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Κύρια Experimental Neurology l-lysine confers neuroprotection by suppressing inflammatory response via microRNA-575/PTEN signaling..
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Experimental Neurology 327 (2020) 113214 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr Research Paper L-lysine confers neuroprotection by suppressing inflammatory response via microRNA-575/PTEN signaling after mouse intracerebral hemorrhage injury T Jing Chenga,1, Jun-Chun Tangb,1, Meng-Xian Panb, Song-Feng Chenb, Dan Zhaob, Ya Zhangb, Hua-Bao Liaob, Yang Zhuangb, Rui-Xue Leib, Shu Wangb, An-Chun Liub, Juan Chenb, ⁎ ⁎ Zhao-Hui Zhangd, Huan-Ting Lic, Qi Wanc, , Qian-Xue Chena, a Department of Neurosurgery, Renmin Hospital of Wuhan University, 99 Zhang Zhidong Rd, Wuhan 430060, China Department of Physiology, School of Basic Medical Sciences, Wuhan University School of Medicine, 185 Donghu Street, Wuhan 430071, China Institute of Neuroregeneration & Neurorehabilitation, Department of Neurosurgery & Pathophysiology, Qingdao University, 308 Ningxia Street, Qingdao 266071, China d Department of Neurology, Renmin Hospital of Wuhan University, 99 Zhang Zhidong Rd, Wuhan 430060, China b c A R T I C LE I N FO A B S T R A C T Keywords: L-lysine Intracerebral hemorrhage PTEN MicroRNA-575 M2 microglia Inflammatory response Neuroprotection L-lysine is a basic amino acid that has been shown to exert neuroprotective effect. However, the underlying mechanism remains to be elucidated. In this study, we investigate how L-lysine exerts its neuroprotective effect in hemin-insulted mouse cortical neurons in vitro and the mouse model of intracerebral hemorrhage (ICH) in vivo. We demonstrate that L-lysine treatment promotes M2 microglial polarization and reduces inflammatory response both in vitro and in vivo, suggesting that L-lysine may play a neuroprotective role in ICH injury. Indeed, we show that L-lysine treatment reduces cortical neuronal death after hemin insult in vitro and decrease the number of degenerating neurons after ICH in vivo. L-lysine also improves the functional recovery of ICH animals in neurobehavioral tests. Consistent with the; role of PTEN in regulating inflammatory response, we find that PTEN inhibition promotes M2 microglial polarization and suppresses pro-inflammatory response in mouse ICH injury, which contribute to the neuroprotective effect of L-lysine. Moreover, our results reveal that microRNA-575 directly suppressed PTEN to promote M2 microglial polarization and mediate the neuroprotective effect of L-lysine in ICH injury. Together, our results suggest that L-lysine confers neuroprotection after ICH injury through enhancing M2 microglial polarization and reducing inflammatory response, which is mediated by microRNA-575 upregulation and subsequent PTEN downregulation. 1. Introduction As a sub-type of stroke, intracerebral hemorrhage (ICH) accounts for 10% to 15% of all strokes in European countries, while accounts for 20% to 30% of those in Asian countries (Askenase and Sansing, 2016; Ziai et al., 2016). ICH is the result of the rupture of small and medium blood vessels in the brain parenchyma, which is subsequently produced and consumed, leading to impaired neurological function (Zhao et al., 2016). The size of hematoma and expansion are associated with poor outcome and early neuron deterioration after ICH (Davis et al., 2006). The incidents of further accelerated brain damage involving destruction of brain cell structure, inflammation of the hematoma, excitotoxicity and release of clot components (Del Bigio, 1996; Keep et al., 2012; Xi et al., 2006). The underlying mechanisms are largely unknown. As an inhabitant immune cell in the central nervous system,microglia is activated after ICH injury (Cunningham, 2013; Taylor and Sansing, 2013). Activated microglia has two phenotypes: pro-inflammatory M1 microglia and anti-inflammatory M2 microglia. However, how M1 and M2 microglia play their roles in ICH injury remain unclear. L-lysine is an essential amino acid that is known to promote protein synthesis (Chang and Gao, 1995). L-lysine has been reported in previous studies to improve neurological function and cerebral blood flow in patients with ischemic stroke through neuroprotective and neurotrophic effects (Kondoh et al., 2010). But the role of L-lysine in hemorrhagic stroke is not investigated. The purpose of this study is to test the effects of L-lysine after ICH injury and explore the possible signaling ⁎ Corresponding authors. E-mail addresses: firstname.lastname@example.com (Q. Wan), firstname.lastname@example.com (Q.-X. Chen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.expneurol.2020.113214 Received 21 September 2019; Received in revised form 10 January 2020; Accepted 24 January 2020 Available online 24 January 2020 0014-4886/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). Experimental Neurology 327 (2020) 113214 J. Cheng, et al. expelled from the uterus of pregnant mice and the brain tissue of the embryos was rapidly isolated. And the pelleted cells were cultured in dulbecco's modified eagle medium (DMEM) (1% antibiotic-antimycotic, 5 ng/ml carrier-free recombinant mouse GM-CS and 10% FBS) incubated at 37 °C. A total of 1.3∗106 cells were added into T75 cell culture treated flasks coated by poly-D-lysine and the flasks were put in incubator of 37 °C. The culture supernatant was replaced with 10 ml of fresh medium every three days, and the cell fusion was observed after about 3 weeks. mechanisms. The phosphate and tension homology deleted on chromosome ten (PTEN) is a tumor suppressor but plays an important role in neurological diseases (Chang et al., 2007; Liu et al., 2010; Zheng et al., 2012). Our previous study for the first time reveals that suppressing PTEN antagonizes ischemic neuronal death through enhancement of phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) signal pathway (Ning et al., 2004). A large amount of work then verify that suppressing is neuroprotective (Farajdokht et al., 2018; Miao et al., 2018; Shabanzadeh et al., 2019; Shi et al., 2011). MicroRNAs (miRNAs), an important non-coding RNAs, can regulate protein expressions (Bartel, 2004). MiRNAs play fundamental roles in cell metabolism, apoptosis, cell development and differentiation (Ketting et al., 2001; Lee et al., 2004). In living organisms, miRNAs direct mRNA interpretation through defective matching with nucleotide arrangements inside the 3′-untranslated locale (3′UTR) of target qualities (Doench and Sharp, 2004). MicroRNA-575 (miR-575) is was firstly portrayed in 2009 (Yao et al., 2009). Recent research has found that part of target genes of miR-575 are related to inflammation. As miR-575 can influence the expression of inflammatory genes, miR-575 is potentially a biomarker for Kawasaki disease diagnosis and prediction of intravenous immunoglobulin treatment outcomes (Zhang et al., 2018). It have been reported that PTEN, a target gene of miR-575, can directly affect inflammatory response (Wang et al., 2014; Wang et al., 2019). We reasoned that PTEN inhibition by miR-575 may suppress the inflammatory response in ICH injury. In this study, we show that L-lysine exerts its neuroprotective effect in ICH injury through regulating miR-575/PTEN signaling. Our results prove that L-lysine increases the expression of miR-575, which subsequently reduces the PTEN expression and thus activates M2 microglial polarization. Our study suggests that the neuroprotective effect of Llysine after ICH injury is mediated through M2 microglial polarization and the suppression of pro-inflammatory cytokines whose release are inhibited by miR-575/PTEN signaling. 2.3. Neuronal viability assays Lactate dehydrogenase (LDH), preserved as a cytoplasmic enzyme in living cells with intact cell membranes, which is released immediately from the cells whose membrane is damaged. In this experiment we use the CytoTox 96 Cytotoxicity Kit (Promega, USA) to measure LDH the release according to the instruction. We use the cultures treated with 10× lysis (supplied by the manufacturer) of the total lysine of the cell to measure the maximum release level of LDH. The 96-well plate reader was used to obtain the absorbance at 490 nm (Molecular Devices, USA). Then we compute the amount of LDH release (%) through calculating the ratio of experimental LDH release to maximum amount LDH according to the manufacturer's instructions (Shan et al., 2009). The important indicators of neuronal cell viability include the ability of neuronal cells to absorb the thiazolyl tetrazolium bromide (MTT). According to the manufacturer's instructions, cells and MTT were incubated at 37 °C for 1 h, then the mixture was lyses with DMSO and overnight in the dark room. The absorbance of the lysates were then read at 540 nm on a plate reader (Qu et al., 2009). 2.4. Quantitative reverse transcription-PCR All RNAs were unwind by utilizing TRIzol reagent (Invitrogen, USA), then reverse transcribed using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo, USA) and the qRT-PCR response using SYBR Green PCR Master Mix (Invitrogen, USA). Preliminary arrangements are portrayed in Table 1. The invert translation was performed on BioRad MJ Scaled down Instrument (Bio-Rad, USA) and QuantStudio 6 K Flex Instrument (ABI, USA) was used for the qRT-PCR. The cycle time value was normalized to the GAPDH level of the same test. And each experiment was repeated three times independently. 2. Materials and methods 2.1. Cortical neuronal culture and treatment As mentioned in our previous reports, we choose C57BL/6 J mice on day 17 of gestation to culture cortical neurons (Brewer et al., 1993; Shan et al., 2009). Pregnant mice were firstly anesthetized with 4% isoflurane containing 30% O2 and 70% N2O and then killed by cervical dislocation. The mice were sprayed with 70% ethanol to remove the embryos. Then the embryos were rapidly decapitated after the meninges removed and the cortex was then placed in plate medium put on the ice board. Which consists of neural basal medium, 25 mM glutamic acid, 0.5 mM L-glutaMAX, 0.5% FBS and 2% B-27 supplement. We suspended the extracted cortical neurons in a petri dish coated with poly-D-lysine. Then one half of the medium was removed and replaced with maintenance medium which includes neural basal medium, 0.5 mM L-glutaMAX and 2% B-27 supplement provided every 3 days. The cultured neurons were used in the experiment after 12 days (Shan et al., 2009). To simulate experimental ICH, the cultures were exposed to 200uM of hemin (Sigma) or to vehicle (0.1% dimethyl sulfoxide) for 3 h in vitro prior to harvesting cell survival assays (Chang et al., 2011; Tao et al., 2016). We dissolved the hemin in the dimethyl sulfoxide (DMSO) and diluted it to the desired concentration with fresh medium. The concentration of DMSO in the medium was 0.1%. 2.5. Constructs and treatment The company of RioBio (China) has provided us with miR-575 agomir, miR-575 agomir control, miR-575 antagomir and miR-575 antagomir control. The 1.0 μM miR-575 agomir, miR-575 agomir control, miR-575 antagomir or miR-575 antagomir control was washed with extracellular solids (ECS) for 10 min and after that treated with serum-free medium for 2 h at 37 °C at 5% CO2, 95% stickiness. The cultures were refined in upkeep medium for 24 h in advance after hatching. 2.6. Luciferase reporter assay The wild-type 3′-UTR arrangement of PTEN (wt PTEN-3′-UTR) associated with miR-575 and site-directed mutagenesis complementary to the miR-575 seed locale (mt PTEN-3′-UTR) was performed to disturb the arrangement inside wt PTEN-3′-UTR. The preliminaries were obtained from the Chinese company named RioBio. We used DNA sequencing analysis to affirm the correspondent builds. An add up to of 5 × 104 PC12 cells were cultured in plates with 24-well for 24 h, then we used Lipofectamine 2000 (Invitrogen, USA) to co-transfect with 100 ng wt PTEN-3′-UTR builds utilizing with 1.0 μM miR-575 agomir or miR-575 agomir control, or with the miR-575 antagomir or miR-575 antagomir control in the PC12 cells. The transfection efficiency was 2.2. Cortical microglia culture We choose C57BL/6 J mice on day 17 of gestation to culture the cortical microglia. The mice were killed by cervical dislocation after anesthesia (4% isoflurane, 70% N2O and 30% O2). The embryos were 2 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. Table 1 Primers for quantitative reverse transcription-PCR polymerase chain reaction. Gene Primer sequence(5′-3′) miR-575 Forward,5′-ACACTCCAGCTGGGGAGCCAGTTGGACAGG-3′ Reverse,5′-TCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCTCCTGT-3′ Forward,5′-ACACTCCAGCTGGGCGCAAATTCGTGAAGC-3′ Reverse,5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAAAATAT-3′ Forward,5′-GAGCGTGCAGATAATGACAAGGAAT-3′ Reverse,5′-GGATTTGACGGCTCCTCTACTGTTT-3′ Forward:5′-GCACCGTCAAGGCTGAGAAC-3′ Reverse:5′-TGGTGAAGACGCCAGTGGA-3′ Forward:5′- ACAGCAAAAGACACCCACGG-3′ Reverse:5′- CTTGTTTCATTCTGAGCCTCCTC-3′ Forward:5′-AATCCTGCCGTTCCTACTGATC-3′ Reverse:5′9-GTGTCACCGTGTCTTCCTTGAG-3′ Forward:5′-TCACCTGAGCTTTGATGTCG-3′ Reverse:5′-CTGAAAGGAGCCCTGTCTTG-3′ Forward:5′-CAAGGAAGGTTGGCATTTGT-3′ Reverse:5′-CCTTTCAGTCCTTTGCAAGC-3′ Forward:5′-CAGGGTAATGAGTGGGTTGG-3′ Reverse:5′-CACGGCACCTCCTAAATTGT-3′ U6 PTEN GAPDH CD86 CD32 Arg-1 CD206 YM-1 hybridization of the Ubc-CreER transgenic line of the Shanghai Model Biological Research Center with the PTENLoxP/LoxP mice of Wuhan University. Genomic DNA was extracted from the tail tissues of F1 and F2 progeny, and then the genotype of each mouse (CreER transgene, focusing on PTEN) was studied. Finally, the genotypes were evaluated by PCR using PTENLoxP and Cre as primers. PTENLoxP forward CTCCT CTACTCCATTCTTCCC, and reverse, ACTCCCACCAATGAACAAAC; Cre forward, GCGGTCTGGCAGTAAAAACTATC, and reverse, GTGAAACA GCATTGCTGTCACTT. At the age of 4–6 weeks, the Pre-mutant mice (PTENLoxP/LoxP Ubc-CreER+) and control mice (PTENLoxP/LoxP UbcCreER−) were given intraperitoneal infusion of 1 mg. Investigation of PTEN mouse phenotype was at the earliest 3 days after the ultimate infusion. All animal experimental protocols were approved by the Animal Experimental Ethics Committee of Wuhan University and carried out in accordance with Hubei Province Laboratory Animal Management Regulations. tested by Renilla luciferase pRL-TK reporter from Promega in USA. The luciferase exercises were evaluated by the dual-luciferase columnist measure framework utilizing the Glomax2020 luminometer (Promega, USA) at 24 h after transfection. We used Renilla luciferase to normalize the data. The test was replicated three times in triplicate wells. 2.7. Western blot As described in previous study (Ning et al., 2004). Primary antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Cell Signaling Technology (CST, MA) and PTEN from Santa Cruz Biotechnology (CA, USA) were incubated on a polyvinylidene difluoride membrane (Millipore, USA). We labeled the primary antibody with a horseradish peroxidase-labeled secondary antibody and protein bands were imaged with Super Signal West Femto Maximum Sensitivity Substrate (Pierce, USA). We obtained blot images from a polyvinylidene difluoride membrane by using an EC3 imaging system (UVP, USA). Finally, western blot data were quantified using Image J software. 2.10. ICH model The ICH model was induced by autologous blood stereotactic injection as previous report (Yang et al., 2015). Briefly, mice were anesthetized with 4% isoflurane containing 30% O2 and 70% N2O placed in a stereotaxic frame (Shenzhen Rewal Life Science and Technology Co., Ltd., Shenzhen, China) and a cranial burr hole (1 mm) was drilled on the right side of the skull (0.8 mm anterior and 2.0 mm lateral of the bregma). Then, 30 μl of autologous blood collected from the right femoral artery was infused into the right striatum by a 30 gauge needle within 60 s from the coordinates relative to the bregma 0.8 mm in the anterior side, 2.0 mm in the lateral surface, and 3.0 mm in the lateral surface. The needle was kept in position for an additional 10 min to prevent backflow before craniotomy was closed with bone wax. During surgical procedures, mice were kept at about 37 °C on an electric heating blanket and then kept separately until the anesthesia was completely restored. Sham operated animals were subjected to needle insertion only. 2.8. Animals Male C57BL/6 J mice (8–10 weeks old) were placed in a temperature and humidity controlled room (23 °C–25 °C) in a 12 h dark cycle. Animals were allowed to acclimate to the environment for at least 7 days prior to the experiment. All animal were used in accordance with the IACUC guidelines. The animal-related protocols were approved and implemented by the Animal Care and Ethics Committee of the Wuhan University School of Medicine. We used a randomized approach when assigning samples, collecting and processing data. Researchers did not know the group to which each animal was assigned. All animal-related studies reported animal experiments have followed the ARRIVE guidelines (Curtis et al., 2015; McGrath and Lilley, 2015). 2.9. Production of PTEN-deficient mice It is embryonically deadly to germline knockout of encoding PTEN. So we embraced a procedure to create mice with tamoxifen-inducible conditional knockout of PTEN. The principle of this method is under the control of the ubiquitin-expressing gene encoding the ubiquitin C (Ubc) promoter, has followed elimination of the PTEN allele on the LoxP side (PTENLoxP/LoxP) with tamoxifen-induced Cre recombinase and the mutated estrogen receptor ligand binding space (CreER). Infusion of tamoxifen into PTENLoxP/LoxP Ubc-CreER+ mice before tamoxifen treatment resulted in excision of the loxP-flanked exon and resulted the knockout of PTEN subsequently. First, we performed an indoor 2.11. FJC staining Mice were anesthetized with excess isoflurane and then perfused intraperitoneally with saline and then fixed with 4% paraformaldehyde. The brain of the mice were removed, taken out and stored in a 4% paraformaldehyde solution at 4 °C overnight. The brain was cut into 20 μm thick slices on the coronal axis by Leica VT1000S vibratome (Leica Micro-systems AG, Nussloch, Germany). The Fluoro-Jade C (FJC) (Sigma Aldrich, USA) mark was made using a modified standard 3 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. the box. The higher the score, the more severe the neurological dysfunction. Adhesive-removal test: A modified sticky-tape (MST) test was performed to assess forelimb function (Sughrue et al., 2006). A yellow paper tape of 3.0 × 1.0 cm was used to make a sleeve, which was then wrapped around the front paw of the mouse so that the tape would stick to the sleeve and the mouse's fingers would protrude slightly from the sleeve. The normal reaction is that the mouse would pull the tape with the mouth or brushes the tape with the contralateral paws, and strongly wanted to pull the sleeve down. The mice were placed in cages for 30 s. The first timer was activated at the start of the experiment and run without interruption. The second timer was only activated when the mice attempted to remove the tape sleeve. We recorded the ratio of left (affected)/right (unaffected) forelimb function in the contralateral and ipsilateral limbs, respectively. The test was repeated three times a day and the average of the two best scores for the day was used as experimental data. The lower the ratio we get, the more severe the neurological dysfunction. 2.13. ELISA analysis TNF-α, IL-1β and IL-6 protein levels were tested according to the manufacturer's protocol of ELISA kits (R & D Systems, Minneapolis, Minn., USA). 2.14. Materials L-lysine and D-lysine purchased from Sigma (USA) was dissolved in normal saline. Bisperoxovanadium (pyridine-2-carboxyl) [BpV(pic)] was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) (Zhang et al., 2017). Fig. 1. L-lysine promotes M2 microglial polarization and reduces inflammatory response in hemin-insulted primary microglia. (A) In vitro studies,qRT-PCR analysis of M2 microglial markers in hemin-insulted primary cultured microglia. (B-D) The levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were measured by ELISA assay in hemin-insulted primary cultured microglia. (n = 6 in each group, *p < .05 or **p < .01compared to Sham, #p < .05 compared to Hemin+vehicle, two-way ANOVA test, followed by Bonferroni post hoc test). 2.15. Statistics The group size for each experiment was based on power analysis (Charan and Kantharia, 2013). The criterion for significance (alpha) was set at 0.05. Student's t-test or ANOVA test was used where appropriate to examine the statistical significance of the differences between groups of data. Bonferroni tests were used for post-hoc comparisons when appropriate. All results are presented as mean ± SEM. Significance was placed at p < .05. protocol. The slides were immersed for 5 min in 1% sodium hydroxide 80% ethanol, then rinsed with 70% ethanol for 2 min, rinsed with distilled water for 2 min, and rinsed with 0.06% potassium permanganate solution for 10 min. After soaking for 2 min in distilled water, the slides were transferred to a 0.0001% FJC solution of 0.1% acetic acid. Rinse 3 times with distilled water for 1 min each time. Then the samples were dried at 50 °C for 5 min and immersed in xylene for 1 min. Sections were mounted with DPX medium (Sigma Aldrich, USA). Slices were taken by researchers using an Olympus fluorescence microscope (IX51, Olympus, Japan). Photomicrographs were taken from three regions of the ipsilateral cerebral cortex using a × 20 target and FJC positive cells were counted by Image J software (Image J, USA). Data were expressed as cells / mm2. 3. Results 3.1. L-lysine promotes M2 microglial polarization and reduces inflammatory response in vitro and in vivo By performing qRT-PCR analysis, we measured the mRNA levels of M2 microglial markers in hemin-insulted primary microglia culture. As shown in Fig. 1A, L-lysine increases the level of Arg-1, CD206 and YM1, the markers of M2 microglia (Herder et al., 2015; Tam and Ma, 2014), and D-lysine does not alter the level of Arg-1, CD206 and YM-1, in the microglia cultures. Consistent with the results in vitro, L-lysine (1 mg/kg, intraperitoneal injection) increases the level of Arg-1, CD206 and YM-1 in the damaged brain tissues at 1 h after mouse ICH (Fig. S1A). In order to determine whether L-lysine regulates the release of inflammatory cytokines in our experimental conditions. We measured the protein level of inflammatory cytokines (TNF-α, IL-1β and IL-6) by ELISA in hemin-insulted primary culture of microglia. In comparison with the sham group, while hemin insult increases the protein levels of TNF-α, IL-1β and IL-6 in hemin-insulted microglia (Fig. 1B–D), L-lysine treatment prevents the increase of TNF-α, IL-1β and IL-6, but D-lysine treatment does not prevent the increase of TNF-α, IL-1β and IL-6 (Fig. 1 B-D). Consistent with the in vitro results, L-lysine treatment prevents ICH-induced increase of TNF-α, IL-1β and IL-6 in the ICH mice model 2.12. Neurobehavioral tests Neurological Severity Scores: As previously reported, mice were subjected to a modified neurological severity score (mNSS) test (Chen et al., 2001). These tests are a series of exercise, sensory, reflex and balance tests similar to those of the human contralateral neglect test. Neurological system was scored of 0–18 points (normal score 0 points, the highest score of 18 points). Beam walk test: Beam walking tests measure the complex neuromotor function in animals (Aronowski et al., 1996; Petullo et al., 1999). The mouse was timed when walking on a (100 × 2 cm) beam. Place a box that will make the mouse feel safe at one end of the beam. A huge sound was created to stimulate the mouse to enter the box (Petullo et al., 1999). The score is based on the time taken for the mouse entered 4 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. Fig. 2. L-lysine has a neuroprotective effect in the model of intracerebral hemorrhage. (A) L-lysine (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM) decreased LDH release in hemin-induced cortical neurons. (B) L-lysine (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM) increased cells viability in hemininduced cortical neurons. (C)The numbers of FJC-positive neurons (green) remarkably increased at 24 h after ICH and L-lysine treatment for 24 h decreased the FJC-positive neurons (n = 6 independent cultures. # p < .05 or ###p < 0.001compared to Con or Sham, *p < .05 or **p < .01 compared to Hemin+vehicle or ICH + vehicle, one-way ANOVA test, followed by Bonferroni post hoc test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) also performed FJC straining to test the effect of L-lysine on neuronal survival at 24 h after ICH. We find that L-lysine treatment decreases the number of FJC-positive degenerating neurons in the cerebral hemorrhage area compared with the vehicle treatment group (Fig. 2 C). In addition, we show that LPS blocks L-lysine-induced decrease of the degenerating neurons in ICH injury (Fig. S2 D). Thus, we conclude that L-lysine is neuroprotective in experimental ICH injury model in vitro and in vivo. (Fig. S1B-D). To further verify the effect of L-lysine on the release of inflammatory cytokines, lipopolysaccharides (LPS) was used to counteract the antiinflammatory response. LPS (5 mg/kg) was administered intraperitoneally at 2 h prior to mouse ICH injury. We show that LPS blocks the effect of L-lysine on the levels of TNF-α, IL-1β and IL-6 (Fig. S2A-C). Together, these results provide the first evidence that L-lysine promotes M2 microglial polarization and reduces inflammatory response in ICH injury. 3.3. L-lysine promotes functional recovery in animals of intracerebral hemorrhage 3.2. L-lysine prevents neuronal death in hemin-induced injury and reduces the number of degenerating neurons after mice ICH To verify the functional outcome of L-lysine-induced neuroprotection, a series of neurobehavioral tests including the MST test, the beamwalking test and the mNSS test were performed. The neurological changes were observed at 1 day before ICH and 1, 3, 7 and 14 days respectively after ICH. L-lysine (1 g/kg, intraperitoneal injection) was injected at 1 h after ICH. Our data show that mice treated with L-lysine has lower scores in the mNSS test compared with mice treated with vehicle (ICH + vehicle group) at 7 and 14 days after ICH (Fig. 3A). Llysine treated mice has lower scores of beam-walking test (Fig. 3B) and The preceding findings led us to reason that L-lysine, through attenuating inflammatory response, might exert neuroprotective effect in ICH injury. To address this possibility, we tested the effect of L-lysine in hemin-insulted cortical neuron cultures. At 6 h after the induction of hemin insult, the cortical neuron cultures were treated with L-lysine at various concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM) for 1 h. Our results show that L-lysine reduces the release of LDH or increases the cell viability in a dose-dependent manner (Fig. 2A-B). We 5 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. Fig. 4. ICH-induced PTEN up-regulation leads to the increase of inflammatory cytokines and the inhibition of M2 microglial polarization. (A) Western blot assay showed that the level of PTEN protein was increased in mice at 24 h after ICH (n = 6 in each group, **p < .01 compared to Sham, Student's t-test). (B) The level of PTEN protein in the hemin-induced cortical neurons was increased (n = 6 in each group, *p < .05 compared to Sham, Student's t-test). (C) Western blot assay showed that the level of PTEN protein was reduced in mutant mice (n = 6 in each group, **p < .01 compared to WT, Student's ttest). (D) In vivo, qRT-PCR analysis of M1 microglial markers and M2 microglial markers in PTEN-mutant mice at 24 h after ICH (n = 6 in each group, *p < .05 or **p < 0.01compared to Sham, #p < .05 compared to ICH + WT, ANOVA test, followed by Bonferroni post hoc test). PTEN-wild-type cells and PTEN-knockout cells. We refer to these mice as "PTEN mutant" mice because PTEN was not removed from all cells. Our data show that the protein expression of PTEN was elevated in mice at 24 h after ICH and in hemin-insulted cortical neurons at 24 h after insult (Fig. 4A-B). PTEN expression was obviously decreased in PTEN-mutant mice (Mut) (Fig. 4C). As shown in Fig. 4D, while ICH injury in WT mice promotes marked increase M1 microglial markers, it also moderately increase the levels of M2 microglial markers. Whereas in PTEN-mutant mice, PTEN deletion inhibits ICH-induced M1 microglial polarization but further increases M2 microglial polarization (Fig. 4D). Similar results were observed in hemin-insulted primary cultured microglia treated with PTEN inhibitor bpV(pic) (200 nM) (Fig. S3). These data suggest that PTEN inhibition increases the M2 microglia polarization but decreases the M1 microglial polarization after ICH injury. Fig. 3. Neurobehavioral tests in ICH mice. (A) Mice with L-lysine injection had lower scores in mNSS test at day 7 and 14 after ICH. (B) Mice with L-lysine injection had lower scores in beam-walking test at day 3, 7 and 14 after ICH. (C) Mice with L-lysine injection had higher ratio in MST test at day 7 and 14 after ICH. (n = 6 for each group; *P < .05 compared to ICH+ vehicle, two-way ANOVA test, followed by Bonferroni post hoc test). higher ratio of MST test compared with mice treated with vehicle at 7 and 14 day after ICH (Fig. 3C). These results provide functional evidence for the neuroprotective role of L-lysine in ICH injury. 3.4. Suppressing PTEN promotes M2 microglia polarization Previous studies have shown that PTEN exerts anti-inflammatory effects by regulating the activation of M2 macrophages (Wang et al., 2014). To explore the possibility by which PTEN regulates microglia and subsequently influence neuronal survival, the mRNA levels of M1 and M2 microglial markers were measured in mice at 24 h after ICH. CD86 and CD32 were selected as the agent markers of M1 microglia (Peng et al., 2017), and Arg-1, CD206, and YM-1 were selected as the markers of M2 microglia (Herder et al., 2015; Tam and Ma, 2014). In this study, we used PTEN transgenic mice (tamoxifen-induced recombination), which usually co-exists with different proportions of 3.5. L-lysine promotes M2 microglia polarization through suppression of PTEN We next test whether PTEN mediates the effect of L-lysine on M2 microglia polarization in ICH injury. We show that L-lysine reverses ICH-induced increase of PTEN in ICH injury of wild-type mice (Fig. 4A; Fig. 5A). L-lysine also increases the levels of Arg-1, CD206 and YM-1, the markers of M2 microglia in ICH injury of wild-type mice (Fig. 5A). 6 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. Fig. 5. L-lysine promotes M2 microglial polarization through inhibiting PTEN. Mice in each group were processed 24 h after ICH. (A) Western blot assay showed that the PTEN protein level was decreased in PTEN-wild-type mice treated with L-lysine, and the level of PTEN did not significantly decreased in PTEN-mutant mice treated with L-lysine. (B) qRT-PCR analysis of M1 microglial marker Arg-1 in each group. (C) qRT-PCR analysis of M1 microglial marker CD206 in each group. (D) qRT-PCR analysis of M1 microglial marker YM-1 in each group. (n = 6 in each group, *p < .05 or **p < 0.01compared to ICH + vehicle in WT group or Mut group, two-way ANOVA test, followed by Bonferroni post hoc test). Fig. 6. miR-575 directly binds the 3′UTR target sites of PTEN in PC12 cells. (A) PTEN-wild-type had the 3′UTR target sites binding to miR-575, and PTENmutant-type had no bingding sites to miR-575. (B) Luciferase reporter assay in PC12 cells was performed after co-transfection with the wild-type or mutanttype of PTEN 3′-UTR reporter plasmids and miR-575 agomir or agomir-control. (C) Luciferase reporter assay in PC12 cells was analyzed after co-transfection with the wild-type or mutant-type of PTEN 3′-UTR reporter plasmids and miR575 antagomir or antagomir-control. (n = 6 for each group; **p < 0.01compared to Con+agomir-control or Con+antagomir-control, two-way ANOVA test, followed by Bonferroni post hoc test). Con: untransfected PC12 cells; Blank: PC12 cells transfected with Luciferase vector carrying PTEN wild-type or PTEN mutant without miR-575 agomir or antagomir. However, in PTEN-mutant mice (Mut) subjected to ICH injury, L-lysine neither decrease the level of PTEN nor increase the levels of Arg-1, CD206 and YM-1 (Fig. 5B–D). These results suggest that L-lysine promotes anti-inflammatory effects of M2 microglia through suppressing PTEN. In the primary cortical neuron cultures treated with miR-575 agomir, the level of PTEN mRNA is decreased, but the level of PTEN mRNA is increased in the cortical neurons treated with miR-575 antagomir (Fig. 7A–B). Furthermore, the level of PTEN protein is decreased in the cortical neurons treated with miR-575 agomir, but the level of PTEN protein is increased in the cortical neurons treated with miR-575 antagomir (Fig. 7C-D). These results indicate that miR-575 inhibits the expression of PTEN mRNA and protein in cortical neurons. 3.6. PTEN is the target gene of miR-575 in cortical neurons PTEN is a potential target gene of miR-575 (Wang et al., 2019). PTEN is considered as a candidate because it has a potential miR-575 binding site in its 3′UTR (Fig. 6A). The luciferase assay show that miR575 agomir causes a significant decrease of luciferase activity in PC12 cell. The mutation of the seed sequence of miR-575 within the 3′UTR of PTEN abrogated the inhibition of luciferase activity by miR-575 agomir in PC12 cells (Fig. 6B). The miR-575 antagomir results in a significant increase of luciferase activity in PC12 cells. The mt PTEN-3′UTR abrogates the increase of luciferase activity by miR-575 antagomir in PC12 cells (Fig. 6C). These results suggest that miR-575 directly binds to the predicted 3′UTR target sites of PTEN. 3.7. L-lysine reduces hemin-induced neuronal injury through up-regulation of miR-575 expression To determine whether miR-575 mediates the neuroprotective effect of L-lysine in ICH injury, we examined the level of miR-575 in L-lysinetreated cortical neurons subjected to hemin insult for 6 h. Treatment of L-lysine (100 μM) for 1 h increases the expression of miR-575 in hemin7 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. Fig. 8. L-lysine increases the level of miR-575 and upregulating miR-575 protects against hemin-induced cortical neuronal death. Each group of cortical neuronal were subjected to hemin for 6 h. (A) miR-575 expression was increased in L-lysine treated group. (B) Treatment of miR-575 agomir increased miR-575 expression. (C-D) LDH assay and MTT assay showed that treatment of miR-575 agomir reduced hemin-induced cortical neuronal death. (n = 6 for each group; *p < .05 or **p < 0.01compared to Sham, #p < .05 or ## p < 0.01compared to Con+agomir-control, two-way ANOVA test, followed by Bonferroni post hoc test). hemin-insulted cultures of cortical neurons (Fig. 9A–B). We further show that the pretreatment of miR-575 antagomir blocks L-lysine-induced increase of the markers of M2 microglia (Arg-1, CD206 and YM1) in hemin-insulted cultures of cortical neurons (Fig. 10A-C). These data suggest that the enhancement of M2 microglial polarization by Llysine treatment in hemin-insulted cultures of cortical neurons is mediated through miRNA-575 upregulation/PTEN downregulation. Collectively, based on the results from this study we conclude that Llysine may confer neuroprotection through promoting M2 microglial polarization and subsequent suppression of inflammatory response in ICH injury, which is in part mediated by miRNA-575 up-regulation/ PTEN down-regulation signaling. Fig. 7. miR-575 regulates PTEN expression in cortical neurons. (A-B) PTEN mRNA level was down-regulated in cortical neurons treated with miR-575 agomir, and PTEN mRNA level was up-regulated in cortical neurons treated with antagomir. (C) The level of PTEN protein in the cortical neurons treated with miR-575 agomir was reduced. (D)The level of PTEN protein in the cortical neurons treated with miR-575 antagomir was increased. (n = 6 for each group; *p < .05 or **p < 0.01compared to Con+agomir-control or Con+antagomir-control, two-way ANOVA test, followed by Bonferroni post hoc test). insulted cortical neurons (Fig. 8A). At 6 h after hemin insult, miR-575 agomir pretreatment for 24 h enhances neuronal viability compared with the treatment of miR-575 agomir control (Fig. 8 B-D). But the treatment of miR-575 antagomir decreases the viability of hemin-insulted cortical neurons (Fig. S4 A-C). Thus, L-lysine attenuates hemininduced neuronal damage through up-regulation of miR-575 expression. 4. Discussion ICH is a common and devastating cerebral disease with higher morbidity and mortality than ischemic stroke (Wang and Dore, 2007). Cell death signaling occurred after ICH injury, leading to a new phase of inflammation. Dead cells can release a series of signals to stimulate the immune system (Zhou et al., 2014). L-lysine is a basic amino acid that is shown to protect against brain damage. Although the neuroprotective effect of L-lysine in ischemic stroke has been reported by previous study (Kondoh et al., 2010), it is not known whether L-lysine is neuroprotective in ICH injury. The cellular and molecular mechanisms mediating the neuroprotective effect of L-lysine remain largely unclear. The present study for the first time demonstrates that L-lysine reduces neuronal 3.8. L-lysine promotes M2 microglial polarization through miRNA-575 upregulation/PTEN downregulation The preceding data thus far suggest that L-lysine exerts its neuroprotective effect through enhancement of M2 microglial polarization that may be mediated by miRNA-575 up-regulation/PTEN down-regulation signaling. To test this possibility, we firstly show that the miR575 antagomir pretreatment attenuates L-lysine-induced increase of miR-575 and prevents L-lysine-induced reduction of PTEN expression in 8 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. Fig. 10. L-lysine promotes M2 microglial polarization through up-regulation of miRNA-575. (A) qRT-PCR analysis of M2 microglial marker Arg-1 in mice at 24 h after ICH in each group. (B) qRT-PCR analysis of M2 microglial marker CD206 in each group. (C) qRT-PCR analysis of M2 microglial marker YM-1 in each group. (n = 6 in each group, *p < .05 compared to Hemin+vehicle in antagomir treated group or antagomir-control treated group, two-way ANOVA test, followed by Bonferroni post hoc test). member of miRNAs which was firstly depicted in 2009 (Yao et al., 2009). A recent study proves that the identified target genes of miR-575 is associated with inflammation response. It is not yet clear whether and how miR-575 is regulated by L-lysine. Our study shows that L-lysine increases miR-575 expression in injured cortical neurons after hemin insult. We also find that increasing miR-575 expression reduces cortical neuronal damage in hemin-insulted conditions. We verify that PTEN is the target gene of miR-575 in cortical neurons. We further show that miR-575 mediates the neuroprotective effect of L-lysine on PTEN downregulation and subsequent suppression of pro-inflammatory cytokine release in ICH injury. Our results provide new evidence for a critical role of miR-575 underlying the molecular mechanisms of neuroprotection. While inhibition of PTEN has been shown to confer neuroprotection through suppressing the function of NR2B-containing N-methyl-D-aspartate receptors and retaining the function of gamma-aminobutyric acid A receptor function (Liu et al., 2010; Zheng et al., 2012), the inhibition of PTEN is recently shown to lead to activation of M2-type cholinergic anti-inflammatory pathways in macrophages in peripheral tissues (Wang et al., 2014). In the present study, we provide evidence that suppressing PTEN promotes M2 microglia polarization. As M2 microglia express anti-inflammatory mediators, such as Arg-1, CD206, and YM-1, which can prevent inflammation and contribute to neuroprotective effects (Xu et al., 2015), we tested whether PTEN inhibition exerts its neuroprotective effect through M2 microglia polarization. We show that PTEN inhibition mediates the effect of L-lysine and miR-575 on the enhancement of M2 microglia polarization, which promotes neuronal survival through enhancing the anti-inflammatory effect in ICH injury. Inflammatory reactions can increase brain damage which usually occurs after ICH injury. The pro-inflammatory responses lead to tissue damage, BBB destruction, secondary edema and brain cell death (Wang Fig. 9. L-lysine down-regulates the expression of PTEN through upregulation of miRNA-575. (A) qRT-PCR shows that L-lysine increased the level of miR-575 in cortical neurons treated with miR-575 antagomir or miR-575 antagomir control. (B) Western blot assay showed that the levels of PTEN protein in L-lysine treated groups was reduced and L-lysine attenuated the ability of miR-575 antagomir improving PTEN expression. (n = 6 in each group, *p < .05 or **p < 0.01compared to Hemin+vehicle in antagomir treated group or antagomir-control treated group, two-way ANOVA test, followed by Bonferroni post hoc test). cell death in the peri-hematomal area and improves functional outcomes after experimental ICH injury. Our results reveal that the enhancement of M2 microglia polarization and subsequent suppression of pro-inflammatory cytokine release contribute to the neuroprotective effect of L-lysine, which is mediated by the miR-575 up-regulation/ PTEN down-regulation signal pathway after experimental ICH injury. The miRNAs play a critical role in many biological processes including cell metabolism, apoptosis, cell differentiation and development (Ketting et al., 2001; Lee et al., 2004). The miR-575 is one 9 Experimental Neurology 327 (2020) 113214 J. Cheng, et al. and Dore, 2007). Activation of the inflammatory response is the precursor of the enlargement of hematoma, edema and secondary neurological impairment (Zhou et al., 2014). We provide the first evidence that L-lysine promotes M2 microglia polarization and subsequently reduces the levels of pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α at 24 h after ICH, which exerts neuroprotective effect in ICH injury. Our study for the first time reveals the role of L-lysine in antiinflammatory responses-mediated neuroprotective effect. (discussion 2319–20). Doench, J.G., Sharp, P.A., 2004. Specificity of microRNA target selection in translational repression. Genes Dev. 18 (5), 504–511. Farajdokht, F., et al., 2018. Inhibition of PTEN protects PC12 cells against oxygen-glucose deprivation induced cell death through mitoprotection. Brain Res. 1692, 100–109. Herder, V., et al., 2015. Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler's murine encephalomyelitis. Brain Pathol. 25 (6), 712–723. Keep, R.F., Hua, Y., Xi, G., 2012. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol. 11 (8), 720–731. Ketting, R.F., et al., 2001. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15 (20), 2654–2659. Kondoh, T., et al., 2010. Lysine and arginine reduce the effects of cerebral ischemic insults and inhibit glutamate-induced neuronal activity in rats. Front. Integr. Neurosci. 4, 18. Lee, Y., et al., 2004. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23 (20), 4051–4060. Liu, B., et al., 2010. Preservation of GABAA receptor function by PTEN inhibition protects against neuronal death in ischemic stroke. Stroke 41 (5), 1018–1026. McGrath, J.C., Lilley, E., 2015. Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br. J. Pharmacol. 172 (13), 3189–3193. Miao, S.Y., et al., 2018. SETD5-AS1 stimulates neuron death in stroke via promoting PTEN expression. Eur. Rev. Med. Pharmacol. Sci. 22 (18), 6035–6041. Ning, K., et al., 2004. Dual neuroprotective signaling mediated by downregulating two distinct phosphatase activities of PTEN. J. Neurosci. 24 (16), 4052–4060. Peng, H., et al., 2017. Increased expression of M1 and M2 phenotypic markers in isolated microglia after four-day binge alcohol exposure in male rats. Alcohol 62, 29–40. Petullo, D., et al., 1999. Model development and behavioral assessment of focal cerebral ischemia in rats. Life Sci. 64 (13), 1099–1108. Qu, Y., et al., 2009. Proapoptotic role of human growth and transformation-dependent protein in the developing rat brain after hypoxia-ischemia. Stroke 40 (8), 2843–2848. Shabanzadeh, A.P., et al., 2019. Modifying PTEN recruitment promotes neuron survival, regeneration, and functional recovery after CNS injury. Cell Death Dis. 10 (8), 567. Shan, Y., et al., 2009. Regulation of PINK1 by NR2B-containing NMDA receptors in ischemic neuronal injury. J. Neurochem. 111 (5), 1149–1160. Shi, G.D., et al., 2011. PTEN deletion prevents ischemic brain injury by activating the mTOR signaling pathway. Biochem. Biophys. Res. Commun. 404 (4), 941–945. Sughrue, M.E., et al., 2006. An improved test of neurological dysfunction following transient focal cerebral ischemia in rats. J. Neurosci. Methods 151 (2), 83–89. Tam, W.Y., Ma, C.H., 2014. Bipolar/rod-shaped microglia are proliferating microglia with distinct M1/M2 phenotypes. Sci. Rep. 4, 7279. Tao, X., et al., 2016. Up-regulation of interferon regulatory factor 3 involves in neuronal apoptosis after intracerebral hemorrhage in adult rats. Neurochem. Res. 41 (11), 2937–2947. Taylor, R.A., Sansing, L.H., 2013. Microglial responses after ischemic stroke and intracerebral hemorrhage. Clin Dev Immunol 2013, 746068. Wang, J., Dore, S., 2007. Inflammation after intracerebral hemorrhage. J. Cereb. Blood Flow Metab. 27 (5), 894–908. Wang, L., et al., 2014. Pten deletion in RIP-Cre neurons protects against type 2 diabetes by activating the anti-inflammatory reflex. Nat. Med. 20 (5), 484–492. Wang, Y.N., et al., 2019. MicroRNA-575 regulates development of gastric cancer by targeting PTEN. Biomed Pharmacother 113, 108716. Xi, G., Keep, R.F., Hoff, J.T., 2006. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol. 5 (1), 53–63. Xu, Y., et al., 2015. Telmisartan prevention of LPS-induced microglia activation involves M2 microglia polarization via CaMKKbeta-dependent AMPK activation. Brain Behav. Immun. 50, 298–313. Yang, F., et al., 2015. Receptor for advanced glycation end-product antagonist reduces blood-brain barrier damage after intracerebral hemorrhage. Stroke 46 (5), 1328–1336. Yao, Y., et al., 2009. MicroRNA profiling of human gastric cancer. Mol. Med. Rep. 2 (6), 963–970. Zhang, Z.F., et al., 2017. Bisperoxovandium (pyridin-2-squaramide) targets both PTEN and ERK1/2 to confer neuroprotection. Br. J. Pharmacol. 174 (8), 641–656. Zhang, X., Xin, G., Sun, D., 2018. Serum exosomal miR-328, miR-575, miR-134 and miR671-5p as potential biomarkers for the diagnosis of Kawasaki disease and the prediction of therapeutic outcomes of intravenous immunoglobulin therapy. Exp Ther Med 16 (3), 2420–2432. Zhao, H., et al., 2016. P2X7 receptor suppression preserves blood-brain barrier through inhibiting RhoA activation after experimental intracerebral hemorrhage in rats. Sci. Rep. 6, 23286. Zheng, M., et al., 2012. Regulation of nuclear TDP-43 by NR2A-containing NMDA receptors and PTEN. J. Cell Sci. 125 (Pt 6), 1556–1567. Zhou, Y., et al., 2014. Inflammation in intracerebral hemorrhage: from mechanisms to clinical translation. Prog. Neurobiol. 115, 25–44. Ziai, W., Nyquist, P., Hanley, D.F., 2016. Surgical strategies for spontaneous intracerebral hemorrhage. Semin. Neurol. 36 (3), 261–268. 5. Conclusion This study demonstrates that L-lysine confers neuroprotection in ICH injury both in vitro and in vivo. Our results suggest that the neuroprotective effect of L-lysine may be mediated in part through its suppression of pro-inflammatory cytokine release. We provide evidence that the suppression of pro-inflammatory cytokine release by L-lysine is mediated through the miR-575 up-regulation/PTEN down-regulation signal pathway in experimental ICH injury. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.expneurol.2020.113214. Author disclosure statement The authors declare no competing financial interests. Acknowledgements This work was supported by National Key Basic Research Program of China (2014CB541606), two National Key R&D Program of China (2018YFC1312300; 2019YFC010167), the Natural Science Foundation of China (NSFC; 81470599), the Key Research and Development Project of Shandong (2019JZZY021010), and the Fund of Collaborative Innovation Center for Brain Science to Q.W. This work was also supported by the National Natural Science Foundation of China (NSFC; 81572489) to Q.X.C. References Aronowski, J., et al., 1996. An alternative method for the quantitation of neuronal damage after experimental middle cerebral artery occlusion in rats: analysis of behavioral deficit. J. Cereb. Blood Flow Metab. 16 (4), 705–713. Askenase, M.H., Sansing, L.H., 2016. Stages of the inflammatory response in pathology and tissue repair after intracerebral hemorrhage. Semin. Neurol. 36 (3), 288–297. Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116 (2), 281–297. Brewer, G.J., et al., 1993. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35 (5), 567–576. Chang, Y.F., Gao, X.M., 1995. L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor. Neurochem. Res. 20 (8), 931–937. Chang, N., et al., 2007. Phosphatase PTEN in neuronal injury and brain disorders. Trends Neurosci. 30 (11), 581–586. Chang, C.F., et al., 2011. Caveolin-1 deletion reduces early brain injury after experimental intracerebral hemorrhage. Am. J. Pathol. 178 (4), 1749–1761. Charan, J., Kantharia, N.D., 2013. How to calculate sample size in animal studies? J. Pharmacol. Pharmacother. 4 (4), 303–306. Chen, J., et al., 2001. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 32 (4), 1005–1011. Cunningham, C., 2013. Microglia and neurodegeneration: the role of systemic inflammation. Glia 61 (1), 71–90. Curtis, M.J., et al., 2015. Experimental design and analysis and their reporting: new guidance for publication in BJP. Br. J. Pharmacol. 172 (14), 3461–3471. Davis, S.M., et al., 2006. Hematoma growth is a determinant of mortality and poor outcome after intracerebral hemorrhage. Neurology 66 (8), 1175–1181. Del Bigio, M.R., et al., 1996. Experimental intracerebral hemorrhage in rats. Magnetic resonance imaging and histopathological correlates. Stroke 27 (12), 2312–2319 10


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subset: commoncrawl


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Magic is in the air e-sesh: Ruby + Ben
Ruby, can we just tell you that we're a wee bit jealous that you're marrying a real magician? Can you guys imagine just how much fun these two must have together? Ben here, is a member of the Magic Castle in Los Angeles and a lover and master of card tricks so it was a simple decision for Chris and Kristen Photography that this was going to be a romantic and whimsical e-sesh. Ruby + Ben's adorableness would do the rest!
The first kiss Ruby gave to Ben was a strawberry flavored Hershey's Kiss at their company's new hire orientation. Ben and Ruby started working at their company around the same time and while working at two different departments in two different floors, their paths were crossed with a chocolately kiss. After dating for a year and half, Ben planned a surprised trip to Ruby's favorite city, San Francisco. Ben took Ruby directly to Crissy Field, a park overlooking the Golden Gate Bridge (magical place on a sunny day). In the middle of the park surrounded by passersby walking their cute pups, Ben proposed to Ruby by playing the ukulele and singing Marry Me, by Train.
Congratulations on your engagement Ruby + Dave, and happy planning!
Photography: Chris and Kristen Photography / Ruby's dress: Forever 21 / Ben's suit: Zara, tie and vest: Ted Baker, fedora hat: Calvin Klein / Makeup + stylist: Shirley Kao
Social Love
dekalb wedding photography on Apr 27, 2012
What a cute couple! Best of luck with the wedding you two : D
Terri on Apr 28, 2012
Ahhh, to be young and in love again :) Congratulations and happy planning!
Roberta on Apr 28, 2012
Cute couple
Tim Duncan on May 19, 2012
They're so cute together! :) And the Magic Castle is so fun!
calderas junkers on Oct 6, 2012
queda espectacular. no sobresale nada


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Yes, Yes It Will Happen (300 wins edition)
I've been sitting on this post for quite awhile, and I know the moment has somewhat passed. Still, I want to reject this commonplace notion that we will never again see a 300 game winner. While we likely won't see one soon, the claims that we will never again witness a 300th win are ridiculous..
First, I thought it would be interesting to see how many wins the last four pitchers to reach 300 had accumulated by the end of the season in which they turned 29. Also included below is their average wins per season.
- Clemens: 152 – 17
- Maddox: 150 – 16
- Glavine: 124 – 15
- Johnson: 75 – 17
Johnson's status as a late-bloomer makes him special but not unique–remember that the great Warren Spahn had only 86 wins when he turned 29; Spahn won 363 games. Two things to take away from this: on average, this last crop had 125.25 wins when they finished the season of their 29th year and averaged 16.3 wins over the course of their careers.
I did some more hunting around Baseball Reference to compare the contemporary crop of elite pitchers to these numbers. For those not yet 29, I projected out how many wins they are likely to accumulate according to their 162 game averages. For those over 29, I reported what they had at that age. Average win totals are based on 162 game expectations as well. Here's what I got:
- CC Sabathia: 122 wins at age 28, averages 16 per season, on pace for 149 wins. Sabathia, playing for the underwhelming Tribe for much of his career, has averaged 16 wins a season. He will win many more games if he stays in the pinstripes. While I think he is overrated, he could be a Bronx win machine for many years. The question is not if he'll reach 300, but whether he'll reach 350.
- Carlos Zambrano: 99 wins at age 28, averages 15 per season, on pace for 126 wins. Admittedly, this one surprised me. I expected to see other names here. The Cubs have been competitive throughout much of Zambrano's career–and although their ownership is in flux, I don't see them completely falling apart as a franchise. Plus, the NL Central is not exactly a payroll murderer's row. The question with Zambrano is likely whether he can control himself as well as he controls his fastball.
- Mark Buehrle: 122 wins at age 29 (-4 off the pace), averages 15 wins per season. Ok, this might seem to support my opposition if Buehrle is third on the list. But he is a big lefty, plays in a strong baseball city, and has been nothing short of a horse his entire career. Given his control, I could see him losing some velocity but still be able to clip the corners well into his 40s.
- Jake Peavy: 92 wins at age 28, on pace for 116 (-10 off the pace), averages 15 wins per season. I think we all know what has to happen for Peavy to have a shot at this: get the hell out of San Diego.
- Johan Santana: 109 wins at age 29 (-17), averages 15 wins per season. You might have expected to see this name sooner, but Santana spent the majority of his first three seasons coming out of the bullpen. Since becoming a starter, his wins per season is 17.2. Although the Mets offense is costing him wins this season, and although the NL East is an extremely competitive division, the Mets figure to be a top salary franchise for his tenure there.
- Roy Halladay: 95 wins at 29 (-31), 141 wins today, averages 17 wins per season. That 17 wins a season is incredible since, like Santana, Halladay spent the first few seasons of his career as a spot starter. Even this far down the list, playing in Toronto (as of today) in the brutal AL East, I think Halladay is the second most likely candidate, after Sabathia, to top 300. He reminds me most of Randy Johnson–a late bloomer who, once developed, couldn't be stopped. The big difference is health–while Johnson has had back issues, he has never missed a single start due to his throwing arm. Halladay cannot make the same claim; the pursuit of 300 is equal parts talent, team, and stamina. As with Peavy, a change in location would likely help his win totals.
I think you can see why most of the ESPN commentators screw this one up–the two most dominant pitchers in recent memory (Santana and Halladay) aren't necessarily the two most likely to challenge the 300 win plateau. But please, stop the sky-is-falling madness talk that no one in the majors is on pace to win 300 games.
To be a homer, here's one more:
- Josh Beckett: 95 wins at age 29 (-31), averages 16 wins per season. My sole reason for considering Beckett is that he will likely spend the next decade playing for Boston. They will win a lot of games. If he can stay healthy, then, like Sabathia, he's got a chance to win those "off" games (well maybe not Beckett's "off" games–but that is another post). Beckett couldn't stay healthy in his youth, and no doubt this will be a big concern moving forward. If he doesn't completely break down, then he will be in an environment conducive to winning.
So, who did I miss?


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Resist the Status Quo. People like to use tools they are used to. Encourage your partners to resist the urge to rely on how they have always done things. Software is more reliable than spreadsheets. And capacity planning software compliments your project management software — it is not a substitute.
Focus on Growth. As a creative team you sell time and skills. Each person adds more complexity. To grow you need a system for planning and tracking that time. You pay for time — better make sure that time is spent on billable projects. Today things might be under control, but with growth it will take more effort. Software allows you to escape spending all of your time managing time. Get ahead of a chaotic future.
Bric is not your typically time tracking system. It allows your team to plan time, track time, view future capacity, and automatically use today's timesheets to make future plans more accurate. Make sure to explain this to your team. Otherwise it is just another time tracking tool.
Here is our guide to selling your team on the value of Bric.
Also, you aren't alone. We are here to help. We have prepared a slide deck that you can use during your all hands meeting.
Download it for PDF, Powerpoint, or InDesign, and modify specifically for your team.
People will resist change. We like what is predictable and routine. However, change is what allows companies to improve and reach goals faster. Bric is part of that necessary but sometime uncomfortable change. Here is how you can make it less uncomfortable.
Tips for Day 5.
- Monitor Usage. Make sure people are filling out their timesheets, and that new opportunities are being planned in Bric. Emphasize how using Bric will help smooth workloads, make sure people have time to do great work, and increase the financial stability of the company.
- Use Project Types. Each project is unique, but that doesn't mean it isn't important to find similarities. Use Project Types to group similar projects together. Then as you complete projects Bric will automatically find patterns in those projects. Patterns that Bric uses to recommend more accurate project plans in the future.


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The Hong Kong-Zhuhai-Macau bridge is built, but not open to the public yet
No official opening date is set but engineers say building work is complete
No official opening date is set but engineers say building work is complete
The construction of the 55km Hong Kong-Zhuhai-Macau bridge (HKZMB) is complete, but no opening date has been officially announced.
The world's longest cross-sea link will connect Hong Kong with Macau and Zhuhai on the mainland, with 22.9km of bridges and a 6.7km undersea tunnel. It's also the longest steel bridge, with its main girders roughly the weight of 60 Eiffel Towers, according to Song Ru'an, deputy commissioner of Beijing's foreign ministry office in Hong Kong.
On a recent trip to the central section of the bridge, senior engineer Gao Xinglin told Young Post that he'd had many sleepless nights over the past 10 years worrying about building the bridge. "We faced many challenges," he said, "but we have done it."
Gao was in charge of planning the project. This included using resources from all over the world, dividing this complicated project to meet goals step by step, and deciding how the bridge would operate.
"We have been building this bridge under 'one country, two systems' [with the help of foreign experts]. So we have faced cultural differences, different operating systems and technical standards," he says.
There have been concerns about the impact of the project on marine life, with large-scale reclamation needed to create several man-made islands. The work has also been dangerous. At least 10 construction workers have died and more than 600 have been injured since work started. The Hong Kong government is paying HK$117 billion for the project and the local link roads, but the final cost remains unknown.


text: The Animal Care and Rescue Center, which officially opened in May 2018, serves as home to off-exhibit animals and animals new to the Aquarium that require quarantine before joining an exhibit, as well as part of our rescue operations. Members of our Animal Husbandry and Animal Care and Welfare staff have stayed busy over the past few months getting a multitude of different species adjusted at the Center. One of the most unique residents of the Center is a South American freshwater stingray confiscated last year at Dulles International Airport by the U.S. Fish and Wildlife Service. Recently, staff performed a full diagnostic work-up on the stingray, where they found he is in great health. After the stingray grows a bit more, he'll be ready to move into the Amazon River Forest exhibit. Two large lungfish and a Gulf saratoga from our Australia: Wild Extremes are also spending time off-exhibit at the Center. And, of course, the Center is not without its reptiles! Two caimans, which were on exhibit in years past, are being cared for at our state-of-the-art facility. Staff continuously work to fine-tune their diets, while also conducting behavior training to prepare them for a possible return to the Amazon River Forest. More than a dozen Australian turtles, including a pig-nosed turtle nicknamed Funzo, also reside at the Center. A few South American turtles that were hatched within the last couple years at the Aquarium's main building can also be found at the Center, where they are using the extra space and attention to grow before heading on exhibit. Two bearded dragon residents are also almost cleared for their time to shine with our Animal Programs team! As previously mentioned, our female zebra shark, nicknamed Zoe, is spending time at the Center as she recovers from a short period of illness. Learn more about the National Aquarium's Animal Care and Rescue Center! Tags: animals, animal-care Categories: Animals, Behind-The-Scenes, Conservation, News Previous Post Next Post


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|B.B. King at the Peabody Opera House in St. Louis on April 4
Imagine this.....you're traveling across the country in a bus, beginning at one end of the country and weaving your way to the other end, stopping every couple of days to make public appearances and give a performance to a huge crowd of people. Imagine that you're also dealing with a few health issues, like Type 2 Diabetes or high blood pressure or poor circulation while you're on this journey. Imagine having to sleep either in the bus or in a different hotel every night while you're doing this. Got it? Good. Now, imagine that you're doing all this, plus you're five months away from YOUR 89th BIRTHDAY!!!
B.B. King made the news last week when a St. Louis Dispatch article was published that reviewed a recent performance at the Peabody Opera House. The article stated that King was nearly 45 minutes into the show before he played a song, talking incoherently and inaudibly at times, flirting with ladies in the front rows, then playing somewhat erratically, forgetting lyrics here and there, and launching into a 15-minute singalong of "You Are My Sunshine." By that time, the article read, some in attendance began to heckle, shout out requests, shout encouragement, and even leave the performance. King rebounded after a few minutes and launched into "The Thrill Is Gone," but he only played a couple more songs before calling it a night.
Looking at comments on some of the various pages that ran the article (which was not a mean-spirited story at all....very fair-minded), most fans were understanding, some were a bit frustrated, and a few were aggravated. King's management released a statement earlier this week, explaining that the Peabody show was King's first in about a month (also pointed out in the article), that he had driven 24 hours from his home in Las Vegas to the gig, and that he had forgotten to take his diabetes medication before he left.
There have been other notes about King in recent months, even a column about a Dallas gig late last year that had some of the same issues, but that's not really what I wanted to talk about today.
Listen, I turned fifty last year and by the end of the work week, I'm ready to go to bed on Fridays at 9:00.....in the MORNING. I can't imagine pulling a 40 hour work week when I'm pushing ninety years old!! Yet, B.B. King still does that during certain times of the year, and his work week sometimes involves driving several hundred miles in a 24-hour period, then playing music for a couple of hours after meeting and greeting fans.
Sure, there's a good chance that King is nearly the end of the road as far as relentless touring goes. He may be to the point where he has to consider sharing a bill with someone, maybe splitting time onstage, maybe cutting back to a couple of shows a month. All that is highly possible.....sad to think about, but highly possible.
Whatever he decides to do, as a blues fan (and a B.B. King fan), I am cool with it. He has more than earned the right to take a step or two back if he wants, just like he's earned the right to have an off-night occasionally, or have a few minutes during a performance that makes you scratch your head. Regardless of what he does at a performance, whether somebody pays $75 to $100 a ticket or whatever......PLEASE DO NOT BOO OR HECKLE B.B. KING!!!! He's EIGHTY-EIGHT YEARS OLD, for goodness sake, and he's gone above and beyond in giving us over 65 YEARS of great music and performances.
King has provided entertainment for blues and music fans since the late 40's and is still showing up for work giving it his all when many people his age (those who are still with us) are either in assisted living or are basically housebound. People need to realize going in that an off night might be in the works, given what we know. It's more about the opportunity to see and interact with a living legend. That alone is worth $75 to $100 to most blues fans. If you're not among them, maybe you should stay at home and listen to a CD of King's instead of sitting in the audience and holding a man up to higher standards than most people hold themselves to.
At the same time, maybe the people who work with King, his management and his support, could take a few things into consideration......his age, his health, his stamina.....and maybe step back a little bit on, say, ticket prices or number of performances or size of venues or length of performances. Maybe the band, who reportedly seemed a bit bewildered by things that were happening last week, could step up and give him a little support, maybe bail him out when he gets bogged down. That would definitely help him a lot. He's earned the right to play as long as he wants and I'm not sure what he would do if he wasn't able to do that anymore. Hopefully, he won't ever get the chance to find out, and neither will we.


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This square lantern is made out of black metal and features a large handle. An LED or regular candle provides subtle light and pretty ambiance in this black votive lantern.
At 3 x 3 x 4.5 inches, this small votive lantern is easy to fit on a shelf or mantel, and can be customized to fit any decor. One square lantern per package.


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Turner Elementary School
Saturday, May 9, 2015
11:00am – 2:00pm
Barnes & Noble at the Waterfront
Join us in celebration of Mother's Day…
Storytimes with very special guest readers, crafts, facepainting fun, scavenger hunt and lots more!
Have lunch in B&N café and enjoy our very own "School Spirit" drink designed 'specially for us!
Best Class attendance wins a special prize.
Invite friends and family for a fun-filled day to celebrate and support our school! You won't want to miss the fun!
All purchases (including B&N Café) help to raise money for our school.


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Reliance securities is a full-service online trading and investment platform and is the broking arm of Reliance Capital. In this blog we look at Reliance Securities Features, reviews, trading account opening and Brokerage Charges.
Reliance Securities started its operation in 2005. It has a strong presence across the country with 1000+ offices serving 8 Lakh customers.
Reliance Securities offers services in:
- Equities
- NCDs/Bonds
- Sovereign Gold Bond
- IPOs
- Mutual Funds
- Derivatives
- Currency
- Corporate FDs
- Insurance Products
- Securities Lending & Borrowing
- Auction Market Participation
Features of Reliance Securities
- Competitive Brokerage: Provides different competitive brokerage plans suited for different categories of investors and traders
- Smart Trading Platforms: Offers a wide range of user-friendly trading platforms from browser-based platform to application based for both computer and mobile devices. It also offers call and trade facility to its customers
- Research: It offers it customers with specialized research report ranging from investment summaries, pre-market reports, futures and options (F&O) calls, options strategies, and its proprietary R-Model Portfolio service.
- Support Services: Get assisted through Relationship Managers in understanding the market dynamics and execution of buying and sell calls
- Reward Programme: It offers its customers with specially designed reward program those use the online platform in equity trading, Investing in Mutual Funds, and select NCDs.
Demat Fees
|Fee Head
|DP Charges
|AMC for Basic Services Demat Account(Only for individual having one Demat Account)
A. Holding Value not greater than Rs 50000
B. Holding Value between Rs 50001- Rs 200000
Rs. 100 P.A
|Regular Demat Account (Non BSDA)
A. For Individual /HUFs / Trust
B. For NRIs / Foreign Nationals
C. For Corporate / Other Non Individuals
Rs. 400 P.A
Rs. 1000 P.A
|Lifetime Free AMC( For Individual /HUFs / Trust)
|Rs. 2500
|Transaction charges:
SELL (Market & Off Market)
0.04 % of transaction value or Minimum of Rs.27/- per ISIN
A. For each request form
B. Extra for each certificate
Rs. 30
Rs. 5
Reliance Securities Brokerage Plan and Charges
Reliance Securities offer three types brokerage plans to its customer depending on the trading and investment pattern. Trading Account opening charges Rs.950 which is a one-time charge. All plans are discussed in details below.
R-Flexi Plan– It offers a sliding scale brokerage plan in which charges decreases with the increase in volume traded. The benefits of this Flexi scheme available in Delivery, Margin, and options. The scheme is best suited for high-value traders.
|Delivery ( NSE, BSE Cash Segment)
|Margin (NSE, BSE Cash Intraday & Futures)
|Monthly Turnover
|Monthly Turnover
|Monthly Turnover
|Brokerage Per Lot (in Rs.)
|More Than Rs. 2.5 crore
|50 Crore or more
|25 lakh or more
|More Than Rs. 2.5 crore
|Rs. 25 cr.- 50 cr.
|25 lakh or more
|Rs 50 lakh- 2.5 crore
|Rs 10 cr.-25 cr.
|Rs 10 lakh-25 Lakh
|Rs.25 lakh- 50 Lakh
|Rs 5 cr.-10 cr.
|Rs 5 lakh-10 Lakh
|Rs. 10 lakh- 25 lakh
|Rs 2 cr.- 5 cr.
|Rs. 2lakh-Rs 5 lakh
RFixed Online Plan: This plan is designed for online customers who themselves operates and executes a trade without any dealer support. The plan has fixed brokerage charges, irrespective of the size of the transaction with seamless trading experience on web platforms.
|Intraday Cash
|Rs. 100 per lot
R Fixed Plan: In this plan, the customer will have the option to pay a fixed cost for all your trades with the ease of having access to branch and a relationship manager who will help to take investment decisions based on in-house generated fundamental and technical research reports.
|Intraday Cash
|Brokerage Rate
|Rs. 100 per lot
* Service tax, Securities Transaction Tax, Stamp Duty, Turnover charges and other government / statutory levies will be charged as applicable.
Trading platforms
- TICK: It is the most advanced trading software offered by Reliance Securities with features like Big Data Analytics, Robo insights, News Analytics, Portfolio & Trade Highlights, Technical Alerts & Signals.
- TICK Pro: This is an App based trading platform with same features of TICK trading software.
- INSTA EXPRESS: It's a free, .exe based trading tool which provides super fast trading experience in equity and derivatives. It allows trading in real time environment with live market rates of scrips in NSE, BSE & NSE Currency.
- INSTA PLUS: It's a web-based trading platform which provides seamless trading experience to retail customers. Some features of the platform are:
Integrated market watch for Equities and Derivatives
Live Streaming Quotes
Know your margins before order entry
Multiple payment gateways
Integrated research with fundamental and technical analysis
Seamless investments in Mutual funds, IPOs, Bonds, etc.
Advanced charting tools.
- Branch Desk Dealing: This service is assisted by your Relationship Manager (RM). The RM guides the investor with all short of market information and executes the trade on the behalf of customer.
- Call and Trade: This facility is available to customer every trading day from 8:30 AM to 4:30 PM. Customers have to call 022-39886000,022-25816000 for all trade related queries. First, 20 calls in a month are free and from 21st call Rs. 20/ call is charged to the customer.
- Trade Express: It's a Java based trading tool to provide world class terminal-like trading experience to customers. Few features are:
Stream quotes from stock exchanges.
Single screen for cash and derivatives to make hassle free trading and investing in stocks
Multiple market watch profiles.
View holdings, check limits on one screen
Pros and Cons Of Reliance Securities
- Offers flexible brokerage plan and trading medium to customers.
- All investment product and financial service are available under one roof
- Spread across all major cities
- Plans with low brokerage charges are available to big traders
- No much difference in brokerage fees with other broking companies for small traders
How to invest in stocks through Insta Express Trading platform
- Login to NEST platform and enter security answers when prompted
- To add stocks to watch list, select NSE/BSE in dashboard
- To allocate funds, Click to secure link and then choose Add funds (it will redirect to your linked account)
- Enter the stock symbol or name and press enter to add
- To buy stock, press F1 on the stock name in market watch
- Enter quantity, price(if limit order) and submit your order
- Cross check your order, then press enter to place the order
- Check order book or Net positions to view or buy
- In order to sell, press F2 on the stock and place your order
|Reliance Securities
|Kotak Securities
|ICICI Direct
|Service Type
|Margins (Equity)
|Full Service
|Full Service
|Discount Broker
|0.05 – 0.55%
|Rs. 20
|Account Opening Cost
|Rs 950
|Rs 750
|Rs 975 for 3-in-1 account
|Rs 200 (couriered)
Rs 100 (form printed)
|Margins (Equity)
|Intraday- 10 times
Delivery- 4 times
|Intraday- 15 times
Delivery- 3 times
|Intraday- 25 times
Delivery- 5 times
|Intraday- 20 times
Delivery- Nil
|Exchange Enabled
|NSE, BSE, MCX, NCDEX, NMCE and MCX-SX
|NSE, BSE, MCX, NCDEX and MCX-SX
|NSE and BSE
|NSE, BSE, MCX, and MCX-SX
Process and Documentation Required
One can open an account in Reliance Securities in three ways
- Through their customer help desk no. : 022 39886000, 022 25816000
- Get yourself registered through online application form available in website(https://dbo.rsec.co.in/diy/home) or Whatsapp to executive at +91 7710078904
- By visiting nearest branch office
For online registration, users should upload scanned copy of PAN copy, Adress Proof, Personalised Cancelled Cheque.
Reliance Security is a reputed stock broking arm and provides a wide array of financial solutions to its customers. Its advanced analytics and Robo-trading software provide an edge over other broking companies to advanced free trading software. It's reasonably priced broking plans are designed keeping in mind of needs of the different types investors. It combines the value of research, big data analytics and long experience in capital markets to provide value to customers.
For more information about products and services of Reliance Securities click on the youtube link.
Frequently Asked questions (FAQs)
How do I check my current brokerage plan on the trading portal?
Please follow the below-mentioned steps after you login to your trading account on Insta Plus Platform:
Click on 'Quick Link' option displayed on the left-hand side of the trading page.
Click on My Profile option and scroll down and check 'Brokerage Details'.
How many bank accounts can be linked to my trading account?
You can link a maximum of three bank accounts with your trading account for making payment.
I want to change my e-mail address and mobile number through the online portal.
Please follow the below-mentioned steps after you login to your account:
Click on 'Quick Link' option displayed on the left-hand side of the trading page.
Click on 'My Profile' option and click on 'Update Primary Email and Phone Number'.
My account is dormant. How do I activate it?
Please follow below mentioned steps to activate account –
Open the website www.rsec.co.in
Click on Trade
Enter User IDand it will redirect to reactivation page;
Enter PAN Number and registered email address and click on submit
Your account will be reactivated immediately and you will be redirected to reset your login password.
How can I generate an online password?
Please follow the steps mentioned below to generate a new password
A. Open www.rsec.co.in
B. Click on, "Trade"
C. Enter User ID
D. Click on, "Forget password"
E. A new window will open. Please enter PAN number and registered email address
F. A new window will open. Please select the desired option and password will be sent to registered email address.
How to place Equity and Derivative orders?
Please follow below mentioned process to place order in Insta Plus
–Equity Order
A. Login to New Insta Plus
B. Click on "Equity"
C. Click on "Place Order"
D. Select "Exchange"
E. Select the scrip name you wish to Buy OR Sell
F. Select the Product Code
G. Enter the Quantity that you wish to Buy OR Sell
H. Select the Order Type Limit, Market or Stop Loss –L (DO not select Stop Loss-M)
I. Select retention type as DAY
J. Click on Submit to complete the order placing process.
Derivative Order
A. Login to New Insta Plus
B. Click on "Derivative"
C. Select the exchange as NFO
D. Select the Instrument Type – OPTSTK, OPTIDX, OPTIGX, FUTSTK, FUTIDX, and FUTIGX
E. Select the Symbol
F. Select the expiry month – 1st Month, 2nd Month
G. Select Option Type – CE, PE (Applicable for Options)
H. Select the Strike Price (Applicable for Options)I. Select the Action (Buy/Sell)
I. Select the Product Code as, "NRML/MIS/CO" (CO for Nifty /Bank Nifty)
J. Enter the Quantity that you wish to Buy OR Sell
K. Select the Order Type Limit, Market or Stop Loss –
L (DO not select Stop Loss-M)L. Select retention type as DAY
M. Click on Submit to complete the order placing process
How does one check trading ledger balance and Demat holdings?
Ledger balance
Please follow below mentioned steps to check Current Ledger in Insta Plus –
A. Login to New Insta Plus.
B. Click on, "Equity"
C. Click on, "Limits"
Demat Holding
Please follow below mentioned steps to check Demat Holding in Insta Plus –
1. Login to Insta Plus
2. Click on Equity
3. Click on DP Holdings
After placing fund withdrawal, when will I receive credit in my bank account?
A. If request is placed before 4.30 PM on working day and correct IFSC code is updated then the amount will credit in his bank account on T+1 day
B. If request is placed after 4.30 PM on working day and correct IFSC code is updated then amount will credit in his bank account on T+2 day
C. If request is placed before 4.30 PM on working day and IFSC code is not updated then cheque for withdrawn amount will be sent to the nearest branch in T+6 working days
*the information provided are sourced from the official website of Reliance Securities here.


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Microsoft held a stealth press event yesterday to unveil a new line of Windows 8-based tablets simply called, for now, Surface. It wasn't entirely unexpected, although some pundits wondered if the secret L.A. location and Microsoft's recent purchase of a large share of Barnes and Noble's e-reader Nook might not signal an Xbox-oriented or Kindle […]
Microsoft held a stealth press event yesterday to unveil a new line of Windows 8-based tablets simply called, for now, Surface. It wasn't entirely unexpected, although some pundits wondered if the secret L.A. location and Microsoft's recent purchase of a large share of Barnes and Noble's e-reader Nook might not signal an Xbox-oriented or Kindle Fire-like consumption tablet release with some Hollywood tie-in. The select group of media invitees continued to speculate on live blogs until seconds before CEO Steve Ballmer took the stage.
What Microsoft showcased was a pair of tablet-notebook hybrids, although presenters steered far from that consumer-repellent term. One version offers some serious enterprise solutions, and one is aimed squarely at Apple's iPad, which commands 62 percent of sales in the $75-billion American tablet market. Ballmer, of course, made no mention of Apple saying the tablet launch's primary purpose is to "prime the pump" for the upcoming release of Windows 8.
Cover Story
Both models have screen sizes almost a full inch larger than the iPad (10.6-inches to the iPad's 9.7-inches), are made of a magnesium "VaporMG" casing, with Gorilla Glass 2 screens, USB ports, and SD card slots. They run an interface called Metro, a user-controlled touch system.
The consumer model, Windows RT, will house an Nvidia Tegra processor and scaled-down versions of Windows 8 and Microsoft's Office productivity suite. It is .1 millimeters thinner than the iPad, a wee bit lighter, and will come with 32 or 64 GB of storage.
The emphasis at the presentation, and much of the media buzz, centered on the cover. It looks like a SmartCover for iPad complete with magnetic clips, but opening it reveals a flat-surfaced, super slim keyboard with touchpad. There is also a kickstand built in, which may seem gimmicky, but will make typing a lot easier. There's a more tactile keyboard option too, with actual buttons that adds a bit of girth.
The Windows 8 Pro model is bigger, has an Intel Core i5 "Ivy Bridge" processor, microSDXC slot, USB 3.0, Mini DisplayPort Video and up to 128GB of storage. It comes with a digital ink stylus, along with a better keyboard and touchpad and will be able to run Windows 8 proper.
Stiff competition
OK, so the hardware looks great. The design is kind of love it or hate it, but it's clearly well thought out. But, can it compete with the iPad? It's a tough question to answer because Microsoft left so much unsaid. There is no word yet on battery life, nor on pricing, although Ballmer insists it will be "competitive" suggesting the RT will start at around $500-$600 and the Pro near UltraBook prices in the $900-$1000 range. No mention was made as to whether Surface is Wi-Fi only or will have cellular connectivity. Even the release date is unspecified, some sources suggesting it will be timed to coincide with the launch of Windows 8, while other predicting a later pre-Christmas roll-out.
The biggest issue though is content, more specifically apps. Tablet owners want Office, and that includes iPad devotees as evidenced by the never-ending rumors of an MS-developed iOS version, and dozens of apps offering some form of Office compatibility (think QuickOffice Pro HD or Office2 HD). Having the real thing, even in a lite version, is going to be a major selling point.
There will be no backwards compatibility with existing Windows apps, however, leaving the Surface facing the same software dearth that put RIM's Blackberry Playbook on its ill-fated course. Developers are already competing on two different platforms (iOS and Android), both with proven track records in sales. It will take some serious incentive to get enough third-party apps ready on an all-new, unproven operating system in time for a 2012 release.
If the question is did Microsoft just preview an iPad killer, or even an iPad contender, the short answer is yes. And no. For enterprise the Windows 8 Pro holds promise. In fact I'm hardly an enterprise user, and I think I want one. The anticipated price point and similarity to UltraBooks and MacBooks begs the question of where it will fall in a Windows ecosystem, but it's pretty sexy even without a fruit logo.
Close, but no cigar?
The RT's future seems lees certain. To beat the iPad and the various Android tablets the Surface with Windows on ARM needs to be better, not merely as good. I don't see it. The keyboard may seem ingenious, but as someone who has tried no less than 10 variations of for-iPad keyboards, I can assure you the typing experience, for the touch typist especially, won't be much better than a touch screen. It's the lack of tactile feedback that plagues both.
It's safe to assume that for digital publications and e-books Microsoft will be well stocked with Barnes and Noble's catalog, but apps make up the biggest and most interesting part of the iPad experience. Without a bevy of them, I don't see this new contender grabbing the youth market or anyone's imagination.
If Microsoft wants a hit, they need to integrate the Surface with their only other successful hardware venture, the Xbox. Microsoft needs to integrate the whole Xbox LIVE experience and use the tablet as some sort of uber-controller.
All in, I think Microsoft is on the right track. While the first iteration of the tablet will be just that, a version one, the company has a history of patience. If they woo developers and produce a second generation of consumer tablets with a full Windows OS, they may fulfil Ballmer's promise of an "intersection between human and machine" that really can improve "all aspects of the experience, hardware and software … working together."
Find great iOS games here