Comparison between endocrine activity assessed using ToxCast/Tox21 database and human plasma concentration of sunscreen active ingredients/UV filters.

Sunscreen products are composed of ultraviolet (UV) filters and formulated to reduce exposure to sunlight thereby lessening skin damage. Concerns have been raised regarding the toxicity and potential endocrine disrupting (ED) effects of UV filters. The ToxCast/Tox21 program, that is, CompTox, is a high-throughput in vitro screening database of chemicals that identify adverse outcome pathways, key events, and ED potential of chemicals. Using the ToxCast/Tox21 database, octisalate, homosalate, octocrylene, oxybenzone, octinoxate, and avobenzone, 6 commonly used organic UV filters, were found to have been evaluated. These UV filters showed low potency in these bioassays with most activity detected above the range of the cytotoxic burst. The pathways that were most affected were the cell cycle and the nuclear receptor pathways. Most activity was observed in liver and kidney-based bioassays. These organic filters and their metabolites showed relatively weak ED activity when tested in bioassays measuring estrogen receptor (ER), androgen receptor (AR), thyroid receptor, and steroidogenesis activity. Except for oxybenzone, all activity in the endocrine assays occurred at concentrations greater than the cytotoxic burst. Moreover, except for oxybenzone, plasma concentrations (Cmax) measured in humans were at least 100× lower than bioactive (AC50/ACC) concentrations that produced a response in ToxCast/Tox21 assays. These data are consistent with in vivo animal/human studies showing weak or negligible endocrine activity. In sum, when considered as part of a weight-of-evidence assessment and compared with measured plasma concentrations, the results show these organic UV filters have low intrinsic biological activity and risk of toxicity including endocrine disruption in humans.

An interim internal Threshold of Toxicologic Concern (iTTC) for chemicals in consumer products, with support from an automated assessment of ToxCast™ dose response data.

Additional non-animal methods are urgently needed to meet regulatory and animal welfare goals. TTC is a broadly used risk assessment tool. TTC based on external dose has limited utility for multi-route exposure and some types of structure activity relationship assessments. An internal TTC (iTTC), where thresholds are based on blood concentration, would extend the applicability of TTC. While work is on-going to develop robust iTTC thresholds, we propose an interim conservative iTTC. Specifically, an interim iTTC of 1 µM, supported by the published experience of the pharmaceutical industry, a literature review of non-drug chemical/receptor interactions, and analysis of ToxCast™ data. ToxCast™ data were used to explore activity versus the 1 µM interim iTTC and recommendations for the analysis and interpretation of HTS data. Test concentration-based points of departure were classified to identify quality of fit to the Hill Model. We identified, for exclusion from the approach, estrogen receptor and androgen receptor targets as potent chemical/receptor interactions potentially associated with low dose exposure to non-pharmaceutical active ingredients in addition to the original TTC exclusions. With these exclusions, we conclude that a 1 µM plasma concentration is unlikely to be associated with significant biological effects from chemicals not intentionally designed for biological activity.

Transcriptional inhibition in Schwann cell development and nerve regeneration.

Schwann cells, the myelinating glial cells of the peripheral nervous system are remarkably plastic after nerve trauma. Their transdifferentiation into specialized repair cells after injury shares some features with their development from the neural crest. Both processes are governed by a tightly regulated balance between activators and inhibitors to ensure timely lineage progression and allow re-maturation after nerve injury. Functional recovery after injury is very successful in rodents, however, in humans, lack of regeneration after nerve trauma and loss of function as the result of peripheral neuropathies represents a significant problem. Our understanding of the basic molecular machinery underlying Schwann cell maturation and plasticity has made significant progress in recent years and novel players have been discovered. While the transcriptional activators of Schwann cell development and nerve repair have been well defined, the mechanisms counteracting negative regulation of (re-)myelination are less well understood. Recently, transcriptional inhibition has emerged as a new regulatory mechanism in Schwann cell development and nerve repair. This mini-review summarizes some of the regulatory mechanisms controlling both processes and the novel concept of "inhibiting the inhibitors" in the context of Schwann cell plasticity.

Zeb2: Inhibiting the inhibitors in Schwann cells.

Development of Schwann cells is tightly regulated by concerted action of activating and inhibiting factors. Most of the regulatory feedback loops identified to date are transcriptional activators promoting induction of genes coding for integral myelin proteins and lipids. The mechanisms by which inhibitory factors are silenced during Schwann cell maturation are less well understood. We could recently show a pivotal function for the transcription factor zinc finger E-box binding homeobox 2 (Zeb2) during Schwann cell development and myelination as a transcriptional repressor of maturation inhibitors. Zeb2 belongs to a family of highly conserved 2-handed zinc-finger proteins and represses gene transcription by binding to E-box sequences in the regulatory region of target genes. The protein is known to repress E-cadherin during epithelial to mesenchymal transition (EMT) in tumor malignancy and mediates its functions by interacting with multiple co-factors. During nervous system development, Zeb2 is expressed in neural crest cells, the precursors of Schwann cells, the myelinating glial cells of peripheral nerves. Schwann cells lacking Zeb2 fail to fully differentiate and are unable to sort and myelinate peripheral nerve axons. The maturation inhibitors Sox2, Ednrb and Hey2 emerge as targets for Zeb2-mediated transcriptional repression and show persistent aberrant expression in Zeb2-deficient Schwann cells. While dispensible for adult Schwann cells, re-activation of Zeb2 is essential after nerve injury to allow remyelination and functional recovery. In summary, Zeb2 emerges as an "inhibitor of inhibitors," a novel concept in Schwann cell development and nerve repair.

Zeb2 is essential for Schwann cell differentiation, myelination and nerve repair.

Schwann cell development and peripheral nerve myelination require the serial expression of transcriptional activators, such as Sox10, Oct6 (also called Scip or Pou3f1) and Krox20 (also called Egr2). Here we show that transcriptional repression, mediated by the zinc-finger protein Zeb2 (also known as Sip1), is essential for differentiation and myelination. Mice lacking Zeb2 in Schwann cells develop a severe peripheral neuropathy, caused by failure of axonal sorting and virtual absence of myelin membranes. Zeb2-deficient Schwann cells continuously express repressors of lineage progression. Moreover, genes for negative regulators of maturation such as Sox2 and Ednrb emerge as Zeb2 target genes, supporting its function as an 'inhibitor of inhibitors' in myelination control. When Zeb2 is deleted in adult mice, Schwann cells readily dedifferentiate following peripheral nerve injury and become repair cells. However, nerve regeneration and remyelination are both perturbed, demonstrating that Zeb2, although undetectable in adult Schwann cells, has a latent function throughout life.

Large scale water lens for solar concentration.

Properties of large scale water lenses for solar concentration were investigated. These lenses were built from readily available materials, normal tap water and hyper-elastic linear low density polyethylene foil. Exposed to sunlight, the focal lengths and light intensities in the focal spot were measured and calculated. Their optical properties were modeled with a raytracing software based on the lens shape. We have achieved a good match of experimental and theoretical data by considering wavelength dependent concentration factor, absorption and focal length. The change in light concentration as a function of water volume was examined via the resulting load on the foil and the corresponding change of shape. The latter was extracted from images and modeled by a finite element simulation.

Terahertz plastic compound lenses.

We present terahertz (THz) lenses made of highly refracting polymeric compounds which provide a better focusing performance and an increased functionality in comparison to conventional THz lenses. Using mixtures consisting of polypropylene (PP) and alumina as well as PP and zinc sulfide allows a significant increase of the refractive index while simultaneously keeping a low extinction and dispersion. With these new material combinations, lenses with an increased focusing capability are realized. This is evaluated by focal plane measurements using a fiber coupled THz time-domain spectrometer.

A role for Schwann cell-derived neuregulin-1 in remyelination.

After peripheral nerve injury, axons regenerate and become remyelinated by resident Schwann cells. However, myelin repair never results in the original myelin thickness, suggesting insufficient stimulation by neuronal growth factors. Upon testing this hypothesis, we found that axonal neuregulin-1 (NRG1) type III and, unexpectedly, also NRG1 type I restored normal myelination when overexpressed in transgenic mice. This led to the observation that Wallerian degeneration induced de novo NRG1 type I expression in Schwann cells themselves. Mutant mice lacking a functional Nrg1 gene in Schwann cells are fully myelinated but exhibit impaired remyelination in adult life. We suggest a model in which loss of axonal contact triggers denervated Schwann cells to transiently express NRG1 as an autocrine/paracrine signal that promotes Schwann cell differentiation and remyelination.

Consensus micro RNAs governing the switch of dormant tumors to the fast-growing angiogenic phenotype.

Tumor dormancy refers to a critical stage in cancer development in which tumor cells remain occult for a prolonged period of time until they eventually progress and become clinically apparent. We previously showed that the switch of dormant tumors to fast-growth is angiogenesis dependent and requires a stable transcriptional reprogramming in tumor cells. Considering microRNAs (miRs) as master regulators of transcriptome, we sought to investigate their role in the control of tumor dormancy. We report here the identification of a consensus set of 19 miRs that govern the phenotypic switch of human dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors to fast-growth. Loss of expression of dormancy-associated miRs (DmiRs, 16/19) was the prevailing regulation pattern correlating with the switch of dormant tumors to fast-growth. The expression pattern of two DmiRs (miR-580 and 190) was confirmed to correlate with disease stage in human glioma specimens. Reconstitution of a single DmiR (miR-580, 588 or 190) led to phenotypic reversal of fast-growing angiogenic tumors towards prolonged tumor dormancy. Of note, 60% of angiogenic glioblastoma and 100% of angiogenic osteosarcoma over-expressing miR190 remained dormant during the entire observation period of ∼ 120 days. Next, the ability of DmiRs to regulate angiogenesis and dormancy-associated genes was evaluated. Transcriptional reprogramming of tumors via DmiR-580, 588 or 190 over-expression resulted in downregulation of pro-angiogenic factors such as TIMP-3, bFGF and TGFalpha. In addition, a G-CSF independent downregulation of Bv8 was found as a common target of all three DmiRs and correlated with decreased tumor recruitment of bone marrow-derived CD11b+ Gr-1+ myeloid cells. In contrast, antiangiogenic and dormancy promoting pathways such as EphA5 and Angiomotin were upregulated in DmiR over-expressing tumors. This work suggests novel means to reverse the malignant tumor phenotype into an asymptomatic dormant state and may provide promising targets for early detection or prevention of cancer.

Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon-glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon-glia metabolic coupling serves a physiological function.

Irradiation-dependent effects on tumor perfusion and endogenous and exogenous hypoxia markers in an A549 xenograft model.

PURPOSE: Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts. MATERIALS AND METHODS: Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion. RESULTS: CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR. CONCLUSIONS: The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged.

Terahertz emission from lateral photo-Dember currents.

The photo-Dember effect is a source of impulsive THz emission following femtosecond pulsed optical excitation. This emission results from the ultrafast spatial separation of electron-hole pairs in strong carrier gradients due to their different diffusion coefficients. The associated time dependent polarization is oriented perpendicular to the excited surface which is inaptly for efficient out coupling of THz radiation. We propose a scheme for generating strong carrier gradients parallel to the excited surface. The resulting photo-Dember currents are oriented in the same direction and emit THz radiation into the favorable direction perpendicular to the surface. This effect is demonstrated for GaAs and In(0.53)Ga(0.47)As. Surprisingly the photo-Dember THz emitters provide higher bandwidth than photoconductive emitters. Multiplexing of phase coherent photo-Dember currents by periodically tailoring the photoexcited spatial carrier distribution gives rise to a strongly enhanced THz emission, which reaches electric field amplitudes comparable to a high-efficiency externally biased photoconductive emitter.

Propagation of spreading depression inversely correlates with cortical myelin content.

OBJECTIVE: Cortical myelin can be severely affected in patients with demyelinating disorders of the central nervous system. However, the functional implication of cortical demyelination remains elusive. In this study, we investigated whether cortical myelin influences cortical spreading depression (CSD). METHODS: CSD measurements were performed in rodent models of toxic and autoimmune induced cortical demyelination, in neuregulin-1 type I transgenic mice displaying cortical hypermyelination, and in glial fibrillary acidic protein-transgenic mice exhibiting pronounced astrogliosis. RESULTS: Cortical demyelination, but not astrogliosis or inflammation per se, was associated with accelerated CSD. In contrast, hypermyelinated neuregulin-1 type I transgenic mice displayed a decelerated CSD propagation. INTERPRETATION: Cortical myelin may be crucially involved in the stabilization and buffering of extracellular ion content that is decisive for CSD propagation velocity and cortical excitability, respectively. Our data thus indicate that cortical involvement in human demyelinating diseases may lead to relevant alterations of cortical function.

The SNARE protein SNAP-29 interacts with the GTPase Rab3A: Implications for membrane trafficking in myelinating glia.

During myelin formation, vast amounts of specialized membrane proteins and lipids are trafficked toward the growing sheath in cell surface-directed transport vesicles. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins (SNAPs) are important components of molecular complexes required for membrane fusion. We have analyzed the expression profile and molecular interactions of SNAP-29 in the nervous system. In addition to its known enrichment in neuronal synapses, SNAP-29 is abundant in oligodendrocytes during myelination and in noncompact myelin of the peripheral nervous system. By yeast two-hybrid screen and coimmunoprecipitation, we found that the GTPases Rab3A, Rab24, and septin 4 bind to the N-terminal domain of SNAP-29. The interaction with Rab24 or septin 4 was GTP independent. In contrast, interaction between SNAP-29 and Rab3A was GTP dependent, and colocalization was extensive both in synapses and in myelinating glia. In HEK293 cells, cytoplasmic SNAP-29 pools were redistributed upon coexpression with Rab3A, and surface-directed trafficking of myelin proteolipid protein was enhanced by overexpression of SNAP-29 and Rab3A. Interestingly, the abundance of SNAP-29 in sciatic nerves was increased during remyelination and in a rat model of Charcot-Marie-Tooth disease, two pathological situations with increased myelin membrane biogenesis. We suggest that Rab3A may regulate SNAP-29-mediated membrane fusion during myelination.

Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system.

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.

NOX4 regulates ROS levels under normoxic and hypoxic conditions, triggers proliferation, and inhibits apoptosis in pulmonary artery adventitial fibroblasts.

The NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the area of vessel lumen, leading to pulmonary hypertension and cor pulmonale. The proliferation of pulmonary artery adventitial fibroblasts (PAFB) is critically involved in this process. In this study, we analyzed the role of the non-phagocytic NADPH oxidase subunits NOX1 and NOX4 in PAFB. NOX4 was predominantly expressed in comparison to NOX1 at mRNA levels. Under hypoxic conditions, NOX4 was significantly upregulated at mRNA and protein levels. Silencing of NOX4 by siRNA caused reduction of ROS levels under both normoxic and hypoxic (24 h) conditions and suppressed the significant hypoxic-induced ROS increase. PAFB proliferation was significantly decreased in cells transfected with NOX4 siRNA, whereas apoptosis was enhanced. Also, the expression of NOX4 was studied in PAFB isolated from the lungs of patients with idiopathic pulmonary arterial hypertension (IPAH). Interestingly, a significant increase of NOX4 mRNA expression was observed under hypoxic conditions in PAFB from the lungs with IPAH compared to healthy donors. In conclusion, NOX4 maintains ROS levels under normoxic and hypoxic conditions and enhances proliferation and inhibits apoptosis of PAFB.

Proteolipid protein is required for transport of sirtuin 2 into CNS myelin.

Mice lacking the expression of proteolipid protein (PLP)/DM20 in oligodendrocytes provide a genuine model for spastic paraplegia (SPG-2). Their axons are well myelinated but exhibit impaired axonal transport and progressive degeneration, which is difficult to attribute to the absence of a single myelin protein. We hypothesized that secondary molecular changes in PLP(null) myelin contribute to the loss of PLP/DM20-dependent neuroprotection and provide more insight into glia-axonal interactions in this disease model. By gel-based proteome analysis, we identified >160 proteins in purified myelin membranes, which allowed us to systematically monitor the CNS myelin proteome of adult PLP(null) mice, before the onset of disease. We identified three proteins of the septin family to be reduced in abundance, but the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 2 (SIRT2) was virtually absent. SIRT2 is expressed throughout the oligodendrocyte lineage, and immunoelectron microscopy revealed its association with myelin. Loss of SIRT2 in PLP(null) was posttranscriptional, suggesting that PLP/DM20 is required for its transport into the myelin compartment. Because normal SIRT2 activity is controlled by the NAD+/NADH ratio, its function may be coupled to the axo-glial metabolism and the long-term support of axons by oligodendrocytes.

Analysis of tumor vessel supply in Lewis lung carcinoma in mice by fluorescent microsphere distribution and imaging with micro- and flat-panel computed tomography.

In lung carcinomas the blood supply varies depending on tumor type and stage and can develop from pulmonary or bronchial circulation, or both. To examine this in vivo, primary bronchogenic Lewis lung carcinoma cells were intratracheally instilled in C57BL/6 mice. Within 7 days, histological examinations showed progressive tumor growth at the peripheral parenchymal region. The relative contribution of tumor blood supply via the pulmonary and systemic arteries was studied in detail using fluorescent microspheres (10 microm). When compared to healthy lung parenchyma (13:1), Lewis lung carcinoma tumor tissue (52:1) showed a fourfold increase in pulmonary to systemic microspheres, indicating that the pulmonary arteries are the predominant tumor-feeding vessels. After filling the vessels with a vascular cast, the microanatomy of vessels being derived from the pulmonary artery was visualized with micro computed tomography. Flat-panel volumetric computed tomography provided longitudinal visualization of tissue bridges between the growing tumor and the pulmonary vasculature. In this model of peripheral parenchymal malignancy, new imaging techniques allowed effective visualization of lung tumor growth and vascularization in living mice, demonstrating a pulmonary blood supply for lung tumors.

Role of hypoxia-inducible factor-1alpha in hypoxia-induced apoptosis of primary alveolar epithelial type II cells.

Hypoxia affects alveolar homeostasis and may induce epithelial injury, which has been implicated in lung diseases such as fibrosis. The underlying cellular and molecular mechanisms are, however, largely unknown. Primary rat alveolar epithelial type II cells (ATII) exposed to graded hypoxia for 24 and 48 h caused a dose-dependent induction of cell cycle arrest and suppression of proliferation, which were comparable to the effects of angiotensin II, a potent inducer of ATII cell death. Hypoxia-induced changes in ATII homeostasis are thought to proceed primarily via activation of hypoxia inducible-factor (HIF)-1alpha, because hypoxia increased HIF-1alpha protein expression, nuclear translocation, and transactivation of its specific DNA binding domain, the hypoxia responsive element (HRE). Under hypoxic conditions, expression of the proapoptotic protein Bnip3L, which belongs to the Bcl 2 family and is known to be one of the HIF-1-dependent target genes, was upregulated. Suppression of HIF-1alpha or Bnip-3L with small interfering RNA (siRNA) fully blocked the hypoxia-induced apoptosis and Bnip3L expression. In line with these data, overexpression of HIF-1alpha by transient transfection enhanced the hypoxia-induced apoptosis. Thus, we conclude that hypoxia suppresses alveolar epithelial cell proliferation and enhances ATII apoptosis through activation of the HIF-1alpha/HRE axis and a mechanism that involves Bnip3L. Targeting HIF-1alpha may represent a new strategy that could impede the alveolar denudation that is observed in several lung diseases.

Axonal neuregulin-1 regulates myelin sheath thickness.

In the nervous system of vertebrates, myelination is essential for rapid and accurate impulse conduction. Myelin thickness depends on axon fiber size. We use mutant and transgenic mouse lines to show that axonal Neuregulin-1 (Nrg1) signals information about axon size to Schwann cells. Reduced Nrg1 expression causes hypomyelination and reduced nerve conduction velocity. Neuronal overexpression of Nrg1 induces hypermyelination and demonstrates that Nrg1 type III is the responsible isoform. We suggest a model by which myelin-forming Schwann cells integrate axonal Nrg1 signals as a biochemical measure of axon size.