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games / Reviews
Microsoft Game Room (360) Review
Title: Game Room
Publisher: Microsoft Game Studio
Developer: Krome
Genre: Various
Players: 1-2
Rated: E10 for Everyone 10+
When it was first announced a few months back there were mixed opinions on Microsoft's newest service, the Game Room. While the concept seemed promising (playing all of your favorite console and arcade games from the past), the pricing method introduced was met with negativity. After a shaky start the Game Room is fully up and running and the results, much like early opinions, are mixed.
The software required to use the service itself is free, and after that you will be required to download game packs, which contains a variety of games in each one (as of this writing, there were two available). You only need one downloaded to being exploring, though downloading them both is recommended to get the full experience. Some of the games included in the initial release are Adventure, Asteroids Deluxe, Lunar Lander, RealSports Tennis, Shao-Lin's Road, Yars' Revenge and more. For review purposes I purchased Asteroids Deluxe, Shao-Lin's Road and Adventure.
But most importantly, Adventure!
One of the biggest complaints about the service initially was the pricing. After playing around with the service for a bit, I've decided that the complaints were a little premature and exaggerated. Players are entitled to one free, ten minute demo of each game found on the service. After this trial period the game can no longer be played for free and can be purchased in three ways. There is a single play option, which costs 40MP (or roughly fifty cents), unlimited play purchases for 240MP for either Windows or Xbox 360, or unlimited play purchases on both platforms for 400MP. While some will argue for cheaper prices per game, and while I understand the outcries I don't think it would really work out. The 40MP single play pricing makes sense and does fit in line with the arcade "experience." From a business standpoint, if you're charging 40MP for a single play and charge 80MP per game (as some people have suggested), why would anyone purchase the single play? I'm not saying I love the prices, but you have to look at things from both sides. You are also given 20 free "tokens" which can be used to play the games, with five tokens equaling one play. These tokens can be "earned," which I'll explain in a little bit.
As of right now, the Atari 2600 and Intellivision are the only consoles supported, though I'd imagine with time more consoles would be supported. Various arcade machines can be found as well. For the Atari 2600 you are able to configure the switches that you would normally find on the console itself via the start button. It provides a more authentic feel and is a nice touch. The Intellivision games feature the numeric keypads, complete with the overlays and everything. Unfortunately, for games that require a lot of button entries, it can become tiresome and irritating, especially if you happen to press the wrong button. For the arcade games you are able to modify the DIP switches for each board. I tested each individual game through the ten minute demo and found nothing that would suggest improper emulation, so everything runs exactly as it should – including graphics, controls and even sound. Each game also features a rewind function, meaning you can back to any point of your game session. If this feature is used in any session it will not be counted on the leaderboards, however.
Clearly some of the best graphics available on the 360
As well as playing these old games, the Game Room allows players to create their own, custom arcade setup. You can choose from a variety of themes including a grave yard, 80's room, and even console-specific rooms. From there decorations can be placed in various locations, along with the arcade machines themselves. The cabinets appear to be spot-on recreations and older fans will love checking out some of their favorite games of yesteryear. Some of the decorations are theme-specific while others include things such as a pool table and snack machines. As fun as it would be, the avatars are unable to interact with anything other than the arcade cabinets, which is a bummer. Having mini-games such as pool or even an animation of the avatar eating a snack would have been nice touches and really added to the atmosphere.
To the atmosphere of the most badass arcade ever!
If the default decorations aren't enough for you, it's possible to level up by earning medals. Earning medals can be done through playing a specific game for a specific duration or getting a high score, etc etc. As you increase the levels more themes and decorations are unlocked, though it doesn't unlock any more games or the like. Some of the medals require you to play the game online either with a friend or solo while going for a high score.
After the perfect arcade is built, you begin inviting friends to come and check it out. When your friends visit your arcade you get more tokens, which can get you free play sessions. But there's one thing to note about the having your friends visit your arcade – you don't get to interact with them. In fact, there will always be people in your arcade, it's just that they won't actually be there. And when you go to other people's arcades, you'll actually be alone. It's kind of a weird feeling when you think about it, but it's not as though it's a make or break experience. You'll also be able to send challenges to your friends, such as attempting a high score or getting to a certain level.
The Game Room, on a whole, is a great idea that could potentially introduce these classic games to a whole new generation of gamers. Despite only thirty titles being made available right now, Microsoft is planning on having over 1000 games on the service in three years, with new games coming out weekly starting sometime in April. There are tons of customization options and seeing as how you're given a three-story arcade to fill up, there should never be any space issues. In addition, the multiplayer support is nice and they did as much as they could to make the virtual arcade feel as authentic as possible. It's not perfect and some people may not be impressed with the launch titles, but I imagine that with time most of the early classics will be covered. When it comes to pricing I think some people are thinking as though they plan on buying every single game made available. When you look at it from the perspective of only buying the games you really like, plus the added benefit of a free ten minute demo (which is more than enough time to beat some of these games), the pricing system isn't all that awful. Even though it's not perfect, the Game Room is a great idea and players should definitely be checking it out, even if it's only to play the demos.
Note: I'm not going to give any individual categories scores, simply because it would be too varied and unfair. The overall score takes into account the games available, pricing, customization and worth of the service.


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RNA expression data reveals that human embryonic stem (hES) cells differ from mouse ES (mES) cells in the expression of RNAs for keratin intermediate filament proteins. These differences were confirmed at the cellular and protein level and may reflect a fundamental difference in the epithelial nature of embryonic stem cells derived from mouse and human blastocysts. Mouse ES cells express very low levels of the simple epithelial keratins K8, K18 and K19. By contrast hES cells express moderate levels of the RNAs for these intermediate filament proteins as do mouse stem cells derived from the mouse epiblast. Expression of K8 and K18 RNAs are correlated with increased c-Jun RNA expression in both mouse and human ES cell cultures. However, decreasing K8 and K18 expression associated with differentiation to neuronal progenitor cells is correlated with increasing expression of the Snai2 (Slug) transcriptional repression and not decreased Jun expression. Increasing K7 expression is correlated with increased CDX2 and decreased Oct4 RNA expression associated with the formation of trophoblast derivatives by hES cells. Our study supports the view that hES cells are more similar to mouse epiblast cells than mouse ES cells and is consistent with the epithelial nature of hES cells. Keratin intermediate filament expression in hES cells may modulate sensitivity to death receptor mediated apoptosis and stress.
Citation: Maurer J, Nelson B, Ceceña G, Bajpai R, Mercola M, Terskikh A, et al. (2008) Contrasting Expression of Keratins in Mouse and Human Embryonic Stem Cells. PLoS ONE 3(10): e3451. https://doi.org/10.1371/journal.pone.0003451
Editor: Thomas Zwaka, Baylor College of Medicine, United States of America
Received: August 4, 2008; Accepted: September 28, 2008; Published: October 20, 2008
Copyright: © 2008 Maurer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the California Institute for Regenerative Medicine RS1-00283-1(RGO), RC1-00132-1(MM), CL1-00511-1(MM), RS1-00466-1 (AT) and the NIH R37HL059592-11(MM). RB was supported by a CIRM postdoctoral training fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Keratin intermediate filament proteins, keratin 8 (K8, Krt8, EndoA) and keratin 18 (K18, Krt18, EndoB) were first identified in liver and as markers of mouse embryonal carcinoma (EC) and embryonic stem (ES) cell differentiation [1]–[3]. Investigation of early mouse embryos confirmed that the differentiation of the inner cell mass of mouse blastocysts to trophoblast derivatives and extra-embryonic endoderm parallels the induction and accumulation of K8 and K18 [4]–[6]. The mouse inner cell mass initially expresses low amounts of K8/K18 intermediate filaments but then represses expression [7], [8]. Many studies have confirmed the low levels of both protein and RNA for K8 and K18 in mouse ES cells in their undifferentiated state. The isolation of human ES cells that express transcription factors associated with pluripotentiality in mouse ES cells (Oct4, Sox2, Nanog) led to the expectation that epithelial keratins K8 and K18 might be expressed at similar low levels as mES. [9]. Recently a new pluripotent stem cell, the epiblast stem cell (EpiSC), derived from post-implantation mouse embryos was isolated and characterized by two different laboratories [10], [11]. While ES cells and EpiSC cells share characteristics of pluripotency such as the expression of Oct4, Sox2 and Nanog, the gene expression profile of EpiSC includes markers of the embryonic epiblast and resembles human ES cells more than mouse ES cells. Our analysis of the published RNA expression profiles of hES, mES and EpiSC reveals that K8 and K18 RNAs are greatly elevated in EpiSC.
Here we show that expression of K8, K18 and K19 is characteristic of hES cell lines in the undifferentiated state and contrasts with mES cells but is similar to EpiSC. The differentiation of hES to neuronal progenitors results in decreased expression of these keratins and elevated expression of neuronal markers while the spontaneous differentiation of hES cells to presumptive extraembryonic endodermal derivatives results in increased accumulation of K18. Expression of K8, K18 and K19 are characteristic of the epithelial nature of undifferentiated hES cells and contrasts with mouse ES cells.
Differential expression of keratin RNA in human and mouse ES cells
The RNAs for simple epithelial keratins K8, K19 and K19 are expressed at low levels in undifferentiated mES and embryonal carcinoma (EC) cells [2], [3]. Figure 1A shows a typical time course of induction of K8, K18 and K19 RNAs as measured by cDNA array analysis, during the differentiation of mES cells as embryoid bodies. The baseline expression of these genes in undifferentiated ES cultures varies with the extent of contamination from spontaneously differentiated cells. In contrast to mES cells, pluripotent epiblast stem (EpiSC) cells have greatly increased levels of K8 (34 fold), K18 (26 fold) and to a less extent K19 (6 fold) while the pluripotency factors Nanog, Sox2 and Oct4 (Pou5f1) vary little between the two different cell type (Figure 1B). The expression of K8 and K18 RNAs appear coordinately regulated (Figure 1C). Furthermore the increased expression of K8 and K18 RNA in EpiSC is directly correlated with increased expression of Jun (Figure 1D), a component of AP1 transcription factor activity and previously identified as a key regulatory of K18 gene regulation [12], [13], [14], [15]. We suggest that epithelial keratin gene expression is consistent with the definitive polarized epithelial nature of the mouse epiblast.
A, time course of K8, K18 and K19 RNA induction in mouse ES cells during embryoid body differentiation plotted from supplementary data of a published report [33]. Data set is identified by the GSE number of the GEO data base. B, contrasting expression of K8, K18 and K19 in mouse ES and epiblast stem cells (EpiSC). RNA expression data [10] for the indicated genes was compared by normalizing the values of the two cell types to the corresponding signals for beta-actin RNA. Note the elevated expression of K8 (34×) and K18 (26×) in EpiSC while RNAs for Nanog, Sox2 and Oct4 are similar in the two cell types. C, coordinate variation of K8 and K18 RNAs in individual array values of ES and EpiSC samples from GSE7902. D, strong correlation between K18 and Jun RNA levels. Jun is a component of the AP-1 transcription factor activity previously identified as important in the induction of K18 RNA in differentiation mouse ES cells.
In contrast to mES cells, publicly available data show that hES cells and human embryonal carcinoma cells express significant levels of K8, K18 and K19 RNAs [9], [16]–[18]. One example is shown in Figure 2A in which keratin RNA expression of undifferentiated hES cells is compared to cells grown as embryoid bodies. K8 and K18 RNAs are easily detected in hES lines and is modestly increased during embryoid body differentiation. These data are supported by a meta analysis of 38 different array experiments. [19] (http://amazonia.montp.inserm.fr/). While K8 and K18 are expressed in hES cells, differentiation of the cells commonly results in higher expression and thus resulted in the identification of K8, K18 and K19 as under expressed in hES cells compared to differentiated cell types.
A, published results of hES cells and embryoid body cultures [17]. Data was downloaded from http://www.stemcellcommunity.org/. Averages and standard deviations of normalized data of six undifferentiated cell cultures (WA09, BG01, BG02, BG03) and three embryoid body (EB) cultures (BG01, BG02, BG03). Keratin RNA expression in EBs was less than 2× greater than stem cultures. B, coordinate levels of K8 and K18 RNAs. Values for single arrays of different conditions are compared. C, coordinate values of K8 and K19 RNAs. D, correlation of K18 RNA values with Jun RNA, a key transcriptional regulator of the K18 gene.
Examination of the data of individual array experiments revealed that K8 and K18 levels are very tightly correlated (Figure 2B). K19 RNA is also correlated with K8 levels (Figure 2C). In addition, like mES and mEpiSC cells, K18 RNA expression is associated with increased Jun RNA expression (Figure 2D). One quantitative difference between hES and mEpiSC is the relative expression level of K19, which is as strong as K18 in hES cells. While comparison of hES and mEpiSC and mES cells depends on similar sensitivities of measuring K19 and K19 RNAs, western blot analysis of the proteins support the view that hES cells may have greater relative contribution from K19 than mES or mEpiSC cells.
K19 is highly expressed in hES cells
To confirm expression of K8 and K18 in hES cells, we performed western blot analysis with antibodies that recognized both mouse and human forms of K8, K18 and K19. Typical results for K8 and K18 are shown in Figure 3. Human ES cells express moderate levels of K8 and K18 proteins (Figure 3B, lanes 5,6) compared to mouse parietal endodermal cells (Figure 3A, B, lane 3). Mouse ES cells express very low levels of the two keratin proteins (Figure 3A, B, lane 4) as expected from the low RNA levels. Human ES cells also express significant levels of K19 (Figure 3C, 3D) while neither mES nor a mouse parietal endodermal cell line had detectable levels of K19 protein. K19 expression is detectable in trophoblast stem cells and increases upon differentiation in culture (data not shown). These results were confirmed with several additional antibodies that were species specific for either mouse or human keratins. While hES cells express significant levels of K8, K18 and K19, human Caco2 colon carcinoma cells and MCF7 breast cancer cells (Figure 3B, 3D, lanes 7 and 8) express 3–20 fold higher levels of these proteins. These results indicate that hES cells differ from mES cells with regard to expression of K8, K18 and K19 RNA and protein. With mES cells, this work confirms previous studies of mES and mEC cells [2], [12], [13], [20].
Western blots for K8 and K18 were performed with antibodies that recognize both the mouse and human forms of K8 (TROMA1) [6] and K18 (1589) [40]. Antibody reactions were detected and quantitated with Infrared dye labeled secondary antibodies and the LI-COR Odyssey imaging system. A, keratin protein signals were divided by the corresponding tubulin signals. K8 and K18 normalized values were set to 100% for mEndo cells. B, digital image of secondary antibody reaction; MEF (1), mouse embryonic fibroblast; csk (2), non-ionic detergent insoluble fraction of mEndo cells; mEndo (3), the HR9 mouse extraembryonic endodermal cells (4) [43]; Human ES cell lines (5,6); CaCo (7), human colon carcinoma cell line; MCF7 (8), human breast cancer cell line. The upper two panels are from one filter and the bottom two panels are from a second duplicate filter. C, ratio of K19 signal to tubulin signal after background subtraction. D, K19 protein expression in hES cells. Western blot reaction with the L2PK mouse monoclonal antibody to K19 [41], [42]. Tubulin was detected after stripping the K19 antibody. Lanes 1–8 are defined as above.
Human ES cell keratin filaments
Both mouse and human ES cultures commonly develop variable numbers of spontaneously differentiated, fibroblast-like and extraembryonic endodermal cells. Immunofluorescent staining of human ES cells was performed to confirm that keratin expression was due to ES stem cells and not the differentiated progeny. Figure 4 shows typical results of K8, K18 and K19 localization in hES cells. At low magnification, a colony of hES cells grown with mouse fibroblast feeder cells is relatively uniformly reactive with antibodies to both Oct4 and K19 (Figure 4A,B). The mouse feeder cells do not react with either Oct4 or K19 antibodies (Figure 4A). Human ES cells express both K18 (Figure 4C) and K19 (Figure 4A,C) as type I keratin filament proteins. K8 is the primary complementary type II keratin expressed in these epithelial cells (Figure 4D). Similar results were obtained for the H9, H14 and Hues7 human ES cell lines (Figure 4F) and in the presence or absence of feeder layers.
A, low magnification of H9 hES cells surrounded by MEF feeder layer double stained for Oct4 (red) and K19 (green). Feeders do not react with either antibody. B, high magnification of colony shown in A, showing single Oct4 positive ES cells contain K19 positive filaments. C, low magnification of H14 hES cell colony grown on Matrigel without feeders and stained for K18. D, high magnification of H14 hES cells showing K8 network. Nuclei are stained with DAPI (blue). E, spontaneous differentiated cells at edge of H14 hES colony have increased size and K18 expression. F, example of K8 staining of Hues7 hES cell line.
Keratin changes associated with the differentiation of hES cells
Spontaneous differentiation of both human and mouse ES cells occurs commonly, even under conditions promoting ES cell self renewal. Large differentiated cells typically found at the borders of ES cell colonies had increased levels of K8, K18 (Figure 4E) and K19 (Figure 4A). Human ES cells, unlike mES cells, spontaneously differentiate to trophoblast lineage cells. K7 has been found to be a useful marker of human trophoblast lineage [21]. While K7 positive cells are relatively rare in hES cultures they can be detected as discrete colonies of presumptive trophoblast derivatives within undifferentiated hES cell neighbors in cultures incubated for a week or longer. In freshly passaged cells K7 positive cells were detected as dispersed single cells, apparently due to the dissociation of rare colonies of presumptive trophoblasts. (Figure 5A, B). Supporting evidence that the K7 expression reflects trophoblast formation is provided by the correlation of K7 RNA with the CDX2 homeobox master regulatory transcription factor (Figure 5C) in a data set that includes both undifferentiated and differentiated hES cells [17]. Furthermore the expression of CDX2 RNA was inversely correlated with OCT4 RNA (Figure 5D), as expected from the known repression of CDX2 by OCT4 [22], [23].
A, K7 positive colony of cells forming within a H14 hES cell colony. Nuclei are stained with DAPI (blue). B, higher magnification showing extended cytoskeletal pattern of K7 with increased densities at intercellular borders. C, K7 RNA expression is correlated with Cdx2 RNA expression in hES cell cultures [17]. Cdx2 is a master regulator of trophoblast differentiation. D, Cdx2 RNA is inversely correlated with Oct4 RNA expression in differentiating cultures of hES cells.
Simple epithelia keratins are expressed only in the ependymal layer of the ventricles of the adult brain. The differentiation of hES to neural tissues can now be performed routinely. We have confirmed that during the accelerated differentiation of hES cells to neuronal progenitor cells expression of K8 and K18 are decreased (Figure 6A). The K8 and K18 RNAs appear coordinately suppressed (Figure 6B). However, this suppression is not due to decreased Jun RNA expression as there is no correlation of K18 and Jun RNAs (data not shown). Repression is correlated with increased expression of Snai2 (Slug) a transcriptional repressor associated with neural crest formation and epithelial-mesenchymal transition (Figure 6C). Snai2 has previously been suggested as active in K8 repression [24]. Unlike the RNAs for K8 and K18, K19 RNA levels do not reveal a simple trend upon the induction of neuronal progenitor differentiation (Figure 6D). Thus the mechanisms for suppression of K8 and K18 RNAs do not appear to extend to K19 regulation.
Averages o fat least two samples for each time point were used to measure K7, K8, K18 and K19 RNAs as a function of hours after starting induction of neuronal progenitor differentiation. A, K7, K8 and K18 RNA levels over time course of 240 hours. Trendline is for K18 data. B, correlation of K8 and K18 RNA levels during neuronal progenitor differentiation. C, negative correlation of K18 RNA and Snai2 RNA levels. D, K19 RNA levels do not reveal a simple trend.
Some investigations of the hES cells have reported unexpected expression of K8 and K18 because these gene products were expected to be differentiation markers. [9]. Some of these early studies suggested that expression might be due to contamination from differentiated cells or possibly translational regulation. However, a meta analysis of the results of over 38 studies of gene expression in hES cells confirms that K8 and K18 RNAs are commonly found in undifferentiated hES cells [19] and are generally increased in differentiated cells from embryoid bodies. We have confirmed that undifferentiated hES cells express simple epithelial keratin RNAs and proteins as filament networks, typical of other simple epithelial cells, and by contrast with mouse ES cells.
The differences in K8 and K18 expression in mouse and hES cells may reflect a fundamental difference between the embryonic equivalent of the mouse and human inner cell masses. The human epiblast is an epithelial structure while the mouse ICM does not adopt an epithelial organization until after blastocyst implantation. Keratin expression in hES cell lines reflects the more flattened epithelial phenotype of hES colonies compared to mouse ES cells. The expression of keratins may reflect differences in the originating cell types of the respective blastocysts or differences in the the state of acquisition of a stable, self-replicative capacity. The suppression of K8 and K18 expression in the mouse inner cell mass is an active process that may correspond to the transcriptional inhibitory activity detected in embryonal carcinoma cells [13]. The increased expression of K8 and K18 in mouse epiblast stem cells (EpiSC) is consistent with the suggestion that both ES and EpiSC cells lines reflect the characteristics of the embryonic tissue of origin. In transplantation experiments human specific K8, K18 or K19 antibodies may aid in identifying both hES cells and their differentiated progeny.
Mouse EpiSC are poorly compatible with embryo chimerism, at least by standard ES cell methods of blastocyst injection and morula aggregation, but retain the ability to differentiate to multiple tissues as judged by teratoma formation and in vitro differentiation [10], [11]. The strong intercellular adhesive and epithelial nature of EpiSC and hES cells may challenge the integration of EpiSC into preimplantation embryos. Thus ES cells are preferable for gene knockout studies. Mouse EpiSC do provide the opportunity of investigating maintenance of the pluripotent state and perhaps model hES cells. Speculative extrapolation of the similarity between hES cells and mouse EpiSC might question the compatibility of hES cells with early embryonic chimerism.
Differences in expression of simple epithelial keratins in mouse and human ES cells may also have direct consequences on hES cells. These keratins have been implicated in resistance against death receptor mediated apoptosis [25], [26], [27] and stress, possibly through the titration of phospho-protein signaling molecules [26], [28]. Phosphorylated K8/18 networks can titrate phospho-protein binding proteins such as 14-3-3 isoforms and thus impact cell proliferation [29]. Furthermore, expression of the relatively insoluble subunits of simple epithelial keratins carries the risk of protein aggregation induced cellular disease [30]in the event of mutation, imbalance of subunit expression, chemical induced aggregation or deficient degradation [31].
While expression of keratins in hES cells is substantial, accumulated expression in some cells can be much higher. For example in the MCF7 human breast cancer cell line, K18 is among the most abundant proteins within the cells. Similarly, spontaneous differentiated cells arising in hES cultures contain substantially higher accumulation of keratin proteins. The stability and abundance of individual keratins makes them excellent cell type or lineage markers. However, the molecular mechanisms responsible for the cell type specific expression are still obscure. The very close correspondence of RNA levels of K8 and K18 very likely reflect their coordinate regulation from adjacent locations at the distal end of the type II keratin locus on chromosome 12. Both genes are regulated by AP-1 and Ets transcription factor families [14], [32]. Jun activates the K18 gene from an enhancer located in the first intron and from a regulatory element embedded within a coding exon [33]. The coordinate regulation of K8 and K18 may reflect the recent identification of CTCF insulator protein binding sites flanking the two genes on chromosome 12 [34] that suggests a chromosomal regulatory domain. In contrast, the coordinate expression of the K8 and K19 genes occurs despite the separate chromosomal locations of K8 on chromosome 12 and K19 on chromosome 17. The basis of coordinate regulation of pairs of type I and II keratins is not known.
Materials and Methods
RNA expression analysis
Primary RNA expression data from mouse ES cells [35] (Agilent platform, GSE3231), human ES cells [17] (Illumina platform), (www.stemcellcommunity.org) and mouse EpiSC and ES cells (Agilent platform, GSE7902) [10] were downloaded and examined for keratin gene expression in Excel. For some comparisons of different cell types, keratin expression was normalized to signals of same arrays for beta-actin although this normalization did not change the results greatly. Confirmation of hES cell expression of keratins was confirmed in array results available through Amazonia! public database [19] (http://amazonia.montp.inserm.fr/).
Differentiating human ES cells RNAs were isolated with the use of Trizol reagent (Sigma Chemical, St. Louis, Mo). Labeled cRNA was prepared from 500 ng RNA using the Illumina® RNA Amplification Kit from Ambion (Austin, TX, USA). The Biotin labeled cRNA (750 ng) was hybridized 18 hr at 58°C to the HumanRef-8 v2 Expression BeadChip. (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. BeadChips were scanned with an Illumina BeadArray Reader and hybridization efficiency was monitored using BeadStudio software (Illumina) and internal controls built into the Illumina system. Gene expression data was imported into Genespring software.
Cell culture
Human ES cell lines H9 and H14, were cultivated on inactivated mouse fibroblast feeder layers or on growth factor reduced Matrigel coated plastic in Knockout DMEM (Invitrogen) supplemented with 200 mM glutamine, 0.1 mM 2-mercaptoethanol [36] 20% Knockout serum substitute (Invitrogen) and 25 ng/ml recombinant human basic FGF (Sigma, St. Louis, MO) as described [37], [38]. Hues7 and Hues13 cells were propagated as described [39]. D9 mouse ES cells, HR9 mouse extraembryonic endodermal cells and mouse trophoblast stem cells were cultivated as previously described [3], [40], [41]. MCF7 and CaCo2 human tumor cells were obtained from ATCC.
Methods and characterization of accelerated hES cell differentiation to neural progenitors, will be described in detail elsewhere (Bajai, R and Terskikh, A, submitted). In short, small clusters (10–100 cells) of hESCs were grown in uncoated dishes (Costar) in 1∶1 ratio of DMEM/ and F12 medium with N2 (Gibco) and B27 (Gibco) factor supplements, 20 ng/ml insulin 20 ng/ml bFGF, 20 ng/ml EGF and 2 mM N-acetyl cysteine (NAC)). The spheres were grown for 6–8 days, with a change in medium every alternate day. Spheres were collected, gently triturated and plated on ornithine coated (5 ng/ml, Sigma) plates in DMEM/F12, 10% BIT 9500 supplement (Stem Cell Technologies, ), 20 ng/ml bFGF, 20 ng/ml EGF, 5 ug/ml fibronectin, 2 ug/ml heparin).
Protein analysis
For western blot analysis of total keratin proteins, 6 cm dishes of PBS washed cells were dissolved in 200 ul of 9.5 M urea, heated to 100c for 5 min, mixed vigorously and cleared of minimal residual debris by centrifugation. Samples were dilute with concentrated SDS sample buffer and separated in low bis acrylamide gels as previously described [3]. Proteins were blotted onto PVDF membranes according to the instructions of the supplier of the secondary antibodies (Li-COR Bioscience) and detected with 680 or 800 CW IR-dye labeled secondary antibodies. Reaction was imaged and quantitated in a LI-COR Odyssey image analyzer. Filters were stripped in 62.5 mM TrisHCL, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol at 55C for 1 hour and re-probed with antibody to beta-tubulin (E7 mouse monoclonal antibody, (Developmental Studies Hydridoma Bank, Iowa City, Iowa). Results are shown for antibodies that detected both human and mouse form of rat monoclonal antibody to K8 (TROMA1) [6] (gift from Rolf Kemler, available from Developmental Studies Hydridoma Bank); rabbit polyclonal antiserum to mouse K18 (1589) [42] and mouse monoclonal antibody to K19 (L2PK) [43], [44] (gift from Dr. Birgit Lane) are shown. Results were also confirmed with the following antibodies that were species dependent: mouse monoclonal to K8, M20 (Sigma); rat monoclonal to mouse K18, TROMA2 [6], mouse monoclonal to human K18, CK5 (Sigma); mouse monoclonal to human K19, K4.62 (Sigma).
Cells were plated on feeder layer or Matrigel coated glass coverslips, fixed in cold methanol for 10 minutes, rinsed and incubated with PBS-T (PBSCa+/Mg++0.1% Tween20) for one hour at room temperature. Cells were blocked for 20 minutes in PBS supplemented with 2% goat serum (Gibco). For combined detection of Oct3/4 and K19, Oct3/4 antibody (H-134, sc-9081 rabbit polyclonal from Santa Cruz) was diluted 1∶250 in 1% goat serum (GS) in PBS and incubated with the cells overnight at 4°C. Cells were washed with PBS-T and PBS. The second primary antibody (K19, LP2K monoclonal cell culture supernatant) was used neat for one hour at room temperature. After washing, secondary antibodies against the species were combined and diluted 1∶500 in 1% GS-PBS (Alexa 488 anti-mouse and Alexa 546 anti-rabbit from Invitrogen). Cells were incubated with the secondary antibodies for one hour at room temperature in the dark. Washes were repeated as described above. Finally cells were incubated with a DAPI solution for 3 minutes followed by two PBS washes. Other keratins were detected by incubating with either TROMA1 (rat antiK8), M20 (mouse antiK8, 1∶200), 1589 (rabbit polyclonal antiK18, 1∶40), RCK105 (mouse monoclonal antiK7, 1∶2, gift from F. Ramaekers) and the appropriate secondary antibody and the same blocking and washing procedures. Images were acquired on an inverted TE300 Nikon or an OlympusIX71 fluorescence microscope with a Diagnostic Instrument cooled color CCD SPOT RT camera or Hamamatsu Digital Camera respectively. Images were combined in either SPOT or MetaVue 188.8.131.52 software and were formatted for publication with Photoshop software.
We thank Jacqueline Lesperance for technical assistance and Dr. Jean Loring (Scripps Research Institute, La Jolla, CA) for providing public access to her hES cell RNA expression data.
Author Contributions
Conceived and designed the experiments: JM BN GC RB AVT RO. Performed the experiments: JM BN GC RB. Analyzed the data: MM AVT RO. Wrote the paper: MM RO. Concept and design: RO. Design and collection of data: JM BN GC RB. Data interpretation: MM. Design and data interpretation: AVT. Financial support: MM AVT.
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- 5. Jackson BW, Grund C, Winter S, Franke WW, Illmensee K (1981) Formation of cytoskeletal elements during mouse embryogenesis: epithelial differentiation and intermediate-sized filaments in early postimplantation embryos. Differentiation 20: 203–216.BW JacksonC. GrundS. WinterWW FrankeK. Illmensee1981Formation of cytoskeletal elements during mouse embryogenesis: epithelial differentiation and intermediate-sized filaments in early postimplantation embryos.Differentiation20203216
- 6. Kemler R, Brulet P, Schnebelen MT, Gaillard J, Jacob F (1981) Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development. J Embryol Exp Morphol 64: 45–60.R. KemlerP. BruletMT SchnebelenJ. GaillardF. Jacob1981Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development.J Embryol Exp Morphol644560
- 7. Chisholm JC, Houliston E (1987) Cytokeratin filament assembly in the preimplantation mouse embryo. Development 101: 565–582.JC ChisholmE. Houliston1987Cytokeratin filament assembly in the preimplantation mouse embryo.Development101565582
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- 11. Brons IG, Smithers LE, Trotter MW, Rugg-Gunn P, Sun B, et al. (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448: 191–195.IG BronsLE SmithersMW TrotterP. Rugg-GunnB. Sun2007Derivation of pluripotent epiblast stem cells from mammalian embryos.Nature448191195
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- 13. Pankov T, Neznanov N, Umezawa A, Oshima RG (1994) AP-1, ETS, and transcriptional silencers regulate retinoic acid-dependent induction of keratin 18 in embryonic cells. Molecular and Cellular Biology 14: 7744–7757.T. PankovN. NeznanovA. UmezawaRG Oshima1994AP-1, ETS, and transcriptional silencers regulate retinoic acid-dependent induction of keratin 18 in embryonic cells.Molecular and Cellular Biology1477447757
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- 16. Liu Y, Shin S, Zeng X, Zhan M, Gonzalez R, et al. (2006) Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines. BMC Dev Biol 6: 20.Y. LiuS. ShinX. ZengM. ZhanR. Gonzalez2006Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines.BMC Dev Biol620
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- 21. Potgens AJ, Gaus G, Frank HG, Kaufmann P (2001) Characterization of trophoblast cell isolations by a modified flow cytometry assay. Placenta 22: 251–255.AJ PotgensG. GausHG FrankP. Kaufmann2001Characterization of trophoblast cell isolations by a modified flow cytometry assay.Placenta22251255
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- 25. Caulin C, Ware CF, Magin TM, Oshima RG (2000) Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis. Journal of Cell Biology 149: 17–22.C. CaulinCF WareTM MaginRG Oshima2000Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis.Journal of Cell Biology1491722
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- 31. Watson ED, Geary-Joo C, Hughes M, Cross JC (2007) The Mrj co-chaperone mediates keratin turnover and prevents the formation of toxic inclusion bodies in trophoblast cells of the placenta. Development 134: 1809–1817.ED WatsonC. Geary-JooM. HughesJC Cross2007The Mrj co-chaperone mediates keratin turnover and prevents the formation of toxic inclusion bodies in trophoblast cells of the placenta.Development13418091817
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subset: commoncrawl


text: Meet Wondo and his bobblehead at Wells Fargo on Friday September 25, 201312:44PM PDTOlivia Curran Follow@ liv_curran Want a chance to meet Chris Wondolowski and his bobblehead? Head to the Wells Fargo location at 1241 S. Mary Avenue in Sunnyvale on Friday from 5 - 7 p.m. Trust us, it's going to be a party. Just check out the invite below:


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More Information
Whether it is for twins, an infant and a toddler, or your baby and his or her playmate, there are times for strolling with a double. The Tango is designed so that it can be used either with two newborns or with children of different ages. The Tango is all about making your life just a little easier. It folds down to a manageable size, is very easy to use and is full of all the features parents want in a stroller. Importantly, the Zooper Tango sports a lightweight aluminum frame and is sized to fit through standard doors while still seating two children comfortably. It's wheels are fitted with Zooper's smart swivel control feature, to make steering a breeze. The Zooper Tango comes with three free accessories: a winter foot cover, a rain cover, and a glare filter. One year limited factory warranty. Features: Compatible with select infant car seats (on one side only), Suitable for newborns and toddlers up to 45 lbs per seat (90 lbs total), Extra-strong die cast front wheel housings, Extra-strong die cast front latch frame lock , Quick release front wheels with one touch auto swivel lock, One-step rear link brake, High-strength aluminum frame design, Compact flat fold, 30" width fits through most standard doors, Convenient one hand backrest recline (each seat), 4-position seat recline including flat position for newborns, Individual napper canopies with sun visor and viewing window and storage pocket, Zip-off hot weather ventilation full coverage canopy, Large easy access basket, Front and rear wheel suspension, Removable front armrest, Free-standing when folded, 5-point adjustable safety harnesses with newborn and toddler settings, Reflective Safety Piping, Auto Frame Lock, Individual adjustable Footrest, Includes UV Sun Shade, Foot muff, Plastic Cup Holder, and Infant Carrier Security Strap.
Product Specifications
At-a-glance details about this product.
Full Reclinable For Infant: Yes
Turns Into Double Stroller: No
Bassinet / Carry Cot Adaptable: No
Car Seat Adaptable: Yes
Child Minimum Weight / Age: Birth
Child Maximum Weight / Age: 55 lbs
Child Length Range: N/A
Product Weight: 32.2 lbs
Dimensions Unfolded: 40 x 30 x 32 inches
Dimensions Folded: N/A
Warranty: 1 year
What's Included
- Stroller


text:
Does a changing economy mean the "End of Men"?
By Anna Louie Sussman
NEW YORK (Reuters) - For all the bluster of her book's title, "The End of Men: And the Rise of Women," Hanna Rosin is surprisingly ambivalent about whether men are, in fact, doomed.
Women are quicker to adapt to the economy's new demands, Rosin says, pursuing higher education in record numbers and dominating fast-growing professions such as nursing and accounting.
At the same time, men watch the shrinking of manufacturing, construction and other traditionally male industries as if paralyzed, on the couch with a beer in hand.
More than two years of research and reporting, though, have left Rosin unconvinced that the end of men is inevitable.
"It's an obnoxious title," she conceded about her book and the 2010 cover story in the Atlantic magazine that launched it. "And I think my argument would have been much easier to make if I believed that women's brains are one way and men's brains are another way and the economy prefers our brains right now."
Rosin draws on data and anecdotes from a wide range of sources to depict a new global matriarchy.
She charts the feminization of pharmacy work, visiting the University of Wisconsin's pharmacy school in Madison, where 62 percent of the freshman class is female. A group of girlfriends there are aiming for six-figure salaries; one envisions a husband who greets her after work "with a freshly baked cookie."
"The economy is incredibly fluid right now. There are always going to be some kinds of new jobs that the economy throws up, so the question is, who is nimble and willing to get with the program and be responsive? And for whatever reason that's not men," she said. Continued...


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Orla Kiely - Silhouette Cutlery Print Poplin Dress
Vintage inspired dress...simple and pretty pretty pretty.
We went all our for our "The Singing Office" Audition - that included crazy 80s earrings. Wanna join our band? Grab yourself some awesome, totally rad star earrings!Tags: jewelry, earrings, blue, stars, ThisNext, singing office, auditions, 80's jewelryAdded 9 Years Ago from 80s Purple
Now that Polaroid is going away, items like this may have a bit more presence. Although, Polaroids have always had a certain mystery about them.Added 10 Years Ago from 80s Purple
Andy Warhol's muse!Tags: t-shirts, art, artists, shirts, tee shirts, warhol, ThisNext, artsy, Andy WarholAdded 10 Years Ago from 80s Purple
It's retro, cinderella meets leggings chic....and it's a nice grey...gotta love it!


text:
Project PJ (Plastic Jeep)
View Single Post
August 23rd, 2011, 10:43 AM
Direct Drive FTW
Join Date: 09-06-08
Location: Caledonia,MI
Posts: 2,341
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Mentioned: 12 Post(s)
Yes, I know how to weld. But, it has been 10 years so I got a 211 to so it would be easier to relearn and work on this.
No, I have not built anything to this degree mostly just restoring and body work. I have welded up some suspensions and fixed some frames and such but this will me my first on a lot of levels. I have always wanted to build a jeep from the ground up but never had the time or the cash.
CAD, modeling, Animation, has always have been a hobby. I am using Sutdiomax because it will allow me to add all the components(not just the frame) see how they all work together, see how they will flex and slide so I can fix things before I build them and find that the drive shaft will rub the suspension arm's drive shaft guard at... a angle or something.
I am a truck driver because I save almost $1500 a month by staying out here not having a car, house, insurance, fuel cost etc to pay for this way. That and I got sick of being told I was not qualified(when applying due to not having a degree despite years of experience) or working jobs that I could end up dead or handed a pink slip the next day. Driving relaxes me, always has even when I was getting 8mph it relaxed me, that's why I drive.
I am saving up for a house while I try and build this which is part of why I don't mind staying out so much. It is not a smart financial move dropping what I estimate will end up around 15k into this(not counting the 4k in tools now). But I DGAF I have always wanted to do it and I am.
I signed up for a new contract that they say is susposto run a year(it pays really well) the beginning of this month. Like a week before he beginning of this month I had 5 days completely off planned and have everything except the steel there(the bender,notcher,welder, etc). It is enough time to do most of the fish plating, install the uprights, and prep the axles but 4 days before I was going to head home they tossed me this contract and started the 1st. It would not be much progress but it would be enough to show everyone on here I was not just talking BS. Despite wanting to tear this down to the frame grind and fish plating the (non rotted) frame to avoid flexing I could not realistically turn down the offer.
I am currently fixing the huge FU in the uprights and have (re re re) drawn up how all the panels and glass will need to slide to be removed while not interfering with the inner cage which is still being reworked.
It is a lot of design, check, design, check. But it is all in a effort to reduce the use of a BFH to remove and rebuild.
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text:
Men's Team Leads C-USA Championships
May 11, 2012
New Orleans, La. - The UTEP track and field men's team takes the lead in the first day of competition at the 2012 Conference USA Outdoor Track and Field Championships in New Orleans, La. on Friday.
The men's squad is in first place accumulating a total of 25 points, followed by Tulsa with 15 points and Memphis with 13 points overall.
Freshman Demetris Elia claimed silver in the men's hammer throw Friday afternoon. Elia put the Miners on the board with a mark of 59.67m (195-09) earning the men's team eight points.
The distance team took the track for the men's 10,000m giving UTEP the lead in the leaderboard. Elkana Rotich finished second in the race with a season-best of 30:50.99. Elphas Maiyo crossed the finish line fourth with a time of 31:00.54, following closely behind was Solomon Gardea taking fifth with a time of 31:07.63.
"Although I was not in the lead at the beginning, I stayed close to Solomon Gardea and Elphas Maiyo" said Rotich. "I wanted to show my teammates and UTEP that together we can accomplish our goal."
Senior Kathya Garcia took second place in the women's 10,000m posting a time of 35:25.52. Sister Karina Garcia registered a time of 36:42.98 taking seventh place overall.
Senior Marine Menez leads the women's team in the heptathlon, finishing second in the 100m hurdles with a personal-best of 14.17. The senior clocked in a time of 25.66 in the 200m taking fourth overall. Menez sits at No. 6 in the heptathlon after only four events.
Freshman Aeisha McDavid currently stands in ninth with 2,918 points and El Paso native Jallycia Pearson is 10th with 2,878 points.
The trio will take the track and field tomorrow in the long jump, javelin throw and 800m run set to begin at 11 a.m. (CT).
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text:
https://groups.diigo.com/group/axisinsurance/content/axis-capital-to-release-second-quarter-financial-results-on-july-29-2014-12457519 PEMBROKE, Bermuda—(BUSINESS WIRE)—Jun. 17, 2014— AXIS Capital Holdings Limited ("AXIS Capital") (NYSE:AXS) today announced that it expects to release financial results for the second quarter ended June 30, 2014 on Tuesday, July 29, 2014, after the close of the financial markets.
Albert Benchimol, President and CEO, and Joseph C. Henry, CFO, will host an investor teleconference, including a question and answer period, on Wednesday, July 30, 2014, at 8:00 AM (Eastern) to discuss the second quarter results as well as related matters.
The teleconference can be accessed by dialing (888) 317-6003 (U.S. callers) or (412) 317-6061 (international callers) and entering the passcode 1-1-1-2-6-8-7 approximately ten minutes in advance of the call. A live, listen-only webcast of the call will also be available via the Investor Information section of the Company's website at www.axiscapital.com.
A replay of the teleconference will be available for three weeks by dialing (877) 344-7529 (U.S. callers) or (412) 317-0088 (international callers) and entering the passcode 1-0-0-4-8-1-3-7. The webcast will be archived in the Investor Information section of the Company's website.
About AXIS Capital
AXIS Capital is a Bermuda-based global provider of specialty lines insurance and treaty reinsurance with shareholders' equity attributable to AXIS Capital at March 31, 2014 of $5.8 billion and locations in Bermuda, the United States, Europe, Singapore, Canada, Australia and Latin America. Its operating subsidiaries have been assigned a rating of "A+" ("Strong") by Standard & Poor's and "A+" ("Superior") by A.M. Best. For more information about AXIS Capital, visit our website at www.axiscapital.com.
Source: AXIS Capital Holdings Limited
Investor: AXIS Capital Holdings Limited Linda Ventresca, 441-405-2727 email@example.com
Media: Kekst and Company Michael Herley, 212-521-4897 firstname.lastname@example.com
For more information: http://www.dailykos.com/news/Specialty%20Insurance%20Axis%20Capital%20Group%20Singapore%20Jakarta%20Indonesia%20Bermuda http://social.technet.microsoft.com/Forums/en-US/d1dd880f-fdee-4dd3-be13-7d7746144ae5/what-does-the-investors-accident-health-axis-capital-group-hartford-jakarta-indonesia-kansas?forum=SCMDM


text:
TennisForum.com (http://www.tennisforum.com/)
- Media (http://www.tennisforum.com/914-media/)
- - R.I.P. Megaupload (http://www.tennisforum.com/914-media/452123-r-i-p-megaupload.html)
R.I.P. Megaupload
all those tennis matches on megaupload :o
Popular file-sharing website Megaupload shut down
McLEAN, Va. (AP) — One of the world's most popular file-sharing sites was shuttered Thursday, and its founder and several company officials were accused of facilitating millions of illegal downloads of films, music and other content.
An indictment accused Megaupload.com of costing copyright holders at least $500 million in lost revenue. The indictment was unsealed one day after websites including Wikipedia and Craigslist shut down in protest of two congressional proposals intended to make it easier for authorities to go after websites with pirated material, especially those with headquarters and servers overseas.
Megaupload is based in Hong Kong, but some of the alleged pirated content was hosted on leased servers in Ashburn, Va., which gave federal authorities jurisdiction, the indictment said.
The Justice Department said in a statement said that Kim Dotcom, 37, and three other employees were arrested Thursday in New Zealand at the request of U.S. officials. Three other defendants are at large.
Before Megaupload was taken down, it posted a statement saying allegations that it facilitated massive breaches of copyright laws were "grotesquely overblown."
"The fact is that the vast majority of Mega's Internet traffic is legitimate, and we are here to stay. If the content industry would like to take advantage of our popularity, we are happy to enter into a dialogue. We have some good ideas. Please get in touch," the statement said.
The indictment may have prompted a response from the loose affiliation of hackers known as "Anonymous," which claimed credit for attacking the Justice Department's website. The site was inaccessible Thursday evening.
Megaupload was unique not only because of its massive size and the volume of downloaded content, but also because it had high-profile support from celebrities, musicians and other content producers who are most often the victims of copyright infringement and piracy. Before the website was taken down, it contained endorsements from Kim Kardashian, Alicia Keys and Kanye West, among others.
The company listed Swizz Beatz, a musician who married Keys in 2010, as its CEO. He was not named in the indictment and declined to comment through a representative.
According to the indictment, Megaupload was estimated at one point to be the 13th most frequently visited website on the Internet. Current estimates by companies that monitor Web traffic place it in the top 100.
The five-count indictment, which alleges copyright infringement as well as conspiracy to commit money laundering and racketeering, described a site designed specifically to reward users who uploaded pirated content for sharing, and turned a blind eye to requests from copyright holders to remove copyright-protected files.
For instance, users received cash bonuses if they uploaded content popular enough to generate massive numbers of downloads, according to the indictment. Such content was almost always copyright protected.
The site boasted 150 million registered users and about 50 million hits daily. The Justice Department said it was illegal for anyone to download pirated content, but their investigation focused on the leaders of the company, not end users who may have downloaded a few movies for personal viewing.
A lawyer who represented the company in a lawsuit last year declined comment Thursday. Efforts to reach an attorney representing Dotcom were unsuccessful.
Megaupload is considered a "cyberlocker," in which users can upload and transfer files that are too large to send by email. Such sites can have perfectly legitimate uses. But the Motion Picture Association of America, which has campaigned for a crackdown on piracy, estimated that the vast majority of content being shared on Megaupload was in violation of copyright laws.
The website allowed users to download some content for free, but made money by charging subscriptions to people who wanted access to faster download speeds or extra content. The website also sold advertising.
The indictment was returned in the Eastern District of Virginia, which claimed jurisdiction in part because some of the alleged pirated materials were hosted on leased servers in Ashburn, Va. Prosecutors there have pursued multiple piracy investigations.
Steven T. Shelton, a copyright lawyer at the Cozen and O'Connor firm in New York, said opponents of the legislation are worried the proposals lessen the burden for the government to target a wide variety of websites. Shelton said he expects to see the government engage in more enforcement in the future, as technology makes it easier to catch and target suspected pirates.
"I think we'll be seeing more of this," he said. "This is just the beginning."
Dotcom, a resident of both Hong Kong and New Zealand, and a dual citizen of Finland and Germany, made more than $42 million from the site in 2010 alone, according to the indictment.
Dotcom had his name legally changed. He was previously known as Kim Schmitz and Kim Tim Jim Vestor. He is founder, former CEO and current chief innovation officer of Megaupload.
Officials estimated it could be a year or more before Dotcom and the others arrested in New Zealand are formally extradited.
The others arrested were Finn Batato, 38, a citizen and resident of Germany, the company's chief marketing officer; Mathias Ortmann, 40, a citizen of Germany and resident of both Germany and Hong Kong, who is the chief technical officer, co-founder and director; and Bram van der Kolk, aka Bramos, 29, a Dutch citizen and resident of both the Netherlands and New Zealand, who oversees programming.
Still at large are Julius Bencko, 35, a citizen and resident of Slovakia, the site's graphic designer; Sven Echternach, 39, a citizen and resident of Germany, head of business development; and Andrus Nomm, 32, a citizen of Estonia and resident of both Turkey and Estonia, head of the development software division.
Several sister sites were also shut down, including one dedicated to sharing pornography files.
Copyright © 2012 The Associated Press. All rights reserved.
Re: R.I.P. Megaupload
This sucks. :o
Re: R.I.P. Megaupload
I know. megaupload was my favorite site for download
Re: R.I.P. Megaupload
Re: R.I.P. Megaupload
16 Attachment(s)
I will miss you buddy. :tears:
Re: R.I.P. Megaupload
Re: R.I.P. Megaupload
:tears: It will be missed badly :tears:
Re: R.I.P. Megaupload
the 90% of matches here were on megaupload
solution is p2p sharing
server sharing brings to these stuffs :tape:
Re: R.I.P. Megaupload
16 Attachment(s)
megaupload used to be SO GOOD. :tears:
Re: R.I.P. Megaupload
Megaupload :sad:
I don't really get how the owners are being charged with so many offences, when you uploaded something to the site you checked a box which acknowledged that you owned the copyright etc.. and have read the rules and regulations....can't see how the owners are to blaim for what the users upload. btw - Their even trying to charge the site's graphic designer!? WTF :help:
The thing that gets me, YouTube is far worse at breaching copyright, whenever you report something...nothing gets done about it...yet because they now having a connection with Google they get away with it :o Doubles standards...
Re: R.I.P. Megaupload
The vast majority of tennis matches uploaded over the past five or six years... gone in an instant. Depressing. :sad:
Re: R.I.P. Megaupload
I guess we are all gonna have to start the painful task of uploading what we have to uploadbox or something....
Re: R.I.P. Megaupload
You have got to be fucking kidding me.
Re: R.I.P. Megaupload
well :sobbing: http://www.mediafire.com/ is much better than Uploadbox though... and stores files as long as Megaupload (I uploaded something more than a year ago and the links still work) only downside is the 200 mb limit for free accounts... so the files will need to be cut to smaller parts. ...This is just horrible to all of you who have ever uploaded anything to Megaupload.
(Mediafire has 150 million visit every month and most of the top 500 companies in the world use it, so it won't be gone just like that within few years)
Re: R.I.P. Megaupload
and so and so
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The Best Films of 2013 | 5 Jan 2014 // 9:13 PM
After a shaky start, 2013 ended up a stellar cinematic year, covering subjects as varied as young love, '70s (and '80s and '90s) con men, our country's horrific history of slavery, and perhaps most importantly, giant robots battling equally elephantine alien monster.
For Infidelity: Reconsidering Aesthetic Anachronism | 17 Sep 2013 // 10:14 PM
Being faithful to the original can require removing it from its context. It's a necessary and paradoxical reversal.
The Riotous Excursions of 'The Great Gatsby' | 27 Aug 2013 // 12:10 AM
Despite the anachronisms, director Baz Luhrmann attempts to not only stay true to the spirit of F. Scott Fitzgerald's book, but also to the way the book was perceived in its own period.
'The Great Gatsby': Excess and Then Some | 10 May 2013 // 2:30 AM
This Jazz Age is presented in the film as so many brightly colored, manic fantasies, in frantic, crowded dance scenes, in speedy, deliriously unconvincing driving scenes, and even conventionally awkward, filtered-light sex scenes.
'The Great Gatsby' Proves That Baz Luhrmann is Our Disco Michael Bay | 23 Aug 2013 // 8:00 AM
One of the year's worst films, Luhrmann's trashy, overhyped, faux- Jazz Age spectacle is so shallow as to be creatively nonexistent.
A "Great" Gatsby? Absolutely! | 9 May 2013 // 7:25 PM
Luhrmann's Gatsby may be the most boisterous downer ever put to film. By playing up the highs, the director increases the depth of the lows.


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As a leading pediatric orthopedic center, TSRHC has treated more than 245,000 children since its inception, with more than 40,000 clinic visits each year. The hospital takes a multidisciplinary approach to care, tailoring treatment to the individual needs of each child and family.
TSRHC is proud of the healing, hope and happiness we have shared with thousands of patients and their families. At TSRHC, every staff member is dedicated to creating a positive hospital experience for our patients – so that they can create their own experiences out in the world.
Help make dreams possible. Your gift allows the hospital to continue its mission of helping children regardless of the family's ability to pay.
Within a unique caring environment, patients at TSRHC benefit from a world-renowned medical staff and the promise of a brighter future, through the research and innovation driven by the hospitals five centers of excellence. TSRHC is consistently recognized as one of the premiere pediatric orthopedic hospitals in the world.
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The word orthopedic refers to the correction or prevention of bone deformities. Orthopedic conditions may be present at birth, such as clubfoot, or may develop over time, such as scoliosis.
TSRHC physicians treat thousands of patients with a wide range of orthopedic conditions each year. Our orthopedic surgeons focus on the treatment, management and rehabilitation of children with orthopedic conditions.
The hospital's long-standing history of treating orthopedic conditions began more than 90 years ago. Today, our physicians are considered leaders in the field of pediatric orthopedics.
The physicians at Texas Scottish Rite Hospital for Children wrote the book on the treatment of orthopedic conditions affecting children. Now in its fifth edition, the Tachdjian's Pediatric Orthopedics: From Texas Scottish Rite Hospital for Children textbook is the standard reference guide for orthopedic surgeons around the world.
Our Fracture Clinic is located in our North Campus in Plano and specializes in the diagnosis, management and treatment of new fractures. Visit our Fracture Clinic page on the website to find out more information.
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Xavier and Debari check-in from the bottom of the world - Mission Antarctic Video Dispatch 2
I just posted about this last week but the Timeline crew has dropped this little nug of a teaser from the action going down right now in the Antarctic and I just had to share. Looks like the boys have found some epic terrain and are already bagging serious lines. They've also let the cat out the bag with a secret filming weapon as it seems like they brought along a (gas-powered?) paraglider for aerial shots. It all looks so insane! Click through for more links and ways to follow the trip.
Check out the article from last week for all sorts of links where you can follow the trip, including a constantly updating blog over at TWSNOW.


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Dead U. Of Chicago Student Goes Unnoticed In Dorm Room For Days
No ones knows how long 20-year-old Nicholas Barnes lay dead in his dorm room before he was discovered.
In fact, it was complaints from other students about a foul smell coming from Barnes' room that lead to his discovery, suggesting the body had been there for some time.
The university indicated that there was no sign of foul play, the Chicago Tribune reports, but a cause of death has not been determined yet.
Photo credit: Flickr.com/Great Beyond


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I recently posted about our search for a new coffee maker, and thought I'd follow up with a quick post about our current equipment. If you love coffee, you need to read this.
For the best taste and texture, coffee must be brewed at a temperature of between 197°F and 205°F (92 – 96° Celsius), and the optimal brew time is around 5 minutes. A typical department store drip coffee maker brews coffee at much lower temperatures than mentioned above – as low as 140°F. Over the years we've used a number of different drip makers, most of them average at best. Our recent experience with a Cuisinart unit was a mixed bag: it made good coffee, but it was the quintessential embodiment of planned obsolescence; it broke after 11½ months.
Our current set up is below – a Technivorm "MoccaMaster" KB-741 and a Capresso Infinity 560 Burr Grinder. We're using filtered tap water, beans from the local Caribou Coffee and Old Bisbee Roasters in Arizona, and are using a SwissGold #4 cone filter.
I'll spare you all most of the flowery prose – suffice it to say, the coffee we're brewing with this combination has a mouthfeel that's silky and lush, is full flavored, and has no bitter aftertaste – or taste – whatsoever. There is a huge gulf between the quality of coffee possible with the right equipment, good water and beans, and what you'll produce with a garden variety $25 department store drip machine and supermarket ground coffee.
I certainly don't want to encourage spending on things that don't matter to you, but if you care about coffee, you ought to look into upgrading your equipment. And for the record, I have no connection whatsoever with the companies mentioned in this post.
4.16.2011 Edit:
Check out this video by George Howell for tips from a coffee guru:
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11 Comments on Our path to great coffee
I have never even heard of cold brewing. Very interesting. Kind of like sun brewed tea.
I use the old style hand filter method. I use Melitta filters and a filter holder that is set directly on top of the coffee pot. I boil filtered water and pour it into the coffee filter. I do wait just some seconds for the boil to subsist, than I pour. It takes about three pours for one liter. I take great care to "wash" the sides of the filter cone because coffee will stick to the sides. So on the second and third pour, I pour the water down the sides to use that coffee. Super simple, super low cost, basically no equipment needed, hardly anything that can break, easy to stow and everybody loves my coffee.
I use either German coffee (Jacobs Kroenung, Dallmayr Prodomo) or coffee from a local coffee specialty shop here in Austin. Pure arabica usually and fair trade to boot.
Some might say that this method takes too long but I disagree. The actual pour time is about 30 seconds. I do something else in the kitchen while the water gets to a boil and while the coffee is infusing. When the coffee is ready so is my breakfast and I sit down and start to eat.
Works the same for tea, of course.
This coffee maker has been our best investment. We have a local coffee shop, who reccomended this and who roasts his coffee just the way you like it, there is nothing better! In case you need the coffee maker or coffee his name is Joe at Storehouse coffee.com
This makes me nostalgic for regular grind and a percolator.


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The Indexer vol 27 No 3 September 2009
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This September 2009 edition of The Indexer is guest-edited by the China Society of Indexers. It traces the history of indexing in China and reflects on how it stands today. Articles include: 'Term... More > selection: the key to successful indexing' Zhang Qiyu; 'GB/T 22466-2008 – Guidelines for indexing: China's new national indexing standard' Wen Guoqiang and Guo Lifang 'Wan Guoding (1897–1963), indexing pioneer' Wang Yage and Hou Hanqing; 'A Chinese typewriter: part of a patent filed by Lin Yutang'; 'Republic of China (1911–49): retrieving the past' Wu Peijuan; 'Example-based text categorization (EBTC); the key to automatic indexing and classification?' Xue Chunxiang and Hou Hanqing; 'Indexing software in China: past, present and future' Wang Yanxiang; 'Around the world' Edited by Glenda Browne; 'A ha'p'orth of tar' John Sutherland; 'Linux and the indexer – a note' Jonathan Jermey< Less
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The Indexer vol 31 No 2 June 2013
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The journal of the Society of Indexers. Articles include: The National Index to Chinese Newspapers and Periodicals (NICNP) in the digital age (Han Chunlei); The Chinese Periodical Full-Text Database... More > (1911-1949): searching the literature of the Republic of China (Xu Shu); Ereaders: exploring how well they work (Pierke Bosschieter); Newcomers: readers' comments (Adam LeBrocq, Sylvia Coates, Ann Hudson, Elena Gwynne and Linda Stumbaugh); XIndex: a cautionary tale (Denise Sutherland); EPUB3 versus HTML5 (Ori Idan); Tools of Change (Pilar Wyman); Portrait of the indexer? (Susan Curran); Letter: A tale of two cultures?; ANZSI conference 2013 (Carol Dawber and Sandy Liddle); Bibliography and the indexer; cullings from the Almanacco bibliografico (Maureen MacGlashan); Plus a Centrepiece featuring: Chinese, Japanese and Korean (CJK) names: resources for the indexer (Lam Lai Heung); Asian names in an English-language context: negotiating the structural and linguistic minefield (Fiona Swee-Lin Price)< Less
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The journal of the Society of Indexers. Articles include: 'Structured data, standards and indexes' (Sally Goodenough); 'The Matrix: creating an active index in all kinds of formats, from all kinds... More > of tools' (Jan Wright, Glenda Browne, Michele Combs, David Ream and Pilar Wyman); 'Working smarter, not harder, with the KPS Indexing Plugins' (Lucie Haskins); 'EPUB indexes and the future of indexing revisited' (Bill Johncocks); 'In defense of multiple indexes: or the index as learning tool' (Florian Ehrensperger); 'On indexing the Argus' (John Hirst); plus a Centrepiece featuring: 'Successful subheadings' (Fred Leise); 'Numbers in indexing' (Max McMaster).< Less
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The journal of the Society of Indexers. Articles include: The secret lives of indexers (Judith Pascoe); Holding hands with the past: indexing historical documents (Kate Mertes); History indexes... More > reviewed (Catherine Sassen); EPUB3 indexes and the future of indexing (Glenda Browne/Jan Wright/Michele Combs); "Bartlett's familiar quotations", 18th edition for mobile app (Kay Schlembach/Seth Maislin); ISC/SCI Conference 2013 (Max McMaster); SI Conference 2013 (Hilary Westwood); South Australia Red Cross Information Bureau Records (Janet Wilkinson/Andrew Piper); plus a Centrepiece featuring: Author citations and the indexer (Sylvia Coates); Tips for newcomers: Wellington 2013 compiled (Jane Douglas); Reflections on the Wilson judging for 2012 (Margie Towery).< Less
The Indexer vol 33 No 1 March 2015
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The journal of the Society of Indexers. Articles include: A banket of South African words and expressions to tax the toughest of indexers (John David Linnegar); ANZSI multi-location Council and... More > proposed restructure (Glenda Browne); DNI ten years on (Jochen Fassbender); Taxonomies, folksonomies, ontologies: what are they and how do they support information retrieval? (Madely du Preez); Linked Data and music: current opportunities (Elizabeth Joan Kelly); Pictures into words (Brian Stewart).< Less
The Indexer vol 30 No 1 March 2012
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The March issue of The Indexer is an extended issue, dedicated to consideration of issues relevant to indexing in the digital age. Articles include: 'digital trends' Jan Wright (ASI); 'The tyranny... More > of the page' Mary Coe (ANZSI); 'New technology and public perceptions' Bill Johncocks (SI); 'The devil is in the details: indexes versus Amazon's X-Ray' Jan Wright (ASI); 'Hand-helds as ereaders: exploratory thoughts on hand-held devices and indexes' Pilar Wyman (ASI); 'Visualizing back-of-book indexes' Ceilyn Boyd and Mitch Wade (ASI); 'The semantic web: an introduction for information professionals' Matt Moore (Australia); 'Publishing, XML and indexers' Nic Gibson (UK); 'XML indexing' Michele Combs (US); 'Web resources for indexers' Pierke Bosschieter (NIN). The reviews section includes a review of Peter Meyer's 'Breaking the page' in which he argues the importance of including indexes in electronic publications.< Less
The Indexer vol 30 No 4 December 2012
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The Indexer vol 34 No 1 March 2016
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The journal of the Society of Indexers. Articles include: TIIARA for an IDOL: an adventure in indexing (Elaine Ménard); Scholarly publications in Latin America: where, oh index, art thou?... More > (Ruby Meraz Gutierrez); Creation, placement, and design of website indexes on university websites in the United States (Ilana Kingsley); Ebook navigation: browse, search and index (Glenda Browne and Mary Coe); Windows 8: what's better, what's worse (Kamm Schreiner); The ANSZI Medal 2012: some thoughts on what makes a prize-winning index (The ANZSI Medal Committee); A publisher's job is to provide a good API for books: you can start with your index (Hugh McGuire); Letter: a response to Bill Johncocks; Obituary: Doreen Blake; Indexes reviewed (Christine Shuttleworth); Book reviews (Christopher Phipps and Michael E. Jackson)< Less
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This will be a guide to most of the undercarriage suspension/powertrain torque specs. I think it's a good idea to go thru these prior and after track sessions (or as part of an annual maintenance program). Things do shake loose, especially after time or stress.
I use rhino ramps- allows me to get to all the "front" bolts/nuts and then in the rear for the back stuff. Most of your suspension stuff needs to be torqued under load ie on ramps, so this accomplishes this requirement.
A couple of other things, many of the rear components are difficult (or impossible) to get to w/ a torque wrench- these you will have to use your best judgement on how to (and how much) to tighten. Many can best accomplished by doubling up w/ combination wrenchs for more leverage. I like to use 6 point stuff (including combination wrenches) for most of the bigger stuff, less chance of rounding off a nut/bolt off.
I use two torque wrenches- a 1/2" drive for the bigger stuff (60 ft lbs +) and a 3/8" for the smaller stuff (under 60 ft lbs). I use the Craftsman digi torq models as you simply dial in the torque number looking into a small viewer. You will appreciate (and need) a assortment of extensions.
IMPORTANT!!! I've tried to carefully edit all of the specs, if you see any that are wrong please let me know. Also note these are from the Impreza 2002 service manual- other models/years may differ.


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Have a 1993 VW Eurovan with the 2.5 litre 5 cylinder. Only 79,xxx original miles, all stock with nothing adulterated. Brand spanking new C rated Michelin tires all around on stock steel 15" wheels with stock clean hub caps. Fixed A/C and it blows super cold all three rows (even on the 100+ degree days!).
Now the nitty gritty: I changed all the fluids and ran Rislone in the Oil and Techron in the fuel to clean everything up. It has sat for over 3 years before I bought it and it has developed several problems in the last 6 weeks. The engine will infrequently blow out blue and or black smoke. I assume from the valve stem seals when it gets loaded up idling at a long stop light, otherwise the engine runs great and strong and has started everytime, no problems since the new battery!
Transmission shifts well. Likes to hang third gear a little and then thump into fourth gear but I figure its a trait of these underpowered vehicles.
Exhaust rattle. Infrequent and only at a certain idle. Have been advised its the Cat. Could get an aftermarket fit piece for under $70 or straight pipe it.
Slight rattle with engine speed increase. Most likely an idler pulley. Worse case scenario, mal adjusted lifters. Not even certain if these engines have the solid or hydraulic lifters. If solid, just needs to be clearanced with new shims.
The big headache: Had a blown boot on the outside passenger axle. Replaced the whole half shaft with no resolve to the following problem:
Intermittent knocking while cruising. AND, only in right turns there is a god awful grinding, humming and slight pulling sound/feeling. Could make left turns at 35+ mph with just a little 'whir' from the tires, but 15 mph in a right turn is catastrophic sounding. Have been told its bearings and could involve some hub chewing.
Coolant and fuel gauge work sporadically. Have been told excessive vibration will shake solder joints loose and its a problem with the gauge set that's easily repairable. Speedo, ODO and tach work fine.
Bottom line, the body and interior are in phenomenal condition regardless of age or mileage and could be even better with a thorough cleaning and buffing. Very few, very minor rust spots (only spots thus far, not pitting) that were spray painted over by PO.
Overall, its nice, but I'm not sure if I feel like putting the time or money into this thing. It drove fine before I bought it and the following week but has developed these problems with continued use. Looking to unload it for $2800 or at the minimum get some advice and hints from the forum about the problems I listed above so I'm not just hunting and randomly putting parts into it, cuz clearly the new axle fixed nothing.
Any help in this scenario or interest to see it are quite welcomed. Uncertain how to host/post pics so if you want some I will email them as requested or if in the Southwest Florida area you can see in person. PM, email, or just post. Thank you!