the chorionic plate L-Trp induced relaxation only after upregulation of IDO1 using interferon gamma and tumor necrosis factor alpha. However, ex vivo placental perfusion of pre-constricted cotyledonic vasculature with L-Trp decreases the vessel back pressure without prior IDO1 induction. Further to this finding, IDO1 protein expression and activity is reduced in IUGR and PE when compared to gestational age-matched control tissue. These data suggest that L-Trp catabolism plays a role in the regulation of placental vascular tone, a finding which is potentially linked to placental and fetal growth. In this context our data suggest that IDO1 deficiency is related to the pathogenesis of IUGR and PE.

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation.

The increased maternal estradiol (E2) concentrations induced by assisted reproductive technology (ART) result in lower birth weight of offspring, which is associated with increased risk of adult diseases. However, the exact mechanism remains unknown. The present study investigated the effect of high E2 exposure on the expression of imprinted genes CDKN1C and IGF2 in human placentas and the DNA methylation status of their differential methylation regions (DMRs).The mRNA expression of CDKN1C and IGF2 in human placentas and the human trophoblast cells (HTR8) treated with E2 were investigated by reverse transcription-real time polymerase chain reaction (PCR). The DNA methylation of their DMRs were investigated by sodium bisulfite sequencing.CDKN1C and IGF2 were significantly up-regulated in ART conceived placentas. The mean birth weight of ART singletons was significantly lower than that of naturally conceived (NC) ones, with the increased percentage of small-for-gestational-age (SGA) birth. The DNA methylation was significantly down-regulated in the DMR of CDKN1C (KvDMR1) and up-regulated in the DMR of IGF2 (H19 DMR) in ART placentas. The treatment of E2 altered the expression of the two genes and the DNA methylation of their DMRs in HTR8 to a similar tendency as in vivo.The maternal high E2 levels after ART up-regulate the expression of imprinted genes in human placentas through epigenetic modifications, which influences the growth potential of the offspring. Further studies are needed to follow up the growth and development of the ART offspring.

The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic adaptation to starvation. Because of its tumor suppressor function, p53 stabilization by starvation could have implications for both metabolic and oncological diseases of the liver.

Implantation and subsequent placental development depend on a well-orchestrated interaction between fetal and maternal tissues, involving a fine balanced synergistic cross-talk of inflammatory and immune-modulating factors. Tumor necrosis factor (TNF)-α has been increasingly recognized as pivotal factor for successful pregnancy, although high maternal TNF-α levels are associated with a number of adverse pregnancy conditions including gestational hypertension and gestational diabetes mellitus. This study describes effects of exogenously applied TNF-α, mimicking increased maternal TNF-α levels, on the secretion profile of inflammation associated factors in human first trimester villous placenta. Conditioned culture media from first trimester villous placental explants were analyzed by inflammation antibody arrays and ELISA after 48 h culture in the presence or absence of TNF-α. Inflammation antibody arrays identified interleukin (IL)-6, IL-8, chemokine (C–C motif) ligand 2 (CCL2), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as the most abundantly secreted inflammation-associated factors under basal culture conditions. In the presence of TNF-α, secretion of GM-CSF, CCL5, and IL-10 increased, whereas IL-4 and macrophage CSF levels decreased compared with controls. ELISA analysis verified antibody arrays by showing significantly increased synthesis and release of GM-CSF and CCL5 by placental explants in response to TNF-α. Immunohistochemistry localized GM-CSF in the villous trophoblast compartment, whereas CCL5 was detected in maternal platelets adhering to perivillous fibrin deposits on the villous surface. mRNA-based in situ padlock probe approach localized GM-CSF and CCL5 transcripts in the villous trophoblast layer and the villous stroma. Results from this study suggest that the inflammatory secretion profile of human first trimester placenta shifts towards increased levels of GM-CSF, CCL5, and IL10 in response to elevated maternal TNF-α levels, whereas IL-6 and IL-8 remain unaffected. This shift may represent a protective mechanism by human first trimester villous placenta to sustain trophoblast function and dampen inflammatory processes in the intervillous space.

An increasingly branched rolling circle amplification (IB-RCA) which contains a padlock probe (PP) and a structurally tailored molecular beacon (MB) was innovatively developed for highly sensitive detection of cancer gene, Kras gene codon 12. In this system, the PP can be circularized after hybridization with the precisely-matched target DNA, while the stem of MB can be also opened by target DNA, resulting in hybridization with the circularized PP to generate a long tandem single-stranded DNA (ssDNA) product. Since the MB is also designed to hybridize with ssDNA product, the newly-opened MBs are able to trigger the next RCA reactions, therapy producing branched rolling circle amplification (RCA) products and in turn leading to the increasingly branched RCA (IB-RCA). This alternately and continuously operates hybridization-based MB opening and opened MBs-triggered RCA. As a result, a great number of MBs are opened that is associated with a dramatically amplified fluorescent signal, enabling to quantify target DNA down to 100 fM. This sensing method demonstrates a new concept of IB-RCA amplification even in a simple way to efficiently transduce the fluorescence signal, accomplishing the highly sensitive and selective detection of cancer gene.

In this study, in order to examine the effect of reactor internal geometry modeling method on the prediction accuracy for PWR (Pressurized Water Reactor) and PHWR (Pressurized Heavy Water Reactor) internal flow distribution, simulations with real geometry modeling were conducted with the commercial CFD (Computational Fluid Dynamics) software, ANSYS CFX R.14 and FLUENT R.14. The predicted results were compared with those of the porous medium assumption and the measured data. Test cases were 1/5 scaled-down APR+ (Advanced Power Reactor Plus) model for PWR and moderator test vessel (Stern Laboratories Inc.) for PHWR. It was concluded that core inlet flow distribution could be predicted more accurately by considering the real geometry of the scale-down APR + internal structures, located in the upstream of core inlet. Therefore, if the sufficient computation resource is available, an exact representation of these internal structures, for examples lower support structure bottom plate and ICI (In-Core Instrumentation) nozzle support plate, is essential for the accurate simulation of PWR internal flow. For PHWR, an approach to consider the real geometry of tubes predicted the local velocity and temperature distribution better than the Porous Medium Model. However, the benefits (e.g. improvements of prediction accuracy) against the computation cost were small; compared with PWR case. The reason may be that relatively simple PHWR internal structure can make it possible to establish a generally applicable correlation for the pressure loss coefficient.

Gene therapy to treat ocular disorders is still starting, and current therapies are primarily experimental, with most human clinical trials still in research state, although beginning to show encouraging results. Currently 33 clinical trials have been approved, are in progress, or have been completed. The most promising results have been obtained in clinical trials of ocular gene therapy for Leber Congenital Amaurosis, which have prompted the study of several ocular diseases that are good candidates to be treated with gene therapy: glaucoma, age-related macular degeneration, retinitis pigmentosa, or choroideremia. The success of gene therapy relies on the efficient delivery of the genetic material to target cells, achieving optimum long-term gene expression. Although viral vectors have been widely used, their potential risk associated mainly with immunogenicity and mutagenesis has promoted the design of non-viral vectors. In this review, the main administration routes and the most studied delivery systems, viral and non-viral, for ocular gene therapy are presented. The primary ocular disease candidates to be treated with gene therapy have been also reviewed, including the genetic basis and the most relevant preclinical and clinical studies.

Placental villous explant culture has been increasingly recognized as suitable model to study secretion of inflammatory and immune modulating factors by human placenta. Most of these factors likely derive from the syncytiotrophoblast, whereas extraplacental sources such as maternal peripheral blood cells are rarely considered. Due to their small size and absence of a nucleus, platelets adhering to perivillous fibrinoid of normal placenta are frequently ignored in routine immunohistochemistry. Here we demonstrate adhering maternal platelets on first trimester placental villi after explant culture and point out that platelet-derived factors must be considered when analyzing the inflammatory secretion profile of human placenta.

The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. Immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. Gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. Fractalkine expression and release were detected in the trophoblast cell line BeWo, in primary term trophoblasts and placental explants. Incubation of BeWo cells and placental explants with metalloprotease inhibitor Batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. These results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. Increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. Aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.

Besides microfilaments and microtubules, intermediate filaments are major components of the cytoskeleton. In epithelial cells intermediate filaments are formed by heterodimers of specific keratins, whose expression pattern highly depends on the type of epithelium and differentiation degree of the cell. During the process of blastocyst implantation and subsequent development of the human placenta a very specialized epithelium appears at the feto-maternal interface. Arising from the trophectoderm of the blastocyst, the epithelium-like layer surrounding the early embryoblast, different trophoblast subtypes differentiate. They either develop into polar cells fulfilling real epithelial functions, or apolar tumor-like cells invading the maternal uterine wall to adapt the maternal tissue to progressing pregnancy. Thus, the whole trophoblast population, with all its subtypes, can be considered as an epithelial compartment and hence expresses keratin filaments. However, differentiation of trophoblast into different phenotypes may be linked to remodeling of the cytoskeletal composition, depending on spatiotemporal requirements of the respective cells. Here, we focus on the keratin composition of different trophoblast subtypes, how these keratins are used in trophoblast research and what is known about placental keratins in pregnancy pathologies.

Neuronal networks are reorganized following brain injury. At the structural level this is in part reflected by changes in the spine turnover of the denervated neurons. Using the entorhinal cortex lesion in vitro model, we recently showed that mouse dentate granule cells respond to entorhinal denervation with coordinated functional and structural changes: During the early phase after denervation spine density decreases, while excitatory synaptic strength increases in a homeostatic manner. At later stages spine density increases again, and synaptic strength decreases back to baseline. In the present study, we have addressed the question of whether the denervation-induced homeostatic strengthening of excitatory synapses could not only be a result of the deafferentation, but could, in turn, affect the dynamics of the spine reorganization process following entorhinal denervation in vitro. Using a computational approach, time-lapse imaging of neurons in organotypic slice cultures prepared from Thy1-GFP mice, and patch-clamp recordings we provide experimental evidence which suggests that the strengthening of surviving synapses can lead to the destabilization of spines formed after denervation. This activity-dependent pruning of newly formed spines requires the activation of N-methyl-d-aspartate receptors (NMDA-Rs), since pharmacological inhibition of NMDA-Rs resulted in a stabilization of spines and in an accelerated spine density recovery after denervation. Thus, NMDA-R inhibitors may restore the ability of neurons to form new stable synaptic contacts under conditions of denervation-induced homeostatic synaptic up-scaling, which may contribute to their beneficial effect seen in the context of some neurological diseases.

GPR55 is a seven-transmembrane G-protein-coupled receptor that has been proposed as a novel type of cannabinoid receptor. Previously, we identified lysophosphatidylinositol (LPI), in particular 2-arachidonoyl-LPI, as an agonist for GPR55. In the present study, we examined whether intracellular phospholipase A1 (DDHD domain containing 1, or DDHD1), previously identified as phosphatidic acid (PA)-preferring PLA1 (PA-PLA1), is involved in the formation of 2-arachidonoyl-LPI. HEK293 cells expressing DDHD1 produced [3H]arachidonic acid-containing LPI after prelabeling with [3H]arachidonic acid and subsequent activation by ionomycin; the formation of [3H]LPI was inhibited by n-butanol and the overexpression of an inactive PLD1 mutant PLD1K898R. DDHD1 was translocated from the cytosol to membranes upon ionomycin treatment. A purified recombinant DDHD1 formed [3H]LPI when incubated with [3H]PI; the Vmax and apparent Km were 190 µmol/min/mg protein and 10 mol% PI, respectively. DDHD1 binds PA, and the addition of PA to DDHD1 increased the affinity for PI (Km ; 3 mol%) and augmented the PI-PLA1 activity. DDHD1 activated by PA was returned to a basal state by its own PA-hydrolytic activity. These results implicate DDHD1 in the formation of 2-arachidonoyl-LPI and indicate that the process is modulated by PA released by phospholipase D. Similar observations for the production of arachidonic acid-containing LPI in neuroblastoma cells suggest the DDHD1-LPI-GPR55 axis to be involved in functions in the brain.