In the central nervous system (CNS), oligodendrocytes extend processes that form highly organized membrane wraps around axons[@b1]. These so-called myelin sheaths form the structural basis for rapid conduction of nervous impulses[@b1] and have important protective and trophic functions[@b2][@b3].

Loss of myelin occurs in a number of neurological disorders. In the most prominent demyelinating disease, multiple sclerosis (MS)[@b4][@b5][@b6][@b7], myelin sheaths are damaged by an autoimmune process[@b8]. With the advent of new drugs that slow the progression of the autoimmune disease, the question of how the regeneration of myelin sheaths can be enhanced is receiving increasing attention. However, so far, no remyelination-promoting therapy is available in a clinical setting.

Failure of remyelination can occur as a consequence of insufficient recruitment of oligodendrocyte progenitor cells (OPCs) into demyelinating lesions[@b9]. Furthermore, intrinsic changes within OPCs and factors that accumulate in MS lesions are able to inhibit OPC differentiation[@b10][@b11][@b12][@b13]. Lesion-associated inhibitors regulate distinct signaling cascades in OPCs, and it may be possible to manipulate them pharmacologically to promote myelin regeneration. For example, we recently demonstrated that inhibition of Phosphodiesterase (PDE)4 is able to promote CNS remyelination[@b14]. Other approaches include inhibiting Leucine rich repeat and Immunoglobin-like domain-containing protein (Lingo)-1[@b15], Wingless/mouse mammary tumor virus integration site (Wnt) signaling[@b16] and Retinoic acid receptor (RXR)-**γ**[@b17].

Previous studies, including our own work, demonstrated that myelin proteins, which accumulate following demyelination, are able to inhibit remyelination by blocking the differentiation of oligodendrocyte progenitor cells (OPCs)[@b18][@b19][@b20]. Investigating the underlying mechanisms we found that myelin proteins inhibit OPC differentiation by modulating RhoA and PKCα signalling[@b21][@b22]. On the basis of these results, we investigated the potential of PKCα inhibitors to overcome the myelin-associated differentiation block to promote OPC differentiation. We found that tamoxifen promoted this most effectively, albeit via an alternative mechanism.

Results
=======

Prompted by our previous findings[@b22], which showed that OPC-differentiation-inhibiting myelin breakdown products activate protein kinase C (PKC)α signalling, we investigated the potential of PKCα inhibitors to promote OPC differentiation in the presence of myelin protein extracts by assessing the expression of O4 ([Fig. 1](#f1){ref-type="fig"}). Of the drugs tested (UCN01, midostaurin, staurosporine, bryostatin-1, rottlerin, and tamoxifen), tamoxifen showed particularly potent differentiation-inducing effects. Addition of tamoxifen to OPCs plated on control substrates or to cells on myelin substrates increased the number of MBP+ and CNP+ cells respectively ([Fig. 1c](#f1){ref-type="fig"}, e--i). Quantitative RT-PCR assessment of *Mbp* relative to *Gapdh* mRNA expression demonstrated that tamoxifen treatment induced *Mbp* expression on the transcriptional level ([Fig. 1d](#f1){ref-type="fig"}). The increase of MBP+ cells in response to tamoxifen was concentration dependent ([Fig. 1e](#f1){ref-type="fig"}).

Tamoxifen is known to modulate estrogen receptor signaling and PKCα activity
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Tamoxifen has been used for the treatment of breast cancer since the early 1970s[@b23]. In addition to inhibiting PKCα activity, tamoxifen is known to modulate estrogen signaling ([Fig. 2a](#f2){ref-type="fig"}). So far, two classes of estrogen receptors have been identified: the nuclear estrogen receptors alpha (ERα) and beta (ERβ), and a membrane-bound G-protein-associated estrogen receptor (GPR30). Whereas ERα and ERβ are estrogen-responsive nuclear receptors that act as transcriptional regulators[@b24], GPR30 is known to modulate several signaling pathways such as the phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways[@b25].

Estrogen receptors are positive regulators of OPC differentiation
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The literature suggests that all three ER receptor types are expressed in oligodendrocyte lineage cells[@b26][@b27][@b28][@b29][@b30][@b31][@b32][@b33][@b34]. To investigate whether sex influences ER expression of neonatal rat OPCs were separately derived from female and male pups. We found that *Er-α*, *Er-β*, or *Gpr30* expression was comparable in both genders ([Supplementary Fig. 1](#S1){ref-type="supplementary-material"}). Based on these results we continued to use OPCs derived from pooled rat pups including both genders.

An interesting property of tamoxifen is that, depending on the cell type, it can act as agonist or antagonist on estrogen receptors[@b35]. For example, it has an *agonistic* effect in the uterus and skeleton but *antagonistic* activity in the breast, where it has been used for several years for the treatment of breast cancer.

We first sought to elucidate how ER or PKCα signaling affects the differentiation of OPCs into oligodendrocytes. For this purpose we transfected OPCs with small interfering (si)RNA directed against individual ERs and PKCα ([Fig. 2](#f2){ref-type="fig"}). Individual silencing of *Er-α, Er-β*, or *Gpr30* resulted in decreased OPC differentiation reflected by reduced mRNA expression of the differentiation markers cyclic nucleotide 3′-phosphodiesterase (*Cnp)* and myelin basic protein *(Mbp)* ([Fig. 2b--g](#f2){ref-type="fig"}).

In contrast, gene silencing of PKCα did not alter the differentiation of OPCs on control substrates ([Fig. 2](#f2){ref-type="fig"}). This was expected as with the culture conditions applied, inhibition of PKCα only shows effects in the presence of inhibitory myelin substrates[@b22]. Taken together these results confirm that ERα, ERβ, and GPR30 have important functional roles during the process of OPC differentiation (see also [Supplementary Fig. 3](#S1){ref-type="supplementary-material"}).

Tamoxifen-induced OPC differentiation depends on the presence of ER
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To investigate the mechanism by which tamoxifen promotes OPC differentiation, we silenced ERα, ERβ, GPR30, and PKCα in cells treated with tamoxifen. Similar to OPCs cultured in the absence of tamoxifen, silencing of ERα, ERβ, or GPR30 negatively affected the differentiation of cells in the presence of tamoxifen, indicating that the differentiation-inducing effects of tamoxifen are dependent on the presence of each of the ERs tested ([Fig. 3a--f](#f3){ref-type="fig"} and see [Supplementary Fig. 4a--h](#S1){ref-type="supplementary-material"} for calculations of the difference in *Cnp* or *Mbp* mRNA expression between "siRNA - tamoxifen" and "siRNA + tamoxifen conditions").

On the other hand, gene silencing of PKCα in combination with tamoxifen treatment resulted in increased *Mbp* expression at early time points as compared to tamoxifen treatment alone ([Fig. 3g,h](#f3){ref-type="fig"}). Tamoxifen therefore seems to exert OPC-differentiation-inducing effects via ERs but not via PKCα.

Assessing the effects of tamoxifen in an *in vivo* model of remyelination
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We next sought to investigate whether tamoxifen can promote OPC differentiation in a model of CNS remyelination. The largest targeted demyelinating lesions in a rodent model have so far been achieved by injection of ethidium bromide (EB) into white-matter tracts of the rat brainstem. Since the efficiency of remyelination is inversely correlated with lesion size, this model is advantageous for studying the kinetics of CNS remyelination[@b36]. Application of EB results in rapid and complete depletion of glial cells, including astrocytes and oligodendrocyte lineage cells, within 3 days with good preservation of axons. As a consequence the axons become demyelinated. This is followed by a highly predictable sequence of events during which the lesion becomes fully remyelinated[@b37]. In young-adult rats, remyelination is complete within 28 days.

Estrogen receptors and active PKCα are expressed by oligodendrocyte lineage cells *in vivo*
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To confirm that ERα, ERβ, and GPR30, as well as activated PKCα, are present in remyelinating lesions, we carried out immunohistochemical staining using antibodies directed against the respective antigens and included co-stainings for the oligodendrocyte lineage cell-specific transcription factor OLIG2. This confirmed the presence of all three ERs and active PKCα in oligodendrocyte lineage cells within remyelinating lesions ([Fig. 4](#f4){ref-type="fig"}).

Tamoxifen induces OPC differentiation in demyelinated lesions
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To investigate whether tamoxifen treatment is able to induce OPC differentiation *in vivo*, animals receiving daily intraperitoneal injections of tamoxifen at two different doses (0.5 mg/day and 2 mg/day) were compared with controls receiving vehicle injections. Several stage-specific molecular markers were used to characterize the OPC response to tamoxifen in remyelinating lesions. We analyzed lesions 7 days post lesion induction (pli) to assess the recruitment of OPCs and lesions 14 days pli to assess the differentiation of OPCs into mature oligodendrocytes ([Fig. 5a--e](#f5){ref-type="fig"}). Whereas the number of immature *Pdgfrα* OPCs or "activated" NKX2.2-expressing OPCs was comparable between the groups 7 and 14 days pli ([Fig. 5f--j](#f5){ref-type="fig"}), a significant increase in cells expressing the mature oligodendrocyte markers adenomatous polyposis coli (APC(CC1)) and proteolipid protein (*Plp*) was detected in animals receiving the higher treatment dose 14 days pli ([Fig. 5k--o](#f5){ref-type="fig"}). These findings indicate that tamoxifen treatment induced the differentiation of OPCs in the lesions.

However, myelin-sheath formation is also regulated at levels downstream of the transcriptional program. Does the enhanced expression of mature markers therefore correlate with an accelerated formation of new myelin sheaths?

Tamoxifen promotes CNS remyelination
------------------------------------

To investigate the extent of CNS remyelination at 14 days, the experiments were repeated using the higher dose of tamoxifen and the tissue was embedded into resin for ultrastructural analysis. Investigator-blinded rank analysis demonstrated that tamoxifen significantly increased the number of remyelinated axons ([Fig. 6a](#f6){ref-type="fig"}). This was confirmed by determining the number of remyelinated axons on high-power digitized micrographs of caudal cerebellar peduncle lesions ([Fig. 6b](#f6){ref-type="fig"}). The relative thickness of newly formed myelin did not differ between the groups ([Fig. 6c--h](#f6){ref-type="fig"}). Taken together, these data demonstrate that tamoxifen treatment resulted in accelerated remyelination at 14 days pli.

Tamoxifen treatment does not alter the macrophage response
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Our *in vitro* data indicate that tamoxifen can directly regulate OPC differentiation. However, it is possible that tamoxifen also modulates the function of other cell types in demyelinated lesions. In particular, macrophages are important determinants for the efficiency of remyelination[@b38][@b39]. We initially assessed the number of osteopontin-positive macrophages in the lesions. No differences were detected between the groups with respect to the overall macrophage presence ([Supplementary Fig. 5](#S1){ref-type="supplementary-material"}). Recent findings highlight the importance of macrophage activation, specifically of M1 and M2 phenotypes[@b40]. Quantification of the presence of CCR7^+^-M1 and Arginase1^+^-M2 macrophages 7 days post lesion induction did not show significant differences between the tamoxifen treated and control animals. Furthermore, assessment of phagocytic activity in remyelinating lesions by staining intracellular myelin-derived neutral lipids with Oil Red-O did not detect differences between the groups. Nevertheless, further indirect effects of tamoxifen cannot be ruled out entirely.

Discussion
==========

Here we have shown that tamoxifen induces OPC differentiation and remyelination *in vitro* and *in vivo*, and that this relies on modulation of the estrogen receptors ERα, ERβ, and GPR30. The effects of tamoxifen do not involve the PKCα pathway.

Estrogen receptors play an important role in OPC differentiation and remyelination[@b31][@b32][@b33][@b41]. Similarly, in our experiments, silencing of ER resulted in impaired OPC differentiation *in vitro*, even estrogen was not specifically added to the medium. However, fetal calf serum (FCS) used in the OPC media does contain several components, which could activate estrogen receptors in a ligand-independent manner. Picard 2003, describes a list of molecules which could cross-talk with the estrogen receptors without physically interacting with them, for example, insulin (which forms an important component of the differentiation medium), IGF and FGF[@b42]. Moreover, testosterone (present in FCS) could be converted by aromatase, which is expressed in low levels in glial cells (<http://web.stanford.edu/group/barres_lab/brain_rnaseq.html>), into estrogen and therefore, activate all estrogen receptors. As a results, silencing of ER is likely to impair OPC differentiation in our culture model.

When estrogen receptors were silenced, the differentiation-inducing effects of tamoxifen were reduced, indicating an involvement of ER (or GPR30) in tamoxifen-mediated effects. In contrast, tamoxifen treatment in combination with silencing of PKCα did not reduce the efficiency of tamoxifen-induced OPC differentiation. In fact we observed transiently increased MBP expression at day 4 compared with tamoxifen treatment alone. While this is only a transient finding at a single time point, it may indicate that combinatorial treatment of tamoxifen and inhibition of PKCα may act synergistically to accelerate OPC differentiation. Nonetheless it can be concluded that differentiation-inducing effects of tamoxifen do not rely on inhibition of PKCα.

Our results also confirm previous reports[@b22] that the inhibition of PKCs does not promote differentiation by itself when cells are cultured in serum containing media. Conversely, in the presence of myelin debris, the inhibition of PKCα neutralizes myelin-associated inhibitors and promotes differentiation[@b22].

The majority of the reported pharmacological (metabolism and toxicology) studies of tamoxifen use the free base form of tamoxifen and not the soluble forms (citrate tamoxifen and/or 4-OH tamoxifen). Therefore, the present *in vitro* and *in vivo* experiments were also conducted with tamoxifen in its free base form. However, a recent paper by Barrat *et al.* evaluated metabolites of tamoxifen (4-hydroxytamoxifen and endoxifen) as well as tamoxifen citrate, a water-soluble version on a rat glia restricted precursor cell line[@b43]. They found that all metabolites promoted the expression of late stage markers of OPC differentiation, such as *Cnp* and *Mbp*, which is consistent with our findings using primary rat OPC cultures. In addition, the effect of tamoxifen was blocked when a pan-antagonist was added to the media, supporting our finding that tamoxifen acts as an estrogen receptor agonist in OPC differentiation.

Apart from oligodendroglial cells, tamoxifen potentially also acts on other cell types. We therefore assessed activated macrophages/microglia, which are known to play a key role in remyelination. We did not observe changes in the number of recruited cells or the myelin debris removal at 7 and 14 pli. Furthermore, no differences were observed regarding the number of the pro-inflamatory (M1) or anti-inflammatory (M2) macrophages. Nevertheless, these results cannot entirely rule out potential indirect effects such as differential release of cytokines or chemokines mediated by tamoxifen. The role of astrocytes was not evaluated since their role during remyelination is less clear.

To limit the biological variation and to increase the challenge of the experimental treatment by using a paradigm of efficient remyelination, only young adult female rats were included in the present *in vivo* experiments. The literature indicates that sex-related differences exist with respect to the efficiency of remyelination: whilst in young rats the rate of remyelination is comparable between the sexes, it is more efficient in aged female rats as compared to male rats[@b44]. Estrogen signalling plays a distinctive role in females. However, investigating OPCs derived from segregated female and male pups with respect to expression of estrogen receptors and their response to tamoxifen did not show any sex-related differences. Nevertheless, it cannot be ruled out that indirect systemic effects exist and that male rats may react differently to tamoxifen treatment. The main reasons why we decided to use female rats in our study were comparability to previous studies and the fact that MS often affects female patients.

In a clinical setting for the treatment of breast cancer tamoxifen was used at doses ranging from 20--40 mg per day. With an estimated average weight of females at 65 Kg, this translates into 0.3--0.6 mg/Kg. The estimated human equivalent in our experiments is 0.27 mg/Kg, which is in keeping with current treatment paradigms in human.

Steroid hormones are known to influence the course of MS and also to modulate demyelination in animal models. During pregnancy, a reduction in the frequency of relapse rate in patients with relapsing--remitting MS (RRMS) has been reported[@b45][@b46][@b47]. Similarly, a decrease in the severity of the symptoms in comparison with a nonpregnant control group was detected in pregnant mice affected by experimental autoimmune encephalomyelitis, a model of the autoimmune destruction of myelin sheaths occurring in MS[@b48]. Interestingly, the administration of estriol to nonpregnant women with RRMS showed a reduction in the number of Th1 cells, which are involved in the autoimmune response, and also in the clinical score of the disease[@b49]. Furthermore, there is evidence that steroid hormones promote myelin repair: administration of progesterone to cerebellar slices after lysolecithin-induced demyelination showed an increase in remyelination[@b50] and recently, the same group has shown that administration of progesterone promotes myelin regeneration in chronic demyelinated lesions in the corpus callosum of mice after being fed with cuprizone[@b51]. These data support the positive effects of steroid hormones and their potential for the treatment of demyelinating diseases.

Considerable efforts are currently being made to develop remyelination-enhancing drugs. The use of tamoxifen has several advantages over the development of new drugs. For example, it has an excellent safety profile that has undergone the test of time by exhaustive application in the clinic. In our study, tamoxifen emerges as a good candidate for testing in an experimental medicine study specifically designed to assess remyelination in MS patients.

Our findings also have important implications for basic research: experiments investigating the function of specific molecules in the CNS on the basis of the widely used CreERT2Lox system rely on the tamoxifen-dependent activation of Cre recombinase for the induction of genetic changes. Our data demonstrate that administration of tamoxifen has a strong influence on CNS stem/precursor cells. To rule out any unintended effects of tamoxifen on the increasingly used inducible CreERT2-rat transgenic models, and potentially also for CreERT2 mice models, the inclusion of adequate controls is advised.

Methods
=======

Preparation of primary OPC cultures
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Primary OPC cultures were isolated from neonatal Sprague Dawley (postnatal day 0--3) rat forebrains following a standard protocol[@b20][@b22] Neonatal pups were euthanized according to "Schedule 1" regulations from the Home Office Animal Procedures Committee UK. Sex-segregated cultures were prepared from female and male only pups. The male-derived OPCs was confirmed by genotyping for the presence of the SRY gene, present only in the Y chromosome (data not shown). Differentiation was induced using Sato's medium supplemented with 0.5% fetal calf serum (FCS). For all *in vitro* experiments, only cultures with ≥94% purity were used.

Preparation of myelin-membrane substrates and myelin-protein extracts
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Myelin was purified following two rounds of centrifugation using a discontinuous density gradient of sucrose and osmotic disintegration as described previously[@b20][@b52]. After centrifugation for 1 h at 100,000 × *g*, the interface between the two sucrose solutions was collected, washed with water and pelleted again at 55,000 × *g* for 10 min. The pellet was dissolved in water and incubated for 1 h on ice to introduce an osmotic shock. The solution was then centrifuged again at 55,000 × *g* for 10 min. The resulting pellet was purified by repetition of the sucrose gradient step described above. The myelin-containing interface was collected and, after two washing steps, a pure myelin pellet was obtained and frozen at −80 °C. The frozen myelin pellets were thawed and resuspended in 1% *N*-octyl-β-D-glucopyranoside, 0.2 M Na~3~PO~4~, pH 6.8, 0.1 M Na~2~SO~4~, and 1 mM EDTA, and incubated at 23 °C for 2 h. Following an ultracentrifugation step (100,000 × *g*, for 30 min at room temperature) the supernatants (which are myelin protein extract, MPE) were collected and stored at 80 °C until further use. To test OPC differentiation, slides were coated with MPE following application of poly-[l]{.smallcaps}-lysine (PLL) to promote cell attachment to the well.

Small molecule experiments in OPC cultures
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Estrogen receptors' agonist/antagonist as well as PKC inhibitors (Tocris Bioscience, Bristol, UK) were dissolved in DMSO, water or ethanol as stated in the according data sheet and added to Sato's differentiation medium in concentrations stated. Dissolved small molecule stock solutions were diluted at least by 1:10000 when added to the cells eliminating eventual side effect of the dissolving agent. Concentrations of the PKC inhibitors were determined in independent dose-finding experiments. Concentrations used for the experiments included in the present study were as followed, 7-hydroxystaurosporine (UCN01): 10 nM, midostaurin: 1 nM, staurosporine: 25 nM, bryostatin-1: 5 nM, rottlerin: 5 nM and tamoxifen: 50 nM.

Immunocytochemical analysis *in vitro*
--------------------------------------

OPCs were seeded onto eight-well chamber slides (2 × 10^4^ cells/well) coated with PLL alone or PLL plus MPE. Following 2 days of differentiation, cells were stained with anti-O4 (1:100; Millipore Corporation, Billerica, MA) and anti-MBP (1:300; Millipore Corporation) antibodies. Secondary antibodies conjugated with Alexa 488 and Alexa 555 were used to visualize positive cells (1:300; Invitrogen, Carlsbad, CA)[@b22]. To assess OPC differentiation, the percentage of O4- or MBP-positive cells relative to \>100 DAPI-stained nuclei per experiment for each condition and each experiment in randomly selected eye fields was determined, with the investigator being blind to treatment group. To assess expression of estrogen receptors in male and female OPCs, OPCs isolated from sex-segregated mixed glia cultures were seeded on PLL-coated glass coverslips on 24-well plates (7 × 10^4^ cells/well). Following 2 and 4 days of differentiation, cells were stained with anti-ERα (1:300; Abcam), anti-ERβ (1:300; Abcam), anti-GPR30 (1:300; Invitrogen) and anti-OLIG2 (1:1000; Millipore) or anti-O4 (1:200; SIGMA), followed by staining with the secondary antibodies indicated above. The percentage of ERα, ERβ or GPR30-positive cells relative to \>100 oligodendrocyte lineage cells (O4-positive or OLIG2-positive) per experiment for each condition was determined in randomly selected eye fields. Following immunocytochemistry, cells were mounted with Prolong gold antifade mounting medium (Invitrogen). Cells were visualized and digitized at ambient temperature on an LSM 700 confocal microscope (Zeiss, Thornwood, NJ) at 20× and 40× magnification using Zen Application software (Zeiss).

RNA extraction and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
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A minimum of 50,000 oligodendrocytes were washed twice with ice-cold phosphate-buffered saline (PBS). OPCs were lysed, and total RNA was extracted and purified using the RNAeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. qRT-PCR was conducted as previously described on an Applied Biosystems 7500HT Fast Real-time PCR system[@b22] (Applied Biosystems, Foster City, CA). Values are represented as target gene/glyceraldehyde-3-phosphate dehydrogenase (*Gapdh*) ratios. Triplicate measurements were made on three biological replicates.

siRNA transfections
-------------------

OPC cultures were kept overnight in Sato's proliferation medium without antibiotic supplement. On the following day, siRNA smart pool (for details of siRNAs used see [Supplementary Table 1](#S1){ref-type="supplementary-material"}) and lipofectamine RNAiMAX were dissolved separately in OptiMem medium (LifeTech, Gaithersburg, MD). Lyophilized ON TARGET plus smart pool siRNA (Dharmacon/ThermoScientific, Waltham, MA) was dissolved in 5 × RNAi buffer, divided into aliquots, and stored at −20 °C. OPCs were transfected using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions and using a 1:400 dilution of lipofectamine and 50 nM of siRNA smart pool per reaction. To determine the silencing efficiencies, cell samples were collected at 48 h and 72 h after transfection for RNA extraction ([Supplementary Fig. 2](#S1){ref-type="supplementary-material"}).

Induction of focal demyelination
--------------------------------

All experiments were conducted in accordance with the UK Home Office's animal-welfare regulations and institutional guidelines for animal care and handling and approved to be performed by UK Home Office's and University Biomedical Support Service under project license number: 70/7715, formerly 80/2228. Female Sprague Dawley rats in estrum (200--220 g) were anesthetized using a combination of 2% isoflurane in oxygen (as carrier gas) and a subcutaneous injection of buprenorphine hydrochloride 0.03 mg/kg (Vetergesic, Animalcare Ltd, Hull, UK). Demyelinated lesions were induced bilaterally by stereotactic injection of EB (0.01%, 4 ml) into the caudal cerebellar peduncle, −10.4 mm caudal, ±2.6 mm lateral, and −7.3 mm ventral from bregma[@b36][@b37]. Tamoxifen (Sigma-Aldrich, St. Louis, MO) was dissolved in 100% ethanol and diluted with corn oil (Sigma-Aldrich) to a final concentration of 10% ethanol. The treatment animals were injected intraperitoneally with tamoxifen equivalent to 0.5 mg/Kg/day or 2 mg/Kg/day, and the control group received an equivalent volume of the vehicle. In both groups, drug and vehicle were injected on the third day post-lesion (dpl) for 11 days until 14th day post-lesion. Animals were sacrificed after 7 and 14 dpl. To rule out any effects attributable to the size of the lesion, the density of *Plp*-positive cells was assessed in PBS-treated animals by linear regression (GraphPad, Prism, San Diego, CA). The inclusion of lesions smaller than 0.2 mm^2^, which were strongly populated with *Plp*-positive cells, resulted in a significant non-zero slope indicating an effect of the lesion size on the cell density, as shown in previous studies[@b14]. This may be explained by the fact that in small lesions, recruitment of OPCs and the clearance of myelin debris are more efficient, and thus remyelination becomes more efficient than with larger lesions. No significant correlation between the size of the lesion and the density of *Plp*-positive cells was observed in lesions larger than 0.2 mm^2^, and so only lesions larger than 0.2 mm^2^ were included in the analysis.

*In situ* hybridization
-----------------------

The expression of a number of marker mRNA species in demyelinated lesions was examined by *in situ* hybridization with digoxigenin-labeled cRNA probes; *Pdgfr-α*, *Plp*, and osteopontin were kindly provided by Dr. Chao Zhao, University of Cambridge. Animals were perfused with 4% paraformaldehyde (PFA) via the left ventricle. The tissue was extracted, postfixed in 4% PFA overnight at 4 °C, and cryoprotected in 20% sucrose. *In situ* hybridizations were carried out on cryostat sections (12 μm) using established protocols[@b19][@b53]. ImageJ 1.44 (Wayne Rasband, National Institutes of Health, Bethesda, MD) was used to determine the number of positive cells within the lesions on digitized sections. All analyses were conducted with the investigator blind to the treatment group.

Immunohistochemical analysis *in vivo*
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Immunohistochemistry was performed on 12-μm-thick sections of 4% PFA-fixed tissue. Antigen was retrieved using 0.01 M citrate buffer (Dako) at pH 6.0 for 10 min in a 95 °C water bath, and slides were rinsed in PBS (pH 7.4) thee times (10 min each at room temperature). Then, sections were incubated with the blocking solution containing 5% inactivated normal donkey serum (Abcam, Cambridge, MA) and Triton X-100 (0.1%) in PBS for 60 min at room temperature. The following antibodies were stained: anti-OLIG2 (1:300; Millipore), anti-APC(CC1) (1:300; Calbiochem, La Jolla, CA), anti-NKX2.2 (1:300; DHSB), anti-ERα (1:300; Abcam), anti-ERβ (1:300; Abcam), anti-GPR30 (1:300; Invitrogen), and anti-PKCα-p (1:300; Santa Cruz Biotech, Santa Cruz, CA). Sections were incubated with primary antibody overnight, washed three times in PBS, and then incubated with appropriate Alexa 488- or 594-conjugated secondary antibodies (Invitrogen) for 3 h at room temperature. They were then counterstained with DAPI (Sigma-Aldrich). Prolong gold antifade mounting medium (Invitrogen) was used to mount the slides. Slides were examined in a fluorescence microscope (Nikon TE-2000; Nikon, Düsseldorf, Germany), and images were taken by confocal microscopy (Zeiss LSM-700). All quantifications were conducted with the investigators blind to the treatment group. Data are given as mean ± SEM and statistically analyzed. Three sections per marker per animal were analyzed, and a minimum of four animals were used.

Oil Red-O staining
------------------

Sections from control and treatment group were left to dry at room temperature and then placed in a Coplin jar containing propylene glycol (100%) for 5 min. Prewarmed Oil Red-O solution (0.5% in propylene glycol, Sigma-Aldrich) was stained at 65 °C for 10 min. Following incubation for 5 min with differentiation solution (85% propylene glycol), slides were rinsed twice with water and then mounted using a jelly-based mounting media. Representative images of Oil Red-O stained lesions were digitized, and, using ImageJ software, the intensity of the lesion area was measured from the red channel. Data are given as mean ± SEM and statistically analyzed. Three sections per marker per animal were analyzed, and a minimum of four animals were used.

Histological analysis of remyelination
--------------------------------------

To assess the extent of remyelination, the tissue was fixed in 4% glutaraldehyde, osmicated, and processed into resin (TAAB Laboratories Equipment Ltd, Reading, UK)[@b29]. Sections (1 mm) were stained with 1% toluidine blue. The extent of remyelination was then assessed by light and electron microscopy (see below). Based on the thickness (or absence) of myelin sheaths, demyelinated axons, axons bearing native myelin sheaths, and remyelinated axons on light and electron micrographs can unambiguously be distinguished[@b54]. Lesions were ranked according to the extent of remyelination by two blinded investigators and statistically analyzed using a two-tailed Mann--Whitney test. Increased remyelination corresponds to a higher rank value. In addition, demyelinated versus remyelinated axons were manually counted on 100× digitized pictures randomly taken from the lesion border. Ratios of remyelinated and demyelinated versus the total number of axons were analyzed using Student's *t*-test.

Electron microscopy
-------------------

Ultra-thin sections (1 μm) containing the lesions were stained with aqueous 4% uranylacetate and lead citrate. The sections were visualized on an electron microscope (Hitachi H-600 electron microscope; Hitachi, Tokyo). A minimum of five micrographs per lesion were analyzed from the border of the lesion. The G-ratios (the ratio of axon circumference to myelin circumference) of oligodendrocyte-remyelinated axons were calculated using ImageJ and statistically analyzed (lower G-ratios indicate thicker myelin sheaths) using Student's *t*-test.

Statistical analysis
--------------------

Data were analyzed using GraphPad software (Prism). Student's *t*-test was performed for single-group comparisons (control vs treatment), and multiple-group comparisons were conducted using one-way ANOVA followed by Dunnett's post-test. qRT-PCR data were analyzed using one-way ANOVA followed by Bonferroni's post-test. For rank analysis, a two-tailed nonparametric Mann--Whitney *U* test was used.

Additional Information
======================

**How to cite this article**: Gonzalez, G. A. *et al.* Tamoxifen accelerates the repair of demyelinated lesions in the central nervous system. *Sci. Rep.* **6**, 31599; doi: 10.1038/srep31599 (2016).

Supplementary Material {#S1}
======================

###### Supplementary Information

This work was supported by grants from Wings for Life and the UK Multiple Sclerosis Society. M.R.N.K. holds an NIHR CL award and a Sir David and Isobel Walker fellowship. G.A.G. was supported by CONICYT (Becas Chile) scholarship, and M.P.H. was supported by a Sir David and Isobel Walker studentship. Research in the author's laboratory is supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. We acknowledge expert technical assistance from Dr. Daniel Morrison for electron microscopy expertise and are very grateful for the support of Prof. Robin J.M. Franklin. We also gratefully acknowledge editorial assistance by Ana Sharman, Cofactor Ltd., Editing, training and consultancy in scientific publishing. This report is independent research arising from a Clinician Scientist Award, CS-2015-15-023, supported by the National Institute for Health Research. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.

The authors declare no competing financial interests.

**Author Contributions** G.A.G., M.P.H. and M.R.N.K. designed the experiments. G.A.G. and J.R. performed the *in vivo* experiments; C.Z. and S.R. assisted with the *in vivo* experiments. M.P.H., Y.A.S. and A.I.A performed the *in vitro* experiments. G.A.G., M.P.H., Y.A.S., A.I.A. and M.R.N.K. analyzed the data. G.A.G., Y.A.S. and M.R.N.K. wrote the paper.

![Tamoxifen promotes OPC differentiation in the presence and absence of myelin associated inhibitors.\
(**a**) Bar graph demonstrating differentiation-promoting effects of PKCα inhibitors (7-hydroxystaurosporine (UCN01): 10 nM, midostaurin: 1 nM, staurosporine: 25 nM, bryostatin-1: 5 nM, rottlerin: 5 nM, and tamoxifen: 5 nM) on OPCs cultured on inhibitory myelin substrates (myelin protein extract, MPE) after 2 days. O4 expression is an early marker of OPC differentiation (n = 4). One-way ANOVA with Dunnett's post-hoc test: MPE vs UCN01, midostaurin, staurosporine, bryostatin-1, rottlerin, and tamoxifen: \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.0001. (**b**) Bar graph evaluating the efficacy of tamoxifen-induced OPC differentiation in the presence of MPE at concentrations ranging from 0.5 nM to 50 μM. (n = 4), One-way ANOVA with Dunnett's post-hoc test: control 2d vs 5, 50, 500 nM: \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.0001. (**c**) Bar graph demonstrating that 5 nM tamoxifen is able to increase the number of MBP-immunopositive cells plated on poly-L-lysine control substrates. In the presence of MPE, tamoxifen is able to increase the number of CNP positive cells after 2 days differentiation. (n = 3); ANOVA: CNP \*\*\*\*p \< 0.0001, MBP \*\*\*\*p \< 0.0001; Dunnett's post-hoc test PLL vs PLL + Tmx: MBP \*\*\*p \< 0.0001; MPE vs. MPE + Tmx: CNP \*\*\*p \< 0.0001). (**d**) Bar graph showing quantification of *Mbp* expression using qRT-PCR for *Mbp* relative to *Gapdh* mRNA. Tamoxifen increased *Mbp* expression in OPCs plated on PLL control substrates after 2 days in dose-dependent manner. One-way ANOVA with Dunnett's post-hoc test: control 2d vs 5, 50, 500 nM: \*\*\*p \< 0.0001. (**e**) Bar graph demonstrating that tamoxifen is also able to increase the number of MBP-positive OPCs plated on poly-L-lysine (PLL) control substrates after 2 days. 1-way ANOVA with Dunnett's post-hoc test: control 2d vs 5, 50, 500 nM: \*p \< 0.05. (**f--i**) Representative images of CNP-MBP-positive OPCs treated with vehicle or tamoxifen (50 nM) in presence and absence of MPE. Error bars: standard error of the mean (SEM). Scale bar: in f-i = 30 μm.](srep31599-f1){#f1}

![ERα, ERβ, and GPR30 are important regulators of OPC differentiation.\
(**a**) Model of tamoxifen signalling in OPCs: (1) tamoxifen binds nuclear ERα/β receptors, which respond by modulating transcriptional events; (2) tamoxifen can also activate membrane bound GPR30 receptors, inducing an increase in intracellular Ca2 + levels. This in turn activates adenylate cyclase (AC), resulting in activation of protein kinase A (PKA) and CREB1. Furthermore, (3) tamoxifen is also able to inhibit PKCα, which is known to modulate cytoskeletal proteins, including MARCKS, and inhibit OPC differentiation. (**b--i**) Line graphs demonstrating impaired OPC differentiation (decreased expression of *Cnp* and *Mbp* mRNA relative to glyceraldehyde-3-phosphate dehydrogenase) when OPCs were transfected with siRNA for *Er-α*, *Er-β* or *Gpr30* as compared with non-targeting scrambled siRNA (Scr, gray line). Gene silencing of *Prkcα* did not negatively affect the differentiation of OPCs on control substrates (n = 3). Oneway ANOVA with Bonferroni's post-hoc tests: \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.0001. dpT, days post siRNA transfection. Error bars: SEM.](srep31599-f2){#f2}

![Tamoxifen-induced OPC differentiation depends on ERα, ERβ, and GPR30, but not PKCα signaling.\
(**a--h**) Line graphs demonstrating that tamoxifen-induced differentiation (+Tmx (50 nM)) of OPCs is impaired when cells are concomitantly transfected with siRNA targeting *Er-α*, *Er-β*, or *Gpr30* as compared with control siRNA (scrambled, Scr, gray line). Gene silencing of *Prkcα* in addition to tamoxifen treatment resulted in transiently increased *Mbp* expression at day 4, demonstrating that tamoxifen-induced OPC differentiation is independent from PKCα (n = 3). One-way ANOVA with Bonferroni's post-hoc tests: \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.001. Error bars: SEM.](srep31599-f3){#f3}

![Expression of estrogen receptors and active PKCα at 14 pli.\
(**a--d**) Representative images taken at 40 × showing expression of estrogen receptors ERα, ERβ and GPR30 (green) and phosphorylated (active) PKCα (white) by OLIG2 + oligodendrocyte lineage cells (red) in CCP lesions 14 days post ethidium bromide injection (pli). White arrows point ERs + or PKCα + /OLIG2 + cells. Scale bar: 20 μm.](srep31599-f4){#f4}

![Tamoxifen promotes OPC differentiation after toxin-induced demyelination in white matter.\
(**a**) Scatter plot showing comparable numbers of immature OPCs expressing *Pdgfr-α* in remyelinating lesions at day 7 and 14 (n ≥ 5), Student's *t*-test 7 days pli: p = 1.000, 14 days pli: p = 0.2468. (**b--e**) Representative sections of tamoxifen- (Tmx) and vehicle treated animals labeled by *in situ* hybridization for *Pdgfr-α* at 7 pli and 14 pli. (**f**) Scatter plot demonstrating comparable numbers of *Pdgfr-α* + /NKX2.2 + activated OPCs in lesions at day 7 and 14 days after demyelination (n ≥ 5), Student's *t*-test 7 pli: p = 0.4206, 14 pli: p = 0.9269. (**g,j**) Representative sections labeled by *in situ* hybridization for *Pdgfr-α* and immunohistochemistry for NKX2.2 at 7 pli and 14 pli. (**k**) Scatter plots demonstrating that tamoxifen treatment significantly promotes OPC differentiation at 14 pli on the basis of increased expression of mature markers (CC1 + /OLIG2 + and *Plp* + cells) 14 pli (n ≥ 5), Student's *t*-test, CC1 + /OLIG2 + : p = 0.0274, *Plp* + : p = 0.0062. (**i--o**) Representative sections of lesions labeled by immunohistochemistry for CC1 and OLIG2, or *in situ* hybridization for *Plp* at 14 pli. White arrows in the insets point to double positive cells. Error bars: SEM. Scale bar: in b-e, g-j, l-o = 200 μm.](srep31599-f5){#f5}

![Tamoxifen treatment accelerates CNS remyelination.\
(**a**) Rank analysis (scatter plot) of remyelination in CCP lesions at 14 pli assessed on toluidine-blue-stained semithin sections. Higher rank scores indicate a greater extent of remyelination (n ≥ 5); Mann-Whitney U test: p \< 0.05. (**b**) Enhanced remyelination was confirmed by manual counts of remyelinated and demyelinated axons on digitized sections (n ≥ 5). Mann-Whitney U test: p \< 0.05. (**c,d**) Representative toluidine blue-stained semithin sections (100x) of vehicle ('Cont.') and tamoxifen-treated animals. (**e,f**) Representative electron micrographs of control and tamoxifen-treated animals (remyelinated axons pseudo-colored). (**g,h**) Scatter plot and box plot of G-ratios demonstrating comparable relative thickness of newly formed myelin sheaths in control and tamoxifen-treated lesions. Student's *t*-test: p \> 0.05. Error bars: SEM. Scale bar: in c, d = 10 μm; in e, f = 5 μm.](srep31599-f6){#f6}

[^1]: These authors contributed equally to this work.


ect to z.
-1
Let u(b) be the first derivative of -b**10/504 - b**6/120 + b**3/3 - 3. Let p(l) be the third derivative of u(l). Find the third derivative of p(d) wrt d.
-1200*d**3
What is the first derivative of 18*c**4 + 28*c**4 + 8 - 104*c - 47*c**4 wrt c?
-4*c**3 - 104
Suppose -2*z - 14 = -5*a, -2*a + 3*z - 1 = -0. Let s be (42/a)/(2/8). Differentiate 7*g - 47 + s - 2*g wrt g.
5
What is the second derivative of -702*n**3 + 96*n + 36*n - 99*n + 17*n + 60*n wrt n?
-4212*n
Let w(d) = d**3 - 17*d**2 + 3*d - 38. Let c be w(17). What is the third derivative of 43*i**3 + 8*i**2 - c*i**2 + 22*i**2 wrt i?
258
Let i(v) be the third derivative of v**6/120 - v**5/60 - v**3/6 + 2*v**2. Let s(k) = 20*k**3 + 2*k**2 + 23. Let l(n) = 2*i(n) + s(n). Differentiate l(q) wrt q.
66*q**2
What is the third derivative of 10*b**2 + 3 - 9 + 6 - 71*b**4 wrt b?
-1704*b
Let n = -68 + 71. Find the second derivative of -15*l + 19*l + 3*l + 30*l**n wrt l.
180*l
Let v = 458/3 - 148. Let j(g) be the first derivative of -v*g**3 - 2*g**4 + 0*g**2 + 4 + 0*g. Find the third derivative of j(y) wrt y.
-48
Let m(n) be the first derivative of 11*n**4/4 + 2*n**3 + 19*n - 8. Let h(a) = 11*a**3 + 5*a**2 + 18. Let t(f) = 6*h(f) - 5*m(f). Differentiate t(x) wrt x.
33*x**2
Find the first derivative of -23*p + 47 - 40 + 143 wrt p.
-23
Let g be -5 - 80/(-6) - (-2)/(-6). Find the third derivative of -11*m**2 + m**6 + 6*m**6 + 2*m**6 - g*m**2 wrt m.
1080*m**3
Let i be (2 - 6 - -4) + 2. Find the third derivative of 1165 - 18*s**4 - 18*s**i - 1165 wrt s.
-432*s
Let r(k) = -404*k**3 - 9*k**2 - 11*k - 710. Let d(m) = -203*m**3 - 4*m**2 - 5*m - 355. Let p(j) = -9*d(j) + 4*r(j). What is the first derivative of p(q) wrt q?
633*q**2 + 1
Suppose -6 = -8*v + 5*v. Let g(o) = 3*o**2 - 2*o + 2. Let j be g(v). Find the third derivative of 8*l**6 - 19*l**6 - l**6 + 0*l**2 + j*l**2 wrt l.
-1440*l**3
Let m(a) = -a**2 - 12*a - 15. Let j be m(-10). Find the second derivative of 1887*l**3 + 2*l - 1887*l**3 - 12*l**j wrt l.
-240*l**3
Let j(m) be the third derivative of m**8/336 - m**7/14 - 61*m**4/24 - 2*m**2 - 8*m. Find the second derivative of j(k) wrt k.
20*k**3 - 180*k**2
Let d(w) be the first derivative of -13*w**4/24 - w**3/3 - 3*w**2 + 5. Let x(a) be the second derivative of d(a). Differentiate x(q) wrt q.
-13
Suppose 3*v = 31 - 25. Suppose 2*f + 2*q = -v*f + 68, 2*f - q - 42 = 0. What is the derivative of 1 + 9*d**3 - 12 - f*d**3 wrt d?
-30*d**2
Suppose 4*r - 35 = -7. Suppose -3*f + 4*f - 2 = 0. What is the second derivative of 3*c**f - 3*c + r*c - 2*c wrt c?
6
Suppose -o = -3*x - 6 - 3, -5*x = -2*o + 16. Find the second derivative of 0*n**3 - 8*n**3 - n**o - n + 20*n wrt n.
-54*n
Suppose 28*b = 26*b + 12. Find the third derivative of 2*h**b - 5*h**3 - 31*h**2 + 11*h**3 - 6*h**3 wrt h.
240*h**3
Suppose -4*h = -10 + 2. Suppose -5*v - 5 = -10*c + 7*c, 0 = 3*v - 6. Differentiate m + 14 + h*m - c wrt m.
3
Let u(d) = -211*d**4 - 3*d**2 - 45*d - 3. Let y(t) = 423*t**4 + 5*t**2 + 90*t + 6. Let w(o) = 5*u(o) + 3*y(o). What is the second derivative of w(f) wrt f?
2568*f**2
What is the first derivative of 886*i + 198 - 14 - 632*i wrt i?
254
Let f(a) = 55*a**2 + 6*a + 21. Let o(j) = 164*j**2 + 17*j + 63. Let u(v) = 8*f(v) - 3*o(v). What is the second derivative of u(s) wrt s?
-104
Suppose -95*h + 426 = -92*h. What is the third derivative of 5*a**2 - h - 2*a**2 + 142 + 19*a**3 wrt a?
114
Let j = 70 - 67. What is the third derivative of -29*k**2 + 9*k**3 + 19*k**3 + 3*k**j - 13*k**3 wrt k?
108
Let y(d) = -d - 1. Let x(i) = 7*i - 2. Let g(h) = -x(h) - 6*y(h). Let v be g(5). Find the third derivative of -5*k**2 + k**3 + 8 + v*k**2 - 8 wrt k.
6
Let m = 43 + -31. Let n = -4 + m. What is the first derivative of -n*l**2 + 31 - 16 - 13 wrt l?
-16*l
Let c(m) be the second derivative of -3*m**3 - 41*m + 7/10*m**6 + 0*m**5 + 0 + 0*m**4 + 0*m**2. Find the second derivative of c(p) wrt p.
252*p**2
Suppose 24 + 6 = 5*j. Let h(v) be the first derivative of 0*v**4 + 7 + 0*v**3 + 2*v**2 - 7/6*v**j + 0*v + 0*v**5. What is the second derivative of h(s) wrt s?
-140*s**3
Let d(k) be the second derivative of k**6/72 + 7*k**5/40 + k**4/12 + 6*k. Let w(j) be the third derivative of d(j). Find the first derivative of w(q) wrt q.
10
Suppose -5*x + 4*y + 377 = 0, -4*x + 3*x + 3*y = -71. What is the third derivative of x*l**5 - l**5 + 15*l**2 - 32*l**5 wrt l?
2640*l**2
Let t(a) be the first derivative of 131*a**4/4 - 273*a + 356. Differentiate t(j) wrt j.
393*j**2
Let l(z) be the first derivative of -18*z**4 + 150*z - 144. What is the derivative of l(v) wrt v?
-216*v**2
Let u = -1927 + 1929. Let y(v) be the third derivative of 0 + 0*v + 8*v**u + 1/3*v**3 + 0*v**4 + 1/20*v**5. Differentiate y(t) with respect to t.
6*t
Let b = -22 + 25. Find the second derivative of -2 - 23*m + 18*m + 2 + 11*m**b wrt m.
66*m
Let j = -1 + 14. Find the second derivative of j*p + 39*p**2 - 36*p**2 - 33*p wrt p.
6
Find the third derivative of -384*o**4 + 148*o**4 - 7*o**2 - 2*o - 155*o**2 + 3*o - 3*o**2 wrt o.
-5664*o
Let p(q) be the second derivative of -32*q + 0 - 17/6*q**3 - 9/2*q**2. What is the derivative of p(t) wrt t?
-17
Let g(s) = -s**3 - 1. Let w(h) = -210*h**3 - 66*h**2 + 12. Let p(j) = -12*g(j) - w(j). Find the third derivative of p(y) wrt y.
1332
Let z(o) be the second derivative of o**7/420 - o**6/60 + 2*o**4/3 - 10*o. Let l(q) be the third derivative of z(q). Find the second derivative of l(n) wrt n.
12
Let t = 33 - 31. What is the third derivative of -t*l**2 + 2*l**3 - 9*l**2 + 0*l**2 + l**2 wrt l?
12
Let j(s) = s**2 - 7*s - 4. Let i(c) = -3 + 9*c - 26*c + 10*c. Let d be 16/6*3/2. Let t(x) = d*i(x) - 3*j(x). Find the second derivative of t(r) wrt r.
-6
Let l(f) be the third derivative of -86*f**7/105 - 5*f**3/2 - 2*f**2 - 4. Find the first derivative of l(j) wrt j.
-688*j**3
Let y(x) = 2*x**2 - 4*x**2 - 7 - 713*x**3 + 714*x**3. Let n(c) = c**3 - c**2 - 5. Let u(v) = -7*n(v) + 5*y(v). Find the third derivative of u(l) wrt l.
-12
Let w(l) = -l**4 - 3*l**3 + l**2 - l - 1. Let q(i) = -2*i**4 - 3*i**3 + 50*i**2 - i - 351. Let c(o) = q(o) - w(o). What is the first derivative of c(x) wrt x?
-4*x**3 + 98*x
Let x(y) be the second derivative of -37*y**8/28 + 7*y**4/6 - 11*y**2 + 520*y. What is the third derivative of x(h) wrt h?
-8880*h**3
Suppose -4*q = -4*h + 72, -5*h - 2*q = -0*q - 90. Find the first derivative of h*s + 40 - 4*s - 16 wrt s.
14
What is the second derivative of -35*t**3 + t + 2*t - 11 - 14*t**5 + 35*t**3 wrt t?
-280*t**3
Suppose -2*g + 3 + 7 = 0. What is the derivative of 7*a**3 + g*a**3 - 4*a**3 + 19 wrt a?
24*a**2
Let o(x) be the first derivative of -4*x**4/3 - 5*x**2/2 - 13*x - 13. Let r(a) be the first derivative of o(a). Find the first derivative of r(s) wrt s.
-32*s
Let n(g) be the second derivative of 17*g**5/20 + g**4/12 - 155*g**3/3 - 2*g - 22. Find the second derivative of n(z) wrt z.
102*z + 2
Let t(k) be the first derivative of 2 + 0*k**2 + 1/5*k**5 + 0*k**4 + 0*k - 5*k**3. What is the third derivative of t(h) wrt h?
24*h
Suppose -13*x = -14*x + 309. What is the second derivative of 4*n**2 + 11*n - x + 309 wrt n?
8
Let s(w) be the third derivative of 1/24*w**4 + 0*w + 1/6*w**5 + 0*w**6 - 1/210*w**7 + 0*w**3 + 0 - 35*w**2. Find the third derivative of s(q) wrt q.
-24*q
Let h(n) be the second derivative of 61*n**7/42 + 13*n**3/3 + 80*n. Find the second derivative of h(r) wrt r.
1220*r**3
Find the second derivative of -154*p**5 - 390416*p**3 + 2 + 105*p + 390416*p**3 wrt p.
-3080*p**3
Suppose 60 = 9*l - 5*l. What is the third derivative of 7*q**2 - l*q**3 + 25*q**3 + 6*q**2 + 2*q**2 wrt q?
60
Let h(z) be the second derivative of 7*z**6/30 + z**4/6 - 82*z**2 - 6*z - 8. What is the first derivative of h(i) wrt i?
28*i**3 + 4*i
Suppose -2*u - 24 = -34. Find the third derivative of -535*q - 12*q**4 + 5*q**2 + 531*q + u*q**2 wrt q.
-288*q
Let i(h) = -711*h**3 + 9*h**2 + 1161. Let z(s) = 179*s**3 - 2*s**2 - 290. Let q(t) = -2*i(t) - 9*z(t). Differentiate q(g) with respect to g.
-567*g**2
What is the third derivative of 39030 - 60*z**3 - 39033 - 87*z**3 - 2*z**2 wrt z?
-882
Let l(r) be the third derivative of 443*r**6/120 - 79*r**5/20 + r**3/3 + 327*r**2. What is the third derivative of l(t) wrt t?
2658
Let k(d) = 32*d**3 + 27*d**2. Let


Last year Nautilus lost the ability to show desktop icons — now GNOME developers plan to drop another familiar feature.

According to a code commit on Gitlab the famous file manager is set to lose the ability to run binaries and launch apps directly.

Or, to put it another way, you won’t be able to double-click on programs, scripts or apps to launch them using Nautilus.

This change could affect:

Binary .bin and .run files

App Images (.appimage)

Program shortcuts (.desktop)

Scripts (e.g., .sh)

It’s a bit of a head-scratcher; why is GNOME doing this?

Why GNOME developers are making this change

GNOME developers has reverted this change since this article was published. Discussions are taking place with community members about different approaches to address the “issue”.

GNOME developer Carlos Soriano made the commit referenced above.

In it, he reasons that without a desktop on which to place app shortcuts and binaries the express need to launch apps using Nautilus is ‘not as useful’ as it once was.

After all, the GNOME Shell desktop comes with a full-screen app launcher by default, and a slate of third-party app menu extensions are available.

Apps should be launched from an app launcher, and the file manager should manage files

Plus, for those who can put up with its slow start-up times — which is being worked on this cycle — the GNOME Software store offers ‘launch’ buttons beside apps you have installed.

“It was [once] common for apps to be delivered in a tarball, nowadays that’s out of question,” Soriano says.

“Not only that, but we are moving towards a more sandboxed system, and we should use the standard and system wide support for launching apps based on users choices.”

It’s a sound reasoning to be fair: Software should handle software and Files should handle, uh, files.

But there is a perhaps a more pressing reason why GNOME want to make this change.

Improving Security

Removing the ability to run binary files from Nautilus will help to improve security

Although it’s easy to joke about GNOME developers do not remove, retire or evolve features for fun or to spite anyone (at least, I hope not). All changes, even potential unpopular ones like this, tend to have one core goal in mind: improve the end-user experience.

In this instance, removing the ability to run binary files helps to improve security.

Containerised apps like Flatpak and Snappy offer system isolation and other security benefits, and Software stores are now common place. Most of us have little real need to install or run random binary blobs we download from the web, and if we do, we’re better off using the terminal to do it.

“As we saw in the past we allowed untrusted binaries to be launched [in Nautilus] and therefore we had a CVE (CVE-2017-14604) […] we are not in a position that we can let this issues slip,” Soriano notes.

By removing the ability to run binaries from Nautilus GNOME is trying to help to protect users machines from rogue and malicious code. The “run” option won’t be shown for untrusted or trusted launchers.

You Can Still Run Binary Apps Though

If you need to launch apps using a file manager there are alternatives

Corner cases exist, naturally.

There will be times when you need to run an app you download from the web, or which is provided in the form of a script, and so on. So while Nautilus (the file manager) is dropping the ability run binary files the GNOME desktop at large isn’t.

The command line is (as always) but a few key presses away, and other GUI tools for managing and launching apps are available.

And for those who really cannot cope without using a file manager to launch their favourite apps there is, as ever, the option to use a Nautilus alternative like Dolphin, Nemo, PCManFM, Caja, etc.

You might not be able to double-click on a binary to run it, but you can still right click > open with…

Another point to keep in mind is that just because Nautilus won’t handle running binaries (like AppImages) directly is perfectly possible that a Nautilus plugin or facilitator app could take over the task instead.

For example, instead of double-clicking on a binary to run it you would right-click > open with… > and select a compatible app.

Which brings me on to the final point I should stress: this change does not affect the “Open With…” option; Nautilus will continue to be able to open files with/in apps.

Will This Affect You?

Do you regularly run/launch software using Nautilus? Do you agree this change will make using GNOME safer? Let us know what you think about this issue in the comments below, but please try to keep things civil and constructive.


203 A.2d 252 (1964)
Edward LIFMANN, Plaintiff,
v.
Harry ARONSON, Lawrence M. Aronson, Morris Draft, Seymour Rady, Clifford L. J. Siegmeister, Ben Cole, Saul Rubin, Donald Pollack, Charles J. Schaniel, Abe Fell, M. F. Lewis, Herman Segall, and Waltham Watch Company, Defendants.
Court of Chancery of Delaware, New Castle County.
August 28, 1964.
N. Maxson Terry and William H. Draper, Jr., Dover & James A. Thomas, Jr., of Lewis, MacDonald & Varian, New York City, for plaintiff.
Irving Morris and Joseph A. Rosenthal, of Cohen, Morris & Rosenthal, Wilmington, for defendants Harry Aronson, Lawrence M. Aronson, Morris Draft, Seymour Rady, and Waltham Watch Co.
MARVEL, Vice Chancellor.
Plaintiff claims to have been a stockholder of Waltham Watch Company since June 1961 and brings this suit derivatively for the alleged benefit of Waltham. He complains that the individual defendants Aronson, Draft and Rady, under the leadership of Harry Aronson, have controlled and improperly *253 managed the affairs of Waltham since 1959 so as to cause it compensable injury as hereinafter set forth. Plaintiff also charges that the above named defendants have conspired with the non-director Segall to conceal the true facts of the transactions complained of. He seeks joint and several accountings from the directors allegedly in control of the corporation as well as from the defendant Segall and the other named director defendants of Waltham.
The individual defendants Aronson, Draft and Rady as well as the corporation have appeared and filed a motion for summary judgment of dismissal of plaintiff's first and second causes of action on the ground that further judicial action on said causes is barred under the doctrine of res adjudicata, the claims therein asserted having been allegedly adjudicated in earlier New York and Delaware actions. Following an approved settlement in New York of the case of Fistel v. Aronson,[1] this Court granted summary judgment of dismissal of the Delaware consolidated Civil Action[2] Nos. 1159 and 1219 after finding that the allegations in the New York action and those asserted in the Delaware actions were identical in all material respects. This is the opinion of the Court on defendants' motion, it being assumed but not decided for the purposes of this motion that plaintiff is capable of bringing this suit.
In his first cause of action plaintiff alleges that from the year 1961 until the present the individual defendants have wrongfully concealed from Waltham and its stockholders the fact that they had improperly caused Waltham to assume certain contractual arrangements entered into by them with ten Swiss suppliers of watch movements. It is claimed that such undertaking was first saddled on Hallmark, Inc. when the individual director defendants were officers and directors of that corporation. The complaint goes on to allege that the individual defendants, having thereafter gained control of Waltham, caused Waltham to assume such improper obligation, said assumption having been designed and carried out in order to repay the Swiss suppliers for moneys advanced by them to the individual defendants to enable the latter to gain control of Waltham. The device used to bring about such repayment is alleged to have been a hidden premium consisting of 2.4 Swiss francs[3] charged on each of one million of watch movements to be purchased by Waltham from the Swiss suppliers over a term of five years. Plaintiff further alleges in his first cause of action that it had been orally agreed between the individual defendants and the Swiss suppliers that the premium referred to above should be hidden by being included on each invoice for watch movements as a payment "for research and development." According to plaintiff, the scheme outlined in his first cause of action resulted in a loss to Waltham of not less than $750,000, exclusive of interest payments, for which the individual defendants are individually and personally liable to Waltham. It is further contended that notwithstanding a purported settlement of litigation bearing on the matters complained of, Waltham has been further damaged in an amount in excess of $200,000 by reason of continuing payments being made under the improper premium arrangement referred to above.
While a second cause of action set forth in the complaint is concerned with the same matter complained of in the first cause of action, it is therein contended that the acts complained of resulted from a conspiracy entered into in January 1959 between the individual defendant directors and officers, *254 the defendant Segall and the Swiss suppliers for the purpose of concealing from Waltham's stockholders the improper premium arrangement alluded to in the first cause of action. Such second cause of action apparently seeks damages from the alleged conspirators in the same amounts sought in the first cause of action, namely $750,000 for damages sustained during the entire period of the allegedly continuing conspiracy and not less than $200,000 for the period from 1962 to date.
First of all, notwithstanding plaintiff's reliance on what he terms a plan of concealment as a basis for his suit, I am satisfied that the basic transaction here complained of was one of the subject matters of earlier stockholder complaints filed and adjudicated both in this Court and in the Supreme Court of New York, said complaints having sought relief from wrongs caused by the allegedly improper transfer of voting control of Waltham from the Axlers to the Aronsons, although such earlier complaints encompassed a number of other alleged breaches of fiduciary duty not mentioned in the present complaint. Thus, it was alleged in an uncontradicted affidavit filed on April 27, 1960 in the New York action of Fistel v. Axler[4] in support of defendants' motion for summary judgment that as an incident to transfer of the Axler stock carried out in connection with Mr. Harry Aronson's efforts to extricate Waltham from serious financial difficulties "* * * a group of approximately twelve Swiss watch suppliers designated Seymour Rady, Esq., an attorney of Chicago, Illinois, to act in their behalf in negotiations with the Axlers to purchase their stock of Waltham for a price not to exceed $650,000, provided that such purchase would be conditioned upon the net worth of Waltham, as determined by an independent audit, being shown to be not less than a stated minimum figure. * * *" (Para. 19, affidavit of Robert Schur). In fact, there is no real disagreement in the papers before me on defendants' motion as to the fact that a group of some ten or twelve Swiss watch manufacturers participated in a 1959 business transaction which led to the disposal by the Axlers of their 120,000 shares of Waltham and the loss on their part of voting control over the affairs of Waltham as part of a complicated arrangement which culminated in an exchange of Hallmark stock for that of Waltham. What plaintiff here seeks to counter, on his theory of concealment, are the inferences to be drawn from the fact that the plaintiffs in the New York actions failed in their efforts to establish that the Aronsons rather than the Swiss manufacturers were the true owners of the 120,000 Waltham shares. And while, to be sure, the first dispositive step in the case in which the Schur affidavit was filed was, perhaps by inadvertence, a severance and dismissal on the merits merely as to the Axlers, the charges concerning the Aronsons were later specifically dealt with judicially by the New York and Delaware courts. Thus, in the Delaware Fistel action, after a similar judgment as to the Axlers had been entered, the complaint was amended so as to plead in detail the same allegedly improper premium arrangement here complained of and such action (Civil Action 1159) was then consolidated with the Pohl action (Civil Action 1219) for the purposes of trial. Thereafter, after discovery had proceeded in both New York and Delaware, the several pending cases involving the remaining charges against the Aronsons were consolidated in the Supreme Court of New York and paragraph 19 concerning the acquisition of the 120,000 shares of Waltham stock owned by the Axlers and the arrangement for their payment through the alleged fraudulent device of a payment of 2.5 francs per movement disguised as a charge "* * * to cover research and development * * *", as set forth in the Delaware Fistel case, was incorporated in haec verba in the New York complaint in a similarly numbered paragraph.
*255 Meanwhile, however, general counsel for plaintiffs in the New York actions reached an agreement with counsel for the defendants whereby in return for dismissal of their consolidated action and the furnishing of releases the Aronson defendants agreed to transfer to Waltham 30,000 shares of Waltham stock, the equivalent of 150,000 shares of Waltham stock as of the time of their exchange for Hallmark shares in April 1959. Such proposed settlement was then referred to a referee for consideration. Having evaluated the various causes of action leveled against the Aronsons, including the one which charged "* * * the Aronsons with a plan to acquire control of Waltham with the use of Waltham's funds * * *", the referee reached the following conclusion and made the following recommendation as to such claim, namely, "* * * I conclude that the plaintiffs' second cause of action is not supported by the evidence submitted to me, nor has there been a showing of fraud * * *". The overall conclusion of the referee was that the proposed settlement was fair and adequate and that it should be approved and confirmed. Thereafter, the referee's report having been mailed to each stockholder of record of Waltham together with notice of a hearing date, such a hearing was held, the settlement was approved, the action dismissed with prejudice, and releases ordered to be given to the Aronson defendants. Finally, as noted earlier, on the basis of such judgment, this Court dismissed Civil Action Nos. 1159 and 1219 with prejudice.
Faced with the defense of res adjudicata, plaintiff does not attempt a collateral attack, professing that he does not "* * * seek to overturn the prior judgment of the New York court, or the subsequent judgment of this Court, based solely on New York's settlement * * *". He rather contends that he merely seeks to obtain relief for claims which were not dealt with in the prior litigation. However, I am satisfied that although plaintiff has made every effort to characterize his claim concerning the so-called hidden premium arrangement as different than that asserted in the earlier litigation, placing reliance on alleged oral contracts and on a so-called different theory of action, what he actually seeks is an opportunity to introduce additional trial evidence concerning a transaction which, in my opinion, has been put to rest in earlier litigation. This he may not do, Epstein v. Chatham Park, Inc., 2 Storey 56, 52 Del. 56, 153 A.2d 180. Plaintiff's grievances about the results of the earlier litigation would, if given the hearing he seeks, destroy the salutary effects of modern procedures for the settlement of stockholder derivative suits after notice to all stockholders of record. See Hornstein, Corporation Law and Practice § 731, and compare Barroway v. Reynolds (Del.Ch.) 176 A.2d 850. Finally, plaintiff's reliance on Darraugh v. Carrington, Sup., 50 N.Y. S.2d 481, aff'd 270 App.Div. 932, 62 N.Y. S.2d 241, is misplaced. In that case stockholders suing derivatively had not had their day in court in California on claims asserted in New York, it being pointed out in the opinion of the Appellate Division that prior to the entry of the California judgment relied on by defendants there was not "* * * an unequivocal calling upon the plaintiff in such action to introduce evidence in support of his claims."
To be sure, there may be error in the prior proceedings here relied on both as to facts and law, and a better settlement might perhaps have been made. Nonetheless, the New York judgment relied on by defendants, having become final after a settlement hearing, stands unimpeached, a final judgment on the merits rendered by a court of competent jurisdiction being conclusive as to the rights of the parties and their privies. There is no doubt but that the same evidence which would have successfully established the claim made in the earlier litigation based on allegedly improper methods used by officers and directors to gain control of 120,000 shares of Waltham stock would be required to establish the claim made here. Plaintiff's grievance *256 simply stated is that plaintiffs in the New York action were not sufficiently diligent and failed to unearth evidence which plaintiff professes to have now or to anticipate. This is no basis for a trial de novo. The point is, of course, that while counsel for plaintiffs in New York were commended by the referee "* * * for the zeal with which they pursued this cause of action by obtaining depositions and documentation in Switzerland * * *", such counsel were not satisfied with the evidence they developed in New York and Delaware discovery as a basis for trial. They thereupon negotiated an overall settlement which the referee agreed was fair and adequate and which the Supreme Court of New York approved. Such a judgment clearly has a res adjudicata effect, Rome v. Archer (Sup.Ct.Del.) 197 A.2d 49.
While the defendants Rady and Draft were not parties to the earlier litigation, Rady become a director of Waltham in February 1959 and Draft was elected a director on April 17, 1959. They could in no possible way be held liable to their corporation on plaintiff's theory of his case other than by reason of their participation in the steps taken towards acquisition of and payment for the Axlers' Waltham shares. Such transaction having been an integral part of the prior complaints and the claims thereon having been dismissed, the judgments therein entered enure to the benefit of the defendants Rady and Draft as well as to the benefit of the Aronsons, Coca-Cola Co. v. Pepsi-Cola Co., 6 W.W. Harr. 124, 36 Del. 124, 172 A. 260.
On notice, an order will be entered granting the motion of the appearing defendants for summary judgment of dismissal of plaintiff's first and second causes of action.
NOTES
[1]  Supreme Court of the State of New York, County of New York, Index No. 9411-1959.
[2]  Fistel v. Axler and Pohl v. Axler.
[3]  At the argument on defendants' motion, counsel for plaintiff stated that the premium was actually 2.5 Swiss francs and that the figure of 2.4 set forth in the complaint was a typographical error. See also affidavit of James A. Thomas, Jr., in opposition to defendants' motion.
[4]  Supreme Court of the State of New York, County of New York, Index No. 9411-1959.


Q:

Headphone jack not working in Windows 7

My headphone jack doesn't work under Windows 7 64 bit.
I tried the following things:

3 different headphones speakers
2 different versions of the Realtek driver (2.7 /  2.6)
Standard Windows 7 drivers

I can't see anything apart from "Speakers" in the playback devices (I have enabled Show hidden / deactivated devices). Under Ubuntu there is no problem whatsoever!
When I used the Realtek configurator I wanted to disable the auto-detection of plugged in speakers, but the option (the little folder icon) wasn't there, in neither version.
I use HDMI to connect my screen from time to time. The audio jack from the screen does work (sometimes!). But I guess that's unrelated, because even when I boot Windows without the screen, it still doesn't work.

A:

Try completely uninstalling the realtek drivers and then reboot and see if Windows propitery audio drivers detect.
Hopefully the audio device has been correctly detected by Windows, to check it go to "Device Manager"


A chromenoquinoline-based fluorescent off-on thiol probe for bioimaging.
A new chromenoquinoline-based fluorescent off-on thiol probe 2 is reported. In aqueous buffer solutions at physiological pH, the probe exhibited 223-fold enhancement in fluorescence intensity by a Michael addition of cysteine to the maleimide appended to a chromenoquinoline. Cell permeability and live cell imaging of thiols are also demonstrated.


Ray Winstone

A strong character actor known for his multi-dimensional portrayals of hard men on the wrong side of the law, Ray Winstone burst onto the scene with a riveting performance in the British-made "Scum" (...
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Russell Crowe was left with bruised ribs on the set of Noah after fellow Hollywood hardman Ray Winstone repeatedly elbowed him in the chest. The pair stars in the upcoming Biblical epic and Crowe has previously told how he developed hypothermia during the tough shoot as he was filming in freezing water for hours at a time.
Now it has emerged it was not the only health issue Crowe suffered during filming, as British actor Winstone took joy in striking him in the ribs during a fight scene.
Crowe tells British magazine Event, "He's acting royalty, that man. All the way back to Quadrophenia and Scum. We did this fight sequence where Ray is lying on top of me - and there's a bit of weight to Ray - and every time we did a take, just prior to 'Action!', he would drive an elbow into my ribs. I said, 'Mate, why are you doing that?'. And he goes, 'Because you're younger and I'm old and you should know to look out for a bloke like me.' It was great."

British actor Ray Winstone has suffered a setback in his attempt to give up smoking after struggling to get to grips with an electronic cigarette. The Departed star has decided to kick his longtime addiction and opted to swap real smokes for a battery-powered device.
However, the actor was dealt a blow when he realised he didn't know how to work his e-cigarette.
Winstone tells Britain's Daily Telegraph newspaper, "I bought one of those false ones and I put the oil in the wrong end. I puffed on it and all the oil went in my mouth. It's like I was not destined to give up (smoking)."

British actor Ray Winstone was furious when he found out about his daughters' secret tattoos. The Indiana Jones and the Kingdom of the Crystal Skull star was apoplectic with rage when he discovered actress Jaime, 28, and her elder sister Lois had gone under the needle and kept the inkings hidden from their protective dad.
He tells Britain's Daily Telegraph newspaper, "They wouldn't tell me, but I found out and went berserk. I actually wanted to find the tattooist and kill him for scarring my daughters."

British actor Ray Winstone struggled with soaring temperatures while shooting biblical epic Noah and almost fell down some stairs after he was overcome on set. The film - in which Winstone stars as Noah's nemesis Tubal-Cain - was shot in New York at the height of summer and he suffered through the heat in a heavy costume and make-up.
His co-star Emma Watson describes his ordeal to Wonderland magazine, "We started filming in the summer in New York. It was so hot that Ray Winstone at one point, who wore a beard and heavy make-up, nearly fell down a flight of stairs. His make-up was literally melting off his face."

Actor Ray Winstone vowed to avoid boozing on the set of new British drama Moonfleet in Ireland after spending weeks in a drunken haze the last time he was shooting on the Emerald Isle. The Departed star plays an 18th Century smuggler in the upcoming two-part Sky TV drama, and he headed to Dublin, County Kildare, and County Wicklow to shoot scenes for the swashbuckling show.
The trip brought back memories of his last time filming in Ireland, when he starred in 2004 movie King Arthur alongside Keira Knightley, and he was determined to avoid the local bars after becoming sucked into the country's legendary drinking culture.
Winstone tells the Irish Mirror, "When I was on King Arthur, we were out all the time. I think everyone was drunk on that job for four and a half months. I actually moved out of Dublin - I was staying at the Four Seasons - and I went: 'I've got to get out of Dublin because I'm just p**sed all the time'.
"I said: 'I've got to go', so they found me a little place... I said: 'I'm going to live there, on an old farm place, I'm going to live there for the duration of filming'. And nothing changed. I was on the p**s (drinking) but the only difference was that I was looking at mountains instead of the town of Dublin... But I'm getting older now, I can't do that so much anymore, can't be out working in the morning if you do that."
Asked if he had been boozing while filming Moonfleet, the 56 year old replies, "Not this time, we haven't had the time... For this film, it was the time (that prevented drinking), but if I'd been younger, I think I would have made the time. That's probably where I made a mistake in the past, I just couldn't wait to get out and probably stay out all night, and go straight to work the next morning. But I just can't do that no more (sic), physically and mentally."

Actor Danny Dyer decided to accept a role in longrunning British soap opera Eastenders over fears his career had become a joke following a string of film flops. The Business star will join the beloved show at Christmas (13) after spending several years snubbing offers from BBC bosses to become a member of the cast.
Now he reveals his floundering film career pushed him to take the leap to the small screen, and he hopes he can turn his fortunes around like his idol Sir Michael Caine.
Dyer tells Britain's The Sun newspaper, "Back in the Eighties, Michael was known for making terrible films and now he's been knighted. I can hope!
"I did too many people favours (accepting film roles). I realised, 'F**king hell, I'm becoming a bit of a joke here.' I'm my own worst enemy. I don't blame anyone but myself."
Dyer's most recent big screen release, Run for Your Wife, which also features Ray Winstone and Girls Aloud singer Sarah Harding, was deemed a massive failure after it earned just $963 (£602) from U.K. ticket sales in February (13).

Hollywood star Sean Penn stopped traffic in London over the weekend (07-08Sep13) as he filmed scenes for new movie The Gunman. The actor spent Sunday (08Sep13) shooting footage for the upcoming thriller on the famed Tower Bridge as a low-flying helicopter zoomed overhead and passers-by crowded round to watch.
Penn stars as Martin Terrier, an international spy trying to clear his name so he can retire to be with his longtime love.
The Gunman, which also stars Idris Elba, Javier Bardem and Ray Winstone, is due in cinemas next year (14).

Ryan Gosling is undoubtedly one of the most crushed on celebrities of the 21st century. Girls' hearts exploded when they watched him kiss Rachel McAdams in the rain in The Notebook, and ever since then their opinions have not wavered. But if you take a closer look, you'll see that since the beginning of his movie career, he has steadily played psychologically unsound weirdos film after film. So what is it about the Gos that makes girls swoon, no matter how creepy he is on film? Oh yeah, it's probably because he's really, really ridiculously good-looking, with or without a pedo 'stache.
GALLERY: Ryan Gosling's Creepy Roles
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These actors have a history of playing bullies, ruthless killers, evil masterminds, and all-around badasses. Their distinct physical appearances (weathered, dark-haired, unsmiling) and general demeanors (quiet, menacing, and angry all the time) make them the perfect villain or antihero type. Unlike some actors who can play both the tough guy and the big, loveable goofball (we're looking at you, The Rock — you, too, Vin Diesel), these guys are just too fearsome to play mannies and tooth fairies. You might want to avoid direct eye contact, as we present to you the six most badass badasses in Hollywood.
GALLERY: 6 Most Badass Badasses in Hollywood
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If presented with a picture of Ray Winstone, you'll probably think to yourself, "Oh yeah, that guy." He looks familiar because there's a good chance you've seen one of his movies. The character actor has been in some major films over the last 10 years, such movies as Cold Mountain, The Departed, Hugo, and Snow White and the Huntsman, but you've probably never heard of him. Well here's your chance to get to know him with six fun facts about the British chameleon, who you'll no doubt be seeing plenty more of on the big screen.
You Probably Know Him Best From Sexy Beast
His most prominent role was in Sexy Beast, in which Winstone played the lead, Gal Dove, opposite villain Ben Kingsley. Winstone's performance in Sexy Beast as a loveable but hapless retired ex-con is solid, but he was unerstandably upstaged by Kingsley's rapid-talking, short-tempered psychopath, a role that garnered Kingsley an Oscar nomination.
Or Maybe The Departed
Martin Scorsese's wildly successful The Departed put Winstone on the map once again as tough guy Mr. French. His character is one of the most memorable in a stellar ensemble cast that includes Jack Nicholson, Leonardo DiCaprio, and Matt Damon. To audiences, he's the guy who slams Billy's (DiCaprio) arm cast on the table so hard it breaks, while Billy screams in pain, and as the guy who wryly repeats the film's infamous line, "What is it, your period?" when Billy orders a cranberry juice.
He Played a Dwarf
In a departure from his tough-guy roles, Winstone played one of the dwarves in 2012's Snow White and the Huntsman. Perhaps it's his physical stature, or just his incredible range as an actor, but he really made his character, Gort, come to life, and he gave him that rough-and-tumble edge that he gives all of his characters. So I guess his dwarf was a tough guy after all.
Scorsese Seems to Love Him
After his turn in The Departed, Scorsese decided to cast Winstone in Hugo, in which he plays the title character's alcoholic uncle Claude. Though this was a smaller role for the British actor, we feel safe betting that the legendary director will seek him out again in the future.
He Does Action Movies Like The Sweeney
Even at the age of 56, he still gets down and dirty in gritty action movies like 2012's The Sweeney, in which he plays Detective Inspector Jack Regan, who leads the London Metropolitan Police department's Flying Squad, aka the armed police. Not only does Winstone beat people up and partake in car chases, but he also gets raunchy with Hayley Atwell's character. Not too shabby.
His Voice
Winstone has the kind of gritty voice and Cockney accent that lends well to the screen, so much so that it's often just his voice. Winstone has done several voice-over roles, including Rango, The Chronicles of Narnia: The Lion, The Witch, and the Wardrobe, and Beowulf. So even if his face doesn't look familiar, you've probably at least heard him on screen.
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Title

Featured as a loan shark in Anjelica Huston's 1967 Dublin-set drama "Agnes Browne"

Cast in the fourth Indiana Jones film series, "Indiana Jones and the Kingdom of the Crystal Skull"

Portrayed a working-class father who, along with his wife, must cope with the accidental death of their young son in the British telefilm "Our Boy" (screened at Toronto International Film Festival)

Starred in "Births, Marriages and Deaths," a four-part BBC TV series

Played recurring role of a gangster in the British series "A Fairly Secret Army"

Cast in Anthony Minghella's ensemble "Breaking and Entering"

Portrayed the title character in Robert Zemeckis' big-budget film version of "Beowulf"

Acted in "My Father, The Liar," a Bob Hoskins-directed segment of the omnibus film "Tube Tales"

Played leading role as a family patriarch in "The War Zone," an incest themed drama marking Tim Roth's directorial debut; screened at both the Sundance and Cannes Film Festivals

Appeared in the romantic comedy "Martha, Meet Frank, Daniel and Laurence" (released in the USA as "The Very Thought of You" in 1999); film starred Monica Potter, Joseph Fiennes, Rufus Sewall and Tom Hollander

Cast as one of the eight dwarfs in "Snow White and the Huntsman" opposite Kristen Stewart and Charlize Theron

Played a CIA officer covering up a murder in the film adaptation of "Edge of Darkness"

Appeared in "Ladies and Gentlemen, the Fabulous Stains," starring Diane Lane as a teenage punk star

Was in the ensemble of the drama "Last Orders"

Voiced Mr. Beaver in "The Chronicles of Narnia: The Lion, the Witch, and the Wardrobe," based on the children's novel by C.S. Lewis

Starred in Patrick Marber's play "Dealer's Choice"; transferred to the West End

Discovered by director Alan Clarke and hired to play reform school terror Carlin in the BBC telefilm "Scum" (shelved by censors for content)

Appeared in Ken Loach's "Ladybird, Ladybird"

Returned to the stage in the Royal Court production of "Some Voice"

Cast in Martin Scorsese's mob drama, "The Departed"

Had title role in the British TV series "Fox"

Starred in the BBC sitcom "Get Real"

Had first real romantic lead opposite Kerry Fox in "Fanny and Elvis"

Starred in "The Proposition," an Australian western written by musician Nick Cave

Reprised his role for Clarke's feature remake of "Scum"

Was a championship boxer, losing only eight of 88 bouts

Starred alongside Robert Carlyle as a career criminal who betrays his cohorts in "Face," helmed by Antonia Bird

Starred in Jez Butterworth's stage play "The Night Heron" at London's Royal Court Theatre

Starred as Will Scarlett in the British TV series "Robin of Sherwood"

Played Teague in the drama "Cold Mountain"

Appeared in "King Arthur" with Clive Owen and Keira Knightley

Portrayed rocker Kevin in "Quadrophenia," a Mods vs Rockers tale inspired by The Who's album of the same name

Starred in "Sexy Beast"; screened at Sundance in 2001

Starred in "Tank Malling" as titular investigative reporter

Attended drama school; was asked to leave because of an incident involving his sabotaging of the headmistress' car

Cast in Martin Scorsese’s family adventure "Hugo"

Made a a triumphant return to form with a starring role as the raging Raymond in Gary Oldman's "Nil By Mouth"

Appeared in the Donmar Warehouse production of "To the Green Fields and Beyond," directed by Sam Mendes

Initial collaboration with Kathy Burke, starring in the stage play "Mr. Thomas," written and directed by Burke

Summary

A strong character actor known for his multi-dimensional portrayals of hard men on the wrong side of the law, Ray Winstone burst onto the scene with a riveting performance in the British-made "Scum" (1979), only to spend the ensuing decade wallowing in lesser roles unworthy of his talents. Forced into bankruptcy and on the outs with acting by the end of the 1980s, Winstone re-emerged with an acclaimed performance in friend Kathy Burke's stage play, "Mr. Thomas," which not only helped relaunch his career, but also invigorated his confidence. Ever since that play, Winstone developed into a highly-sought performer who - with age and experience - gravitated towards more complex characters, portraying often violent men with a strong degree of sympathy, as he did playing an abusive alcoholic in his breakthrough performance, "Nil By Mouth" (1997). But it was his turn in "Sexy Beast" (2001), as a reticent ex-con beset by a rabid ex-colleague trying to recruit him for a job, which earned Winstone some of the highest praise of his career. From there, he entered into the realm of the high-profile Hollywood feature, appearing in "King Arthur" (2004) and "The Departed" (2006), before co-starring in perhaps one of the most anticipated summer movies of all time, "Indiana Jones and the Kingdom of the Crystal Skull" (2008). Acclaimed projects like "Hugo" (2011) and "Great Expectations" (PBS, 2012) only solidified Winstone's reputation as one of the most prolific and acclaimed actors of his generation to cross the Atlantic.

Name

Role

Comments

Elaine Winstone

Wife

Married c. 1978; separated briefly in the early 1990s when Winstone declared bankruptcy

Education

Name

Edmonton County School

Corona Stage Academy

Notes

Winstone on the aftermath of his intense and startlingly realistic portrayal of a violent husband in Gary Oldman's "Nil By Mouth": "Well, I didn't emerge unscathed. That's the truth of it. I mean, 'Nil By Mouth' is a very harrowing film. But it wasn't harrowing to make. We had a ball doing it. Lots of laughs. It was only when the filming was over that I started asking myself was there something in me that was like the character? Was I capable of showing that kind of rage myself? It can be quite frightening when you start worrying about stuff like that. But it soon passes. You get offered another role and start getting into that." – quoted in Loaded, December 1997

"You know directing is a hard game and life is too sweet for all that aggravation. When I finish a film I go home. Sit in an editing suite for three months? Sod that. I've been paid, thank you very much, see you at the premiere." – Winstone on following in the footsteps of actor-directors Gary Oldman and Tim Roth, quoted in The London Times, Jan. 8, 1999

Winstone on his character on the BBC's "Births, Marriages and Deaths": "Even though he's the baddie, I play him as the goodie. I think it's more interesting to play it the opposite way. Even Hitler had someone who loved him" – quoted in Inside Soap, Feb. 19-March 5, 1999


Space enthusiasts in certain regions of the world will get free seats to a potentially once-in-a-lifetime event on the celestial stage later this month.

On July 27, lucky stargazers will see the longest total lunar eclipse of the 21st century ― also known as a blood moon. The nearly two-hour total eclipse will be visible to people in parts of Africa, the Middle East and Asia, according to NASA.

Blood moons are not unusual occurrences. Still, these dramatic celestial events have inspired both awe and alarm throughout the centuries ― including among some fringe segments of American Christianity that take the appearance of blood moons to be a sign that the apocalypse is drawing nearer.


1. Field of Invention
This invention relates to abrasion resistive packaging, such as packaging to protect a photoreceptor of a print device.
2. Description of Related Art
In current printing applications, print cartridges are incorporated in printing devices. The print cartridges contain a printing material, such as toner. Typically, the print cartridges are replaceable, and the print cartridges are discarded when they run out of toner, and a new print cartridge is inserted into the print device. The disposability of the print cartridges requires that new print cartridges be shipped.


/*
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 * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
 * See the License for the specific language governing permissions and
 * limitations under the License.
 */
package com.gargoylesoftware.htmlunit.svg;

import java.util.Map;

import org.apache.commons.lang3.StringUtils;

import com.gargoylesoftware.htmlunit.SgmlPage;
import com.gargoylesoftware.htmlunit.html.DomAttr;
import com.gargoylesoftware.htmlunit.html.ScriptElement;
import com.gargoylesoftware.htmlunit.html.ScriptElementSupport;

/**
 * Wrapper for the SVG element {@code script}.
 *
 * @author Ahmed Ashour
 */
public class SvgScript extends SvgElement implements ScriptElement {

    /** The tag represented by this element. */
    public static final String TAG_NAME = "script";
    private boolean executed_;

    /**
     * Creates a new instance.
     *
     * @param namespaceURI the URI that identifies an XML namespace
     * @param qualifiedName the qualified name of the element type to instantiate
     * @param page the page that contains this element
     * @param attributes the initial attributes
     */
    SvgScript(final String namespaceURI, final String qualifiedName, final SgmlPage page,
            final Map<String, DomAttr> attributes) {
        super(namespaceURI, qualifiedName, page, attributes);
    }

    /**
     * {@inheritDoc}
     */
    @Override
    public boolean isExecuted() {
        return executed_;
    }

    /**
     * {@inheritDoc}
     */
    @Override
    public void setExecuted(final boolean executed) {
        executed_ = executed;
    }

    /**
     * {@inheritDoc}
     */
    @Override
    public final String getSrcAttribute() {
        return getSrcAttributeNormalized();
    }

    /**
     * Helper for src retrieval and normalization.
     *
     * @return the value of the attribute {@code src} with all line breaks removed
     * or an empty string if that attribute isn't defined.
     */
    protected final String getSrcAttributeNormalized() {
        // at the moment StringUtils.replaceChars returns the org string
        // if nothing to replace was found but the doc implies, that we
        // can't trust on this in the future
        final String attrib = getAttributeDirect(SRC_ATTRIBUTE);
        if (ATTRIBUTE_NOT_DEFINED == attrib) {
            return attrib;
        }

        return StringUtils.replaceChars(attrib, "\r\n", "");
    }

    /**
     * {@inheritDoc}
     */
    @Override
    public final String getCharsetAttribute() {
        return getAttributeDirect("charset");
    }

    /**
     * Executes the <tt>onreadystatechange</tt> handler when simulating IE, as well as executing
     * the script itself, if necessary. {@inheritDoc}
     */
    @Override
    public void onAllChildrenAddedToPage(final boolean postponed) {
        ScriptElementSupport.onAllChildrenAddedToPage(this, postponed);
    }
}