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Over the last years, the critical role of cytoskeletal proteins in cortical development including neuronal migration as well as in neuronal morphology has been well established. Inputs from genetic studies were provided through the identification of several mutated genes encoding either proteins associated with microtubules (DCX, LIS1, KIF2A, KIF5C, DYNC1H1) or tubulin subunits (TUBA1A, TUBB2B, TUBB5 and TUBG1), in malformations of cortical development (MCD). We also reported the identification of missense mutations in TUBB3, the postmitotic neuronal specific tubulin, in six different families presenting either polymicrogyria or gyral disorganization in combination with cerebellar and basal ganglial abnormalities. Here, we investigate further the association between TUBB3 mutations and MCDs by analyzing the consequences of Tubb3 knockdown on cortical development in mice. Using the in utero-electroporation approach, we demonstrate that Tubb3 knockdown leads to delayed bipolar morphology and radial migration with evidence, suggesting that the neuronal arrest is a transient phenomenon overcome after birth. Silenced blocked cells display a round-shape and decreased number of processes and a delay in the acquisition of the bipolar morphology. Also, more Tbr2 positive cells are observed, although less cells express the proliferation marker Ki67, suggesting that Tubb3 inactivation might have an indirect effect on intermediate progenitor proliferation. Furthermore, we show by rescue experiments the non-interchangeability of other beta-tubulins which are unable to rescue the phenotype. Our study highlights the critical and specific role of Tubb3 on the stereotyped morphological changes and polarization processes that are required for initiating radial migration to the cortical plate.
During brain development, the radial migration of newborn pyramidal neurons is critical for the formation of a laminar cortex in mammals. Post-mitotic neurons exiting from the proliferative ventricular zone (VZ) start their journey by acquiring a succession of bipolar, multipolar and then again bipolar morphologies in the subventricular (SVZ) and intermediate zones (IZ). They migrate long distances to reach their final destination in the cortical plate (CP) forming an 'inside-out' layered cortex (1–3). These steps are followed by dendritic and axonal extensions and the implementation of neuronal circuitry.
Over the last years, the critical role of cytoskeletal proteins in cortical development including steps of neuronal migration and neuronal morphology has been well established. Genetic studies demonstrated the implication of several genes encoding either microtubule-associated proteins (MAPs), other microtubule-binding proteins or motors, or tubulin subunits in malformations of cortical development (MCD). Mutations in the DCX and LIS1 genes, encoding proteins that modulate microtubule (MT) homeostasis, were shown to be associated with a spectrum of lissencephaly neuronal migration disorders encompassing agyria, pachygyria and laminar heterotopias (4–8). More recently, mutations in genes encoding MT-related motor proteins, including kinesins (KIF2A and KIF5C) and dynein (DYNC1H1) were also found in MCD patients (9). Moreover, the importance of MT related functions were further demonstrated by the direct association of mutations in tubulin genes and these cortical developmental disorders (10). Indeed, alpha-tubulin (TUBA1A and TUBA8), beta-tubulin (TUBB2B) and gamma-tubulin (TUBG1) genes were shown to be involved in lissencephalies and polymicrogyria (11–14), while mutations in TUBB5 were identified in cases presenting microcephaly associated with cortical gyration abnormalities (15). Furthermore, we identified missense mutations in TUBB3, a postmitotic neuronal tubulin, in six different families presenting either polymicrogyria or gyral disorganization in combination with cerebellar and basal ganglial abnormalities (16) (OMIM#614039). In this study, we also reported a de novo heterozygous p.Met388Val mutation in a fetal case with microlissencephaly. TUBB2B and TUBB3 were also found mutated in congenital fibrosis of the extraocular muscle disorders (17,18). Finally, mutations in the TUBB4A gene were recently reported in families presenting whispering dysphonia associated with dystonia (19,20) and in a leukoencephalopathy hypomyelination syndrome (21).
Tubulin subunits are highly conserved during evolution and alpha–beta tubulin heterodimers are the structural components of MTs. There are multiple copies of the tubulin genes in human and rodent genomes. Alpha- and beta-tubulin subunits are thus encoded by 19 genes (10 alpha and 9 beta) in human and eight alpha- and eight beta-tubulin genes have been isolated in mice (http://genome.ucsc.edu/). Each alpha/beta-tubulin shows >90% amino acid identity with other alpha/beta-tubulins (22). Tubulin isotypes expression and distribution in different cells or tissues is highly regulated, and their apparent redundancy more probably underlies cellular and functional diversity requirements (23). Whereas any of the isotypes could ensure basic structural functions, such as forming an interphase cytoskeleton network (24), convergent studies tend to confirm the functional specificity of the different tubulin isoforms. For instance, specific knockdowns of a set of beta-tubulins in cell cultures showed, for each of the targeted subunits, different cellular phenotypes in differentiating neuronal cells (25). Though further in vivo studies are required, these results highlighting the importance of specific combinatorial expression of different tubulin isoforms, are in favor of the much debated multi-tubulin hypothesis (26).
Here, we investigated the association between TUBB3 mutations and MCDs by analyzing the consequences of either Tubb3 knockdown or overexpression of TUBB3 mutations on radial neuronal migration in the mouse cortex, using an in utero-electroporation approach. We also demonstrate, by rescue experiments, the non-interchangeability of certain beta-tubulins during brain development, confirming their functional specificity.
Tubb3 inactivation perturbs radial migration
In order to assess the consequences of Tubb3 loss of function on the cortical development, we used an in utero-electroporation approach to deliver specific short hairpin RNAs (shRNAs) into cortical progenitor cells of the mouse brain VZ. Two specific anti-Tubb3 shRNAs targeting the 3′UTR sequence were cloned in a psiStrike vector under the control of the U6 promoter. Cellular experiments in P19 neuroblastoma cells showed an extinction of Tubb3 at the transcript (sh1:−44%; sh2: −67%) and protein (sh1:−34%; sh2: −67%) levels and only a minor effect of a scrambled sequence compared with the untransfected control (Fig. 1A and B). The validated shRNAs and scramble constructs were then individually electroporated in utero, together with an RFP reporter construct into the mouse cortical VZ at embryonic day E14.5, a time coincident with extensive neuronal production and migration. Control electroporated cells gave rise to red fluorescent young neurons migrating toward the CP. Embryos were harvested 4 days later (E18.5) and the migration pattern of the electroporated cells was determined by the quantification of RFP+ cells in the different cortical regions: VZ/SVZ, IZ and CP. We checked the efficiency of the shRNAs in vivo by immunolabeling the Tubb3 protein in brain sections and confirmed the large reduction of Tubb3 expression in electroporated cells (Fig. 1C). In cortices transfected only with the RFP plasmid, nearly all the neurons were found as expected in the CP (VZ: 5.6 ± 3.4%, IZ: 6 ± 3.3%, CP: 88.4 ± 6.7%), while the scrambled construct showed slightly lower number of cells in the CP with a proportion still remaining in the IZ, although this did not reach significance (VZ: 8.86 ± 3.6%, IZ: 18.8 ± 5.1%, CP: 74.1 ± 8.3%) (RFP versus scramble Mann–Whitney test; P-value: VZ 0.59; IZ 0.11; CP 0.26). In contrast, neurons transfected with the shRNAs against Tubb3 displayed a severe and significant arrest in radial migration. A majority of them were blocked in the IZ with only a few neurons found in the CP (Sh1; VZ: 11.5 ± 5.2%, IZ: 78.2 ± 7.8%, CP: 10.5 ± 4.5%; Sh2; VZ: 23.3 ± 3.5%, IZ: 53.7 ± 3.5%, CP: 22.3 ± 0.2%) (sh1 versus scramble P-value: VZ 0.35; IZ**; CP**; sh2 versus scramble P-value: VZ*; IZ**; CP**). To validate the specificity of this result, we carried out a rescue experiment by co-electroporation of the anti-Tubb3 sh1 construct and the sh-resistant human TUBB3-coding sequence (NM_006086.3) and revealed a significant derepression of the shRNA blocking effect (VZ: 1.4 ± 0.7%, IZ: 16.3 ± 6.3%, CP: 82.3 ± 6.8%) (sh1 versus sh1+cTUBB3 P-value: VZ 0.06; IZ*; CP*) confirming the specificity of the shRNAs and the importance of Tubb3 for radial migration to the cortex (Fig. 1D–F).
TUBB1, TUBB2B and TUBB4A do not fully rescue the Tubb3 knockdown migration defect
To assess the possible functional redundancy of tubulin subunits and the capacity of other beta-tubulins to substitute for the Tubb3 loss of function, we co-electroporated the Tubb3 shRNA with the human-coding sequence of three other beta-tubulins. We selected one that is weakly expressed in human fetal brain (TUBB1, NM_030773) and two which are highly represented in this tissue (TUBB2B, NM_178012 and TUBB4A, NM_006088) (data not shown). TUBB1 cDNA was not able to rescue the Tubb3 knockdown phenotype as a large majority of electroporated cells remained blocked in the VZ and IZ (VZ: 21.5 ± 6%, IZ: 68.2 ± 5.2%, CP: 10.3 ± 4.1%) (sh1 versus sh1+cTUBB1 P-value: VZ: 0.29; IZ: 0.41; CP: 0.73). On the other hand, TUBB2B and TUBB4A expression led to a partial rescue as two equal populations of neurons were observed in the IZ and CP at day E18.5 (sh1+cTUBB2B VZ: 18 ± 3.1%, IZ: 47.1 ± 8.4%, CP: 34.9 ± 9.6%; sh1+TUBB4A VZ: 13.8 ± 3.7%, IZ: 44.5 ± 9.7%, CP: 41.7 ± 12.5%) (sh1 versus sh1+cTUBB2B P-value: VZ 0.24; IZ*; CP 0.07; sh1 versus sh+cTUBB4A P-value: VZ: 0.60; IZ: 0.09; CP: 0.26). In view of this result, we checked the expression efficiency of these beta-tubulins after electroporation. Indeed, expression analysis of the transgenes by real-time RT–PCR experiments after in utero electroporation and dissection of the electroporated cortical region, showed an efficient and significant expression of these beta-tubulin transgenes (i.e., 177–564 times higher in electroporated regions compared with non-electroporated brains, data not shown).
Although a partial rescue of the migration phenotype was obtained by these two beta-tubulin subunits, the diffused distribution of electroporated cells still remained substantially different from that of the organized cells expressing scramble RNA and RFP alone (Fig. 2A and B), supporting the non-interchangeability of these beta tubulins during cortical development and the functional specificity of Tubb3.
TUBB3 mutants do not rescue the migration impairment
We sought to test whether the expression of MCD-related TUBB3 mutants could complement the phenotype caused by knockdown of Tubb3. We selected therefore TUBB3 variants associated with variable phenotypes in patients and tested p.Gly82Arg and p.Met388Val TUBB3 mutations, responsible for multifocal polymicrogyria in one patient and fetal microlissencephaly, respectively (16). We electroporated each of these two constructs, in combination with the Tubb3-shRNA-1 and analyzed the position of electroporated cells within the cortex. In both experiments, the mutants did not suppress the shRNA effect: while some of the cells reached the CP (sh1+p.Gly82Arg: 26.5 ± 11.4%; p.Met388Val: 23.6 ± 4.5%), a majority of them were blocked in the VZ and IZ (p.Gly82Arg VZ: 20.3 ± 6.6%, IZ: 53.2 ± 7.5%; p.Met388Val VZ: 21.5 ± 4.8%, IZ: 55 ± 5.1%), quite similar to the shRNA-1 alone and significantly different from the scramble shRNA (Fig. 2C and D) (sh1 versus sh1+ p.Gly82Arg P-value: VZ: 0.35; IZ*; CP: 0.48; sh1 versus sh1+ p.Met388Val P-value: VZ: 0.3; IZ*; CP: 0.16) (scramble versus sh1+ p.Gly82Arg P-value: VZ: 0.09; IZ*; CP*; sh1 versus sh1+ p.Met388Val P-value: VZ:*; IZ**; CP**).
We also overexpressed the wild type and the two TUBB3 mutants alone and observed that the electroporated neurons reached the CP normally. However, when examining more precisely neuronal position it seemed that while control and p.Gly82Arg electroporated neurons reached the upper layers of the CP in a comparable way, the p.Met388Val transfected neurons were arranged in a more diffuse manner in the CP compared with the wild type which may suggest a minor dominant effect on migration (Fig. 2E and F).
The migrating defect related to Tubb3 knockdown is a transient phenomenon
We looked at the effect of the Tubb3 shRNA electroporated at E14.5, on neuron positioning at different embryonic and postnatal stages (E16.5, E17.5, E18.5, P0, P2 and P7). At E16.5 the distribution of RFP+ neurons was quite similar comparing sh and scramble conditions, most of them were found in the VZ and the lower IZ, except for a small proportion of neurons that had already reached the upper IZ. The migration delay of the sh electroporated neurons appeared more evident at E17.5, the effect culminating at E18.5. At the neonatal stage P0 some of the sh electroporated neurons reached the CP and at postnatal stages P2 and P7, no particular differences between the scramble and sh conditions were seen and fluorescent neurons formed a thin layer in the upper part of the cortex (Fig. 3A). In order to verify this result, we checked by immunolabeling of postnatal (P2) brain sections that silencing by shRNA is not transient in neuronal cells that reach the CP. Anti-Tubb3 antibodies showed the presence in the CP of neuronal cells positive for the RFP reporter marker showing a downregulation of Tubb3 (data not shown). Furthermore, brain slices were also stained with an antibody against caspase 3 to assess for an apoptosis of cells arrested in the IZ by immunolabeling of brain sections at E16.5, E18.5 and P2 stages (2, 4 and 6 days after electroporation). No differences were observed between scramble and shRNA conditions (data not shown). These data suggest that the migration phenotype of the neuronal population deficient for Tubb3 is a transient phenomenon and arrested cells maintained their ability to reach the CP 6 days after electroporation.
Tubb3 inactivated cells displayed an abnormal morphology
To better define the defect observed during radial migration, we examined the morphology of cells accumulated in the SVZ/lower IZ in control and sh1 conditions at E16.5. While the migration pattern did not seem to be widely altered, the shape of Tubb3 deficient cells appeared to be different when compared with control. Indeed, a great majority of control cells showed a multipolar morphology with visible processes (scramble: 60 ± 5.5%, sh: 42.4 ± 2.5%; t-test, P* < 0.05), compared with the inactivated cells which showed more round shape with no or very short processes (scramble: 8.1 ± 2.6%, sh: 26 ± 3.8%; t-test, P* < 0.01) (Fig. 4A and B). Around 30% (scramble: 31.9 ± 3.6%, sh: 31.6 ± 3.1%) of cells in both control and knockdown conditions displayed a typical migrating uni- or bipolar morphology. Similar morphological states were still visible at E18.5 in shRNA electroporated cells blocked in the lower IZ, whereas the control cells had already reached the CP. Approximately 40% of shRNA cells remained multipolar (20.4 ± 8.6%) or displayed round shapes with no or short processes (20.3 ± 9%) (Fig. 4B). Thus, Tubb3 shRNA cells may be retarded in their morphological development.
In addition, we examined the orientation of the main process of cells harboring a bipolar shape at E16.5 and E18.5 days. We divided them into three main categories according to the orientation of the main process with respect to the surface of the ventricle (radially, tangentially or inversely oriented). shRNA and scramble conditions showed similar distributions at E16.5 with a majority of cells oriented radially with an angle of migration between 60° and 90° (scr: 65.1 ± 5%, sh: 51.4 ± 8.4%), a small proportion of cells moved tangentially with an angle between 0° and 30° (scr: 10.1 ± 2.4%, sh: 14.1 ± 4%) and ∼20% of them migrated towards the ventricle (scr: 15.2 ± 4.9%, sh: 22.9 ± 3.6%) (n: scr = 262, sh v = 206). At E18.5, whereas control cells had already reached the CP, knockdown cells displayed similar orientations as at E16.5. Half of them (48.3 ± 4.8%) were radially oriented, 5.8 ± 4.2% were tangentially oriented and 35 ± 4.5% moved toward the ventricle (Fig. 4C). Altogether, these results showed that Tubb3 inactivation has an impact on neurons morphological changes at E16.5 but not on the orientation of their main process.
Effect of Tubb3 inactivation on intermediate progenitors
We then sought to determine whether Tubb3 inactivation has an impact on the intermediate progenitor (IP) population in the SVZ. We therefore examined the number of these progenitors by labeling with the IP specific marker, Tbr2, at the E16.5 stage in brains electroporated 2 days earlier with shRNA and scramble constructs. We found a significant increase of Tbr2+ electroporated cells in the shRNA condition compared with the control (scramble: 27.8 ± 1.7%; sh1: 36.5 ± 2%; t-test, P* < 0.05) (Fig. 5A). This result suggesting a transition defect and/or a failure of these cells to differentiate into postmitotic neurons prompted us to further investigate consequences of Tubb3 downregulation on IP proliferation.
We checked the expression of Tubb3 in IPs by immunolabeling embryonic brain sections and primary cultures of dissociated progenitors. Double labeling of brain sections suggests that Tubb3 is expressed in a subset of Tbr2 positive cells which are enriched in the basal layer of the SVZ. Because Tbr2 and Tubb3 are localized in different cell compartments, it remained difficult to unambiguously ascertain that these cells express both markers. To further confirm this result, we generated cultures of neuronal progenitor cells and assessed the expression of the two markers. Analysis of the results showed a minor population co-expressing Tubb3 and Tbr2, corresponding to ∼10% of the total population of IPs (data not shown).
We also performed quantitative comparative analysis of the proliferation of these cell populations following in utero electroporation of scramble and Tubb3-targeting shRNAs. In the VZ no quantitative difference for PH3+, nor for KI67+ cells (RFP electroporated cells) was observed. However, in the SVZ, we found a reproducible significantly reduced number of Ki67+ cells for the Tubb3 shRNA in comparison with the scramble control. For PH3+ cells in the SVZ, no difference between the scramble and shRNA was observed, the scarcity of PH3-positive cells did not allowed reliable statistical comparison (Fig. 5B and C). In independent subsequent in utero-electroporation experiments and quantitative comparative analysis using double immunolabeling of brain sections with anti-Tbr2 and anti-Ki67 antibodies, we confirmed the observed decrease of cells in the SVZ co-expressing Tbr2 and Ki67 in the shRNA condition compared with the control (scr: 4.9 ± 1%, sh: 2.4 ± 0.9%; n: scr = 453, sh = 372, χ2-test, *P < 0.05; data not shown).
Altogether these data show the critical role of Tubb3 in cortical development, especially in the morphological changes undertaken by cells at the beginning of migration and in the regulation of the IP transition towards their differentiation into neuronal cells.
The subunit tubulin beta 3 has been considered as a specific marker of neurons for decades and its implication in brain development was more recently emphasized by its involvement in MCDs (16). Here, we used an in utero-electroporation approach to explore more precisely the role of Tubb3 in mouse brain during cortical development. We describe a delay of migration that culminates at E18.5 in neurons electroporated at E14.5 with shRNA constructs suppressing Tubb3 expression. These results show that Tubb3 plays an important role in the correct migration of pyramidal neurons in fitting with the MCD phenotypes. Similar studies previously demonstrated a deleterious effect of shRNAs against Tubb2b and Tubb5 during cortical radial migration in the rodent. While the knockdown of Tubb2b disrupt the radial migration in rat (13), anti-Tubb5 shRNA led to a blocking of the migration in mouse cortices with a specific impact on the length of the neurogenic cell cycling (15). We show here delayed initiation of migration in Tubb3 mutant cells which take a longer time to develop a bipolar morphology. These combined results confirm the importance of the MT during various steps of cortical development.
Interestingly, in this experimental design the Tubb3-related migration defect seems to be a phenomenon that disappears 4–5 days later in postnatal stages. The result showing a continued downregulation of Tubb3 in neuronal cells that had reached the CP suggest that Tubb3-related migration defect is transient. Though additional investigations are required to address this observation, these data suggest that neurons that do not express Tubb3 maintain migratory potential in early stages after birth. This has been also shown in Dcx knockdown condition, in which blocked neurons in the VZ/ IZ resumed their migration after overexpression of Doublecortine transcript at P0 (27). Also, as expression of TUBB2B, TUBB1 and TUBB4A cDNAs did not rescue the migration phenotype, it suggests that other tubulins could be involved in the adaptive phenomenon and compensate the lack of Tubb3 allowing neuronal cells to recover their migration ability. Similarly, expression profiles during neuronal development of MT-interacting proteins could also contribute to the observed recuperation of migration. Tubb3 downregulation leads to a significant reduction of multipolar processes in E16.5 SVZ. Moreover, silenced cells still display multipolar or round shapes at E18.5 (morphologies which are not observed at this stage in control cells). These results are reminiscent of previous studies showing that RNAi in utero-electroporations-targeting Filamin A, Lis1 and Dcx, three other genes responsible for MCDs and interacting directly or indirectly with the cytoskeleton, lead to an accumulation of multipolar cells in the SVZ or the IZ (28). They also suggest that Tubb3 appears necessary, through its structural role, for proper MT dynamics and therefore neuronal morphology organization and process elongation.
Concerning the other beta-tubulins tested here (TUBB1, TUBB2B and TUBB4A), TUBB1 that is weakly expressed in human fetal brain is not able to replace Tubb3 function, leading to the hypothesis that these two beta subunits ensure completely different functions, perhaps involving different protein partners. Beta-tubulin-2B and -4A, two subunits expressed in human fetal brain, overcome partially the Tubb3 related migration defect showing that they share some, but not all brain specific functions. This result tends to support the idea of the non-interchangeability of tubulin subunits. Our data suggest that even if they share significant homology and common functions, each tubulin subunit may have a specific role that cannot be replaced by other subunits during cortical development. These findings are also consistent with the existence of specific syndromes in human caused by mutations in different tubulin subunits. The complexity is also such that different mutations in one tubulin gene can lead also to several different phenotypes (13,16–18).
In this in utero-electroporation-based study, we tested two TUBB3 MCD-related mutations: (i) p.Gly82Arg, responsible for a moderate form of MDC within the TUBB3-related spectrum, and predicted to be adjacent to the lateral beta–beta interaction domain of tubulins and (ii) p.Met388Val, which leads to a dramatic fetal form of microlissencephaly. This latter mutation resides on the outer surface of the MT and could interact with MAPs and motor proteins or be involved in protein folding (29). We established that the two mutations fail to complement the Tubb3 shRNA phenotype, although the two mutations could act in slightly different ways in an overexpression context. While the p.Gly82Arg mutant had no major overexpression effect, p.Met388Val led to a mild dispersion of neurons in the cortex that could have repercussions in the establishment of a proper neuronal connectivity. These combined observations may suggest that the first mutation acts as a loss of function and the second could have potentially a dominant-negative effect during mouse radial migration, consistent with the different phenotype observed in patients. Given that all mutations identified in the tubulin isotypes (TUBA1A, TUBB2B, TUBB3 and TUBB5) are missense mutations, it is likely that some may act in a dominant-negative fashion via an altered protein function, thus in addition to haploinsufficiency, contributing to the mechanisms underlying MCDs. Though recurrent mutations are consistently associated with almost identical MCDs, at the current level of our understanding of tubulin functions and their cellular roles, and the potential multi-level effects of tubulin mutations assessed by different assays, it is difficult to predict molecular mechanisms (haploinsufficiency or dominant-negative effect) and/or disease phenotype resulting from a given mutated residue or isoform.
Finally, we showed that Tubb3 downregulation is associated with a significant reduction of Ki67+ cells in the SVZ. This finding that is apparently in contradiction with the increased number of Tbr2 cells is also difficult to reconcile with the assumption that Tubb3 is mainly expressed in postmitotic neuronal cells. However, we can hypothesize that Tubb3 inactivation might have an indirect effect on Tbr2 progenitor proliferation. Indeed, Tubb3 inactivation and the subsequent disruption of early differentiation and migration steps of cells that are still Tbr2+ and their accumulation in the SVZ might indirectly trigger through cell–cell contacts a signal that leads to a reduction of proliferation of IPs (Fig. 6). In view of these findings, we are also attempted to hypothesize that normal cortical development may also involve a cross talk between CP and VZ/SVZ. It is well established that position of pyramidal neurons in the CP is dependent of their date of birth, and here we propose that cells that reach their final position in the CP 'talk' to the VZ, via radial glia fibers, to allow a fine tuning of progenitor proliferation.
Although many questions remain to be addressed, our study emphasizes the crucial role of the MT network in different steps of neuronal migration and especially in the critical stereotyped morphological changes and polarization processes that are required for triggering and regulating radial migration towards the CP. From our study, it appears clear that beta-tubulins are non-interchangeable in the context of this neuronal radial migration assay, providing therefore important information concerning the debate of their potential redundancy.
We designed two different shRNA oligonucleotides targeting the 3′UTR of the mouse Tubb3 transcript (Sh1 specifically recognizes nucleotides 1427–1451, Sh2, nucleotides 1435–1459, NM_023279). Annealed oligonucleotides were cloned in the psiSTRIKE U6 promoter-driven vector. As control, we used a scrambled oligonucleotide corresponding to Sh2. Human beta-tubulin cDNAs were inserted in the pcDNA vector (Life Technologies, Carlsbad, CA, USA) under the CAG (chicken beta-actin promoter with CMV enhancer) promoter for the rescue experiments.
Quantitative PCR and western blot
Plasmids used for in utero-electroporation were prepared using the Endotoxin-Free Plasmid kit (Macherey Nagel, Duren, Germany). Efficiency of the shRNAs against Tubb3 was assessed at the transcript and protein levels by transfection in murine P19 cells with the Neon System (Life Technologies). RNAs were purified by the RNAeasy Kit (Qiagen, Venlo, Netherlands), cDNA were prepared with Maxima First Strand cDNA synthesis Kit (Thermo Fisher Scientific, Watham, MA, USA), and then RT–PCR products amplified from cDNA were quantified with the LightCycler 480 FastStart DNA SYBR Green I Master kit in a LightCycler 480 System (Roche, Manheim, Germany). Protein explorations were performed by loading equal quantities of lysates on polyacrylamide gels, transferred to nitrocellulose membrane. After blocking with a 5% milk solution (w/v), blots were incubated with the primary antibody overnight at 4°C. Mouse Tubb3 antibody from Covance (Princeton, NJ) was used at 1:1000 and goat Actin antibody (SC-1616, Santa Cruz Biotechnologies, Dallas, TX, USA) was diluted at 1:1000. HRP (horseradish peroxidase) secondary antibodies from Dako-Agilent Technologies (Santa Clata, CA, USA) were used diluted at 1:5000.
In utero electroporation
In utero electroporations were performed as described previously (30) using Swiss timed pregnant mice. Briefly, 1–2 µg of plasmid DNA combined with fast green (2 mg/ml; Sigma) were injected into the lateral ventricles of E14.5 brains and then electroporated into VZ progenitor cells by delivering five electric pulses at 45 V for 50 ms at 950 ms intervals through the uterine wall using a CUY21EDIT System (Nepagene, Chiba, Japan). Embryos or pups were allowed to develop for 2–13 days, brains were harvested, and brain sections of 80 µm were prepared using a vibratome (VT1000S, Leica, Solms, Germany). The positions and morphology of transfected cells were analyzed by fluorescence microscopy after fixation of the sections with 4% paraformaldehyde using a DMRA2 microscope (Leica Microsystems, Solms, Germany).
Free-floating sections were blocked with 2% goat serum in phosphate buffered saline (PBS) and 0.2% Triton-X 100. The primary antibodies used were anti-TBR2 (rabbit polyclonal, 1:200, Abcam, Cambridge, UK), anti-beta 3 tubulin Tuj1 (mouse, 1:500, Covance, Princeton, NJ, USA), anti-phospho Histone H3 (rabbit, 1:500, Millipore, Billerica, MA, USA), anti-Ki67 (mouse, 1:200, Bethyl Laboratories, Montgomery, TX, USA) overnight at 4°C. After three washes in PBS, sections were incubated with either FITC- or Cy5-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) in a 1:300 dilution at room temperature, washed and mounted with Fluoromount (Sigma-Aldrich, St. Louis, MO, USA). Images were acquired using a spinning disk system (Leica Microsystems, Solms, Germany) for morphological analyses or a TCS SP5 II (Leica Microsystems, Solms, Germany) for immunolabeling analysis. All cells were counted for statistical analyses using ImageJ software (NIH).
This work was supported by fundings from Institut National de la Santé Et de la Recherche Médicale, the Fondation pour la Recherche Medical (J Chelly—Equipe FRM 2013: DEQ2000326477), the Fondation JED, the Agence Nationale pour la Recherche (ANR Blanc1103-01 projet R11039KK, ANR E-RARE-012-01 projet E10107KP, ANR 08-MNP-013) and the EU-FP7 project GENECODYS (grant number 241995). We thank the INSERM Avenir program and the Fondation Bettencourt Schueller for grants awarded to F.F., the Région Ile-de-France for the NERF postdoctoral grant awarded to EBJ/FF and for their support of animal house and imaging facilities. The team of F.F. is part of the Ecole des Neurosciences de Paris Ile-de-France network.
We thank Katia Boutourlinsky, Reham Khalaf and Audrey Roumegous for their technical support and Pierre Billuart for his helpful comments and discussions.
Conflict of Interest statement. None declared.


text:
Water Resources Management
An International Journal - Published for the European Water Resources Association (EWRA)
Water Resources Management is an international, multidisciplinary forum for the publication of original contributions and the exchange of knowledge and experience on the management of water resources. In particular, the journal publishes contributions on water resources assessment, development, conservation and control, emphasizing policies and strategies. Contributions examine planning and design of water resource systems, and
operation, maintenance and administration of water resource systems.
Coverage extends to these closely related topics: water demand and consumption; applied surface and groundwater hydrology; water management techniques; simulation and modelling of water resource systems; forecasting and control of quantity and quality of water; economic and social aspects of water use; legislation and water resources protection.
Water Resources Management is supported scientifically by the European Water Resources Association, a scientific and technical nonprofit-making European association.
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There has traditionally been significant engineering overhead required for the integration of multi-vendor tool and IP design methodologies. Making design-chain integration efficient is the key objective of the SPIRIT Consortium. This Special Session paper provides an insight into how the specifications of the SPIRIT Consortium are being adopted in the… (More)
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Adult Day Care Activity Volunteers Needed
36.181007, -115.2242)
Cause Areas
WhenWed Aug 31, 2016 07:00 AM - Tue Feb 28, 2017 05:00 AM
Where901 N. Jones Blvd.Las Vegas, NV 89108
The Adult Day Care Centers of Las Vegas and Henderson are in need of activity volunteers to work with our senior population. Opportunities include but are not limited to Creative Dance, Tai Chi and Crafts. Most activities are on Tuesday, Thursday, Friday and Saturday mornings at 10 am but other times may be available as well.
- Verbal / Written Communication
Good Match For
People 55+ Group
Requirements & Commitment
- Must be at least 16
- Orientation or Training
- 3 hours


text:
- Solutions
- Library
- About Us
- Security Center
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The Cyber Security Start Up's Answer to Roaming Security
CARLSBAD, CA: Feb 8, 2017: Cyber security company ThreatSTOP announced today a Cloud-based offering that quickly detects and automatically blocks DNS attacks on laptops outside a secured company network, without using external 3rd party DNS servers or requiring a VPN connection. This new SaaS offering, Roaming Endpoint, is ThreatSTOP's answer to a growing mobile workforce, protecting devices when they leave the corporate network, anywhere and anytime.
Locky, the infamous ransomware plaguing computers worldwide since it was first seen early last year, has recently made a comeback after a severe drop in activity over the holiday season. The Necurs botnet, which is Locky's primary distributor, was offline for the final weeks of 2016, equating to an 81% decrease in the number of Locky attacks.
We often analyze indictors of phishing-related compromise from techhelplist.com. These lists contain a large number of indicators, usually not all related to one campaign, but to countless ones that have already spread before the lists were updated.
In December, we introduced a target list of more than 20 malware family DGAs provided by our friends over at 360 Research Team. Continuing their great work, we are happy to integrate 7 new malware DGAs:
The "Digital Plagiarist" campaign, dubbed by researchers at the tr1adx team, was run by the "TelePort Crew" and appears to be an evolution of the Carbanak cybercrime group. This group is infamous for a large-scale campaign against banks, leading to the 2015 theft of hundreds of millions of dollars and the Carbanak/Anunak malware that targets point of sale machines.
Sure, just any old threat feed will do. Like those one-size-fits-all "I Heart NY" shirts in Times Square. Just like Chipotle without guac (if you're obsessed with both Chipotle and guac, like me) or Caesar salad with no… dressing. Laverne without Shirley, Biden without Ray-Bans, or maybe the internet without a politically topical meme. I'm going somewhere with this…. I promise.
Bi-weekly Security Update
Malicious content identified and inserted:
Where do security professionals draw the line between protecting their company's network, and delivering a free-range internet experience for their fellow employees? This quandary came up at ThreatSTOP recently, spurred by a support request we received from a customer who posed this very question to himself, his peers, and to us. It got us thinking, and made us wonder what the consensus is among security professionals who constantly wrestle with balancing the scales of security and user friction.
One of the most recent campaigns highlighting the importance of router security is Mirai (The botnet that had large scale attacks by infected IoT devices). Even before this, reports emphasized the importance and vulnerability of these devices. For example, Report by Malware Researcher Kafeine revealed the use of an exploit kit aimed to exploit routers. This method showed Google Chrome users were redirected to a malicious server that loaded code designed to determine router models. (While changing the DNS servers configured to the router)
"Crime as a Service" (CaaS): It's not just a recently ramped up buzzword, it has actual backing and won't quietly fade into the night anytime soon. It's a service that has the potential to mature into a larger organizational unit, which is telling of the cyber security issues we'll be up against in the future.
Come see the inventor himself, Paul Mockapetris, deliver the keynote presentation at NamesCon 2017:
Received a notice from Infoblox lately?
If you're a DNS Legacy Firewall customer, you've probably gotten a warning to migrate to ActiveTrust by end of January….. or else. However, that's not the case. The Threat Intelligence/RPZ Feed you've been utilizing with Infoblox is a ThreatSTOP powered service and it's still operational. We've also been developing and improving our product, now offering our new, Next Generation DNS Firewall Service to active subscribers without any added charges.
Malicious content identified and inserted:
Target lists updated:
Everyone knows you need to block the bad stuff from getting onto your network and calling home to its masters. However, what happens when something good gets incorrectly flagged as malicious? You've been hit with a false positive, and in some cases, this can be just as bad as letting something truly dangerous get through.


text: Booked a trip today for April. That means 5 trips in queue . Oct, Nov, Dec, Feb & April. Then shock of shocks, my wife said last week that maybe in 2018 we should consider becoming snowbirds for 3 months of the Wisconsin winters. She is on 2 golf leagues and golfs with so many people who either have 2nd homes in AZ or NV or rentals. Never say never, but I said I wouldn't live in Vegas again, but hell, I already know all the streets, restaurants and VP Bars, why not Vegas again, part time?


text: I remember the time when in my country, they played the national anthem before cinema begins and after, and everybody stood up in respect. I remember the time when the same was done by Americans in respect for the national anthem. here's a Jamaican who cuts short an interview to honor the anthem. they say not many do that anymore, and I don't know about that, but this guy got my respect. http://theblacksphere.net/2014/2/usain-bolt-national-anthem


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Man and van removals Nottingham
van, waste disposal, Van Hire
Whether clearing the home of a deceased relative, preparing a property for sale or letting, or whether you are up dating the furniture and equipment of your office/business.
We at Man and van 4 moving are here to provide you a professional, friendly, quick and efficient service, making your property clearance a stress and trouble free experience. You can also rest assured that the items removed are disposed properly and where ever possible making our utmost effort to recycle/reuse.
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My Favorite Movies
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The Lord of the Rings: The Fellowship of the Ring (2001)
A meek Hobbit from the Shire and eight companions set out on a journey to destroy the powerful One Ring and save Middle Earth from the Dark Lord Sauron. (178 mins.)
Stars: Elijah Wood, Ian McKellen, Orlando Bloom, Sean Bean
The Lord of the Rings: The Return of the King (2003)
Gandalf and Aragorn lead the World of Men against Sauron's army to draw his gaze from Frodo and Sam as they approach Mount Doom with the One Ring. (201 mins.)
Stars: Elijah Wood, Viggo Mortensen, Ian McKellen, Orlando Bloom
Titanic (1997)
A seventeen-year-old aristocrat falls in love with a kind but poor artist aboard the luxurious, ill-fated R.M.S. Titanic. (194 mins.)
Director: James Cameron
Stars: Leonardo DiCaprio, Kate Winslet, Billy Zane, Kathy Bates
The Curious Case of Benjamin Button (2008)
Tells the story of Benjamin Button, a man who starts aging backwards with bizarre consequences. (166 mins.)
Director: David Fincher
Stars: Brad Pitt, Cate Blanchett, Tilda Swinton, Julia Ormond
Life Is Beautiful (1997)
When an open-minded Jewish librarian and his son become victims of the Holocaust, he uses a perfect mixture of will, humor and imagination to protect his son from the dangers around their camp. (116 mins.)
Director: Roberto Benigni
The Lord of the Rings: The Two Towers (2002)
While Frodo and Sam edge closer to Mordor with the help of the shifty Gollum, the divided fellowship makes a stand against Sauron's new ally, Saruman, and his hordes of Isengard. (179 mins.)
Stars: Elijah Wood, Ian McKellen, Viggo Mortensen, Orlando Bloom
Se7en (1995)
Two detectives, a rookie and a veteran, hunt a serial killer who uses the seven deadly sins as his modus operandi. (127 mins.)
Director: David Fincher
The Green Mile (1999)
The lives of guards on Death Row are affected by one of their charges: a black man accused of child murder and rape, yet who has a mysterious gift. (189 mins.)
Director: Frank Darabont
Stars: Tom Hanks, Michael Clarke Duncan, David Morse, Bonnie Hunt
The Prestige (2006)
Two stage magicians engage in competitive one-upmanship in an attempt to create the ultimate stage illusion. (130 mins.)
The Help (2011)
An aspiring author during the civil rights movement of the 1960s decides to write a book detailing the African American maids' point of view on the white families for which they work, and the hardships they go through on a daily basis. (146 mins.)
Director: Tate Taylor
The Dark Knight (2008)
When the menace known as the Joker wreaks havoc and chaos on the people of Gotham, the caped crusader must come to terms with one of the greatest psychological tests of his ability to fight injustice. (152 mins.)
Stars: Christian Bale, Heath Ledger, Aaron Eckhart, Michael Caine
Eternal Sunshine of the Spotless Mind (2004)
When their relationship turns sour, a couple undergoes a procedure to have each other erased from their memories. But it is only through the process of loss that they discover what they had to begin with. (108 mins.)
Director: Michel Gondry
In the distant future, a small waste-collecting robot inadvertently embarks on a space journey that will ultimately decide the fate of mankind. (98 mins.)
Director: Andrew Stanton
Stars: Ben Burtt, Elissa Knight, Jeff Garlin, Fred Willard
The Dark Knight Rises (2012)
Eight years after the Joker's reign of anarchy, the Dark Knight, with the help of the enigmatic Selina, is forced from his imposed exile to save Gotham City, now on the edge of total annihilation, from the brutal guerrilla terrorist Bane. (164 mins.)
Stars: Christian Bale, Tom Hardy, Anne Hathaway, Gary Oldman
Life of Pi (2012)
A young man who survives a disaster at sea is hurtled into an epic journey of adventure and discovery. While cast away, he forms an unexpected connection with another survivor: a fearsome Bengal tiger. (127 mins.)
Director: Ang Lee
Stars: Suraj Sharma, Irrfan Khan, Adil Hussain, Tabu
Forrest Gump (1994)
Forrest Gump, while not intelligent, has accidentally been present at many historic moments, but his true love, Jenny Curran, eludes him. (142 mins.)
Director: Robert Zemeckis
Stars: Tom Hanks, Robin Wright, Gary Sinise, Sally Field
Amélie (2001)
Amélie is an innocent and naive girl in Paris with her own sense of justice. She decides to help those around her and, along the way, discovers love. (122 mins.)
Director: Jean-Pierre Jeunet
Stars: Audrey Tautou, Mathieu Kassovitz, Rufus, Lorella Cravotta
Inception (2010)
A thief, who steals corporate secrets through use of dream-sharing technology, is given the inverse task of planting an idea into the mind of a CEO. (148 mins.)
The Fountain (2006)
As a modern-day scientist, Tommy is struggling with mortality, desperately searching for the medical breakthrough that will save the life of his cancer-stricken wife, Izzi. (96 mins.)
Director: Darren Aronofsky
Big Fish (2003)
A frustrated son tries to determine the fact from fiction in his dying father's life. (125 mins.)
Director: Tim Burton
Stars: Ewan McGregor, Albert Finney, Billy Crudup, Jessica Lange
Rango (2011)
Rango is an ordinary chameleon who accidentally winds up in the town of Dirt, a lawless outpost in the Wild West in desperate need of a new sheriff. (107 mins.)
Director: Gore Verbinski
Léon: The Professional (1994)
Mathilda, a 12-year-old girl, is reluctantly taken in by Léon, a professional assassin, after her family is murdered. Léon and Mathilda form an unusual relationship, as she becomes his protégée and learns the assassin's trade. (110 mins.)
Director: Luc Besson
Stars: Jean Reno, Gary Oldman, Natalie Portman, Danny Aiello
Braveheart (1995)
When his secret bride is executed for assaulting an English soldier who tried to rape her, Sir William Wallace begins a revolt against King Edward I of England. (178 mins.)
Director: Mel Gibson
Anonymous (2011)
The theory that it was in fact Edward De Vere, Earl of Oxford, who penned Shakespeare's plays. Set against the backdrop of the succession of Queen Elizabeth I and the Essex rebellion against her. (130 mins.)
Director: Roland Emmerich
Troy (2004)
An adaptation of Homer's great epic, the film follows the assault on Troy by the united Greek forces and chronicles the fates of the men involved. (163 mins.)
Director: Wolfgang Petersen
Stars: Brad Pitt, Eric Bana, Orlando Bloom, Julian Glover
Gladiator (2000)
When a Roman general is betrayed and his family murdered by an emperor's corrupt son, he comes to Rome as a gladiator to seek revenge. (155 mins.)
Director: Ridley Scott
Children of Heaven (1997)
After a boy loses his sister's pair of shoes, he goes on a series of adventures in order to find them. When he can't, he tries a new way to "win" a new pair. (89 mins.)
Director: Majid Majidi
Avatar (2009)
A paraplegic marine dispatched to the moon Pandora on a unique mission becomes torn between following his orders and protecting the world he feels is his home. (162 mins.)
Director: James Cameron
Toy Story 3 (2010)
The toys are mistakenly delivered to a day-care center instead of the attic right before Andy leaves for college, and it's up to Woody to convince the other toys that they weren't abandoned and to return home. (103 mins.)
Director: Lee Unkrich
Stars: Tom Hanks, Tim Allen, Joan Cusack, Ned Beatty
Fantastic Mr. Fox (2009)
An urbane fox cannot resist returning to his farm raiding ways and then must help his community survive the farmers' retaliation. (87 mins.)
Director: Wes Anderson
The Truman Show (1998)
An insurance salesman/adjuster discovers his entire life is actually a television show. (103 mins.)
Director: Peter Weir
Stars: Jim Carrey, Ed Harris, Laura Linney, Noah Emmerich
The Sixth Sense (1999)
A boy who communicates with spirits that don't know they're dead seeks the help of a disheartened child psychologist. (107 mins.)
Director: M. Night Shyamalan
The Departed (2006)
An undercover cop and a mole in the police attempt to identify each other while infiltrating an Irish gang in South Boston. (151 mins.)
Director: Martin Scorsese
The Secret of Kells (2009)
A young boy in a remote medieval outpost under siege from barbarian raids is beckoned to adventure when a celebrated master illuminator arrives with an ancient book, brimming with secret wisdom and powers. (75 mins.)
Director: Tomm Moore, Nora Twomey
Stars: Evan McGuire, Brendan Gleeson, Mick Lally, Christen Mooney
Lucky Number Slevin (2006)
A case of mistaken identity lands Slevin into the middle of a war being plotted by two of the city's most rival crime bosses: The Rabbi and The Boss. Slevin is under constant surveillance by relentless Detective Brikowski as well as the infamous assassin Goodkat and finds himself having to hatch his own ingenious plot to get them before they get him. (110 mins.)
Director: Paul McGuigan
Stars: Josh Hartnett, Ben Kingsley, Morgan Freeman, Lucy Liu
Flightplan (2005)
A bereaved woman and her daughter are flying home from Berlin to America. At 30,000 feet, the child vanishes, and nobody will admit she was ever on the plane. (98 mins.)
Director: Robert Schwentke
Stars: Jodie Foster, Peter Sarsgaard, Sean Bean, Kate Beahan
Inglourious Basterds (2009)
In Nazi-occupied France during World War II, a plan to assassinate Nazi leaders by a group of Jewish U.S. soldiers coincides with a theatre owner's vengeful plans for the same. (153 mins.)
Director: Quentin Tarantino
Stars: Brad Pitt, Diane Kruger, Eli Roth, Mélanie Laurent
Shutter Island (2010)
In 1954, a U.S. marshal investigates the disappearance of a murderess who escaped from a hospital for the criminally insane. (138 mins.)
Director: Martin Scorsese
Unknown (2011)
A man awakens from a coma, only to discover that someone has taken on his identity and that no one, (not even his wife), believes him. With the help of a young woman, he sets out to prove who he is. (113 mins.)
Director: Jaume Collet-Serra
Stars: Liam Neeson, Diane Kruger, January Jones, Aidan Quinn
Mother India (1957)
In this melodrama, a poverty-stricken woman raises her sons through many trials and tribulations. But no matter the struggles, always sticks to her own moral code. (172 mins.)
Director: Mehboob
Stars: Nargis, Sunil Dutt, Rajendra Kumar, Raaj Kumar
Finding Nemo (2003)
After his son is captured in the Great Barrier Reef and taken to Sydney, a timid clownfish sets out on a journey to bring him home. (100 mins.)
Director: Andrew Stanton, Lee Unkrich
A Series of Unfortunate Events (2004)
When a massive fire kills their parents, three children are delivered to the custody of cousin and stage actor Count Olaf, who is secretly plotting to steal their parents' vast fortune. (108 mins.)
Director: Brad Silberling
Stars: Jim Carrey, Jude Law, Meryl Streep, Liam Aiken
Red Cliff (2008)
The first chapter of a two-part story centered on a battle fought in China's Three Kingdoms period (220-280 A.D.). (146 mins.)
Director: John Woo
Red Cliff II (2009)
In this sequel to Red Cliff, Chancellor Cao Cao convinces Emperor Xian of the Han to initiate a battle against the two Kingdoms of Shu and Wu... (142 mins.)
Director: John Woo
The Lovely Bones (2009)
Centers on a young girl who has been murdered and watches over her family - and her killer - from purgatory. She must weigh her desire for vengeance against her desire for her family to heal. (135 mins.)
Stars: Rachel Weisz, Mark Wahlberg, Saoirse Ronan, Susan Sarandon


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I was about to say that it had been a while since I'd blogged about my characters' faces, but apparently it was only last year! The full set of character images are here.
I wish now that I hadn't looked at that post, as I see it's taken me a year and a half to accomplish the goals I'd set for myself in January 2015, and two or three (involving being published) haven't even happened yet. Oh dear. On the other hand, those were simply the writing-related goals, and didn't include hobbies or life projects or anything else. So there is a positive side!
In keeping with all that, and going back to character faces, I thought I'd share some pretty things today, one of which is a set of drawings by the talented illustrator and current UK Children's Laureate, Chris Riddell. He sketches all the time, and shares a lot of his work on social media. Sometimes he sketches real people, and I find it interesting to compare the expression of the person in the photo with the expression he gives them in his sketch.
One of his recent illustrations for Literary Review featured a couple that straightaway reminded me of Rosa and Baha from Out of the Water (which I'm supposed to be submitting to agents, as part of my ongoing goals).
Out of the Water (historical romance, set in 1492)
Book One of the Middle Sea series
After she becomes separated from her family as they flee their Spanish homeland - and the Inquisition - the last thing eighteen-year-old Rosa expects to find is love.
Her one hope of reaching Constantinople and finding her family lies with a stranger, Baha, a Muslim artist from the Ottoman Empire. Rosa's drive to find her loved ones is matched by a deepening desire for the man at her side. Yet despite all his help in reuniting them, her family rejects this man of a different faith, forcing her to choose between them.
Constantinople was meant to be her family's refuge, but when janissaries arrest her father and brother, Rosa and Baha risk their lives on a daring rescue. Together they will prove that their love can withstand their differences... if the Sultan's Grand Vizier doesn't throw them both in the dungeons first.
Riddell shared images of the sketch as it developed:
Also pretty is the cover of Theresa Milstein's forthcoming book!
And then there's Milushka's pretty jewellery. I discovered her through Caramella's blog, and can't wait to get one of her creations for myself -- I've already bought a few as gifts!
Aren't they sweet?
As I used his song title for my blog title, I thought I'd better share a Bowie clip:
What pretty things have you come across recently?


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Zimin And Luhan Are Siblings But You Are Not Blood Related Because When Luhan Was 6,His Parents Decided To A Adopt A Daughter,When Zimin Was 8 And Luhan Is 14 Your Parents Got Into A Accident,You Both Lived Together...And Something Grew In To Love...REAL LOVE...
BeachyCreek Aug 20, 2016
Hello, almost an I love you, and sorry. Gull don't be sorry!
TheJaebummies Nov 04, 2016
*grabs Mina phot and puts on TT beginning* GEENEE MUT TUN NA DE NANO WUT TIE KE EEJIMO BA-BA-BA-BABYY~~
blinkeuuu Dec 10, 2016
Lemme see how you gon' treat me I ain't no easy, better think about it TWICE~~~
Elenix_Viral06 Dec 15, 2016
I was thinking of jimin of aoa, and the irony is ....a minute ago I was rewatching "Unpretty rapstar season 1" xD
TheJaebummies Nov 04, 2016
TT?!?! STAWP WITH THE TWICE JOKES BUT STILL..*puts on TT* KE IN DE NA SINGO CAN DE JE UUMA DE EUN BUNO DWE NA NE OHSACO NAN NIN INDWE
Acupoftaeandsuga Nov 27, 2016
It's not okay unless you burn all the documents and change her last name then it's okay but you didn't do that so INCEST


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Introducing: Carol Dyar, HCHS staff member of the month
by Judy Bushy
January, Carol Dyar was presented with a certificate for being the Happy Camp High School Staff Memeber of the month. In presenting the certificate, Diane Oliver said,
"The Staff? Member of the Month of January, I believe, is the hardest-working member of our staff. In a thousand ways, every day! She helps each and every one of us, and her jobs are endless. She processes a mountain of paperwork, takes and delivers messages, creates and prints transcripts, makes sure we have nutrition and lunch, #elds calls and requests from parents, updates CUM files, makes sure we have the right forms to fill out, watches over students sent to the office, makes sure we're paid, fills in for others when needed, works late hours so things run smoothly, updates Aeries, orders supplies, keeps us on track, bandages our hurts, and gives us candy.
"And she does it all with a smile and a gracious manner. There is no way to list all the ways she is invaluable to us every single day. For all these reasons and more, I am proud to present the Staff? Member of the Month of January to our secretary, Carol Dyar."
We are very grateful for all the staff at Happy Camp High School! At present this included our teachers, Denise Bearding teaches Science classes, Honors English and also the art class. She plans to take students to plays in Ashland, Oregon Shakespeare Theater next month.
Diane Oliver teaches computer literacy, yearbook and helps us out when we have computer questions or problems. Erica Mitchell teaches World History, Economics, German and Health and Physical Education classes. Carissa Bussard teaches English, Drama and helps out the Potter Club. Ed Sherman teaches math classes, from Academy math, Algebra, Geometry and all sorts of number maneuvering accomplishments! David Timbrook teaches woodshop and welding.
Steve VanErt is our principal.
We also have bus drivers, food servers, tutors, janitor, buildings and ground keepers and aides besides Carol in the office. Jeanette Quinn teaches the students next door at Jefferson High School. Takes TEAM work to educate the students and we have a great group of students who we are working to help them reach their highest potential!!
HCHS Students go to Washington!!!
bu Judy Bushy
(Further information on the Trip to Washington for the Presidential Inauguration after January 24th.)
Last week was time for semester final exams AT hc High School! Snow storms, electric outages, and bad road conditions resulted in some high school students missing some final exams!
Some of the students were missing them because they were a very long way away! About three thousand miles away!
It was great that fourteen of our students from Happy Camp High Tashawna Brink, Madison Carroll, Wren Crapser, Kai Crockett, Estalita "Ella" Galindo, Rudolf "Rutz" Galindo, Adriana Garcia, Angelica Garrison, Madison "Maddy" Offield, Royale Pinassi, Elijah"E.J." Triance, Noah Triance, Sophia Triance, Damian Valenzuela, and Silas Yamamoto, along with their history teacher, Erica Mitchell went to Sacramento to fly to Washington D.C. for the Inauguration.
Snowstorms and Power Outages for the New year….
Neighbor helping Neighbor! That is the most wonderful thing our recent difficulties have shown us! New Year has brought with it snow storms and rain, electrical and heat outages.
Some of our neighbors, some who are elderly or disabled, not only had no heat or way to make a hot meal with the electricity off, but were unable to physically get out because show blocked streets and drives!
The electric hasn't been off completely for the past three weeks, but much of the time! After being off 18 hours, so when it came on there was the mad rush to do all one could while it was on, and then off again.
Sometimes it was on downtown and there was school, but off at our house and couldn't get out of the drive anyway…so we were stuck!
I didn't see all who were out there clearing snow or helping out, but Siskiyou Road workers, CalTrans, and Pacific Powers have been working long hours and doing a great job and we want to thank them! Tha nks to all our neighborly neighbors who did so much to help!!
Make Someone's Christmas!
by Judy Bushy
What do you think of when you remember Christmas as a child, a youth, and through the years?
My early Christmases were spent at the large two story home that my father had built over fifty years before as home for my grandmother as a new bride. There was plenty of space for five daughters and son and all the spouses and at least a dozen children for breakfast. No one could do anything until breakfast was eaten together and the dishes all washed except look at the beautifully lit tree, We'd first seen the tree that morning as the adults decorated late Christmas eve. After dishes we opened presents and then my mother would gather us to leave for church. I'd put on a red choir robe for the junior children's choir. The organ would play out the joyous music as the bells in the tower rang and we began the processional singing down the aisle.
It is such fun to see the joyous anticipation of children at this time of year. At a department store in Minneapolis while in High School— two Christmas seasons! It was especially fun when I got to help the elves make handmade candy canes that were given out by Santa Claus! Cleaning up the orange soda that was stuck everywhere from the tikes dropping them wasn't as such fun, but it was so neat to see people thoughtfully buying gifts for the children in their lives!
After meeting my husband the week of high school graduation at seventeen, we were busy driving from Minneapolis to Bayfield, Wisconsin each Christmas Eve. After time with my family we drove 300 miles to Dan's family on the shores of Lake Superior for Christmas morning. Usually there was a blizzard. It was always beautiful and we loved the snow. The years went by and we added pur little ones, and they kept growing up fast.
As they years went by I'd usually have first five or six and then ten or more children every day for daycare. We'd make something new, ornaments, gifts, wrapping paper and cards; projects of artwork and stories, songs and games each day in December.. That was always fun!
But sometimes this isn't such a happy time of year. There was 1976, when my mother was in the hospital, mostly unconscious from before Thanksgiving to New Years eve.. We were grateful for the years the Lord had given us with her as she was expected to leave us six months after the diagnosis/ Instead, by the Lord's mercy, was able to be present for four grandchildren thirteen years later.
I remember in 1990, crying for months solid. It is unbelievable how much tears a person has capacity to shed. Anytime "I'll be home for Christmas" was sung or another program suggested families all being together, would bring on another cloudburst since our oldest son, Michael, had gone home to be with the Lord October 26th that year.
Even when it isn't a time of great grief or sorrow this time of year it just seems like there should be gatherings of friends and family, When that isn't possible, missing them is hard. So sometimes this time of year isn't the joy and excitement as it is at other stages of life. Pray for the opportunity to help other's for whom Christmas will be lonely or sad or very difficult this year. Pray that you'll forgive any enemies you need to forgive. Pray that the Lord will use you to bring a Christmas peace nd joy to their life.
January 2017: Pioneering on the Klamath
By Judy Bushy
The first week of the New Year, and I think we have 3 to 4 feet of snow in most areas. Plowed up in heaps to the rooftops in some places, as there is plenty of snow to clear!! It reminds me of childhood in Minnesota. How kids used to enjoy ice skating near our home, making snow angels and playing fox and goose as we chased around circles in the snow. We dressed for it every day, snow pants, warm coats, hats, scarves and mittens!
The complicating factor, when we get snow in Happy Camp, is that the electricity goes off. At our home it went off Monday night about 6:20 p.m. The generator didn't want to start. Since we'd had dinner, sensible thing to do is turn in for the night.
The electricity came back on Tuesday afternoon, but only for a few hours. About 6:30 in the evening, off it went again. Curly Jack Road hadn't been plowed, so that's one way to get a good night's sleep! Wednesday and Thursday evening, being under blankets and quilts was the only place to be, it was getting a little old! Some people had electricity and we didn't.
We have good neighbors. Craig Lighthill was driving his truck to the rescue of stranded motorists and others, and made a path in our driveway. Our good neighbor, Carol Day had two strong men, son T.J. and grandson, who came to help.
Dan shoveled out and put chains on the pickup truck. Off he went to see Kingfisher Market, get the mail, and brought a pizza from Pizza House to eat by candlelight. In the middle of the night, our son, Stephen arrived from Yreka with the neatest lantern, it will even charge my cellphone! How amazing! So of course, the next day the electricity was back on, for awhile!
We are experienced in pioneering skills in Happy Camp! We have good neighbors, and it is kind of nice to have a quiet spell for a change. Later, Stephen got the generator going as well so we will do better next outage.
Saturday morning we woke up to electricity blinking off again!
Thanks to Kathy Purvis standing at the cold gas pumps helping people get gas. Linda is quilting. Friends are shoveling neighbor's driveways, bringing firewood, picking up supplies, checking on neighbors. You really feel the warmth and gratitude for living here along the wild and beautiful Klamath River with the best neighbors ever!
Sure appreciated when the oven was working awhile that Dan made oatmeal cookies and hot chocolate! Some use this time to catch up on a few things, spend time with friends and family, play a game by candlelight, nap, cook up something special on the woodstove and count your many blessings. Debbie Bickford says, "Count your blessings, Name them one by one…You will be surprised what the Lord has done" is one of her favorite hymns and it's a good way to live as well.
There was no Happy Camp Christian Fellowship gathering Sunday, snow still filled the parking lot.
Rogue Weather report for our area said that the East locations will take longer to change over from snow to rain but will eventually have rain (we are!) Ice may accumulate in areas where freezing rain develops and the impact on travel could be severe, so travel is NOT advised. Snow packed roads create hazardous driving conditions especially when freezing rain is on top of the ice as it is extremely slippery. If you must travel, take tire chains, a flashlight, food, water, any necessary medications and a fully charged phone with you. Drive with care. Keep safe! Stay inside and warm, when possible, in hazardous conditions!
Glad and Grateful New Year!
by Judy bushy
Sunday morning we hoped to welcome our first sunrise of the new year. It's a beautiful sight to see, if the clouds don't obscure the view. This year, however, we looked out to beautiful light fluffy new snow!! Not only that, but my dear red Australian Shepherd was enjoying it! He runs around pushing his nose in the snow and shaking it off on occasion and seemed thrilled to have the new play matter all around.
Celebrating such beauty and hope, from the inside of a warm home, reminds us how grateful we are to have that nice warm shelter, but also to be able to look around at such beauty on a daily basis.
Bruce Harlow posted his New Year's Grateful List! The things that he is grateful for, living in Happy Camp were, first of all our small market, Kingfisher Market, that packs so many things in it. He also appreciated that they even have a butcher. He is thankful for our water service, Happy Camp Community Services District, as well as sewer which is through the Happy Camp Sanitary District.
We are very thankful for Pacific Power and Light and I will add that with snow coming down, that it hasn't gone off! Sometimes snow on trees causes' disruption, but that makes us more grateful when all is working well. With snow on the roads today, we appreciate CalTrans and the County Road workers who help to keep the roads safe for travel.
Bruce also mentioned being grateful to the Karuk Tribe, who do so much for the entire community, not only their natives and descendants, and the Forest Service. I'm especially grateful for Jim Elison, District Recreation Officer who made a map and listed trails that are convenient for hiking around town.
Everyone is grateful to the Post Office that brings us our mail, and for those without computers who want email get it at the Happy Camp Computer Center. Packages are also brought by FedEx and Tom with UPS. Our schools are appreciated and the teachers, bus-drivers, and all the staff.
What would we do without the businesses, Double J, Pizza House, Clinic Pharmacy, Rick's Auto Supply and Al's Garage that keep things humming along when we need things. We have a large number of churches and non-profit organizations for which we are grateful, and youth programs. To his list, Bruce added, "Everyone who cares and keeps on working to make it better", a "Thank You, and Happy New Year!"
To that list, Scott Grandstaff added, "Don't forget Fire and Ambulance; Selfless heroes out there risking their lives in the middle of the night, for us. The Highway Patrol and the Sheriff's Deputies also serve our community.
With grateful hearts, we look forward to the new calendar year and it is exciting to think of all the opportunities that await us, some planned, and some we probably don't even know about yet. While preparing the Chamber calendar for the coming year I noticed Saturday is a Mid Klamath Food Shed and the Karuk DNR is having a Pruning Workshop at Sandy Bar Ranch from One o'clock to four O'clock, rain or shine. They ask you to dress accordingly. Give them a call at MKWC 627-3202 or email email@example.com for details of the workshop.
Basketball coming
The High School is looking forward to beginning more Basketball, but not this week. The first games are the 10th at Big Valley and 14th when Dunsmuir comes here. Homecoming is planned for February 3rd. In only 17 days, some of our high school students will be attending the Inauguration of President Donald Trump in Washington, D.C., that will be something to tell their children and grandchildren about!


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The next Hamish Macbeth novel will be Death of a Ghost released on the 21st of February 2017, but we have a first look at the novel to follow.
The Agatha Raisin TV Series, starring Ashley Jensen, has finally come to the US, and we have the show times below.
M.C. Beaton's Hamish Macbeth series and The Poor Relation, an historical romance series, have both been optioned by Free@Last TV, currently producing the Agatha Raisin series, adapted from her best-selling novels and starring Ashley Jensen.
If you want to be kept up-to-date on all of the latest news, events, and book releases, sign up for our newsletter.


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Classic Malaysia
Oh Dear!
This particular product has expired and is not available right nowSee similar Journeys
... Malay, Indian and Indo-Chinese cultures have combined to give the nation its unique flavour.
Some of the highlights included are:
- Soak up the modernity of Kuala Lumpur
- Follow the footsteps of old-world traders in Melaka
- Relish the serenity of Pulau Langkawi's beaches
- Peer into the past in old Penang
Holiday Type
Not what you're looking for? Use our Inspiration tool to find the holiday you want.
What's included
- Meals: 9 breakfasts, 1 lunch, 2 dinners
- Accommodation: Hotel (6 nights), Resort (3 nights)
- Transport: Public bus, Trishaw
- Activities: Kuala Lumpur - City tour, Kuala Lumpur - KL Tower, Langkawi - Cable Car, Langkawi - Island Tour, Melaka - Trishaw Tour, Penang - Fort Cornwallis, Penang - Malaysian Cooking Class
Need to know
- Group size: Min 1, Max 16
- Ages: Min 15
Your route
With flourishing retail hot spots, a booming, cross-cultural culinary scene and one of the world's best airports, it's not hard to see why Singapore acts as a launching (and finishing) point for many of our Asian odysseys. From the traditions and heritage of Chinatown to the aromatic delights of Little India and the modernity of glitzy hotels, a few days in sizzling Singapore is the perfect bookend to any Intrepid adventure. Singapore has few areas untouched by modernisation, but wandering the streets of Little India and Chinatown is a great way to see the old parts of the city. To explore further, check out the renowned shopping precinct of Orchard Road, or stop by Raffles to experience the old world charm of a Singapore Sling. An evening in Singapore is not complete without an evening stroll along Clarke Quay or take a river cruise to learn more about the fascinating history of this city.
A public bus journey (approx. 5.5 hrs) takes us to the World Heritage-listed port town of Melaka. Once the most important trading port of Malaysia, Melaka attracted Chinese, Indian, Dutch, Portuguese and British traders, all seeking wealth or an opportunity to control the Straits of Malacca, a vital trading route between the Indian and Pacific oceans. Today, Melaka's wealth of historical sites remind visitors of times gone by and its multicultural past is revealed in its distinctive cuisine. Take a quaint trishaw tour along the narrow winding streets to get a taste of old Melaka and learn about the ancient spice trade. Take in sites such as Harmony Street, a mosque, Chinese Buddhist Temples and a traditional Muslim house-museum.
Kuala Lumpur
Kuala Lumpur
Transfer from Melaka to Kuala Lumpur by minivan (approx. 2 hours). Kuala Lumpur, the cosmopolitan capital of Malaysia, is affectionately known as KL and you'll almost always hear locals refer to it by its acronym. Communities of Indian, Chinese and ethnic Malay people mingle peacefully, allowing travellers to get the best of all three worlds. Take a half-day tour and experience the fascinating cultural mix that gives it such a uniquely cosmopolitan feel. Visit some of the city's most impressive architecture such as the National Monument, National Mosque, Istana Negara (National Palace), Batu Caves and Royal Selangor Pewter. Enjoy an included dinner at the Old China Cafe in KL.
Transfer by private minivan from Kuala Lumpur to Penang. Penang Island was the first destination of the colonial British in 1786. They invited Chinese, as well as others from all over Asia, to live on this island in the tropics. Today, this exotic blend of cultures still exists and the World Heritage site of Georgetown contains a fascinating blend of religious places of worship. Discover Penang's Chinese and British colonial history around Georgetown. Explore Little India taking a glimpse of the ancient Hindu traditions in its decorative temples and tasting freshly made delicious Indian snacks. Don't miss a visit to the Chinese stilt houses or the chance to have high tea at the legendary Eastern and Oriental Hotel. A tour of Penang takes in Fort Cornwallis, the site of the original British settlement. You also take a drive to Kek Lok Si, the largest Buddhist temple in South East Asia. Discover the flavours of Malay cuisine with a cooking class in Penang.
Pulau Langkawi
Pulau Langkawi
Fly from Penang to the island of Langkawi. Pulau Langkawi is only 487.5 sq km in area but it's one of Malaysia's premier tourist destinations, featuring stunning beaches and some of the best sunsets you'll find in the country. It's essential to treat yourself to a swim, snorkel or sunbake on the sultry exotic white sand beaches or perhaps unwind in a boutique spa. Take a day trip to explore some of the smaller islands. Please note, if the weather is too wet we'll do a day tour of the main island instead. Not to be missed is a trip on the cable car with spectacular views over the Andaman Sea and some of the 99 tropical islands of Langkawi. Enjoy a delicious seafood barbecue dinner. There are no activities planned for the final day and you are able to depart the accommodation at any time.


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tag:blogger.com,1999:blog-1182128440772477840.post3198150529056893195..comments2017-02-27T19:56:52.168+05:30Comments on OpenCV Tutorial C++: Change Contrast of Image or VideoShermal email@example.com:blogger.com,1999:blog-1182128440772477840.post-75041690129271755552015-06-04T16:00:29.190+05:302015-06-04T16:00:29.190+05:30Can anyone tell me the equivalent of "im2doub...Can anyone tell me the equivalent of "im2double" function of MATLAB. How can we do it using opencv

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Abdul WahabAbdul Wahabhttp://email@example.com:blogger.com,1999:blog-1182128440772477840.post-63599378923352207882015-02-21T20:43:43.131+05:302015-02-21T20:43:43.131+05:30It is indeed a contrast transformation because the...It is indeed a contrast transformation because the distance between dark and bright pixels increases by the multiplication factor.

Let's say you have a brighter pixel with value 100 and a darker pixel with value 10.
Then: distance = 100 - 10 = 90
However if you multiply all pixels by e.g. 2
Then: new distance = 200 - 20 = 180 = 2 * old distance
So you've actually doubled the distance between dark and bright pixels.

The problem with this approach is that you increase all pixel values which means that more pixels reach a value > 255.
In this example all values > 255 are set to 255 (i.e. white).
That's why we can't see the river in the transformed example image.

If you increase your factor (because you want more contrast) you'll get a completely white image very soon. (Instead of an image with high contrast)

Instead of making all pixels brighter (some more some less), you could indeed separate pixels into dark and bright pixels (depending on a threshold or formula like you suggested) and then make the bright pixels brighter and the dark pixels darker.

firstname.lastname@example.com:blogger.com,1999:blog-1182128440772477840.post-47777867678521777482014-12-12T00:40:27.998+05:302014-12-12T00:40:27.998+05:30This is not a contrast transformation, simply brig...This is not a contrast transformation, simply brightness by multiplication.
When you increase the contrast, a dark pixel will get darker and a white pixel will get whiter.
In other words you would some kind of formula like:
pixel x; (range 0-255)
x'=x*(1+(x-128)/256))
Please dont misinform people on the firstname.lastname@example.com:blogger.com,1999:blog-1182128440772477840.post-71870346771390802752014-03-14T11:00:20.474+05:302014-03-14T11:00:20.474+05:30Its simply great tutorial Can you right a tutorial...Its simply great tutorial
Can you right a tutorial about depth calculation using single camera. using simple pinhole camera model.
it will be helpful for me.musafirhttp://firstname.lastname@example.com:blogger.com,1999:blog-1182128440772477840.post-21975696607790489912013-11-07T15:30:54.556+05:302013-11-07T15:30:54.556+05:30(you forgot to adjust the brightness to compensate...(you forgot to adjust the brightness to compensate for the multiplication)

#define NA -1 // indicator that value is [N]ot [A]vailable or [N]ot [A]pplicable
#define MIX(v1,v2,ratio) (v1+(v2-v1)*ratio) // Mix two numbers according to a floating point ratio 0..1 (being 0%..100%)
#define MINMAX(min,max,v) ((v)<(min) ? (min) : (v)>(max) ? (max) : (v)) // Limit value inside given valid range

double multiplier = (double)MINMAX(-100,100,contrast)/100.; // convert range -100..100 -> -1..1
double alpha = multiplier>0 ? MIX(1.0, 2.0, multiplier) : MIX(1.0, 0.5, -multiplier); // 0.5 will halve the contrast while 2.0 will double the contrast
double beta = multiplier>0 ? MIX(0.0, -0.5, multiplier) : MIX(0.0, 0.25, -multiplier); // compensate brightness accordingly to get the original average
cv::Mat(src).convertTo(cv::Mat(dst), NA, alpha, beta);
stephan verbeeckhttp://firstname.lastname@example.com:blogger.com,1999:blog-1182128440772477840.post-79631837682933406802013-11-07T15:16:07.279+05:302013-11-07T15:16:07.279+05:30This comment has been removed by the author.stephan verbeeckhttp://email@example.com:blogger.com,1999:blog-1182128440772477840.post-41206689440909607312013-10-28T03:20:56.973+05:302013-10-28T03:20:56.973+05:30This has been extremely helpful. All other program...This has been extremely helpful. All other programs that I encountered to change contrast of the image involved accessing each pixel of the image and applying the formula new_img (i, j) = img(i, j) * c .
Even in opencv docs uses the formula mentioned earlier. The formula approach didn't work for me, but this did and I'm delighted. Thanks! firstname.lastname@example.com


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"Shoot for the moon. Even if you miss it you will land among the stars." The ideal combination of inspiration and pragmatism, this Black Rock journal is the perfect place to record your everyday thoughts and ideas, or jot down favorite quotations and poems.
- 160 lined pages
- Ultra stiff black cover serves as a writing board
- Crush-proof rounded corners
- Attached elastic band place-marker
- Measures 6" x 8"
- Made in Canada
Product Details
- 9781593593803
- Manufacturer:
- Peter Pauper Press, Incorporated
- Publication date:
- 02/28/2005
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Brine King III Lacrosse Shoulder Pad Liner
#1 Customer Rated Lacrosse Website Learn MoreWhy We're Top Rated Learn More
Increased foam density in "Hot Zones" throughout the pad keep players protected in key areas of constant contact.
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Is The Bunker Restaurant & Bar incorrect or missing key business information? Update your Hotfrog listing today.
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text:
Harriers The Making of a Championship Cross Country Team
Written by Joseph Shivers, Paul Shivers
Illustrated by
Lovereading4kids Price £4.79
£5.99 saving £1.20 (20%)
when in stock
As soon as this book is back in stock we will send you an email.Compare book price We'd love you to buy your books from Lovereading - especially if we helped you to choose them! But as our mantra is guidance, and that includes guidance on price, please do use our 'Compare Book Price' service to discover if the best value deal is from Lovereading or another specialist bookstore.
Harriers The Making of a Championship Cross Country Team by Joseph Shivers, Paul Shivers
About the Author
Other Formats
Book Info
180 pages
Joseph Shivers, Paul Shivers
More books by Joseph Shivers, Paul Shivers
Holy Macro! Books
Publication date
1st March 2006
I love 'LoveReading 4 kids' because they let you read and learn things you'd never dreamed of learning before.Emily Horncastle – age 11
A great way to introduce kids to great books, authors & genres. Parents can find age-appropriate books to share with their children.Judi Davies – Aberdare Girls Sch
We love Lovereading4kids because it promotes reading choices, new authors and a sense of community for children of all ages!Rachel Bridgeman
I love Lovereading4kids because I get an opportunity to put my opinion forward and to try new booksElosie Clarkson – age 11
It is fantastic, you get to read lots of books and you always find something new and amazing in them.Erica Motoc, age 7
Love "Lovereading4kids" as my son gets to hear about & read new books before his mates which keeps him interested in reading=a very happy MumLiz Evans
We love Lovereading4kids because they put books in front of us we wouldn't otherwise have read. They make us more adventurous readers!Emily Jacques
Lovereading is just a convenient way to find new books and hear others opinions on them.Sarah Murray – age 15


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