prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What is the scaling form for the alternative order parameter O?

Read the following text and answer briefly.

Context 1: \section*{Dynamical Behaviour of $O$ in Lattice Gases}

The dynamical behaviour of the anisotropic order parameter  $m$ [see Eq ~\eqref{eq:def-m} in the Letter] following a quench to the critical point is well described by
the Gaussian theory for all the three lattice gas models studied, $i e ,$ driven lattice gas with either constant (IDLG) or random (RDLG) infinite drive and equilibrium lattice gas (LG)  In other words, in the short-time regime, $m \sim t^{1/2}$ [see Eq  \eqref{eq:mt}] and the Binder cumulant $g$ of the lowest transverse mode [defined in Eq  \eqref{eq:binder}] is zero in this regime  The alternative order parameter $O,$ however, distinguishes between the driven (IDLG, RDLG) and the equilibrium (LG) lattice gases  

In order to understand  this, we first write the phenomenological scaling form for $O$,  analogous to Eq  \eqref{eq:scalingass} in  the Letter,
\begin{eqnarray}
O (t, L_{\parallel} ; S_\Delta) = L_{\parallel}^{-\beta/[\nu(1+\Delta)]} \tilde f_O (t/L_{\parallel}^{z/(1+\Delta)} ; S_\Delta) \quad
\label{eq:Oscalingass}
\end{eqnarray}
We already remarked that, in the LG, this scaling form is not compatible with the prediction $O \sim t^{1/8}  L_{\parallel}^{-1/2}$ of the Gaussian theory   However, following Ref  \cite{AS2002}, it can be argued that, at short times, the only dependence of $O$ on the system size $L_{\parallel}$ is of the form $O \sim L_\parallel^{-1/2}$ which is very well confirmed by numerical simulations  Accordingly,  the generic behaviour of $O$ can be assumed to be
\begin{eqnarray}
O \sim t^{\alpha} L_\parallel^{-1/2}, \label{eq:O}
\end{eqnarray}
where $\alpha$ is a phenomenological exponent to be determined  This, along with Eq  \eqref{eq:Oscalingass}, implies $\tilde f_O(x) \sim x^{\alpha} $ Comparing the finite-size behaviour in Eq ~\eqref{eq:O} with Eq ~\eqref{eq:Oscalingass} one actually infers,
\begin{eqnarray}
\alpha &=& \frac{1+ \Delta -2 \beta/\nu}{2 \, (4- \eta)}  \label{eq:alpha}
\end{eqnarray}
This equation, together with the hyperscaling relation $\Delta - 2 \beta/\nu= - \eta$ in two spatial dimensions, shows that the prediction $\alpha = 1/8$ of the Gaussian theory [see Eq  \eqref{eq:Ot}] can be obtained only when $\eta=0,$ which is the case for the IDLG (exactly) and the RDLG (approximately) but not for the LG  

On the other hand,  Eq ~\eqref{eq:alpha} predicts $\alpha = 1/10$ upon substituting the values of the critical exponents corresponding to the Ising  universality class (LG)  This is consistent with the numerical simulation results presented in the main text, see Fig  \ref{fig:ising}(b) therein 

\begin{figure}[th]
\vspace*{0 2 cm}
 \centering
 \includegraphics[width=10 cm]{ /compare_binder pdf}

\caption{Comparison between the temporal evolution of the Binder cumulants $g$ corresponding to the $12^{th}$ transverse mode, $i e ,$ with $n_\perp =12,$ in the LG (lowest curve), IDLG and RDLG (two upper curves) on a $32 \times 32$ lattice  \label{fig:b}}
 \label{fig:binder}
\end{figure}


The emergence of this new value $1/10$ of the exponent $\alpha$ must be traced back to the non-Gaussian nature of higher fluctuating modes in the LG  In fact, even though the lowest mode behaves identically in all the three models we considered,  characterized by the same behaviour of $m$, higher modes show a significant difference in the non-driven case   


To illustrate this, we measured the Binder cumulants of higher modes which is defined  analogously to Eq ~(11), using transverse modes other than the first, i e , with $\mu=\tilde \sigma(0,2 \pi n_\bot/L_\bot)$ and $n_\bot>1 $  
 Figure \ref{fig:b} compares the same for all the three lattice gases for the mode with $n_\perp =12$ on a $32 \times 32$ lattice  Clearly, the curve corresponding to the LG (lowest, blue) departs from Gaussian behaviour $g=0$ (in practice, $e g ,$ $|g| \lesssim 0 005,$ corresponding to the shaded gray area) much earlier than it does for the IDLG  or RDLG (two upper curves, red and green respectively) 

Accordingly, the different dynamical behaviour of $O$, which involves a sum over all modes, can be attributed to the non-Gaussian nature of the higher modes in the LG  
Such a departure is not entirely surprising  In fact, for higher modes, mesoscopic descriptions such as the ones in Eqs  \eqref{eq:L-DLG} or \eqref{eq:g_evol} are not expected to hold, while the anisotropy at the microscopic level could be the mechanism leading to the Gaussianity of higher modes in the driven models 


Context 2: The future of mobile CPUs, part 1: Today’s fork in the road | Ars Technica
2013 may be a big year for the evolution of smartphones and tablets 
Mobile computing's rise from niche market to the mainstream is among the most significant technological trends in our lifetimes  And to a large extent, it's been driven by the bounty of Moore’s Law—the rule that transistor density doubles every 24 months  Initially, most mobile devices relied on highly specialized hardware to meet stringent power and size budgets  But with so many transistors available, devices inevitably grew general-purpose capabilities  Most likely, that wasn't even the real motivation  The initial desire was probably to reduce costs by creating a more flexible software ecosystem with better re-use and faster time to market  As such, the first smartphones were very much a novelty, and it took many years before the world realized the potential of such devices  Apple played a major role by creating innovative smartphones that consumers craved and quickly adopted 
To some extent, this is where we still stand today  Smartphones are still (relatively) expensive and primarily interesting to the developed world  But over the next 10 years, this too will change  As Moore’s Law rolls on, the cost of a low-end smartphone will decline  At some point, the incremental cost will be quite minimal and many feature phones of today will be supplanted by smartphones  A $650 unsubsidized phone is well beyond the reach of most of the world compared to a $20 feature phone, but a $30 to $40 smartphone would naturally be very popular 
In this grand progression, 2013 will certainly be a significant milestone for mobile devices, smartphones and beyond  It's likely to be the first year in which tablets out-ship notebooks in the US  And in the coming years, this will lead to a confluence of high-end tablets and ultra-mobile notebooks as the world figures out how these devices co-exist, blend, hybridize, and/or merge 
Against this backdrop, in this two-part series, we'll explore the major trends and evolution for mobile SoCs  More importantly, we'll look to where the major vendors are likely going in the next several years 
Tablet and phone divergence
While phones and tablets are mobile devices that often share a great deal of software, it's becoming increasingly clear the two are very different products  These two markets have started to diverge and will continue doing so over time 
From a technical perspective, smartphones are far more compact and power constrained  Smartphone SoCs are limited to around 1W, both by batteries and by thermal dissipation  The raison d’etre of a smartphone is connectivity, so a cellular modem is an absolute necessity  For the cost sensitive-models that make up the vast majority of the market, the modem is integrated into the SoC itself  High-end designs favor discrete modems with a greater power budget instead  The main smartphone OSes today are iOS and Android, though Windows is beginning to make an appearance (perhaps with Linux or BlackBerry on the horizon)  Just as importantly, phone vendors like HTC must pass government certification and win the approval of carriers  There is very much a walled-garden aspect, where carriers control which devices can be attached to their networks, and in some cases devices can only be sold through a certain carrier  The business model places consumers quite far removed from the actual hardware 
In contrast, tablets are far more akin to the PC both technically and economically  The power budget for tablet SoCs is much greater, up to 4W for a passively cooled device and as high as 7-8W for systems with fans  This alone means there is a much wider range of tablet designs than smartphones  Moreover, the default connectivity for tablets is Wi-Fi rather than a cellular modem  The vast majority of tablets do not have cellular modems, and even fewer customers actually purchase a wireless data plan  As a result, cellular modems are almost always optional discrete components of the platform  The software ecosystem is relatively similar, with Microsoft, Apple, and Google OSes available  Because tablets eschew cellular modems, the time to market is faster, and they are much more commonly sold directly to consumers rather than through carriers  In terms of usage models, tablets are much more PC-like, with reasonable-sized screens that make games and media more attractive 
Looking forward, these distinctions will likely become more pronounced  Many tablets today use high-end smartphone SoCs, but the difference in power targets and expected performance is quite large  As the markets grow in volume, SoCs will inevitably bifurcate to focus on one market or the other  Even today, Apple is doing so, with the A6 for phones and the larger A6X for tablets  Other vendors may need to wait a few years to have the requisite volume, but eventually the two markets will be clearly separate 
Horizontal business model evolution
Another aspect of the mobile device market that is currently in flux and likely to change in the coming years is the business model for the chip and system vendors  Currently, Apple is the only company truly pursuing a vertically integrated model, where all phones and tablets are based on Apple’s own SoC designs and iOS  The tight integration between hardware and software has been a huge boon for Apple, and it has yielded superb products 
Samsung is one of the few others companies that takes a vertically integrated approach to phones and tablets, although in truth its strategy seems to be ambivalent on that point  Unlike Apple, Samsung’s SoCs are readily available to third parties, and some Samsung devices, such as the S7562 Galaxy S Duos, use SoCs from competitors  More recently though, there has been a trend of Samsung devices using Samsung SoCs, at least for the premier products  For the moment, Samsung’s approach is best characterized as a hybrid, particularly as the company lacks a bespoke OS 
The rest of the major SoC vendors (e g , Intel, Qualcomm, Nvidia, TI, Mediatek, etc ) have stayed pretty far away from actual mobile devices  These companies tend to focus on horizontal business models that avoid competing with customers or suppliers 
In the long term, mobile devices are likely to evolve similarly to the PC and favor a horizontal business model  The real advantage is one of flexibility; as costs drop and the market expands, it will be increasingly necessary for vendors like HTC to offer a wide range of phones based on radically different SoCs  While a vertically integrated company like Apple can focus and maintain leadership in a specific (and highly lucrative) niche, it would be very difficult to expand in many growing areas of the market  The differences between an iPhone 6 and a $20 feature phone are tremendous and would be very difficult for a single company to bridge 
However, SoC vendors will attempt to reap the benefits of vertical integration by providing complete reference platforms to OEMs  Conceptually, this is a form of "optional" system integration, where the phone vendor or carrier can get the entire platform from the SoC supplier  This has the principal advantages of reducing time to market while also providing a baseline quality and experience for consumers  Currently, this approach has mostly been tested in emerging markets, but it's likely to become more common over time  There is a crucial distinction between reference platforms and vertical integration  Namely, OEMs can always choose to customize a platform to differentiate, and the SoC vendor avoids dealing with consumers directly  Typically, most of the customization is in terms of software on top of a base operating system 
Quote:Moreover, that will make the transition to a 10nm node even more difficult, as the foundries will have to move from 20nm interconnects to 10nm interconnects and skip a generation The advances in technology lately allowing components on such a small scale to even be envisioned, much less planned for, are truly amazing 
Off topic: show
I present the first generation 'non-technical' rock:
I don't think your horizontal market development theory is supported by facts  Samsung and Apple are more vertically oriented than their competition, for starters  I know this article is narrowly focused on the hardware, but MS and Intel getting into hardware, Amazon getting into hardware, Google buying Moto, this is all vertical integration  How can you support the idea that this trend will be reversed with no real justification? I'm sure mobile chips will continue to specialize, but I don't think this means what you think it means  Automobile companies started making their own engines and with rare exceptions, never went back to being more horizontal  Same with retail and their store brands  Same with cloud companies and their servers  Same with mobile companies and their OSs  The horizontal market of PCs created by long-lasting standards and loose hegemony is the exception, not the norm 
Why wouldn't the foundries be able close the process gap with Intel? Is it a matter of money? Scale?
I'm not so sure about several things:1- Moore's law's relevance  Moore's Law is about ICs  ICs are not as big a part of mobile computers as they are of desktops, even of laptops: screens, batteries, radios are a huge part of tablets' and phones' costs, as opposed to the bare SoC + RAM 2- The tablet vs phone dichotomy  For some reason (probably price insensitivity due to subsidies), Phones have a tendency to be more powerful than Tablets, ie phone SoCs are more than good enough for tablets  Since the OS and peripherals are the same, it makes more sense to design and build just one type of SoC, and just disable the phone-modem part of it (even the other radios are still required: BT, Wifi, GPS   ), same as Intel disable cache and cores for their entry-level CPUs  Once you're fabbing a SoC, it makes more sense to make more of the same than to setup a separate run of a cut-down SoC on an older process, unless volumes are huge  We might still be getting previous-generation, well amortized SoCs in cheaper tablets, though 3- On the contrary, I see a tablet and phone convergence (the ugly phablet)  I'm patiently waiting for the new 6"+ phones to replace my Nook Color and Galaxy Note 1 with a single device 4- The advantage of diversity ? Software is becoming ever more important than hardware  Multiplying SoCs means multiplying product development costs, making support and updates more difficult    Again, unless volumes are huge, OEMs are probaly better off going the way of the car industry and using modular "platforms" housed in different chassis with various screen sizes, keyboards, radios, digitizers   I'm wondering why the "single device" trend does not figure in your analysis  Is it stillborn ? Does it have no impact nor dependency on/with SoCs ?
Samsung has its own bespoke OS: Bada and it is used on an extensive line of devices  I think there are numbers somewhere that it outsold Windows Phone 7 for a time 
gypsumfantastic wrote:Why wouldn't the foundries be able close the process gap with Intel? Is it a matter of money? Scale?First mover advantage 
SoC? System on a Chip I guess?
You're way off on the Moore's Law/cost of smartphones point  The processors used in today's high-end smartphones are already cheap, around $25  And there are less expensive options if you want a lower end product  In fact, the hardware in the whole smartphone is relatively cheap  Analyst's estimate the Z10's materials cost around $160, the iPhone 5 around $140  They're using expensive glass and metals, then there's the battery, memory, etc  which means the processor is a small factor of the cost And then there's the jump from $140 in materials to the unsubsidized costs  The reason these phones cost $650 is because of the high margins these companies are able to get and the high cost of hardware design and/or software development  But the point is that making the processors 4 times better/cheaper isn't going to change the economics of the smartphone  What will change the economics is commoditized designs and software and cheaper materials all around  Then you'll have a $50 smartphone that's decent 
Last edited by ggeezz on Wed Feb 13, 2013 9:17 am
bigterp wrote:SoC? System on a Chip I guess?Yup 
gypsumfantastic wrote:Why wouldn't the foundries be able close the process gap with Intel? Is it a matter of money? Scale?Money and momentum, the x86 market is a huge money maker for Intel so it is able to recoup its huge investments for advanced foundries 
Quote:Currently, the only products using 3D integration are FPGAs from Xilinx,Doesn't Sony use it in the PS Vita? I thought I read somewhere that they had the CPU, main memory (2 dies) and video memory, so 4 dies in total, sitting on top of each other all on the same chip 
renoX wrote:gypsumfantastic wrote:Why wouldn't the foundries be able close the process gap with Intel? Is it a matter of money? Scale?Money and momentum, the x86 market is a huge money maker for Intel so it is able to recoup its huge investments for advanced foundries Exactly and I would clarify that it's all about margins, the difference between what it costs to make a chip and what it sells for  The margins for desktop and server processors is huge because a) the whole product is expensive so $200 to $1000 for the chip is acceptable, and b) Intel has huge advantages in that space and little competition So Intel can afford to do the R&D to stay ahead of the curve and keep their position  When your smartphone chip sells for $25 you can't do the R&D to leapfrog a company that sells Xeons for $1000 and Core i5's for $200 
I am happy to see Kanter here at Ars, I like his writing and he maintains Real World Tech, where Linus Torvalds often shows up to comment on CPU arch and other interesting topics 
ggeezz wrote:renoX wrote:gypsumfantastic wrote:Why wouldn't the foundries be able close the process gap with Intel? Is it a matter of money? Scale?Money and momentum, the x86 market is a huge money maker for Intel so it is able to recoup its huge investments for advanced foundries Exactly and I would clarify that it's all about margins, the difference between what it costs to make a chip and what it sells for  The margins for desktop and server processors is huge because a) the whole product is expensive so $200 to $1000 for the chip is acceptable, and b) Intel has huge advantages in that space and little competition So Intel can afford to do the R&D to stay ahead of the curve and keep their position  When your smartphone chip sells for $25 you can't do the R&D to leapfrog a company that sells Xeons for $1000 and Core i5's for $200 Spot on Intel are able to piggyback other development efforts off the highly lucrative mainstream x86 market which generates the huge sums of money to fund their amazing fab technology The question for the future is how the economics will stack up when overall device costs fall significantly and there is a big downward pressure on SoC prices  In that situation, can Intel still justify bringing their A-game to a market where products are essentially commoditised and you have processors selling for a only a few dollars each?The lesson from their previous involvement in the DRAM market is that they probably won't want to be involved because there isn't enough money to be made to justify manufacturing phone SoCs on a cutting edge, or near cutting edge process  In that scenario, Intel may not totally abandon the market but they might just stick to manufacturing SoCs on nodes that are a step or two behind the state of the art 
solomonrex wrote:I don't think your horizontal market development theory is supported by facts  Samsung and Apple are more vertically oriented than their competition, for starters  I know this article is narrowly focused on the hardware, but MS and Intel getting into hardware, Amazon getting into hardware, Google buying Moto, this is all vertical integration  How can you support the idea that this trend will be reversed with no real justification? I'm sure mobile chips will continue to specialize, but I don't think this means what you think it means  Automobile companies started making their own engines and with rare exceptions, never went back to being more horizontal  Same with retail and their store brands  Same with cloud companies and their servers  Same with mobile companies and their OSs  The horizontal market of PCs created by long-lasting standards and loose hegemony is the exception, not the norm Yea, each year Amazon, MS, Apple and Google look more and more the same 
Intel cannot abandon the phone/tablet market  Desktop/laptop sales are stagnating/decreasing and phones/tablets are on the rise  This trend is only going to increase going forward But you're right, they're going to have use their fabs that are a step or two behind the cutting the edge  But they're going to have to up their game in the tablet space to even be able to do that 
gypsumfantastic wrote:Why wouldn't the foundries be able close the process gap with Intel? Is it a matter of money? Scale?Intel's called Chipzilla for a reason up
Lagrange wrote:The question for the future is how the economics will stack up when overall device costs fall significantly and there is a big downward pressure on SoC prices  In that situation, can Intel still justify bringing their A-game to a market where products are essentially commoditised and you have processors selling for a only a few dollars each?The lesson from their previous involvement in the DRAM market is that they probably won't want to be involved because there isn't enough money to be made to justify manufacturing phone SoCs on a cutting edge, or near cutting edge process  In that scenario, Intel may not totally abandon the market but they might just stick to manufacturing SoCs on nodes that are a step or two behind the state of the art I think the processing is a bigger advantage than many realize  If Intel can stay ahead in process design - which this article seems to indicate - they should have a major advantage  All else being equal a 14nm chip should be significantly faster and more efficient than the same chip at 22nm  Add in the fact that yields increase geometrically - you can fit a lot more 14nm chips on a given wafer size vs 22nm (or 32nm for the other manufacturers ) and you have a very appealing proposition  And then add in the fact that Intel actually has a pretty good graphics stack and IP  It's not a sure thing by any means, but I suspect ARM may have just prodded a sleeping giant edit: Also worth noting, Intel, TSMC, and Samsung are the only manufacturers who are building out 450nm wafers  This will increase yields dramatically  Of course Samsung and TSMC will build ARM out, but it definitely puts quite a bit of pressure on all other manufacturers  As the article mentions Intel and Samsung are the only ones who control production top to bottom, and Samsung must share some of the benefits with ARM 
As someone who has worked in the semiconductor industry for longer than contemporary fanboys have existed, I'm getting a bit fed-up seeing many of these articles which rewrite history with analyses distorted by the lens of the contemporary Apple or Samsung fanboy The mobile industry moved to horizontal integration a long time ago  Better indicators than these odd contemporary obsessions with the relatively non-innovative Samsung and Apple where when Motorola spun out its semiconductor division as Freescale, Nokia stopped making it's own custom designs with TI and ST, and Ericsson spun out it's Ericsson Mobile Platforms division and formed ST-Ericsson with ST The true innovation in the mobile market was done a decade or more ago mainly by Moto/Nokia/Ericsson/TI/Qualcomm, and Samsung and Apple had little to do with it  Under the hood most stuff has been on a simple linear evolutionary path by the SoC providers since then  The phone manufacturers have then mainly been simply sticking these off-the-shelf SoCs (and their included low-level software stacks) in a box, a job made all the easier with the SoC manufacturer collaboration providing the bulk of the work for the AOSP 
Last edited by paul5ra on Wed Feb 13, 2013 11:06 am
introiboad wrote:I am happy to see Kanter here at Ars, I like his writing and he maintains Real World Tech, where Linus Torvalds often shows up to comment on CPU arch and other interesting topics Indeed  Most tech writing in this area is atrocious  This piece is one of the few well informed articles I've read in a long time 
ggeezz wrote:Intel cannot abandon the phone/tablet market  Desktop/laptop sales are stagnating/decreasing and phones/tablets are on the rise  This trend is only going to increase going forward But you're right, they're going to have use their fabs that are a step or two behind the cutting the edge  But they're going to have to up their game in the tablet space to even be able to do that The word you're looking for is Haswell, as far as I know 
Mabsark
ggeezz wrote:Intel cannot abandon the phone/tablet market  Desktop/laptop sales are stagnating/decreasing and phones/tablets are on the rise  This trend is only going to increase going forward But you're right, they're going to have use their fabs that are a step or two behind the cutting the edge  But they're going to have to up their game in the tablet space to even be able to do that Actually, that trend will not simply keep increasing going forward  The reason desktop/laptop sales are stagnating/decreasing is due to the fact that most people already have one and therefore don't need to buy another one  The exact same thing will happen with tablets as well  Sales are increasing now because people without tablets are buying them  When most people already own a tablet, they won't be buying a new one every year and therefore sales will stagnate/decrease The PC market is saturated and in a couple of years, the tablet market will be saturated too  Basically, in order to increase sales in a saturated market, you need to increase the population growth or decrease the longevity of the product 
gypsumfantastic wrote:Why wouldn't the foundries be able close the process gap with Intel? Is it a matter of money? Scale?Probably a mix of a lot of things  One big thing was during this recession, Intel was the ONLY fab company that didn't scale back their R&D  That alone gave Intel a large advantage Intel has almost always been ahead  One of the reasons could be that Intel works with much higher margins than many of the commodity companies like Samsung and TSMC Outside of the P4 flop and some of the monopolistic abuses, Intel has typically been selling to high end customers that are willing to pay a premium for "the best" Intel has a large benefit of having a relatively "good name" when it comes to CPUs, so they can effectively charge a brand-name premium I'm sure there are other reasons, and probably better reasons, but these are the main ones that I think of 
Mabsark wrote:Actually, that trend will not simply keep increasing going forward  The reason desktop/laptop sales are stagnating/decreasing is due to the fact that most people already have one and therefore don't need to buy another one  The exact same thing will happen with tablets as well  Sales are increasing now because people without tablets are buying them  When most people already own a tablet, they won't be buying a new one every year and therefore sales will stagnate/decrease The PC market is saturated and in a couple of years, the tablet market will be saturated too  Basically, in order to increase sales in a saturated market, you need to increase the population growth or decrease the longevity of the product That's true as long as most people are still buying both a tablet and a laptop when each needs to be replaced  I think the assumption is that, as you say, the tablet market will saturate, with people just replacing existing ones, but the desktop/laptop market could decrease much farther than that, if most people stop replacing them at all  I'm not sure of the likelihood of that, but I think that's where this idea comes from 
ggeezz wrote:Intel cannot abandon the phone/tablet market  Desktop/laptop sales are stagnating/decreasing and phones/tablets are on the rise  This trend is only going to increase going forward But you're right, they're going to have use their fabs that are a step or two behind the cutting the edge  But they're going to have to up their game in the tablet space to even be able to do that The upcoming Haswell chip is showing to consume 1/3 the power of IvyBridge at peak, consumes 1/20th the power at idle, all the while maintaining Identical or better performance This chip should actually compete with ARM CPUs on both power/performance and idle I am expecting a large war 
Apple once again is dictating the performance in the mobile industry  Nice to see others being able to keep the pace, as well 
paul5ra wrote:As someone who has worked in the semiconductor industry for longer than contemporary fanboys have existed, I'm getting a bit fed-up seeing many of these articles which rewrite history with analyses distorted by the lens of the contemporary Apple or Samsung fanboy The mobile industry moved to horizontal integration a long time ago  Better indicators than these odd contemporary obsessions with the relatively non-innovative Samsung and Apple where when Motorola spun out its semiconductor division as Freescale, Nokia stopped making it's own custom designs with TI and ST, and Ericsson spun out it's Ericsson Mobile Platforms division and formed ST-Ericsson with ST The true innovation in the mobile market was done a decade or more ago mainly by Moto/Nokia/Ericsson/TI/Qualcomm, and Samsung and Apple had little to do with it  Under the hood most stuff has been on a simple evolutionary path by the SoC providers since then Yeah, and most of the innovation in the automobile industry came about before Henry Ford came into the business  Doesn't change the fact that cars would probably have been an asterisk in the history books under "toys for rich people" if it weren't for him The same applies to to mobile computing for Apple, Samsung, et al 
SheldonRoss wrote:Lagrange wrote:The question for the future is how the economics will stack up when overall device costs fall significantly and there is a big downward pressure on SoC prices  In that situation, can Intel still justify bringing their A-game to a market where products are essentially commoditised and you have processors selling for a only a few dollars each?The lesson from their previous involvement in the DRAM market is that they probably won't want to be involved because there isn't enough money to be made to justify manufacturing phone SoCs on a cutting edge, or near cutting edge process  In that scenario, Intel may not totally abandon the market but they might just stick to manufacturing SoCs on nodes that are a step or two behind the state of the art I think the processing is a bigger advantage than many realize  If Intel can stay ahead in process design - which this article seems to indicate - they should have a major advantage  All else being equal a 14nm chip should be significantly faster and more efficient than the same chip at 22nm  Add in the fact that yields increase geometrically - you can fit a lot more 14nm chips on a given wafer size vs 22nm (or 32nm for the other manufacturers ) and you have a very appealing proposition  And then add in the fact that Intel actually has a pretty good graphics stack and IP  My point was that Intel might have a one or two process advantage over the rest of the industry at the cutting edge but that doesn't mean that they can afford to manufacture on those processes for very low margin parts  If the SoC market becomes increasingly commoditised, there isn't going to be the money to justify making them in a state of the art fab Remember that one of the big selling points of Itanium was that it would make use of process advantages that were effectively paid for by the mainstream x86 market  That didn't quite work out in practice and Itanium processors were often well behind Xeons in process technology 
paul5ra wrote:As someone who has worked in the semiconductor industry for longer than contemporary fanboys have existed, I'm getting a bit fed-up seeing many of these articles which rewrite history with analyses distorted by the lens of the contemporary Apple or Samsung fanboy The mobile industry moved to horizontal integration a long time ago  Better indicators than these odd contemporary obsessions with the relatively non-innovative Samsung and Apple where when Motorola spun out its semiconductor division as Freescale, Nokia stopped making it's own custom designs with TI and ST, and Ericsson spun out it's Ericsson Mobile Platforms division and formed ST-Ericsson with ST The true innovation in the mobile market was done a decade or more ago mainly by Moto/Nokia/Ericsson/TI/Qualcomm, and Samsung and Apple had little to do with it  Under the hood most stuff has been on a simple linear evolutionary path by the SoC providers since then  The phone manufacturers have then mainly been simply sticking these off-the-shelf SoCs (and their included low-level software stacks) in a box, a job made all the easier with the SoC manufacturer collaboration providing the bulk of the work for the AOSP Just goes to show that people who have worked in an industry for a long time don't always understand what that industry is doing You haven't been working in it long enough to seem to know that it was Acorn and Apple that invented the mobile ARM CPU in the first place  All those companies you've mentioned have just been working off Acorn and Apple's pioneering work  Now, Apple is back at it again, very successfully, and all the companies you mentioned that produce chips with ARM IP in them are licensing them from the company Acorn and Apple formed—ARM 
Last edited by melgross on Wed Feb 13, 2013 11:13 am
Mark Havel wrote:ggeezz wrote:Intel cannot abandon the phone/tablet market  Desktop/laptop sales are stagnating/decreasing and phones/tablets are on the rise  This trend is only going to increase going forward But you're right, they're going to have use their fabs that are a step or two behind the cutting the edge  But they're going to have to up their game in the tablet space to even be able to do that The word you're looking for is Haswell, as far as I know If tablets move into the $100-200 range, is there going to be room for Haswell?So long as there is a higher-end tablet market, then Haswell will be able to shine, but it's going to be a much more powerful and costly part than the sort of ARM based hardware that often runs tablets  If we see a race to the bottom where price is the dominant motivator behind purchases, then a high performance SoC will struggle to make its mark 
melgross wrote:paul5ra wrote:As someone who has worked in the semiconductor industry for longer than contemporary fanboys have existed, I'm getting a bit fed-up seeing many of these articles which rewrite history with analyses distorted by the lens of the contemporary Apple or Samsung fanboy The mobile industry moved to horizontal integration a long time ago  Better indicators than these odd contemporary obsessions with the relatively non-innovative Samsung and Apple where when Motorola spun out its semiconductor division as Freescale, Nokia stopped making it's own custom designs with TI and ST, and Ericsson spun out it's Ericsson Mobile Platforms division and formed ST-Ericsson with ST The true innovation in the mobile market was done a decade or more ago mainly by Moto/Nokia/Ericsson/TI/Qualcomm, and Samsung and Apple had little to do with it  Under the hood most stuff has been on a simple linear evolutionary path by the SoC providers since then  The phone manufacturers have then mainly been simply sticking these off-the-shelf SoCs (and their included low-level software stacks) in a box, a job made all the easier with the SoC manufacturer collaboration providing the bulk of the work for the AOSP Just goes to show that people who have worked in an industry for a long time don't always understand what that industry is doing You haven't been working in it long enough to seem to know that it was Acorn and Apple that invented the mobile ARM CPU in the first place  All those companies you've mentioned have just been working off Acorn and Apple's pioneering work  Now, Apple is back at it again, very successfully, and all the companies you mentioned that produce chips with ARM IP in them are licensing them from the company Acorn and Apple formed—ARM Of course I realise ARM IP has indeed been a major driving factor too (though only one if several architectures before ARM became dominant), though I see ARM's influence on the mobile industry as having nothing to do with modern day Apple and only one small piece of the puzzle  My point is that the hard electrical engineering, mathematics, DSP, semiconductor physics/chemistry, RF engineering, analogue design, CAD etc  that make modern telecommunications possible has very little to do with the fashion companies who consumers (and unfortunately much of the tech media) associate with it and give the credit (though in this respect Samsung does deserve a bit more credit for their work on NAND flash and displays)  The industry simply would not exist TODAY without the overwhelming horizontal integration that already dominates 
Quote:In the long term, mobile devices are likely to evolve similarly to the PC and favor a horizontal business model  The real advantage is one of flexibility; as costs drop and the market expands, it will be increasingly necessary for vendors like HTC to offer a wide range of phones based on radically different SoCs  You don't mention in the article that each SoC necessarily requires a bit of parallel dev work unlike the PC  In the PC world there is a standard BIOS and HW architecture that allows for pluggable designs  On a highly integrated SoC this is untrue  HTC suffers because it has to support radically different SoCs, their drivers and boot loaders, etc  Quote:While a vertically integrated company like Apple can focus and maintain leadership in a specific (and highly lucrative) niche, it would be very difficult to expand in many growing areas of the market  The differences between an iPhone 6 and a $20 feature phone are tremendous and would be very difficult for a single company to bridge It's only difficult because Apple chooses to ignore that market, not because they can't  If they can release a $99 Apple TV, they can surely cobble together a $20 feature phone if they chose to eschew 8GB of NAND, BT, WiFi, a specialized dock connector, LTE, and their specialized processors  In other words, build the equivalent of an iPod shuffle with a horrible screen and no OS to speak of 
paul5ra wrote:melgross wrote:paul5ra wrote:As someone who has worked in the semiconductor industry for longer than contemporary fanboys have existed, I'm getting a bit fed-up seeing many of these articles which rewrite history with analyses distorted by the lens of the contemporary Apple or Samsung fanboy The mobile industry moved to horizontal integration a long time ago  Better indicators than these odd contemporary obsessions with the relatively non-innovative Samsung and Apple where when Motorola spun out its semiconductor division as Freescale, Nokia stopped making it's own custom designs with TI and ST, and Ericsson spun out it's Ericsson Mobile Platforms division and formed ST-Ericsson with ST The true innovation in the mobile market was done a decade or more ago mainly by Moto/Nokia/Ericsson/TI/Qualcomm, and Samsung and Apple had little to do with it  Under the hood most stuff has been on a simple linear evolutionary path by the SoC providers since then  The phone manufacturers have then mainly been simply sticking these off-the-shelf SoCs (and their included low-level software stacks) in a box, a job made all the easier with the SoC manufacturer collaboration providing the bulk of the work for the AOSP Just goes to show that people who have worked in an industry for a long time don't always understand what that industry is doing You haven't been working in it long enough to seem to know that it was Acorn and Apple that invented the mobile ARM CPU in the first place  All those companies you've mentioned have just been working off Acorn and Apple's pioneering work  Now, Apple is back at it again, very successfully, and all the companies you mentioned that produce chips with ARM IP in them are licensing them from the company Acorn and Apple formed—ARM Of course I realise ARM IP has indeed been a major driving factor too (though only one if several architectures before ARM became dominant), though I see ARM's influence on the mobile industry as having nothing to do with modern day Apple and only one piece of the puzzle  My point is that the hard electrical engineering, mathematics, DSP, semiconductor physics/chemistry, RF engineering, analogue design,etc  that make modern telecommunications possible has very little to do with the fashion companies who consumers (and unfortunately much of the tech media) associate with it and give the credit (though in this respect Samsung does deserve a bit more credit for their work on NAND flash and displays)  The industry simply would not exist TODAY without the overwhelming horizontal integration that already dominates Yes the efforts of these companies getting cellular communications standardized were immense  And the technology matured  And then they didn't do much with it  It took some youngin's to look at the problem fresh and add the UI that make today's smartphones work  As we have all seen, the moment your technology has matured is the moment you are screwed because someone else now has the opportunity to look at it as a black box and make something new  Each of those manufacturers knew that smartphones would eventually be awesome, but none of them had the UI and software design to make a truly breakout product  Imagine if Motorola would have been smart enough to buy the Android guys instead of Google  Instead, Google bought a bunch of patents on that cellular black box to try to defend it's platform And when you think about it, which consumes more man years of engineering effort per year at this point     iterating that cellular black box or developing the OS, services and apps that power today's smartphones?
Intel had better decide that they are competing in this space "for real", or they are screwed  They've already let the Atom languish for five years, during which ARM has completely caught up in performance Just like Tim Cook said, if you don't cannibalize your own markets someone else will do it for you Whether Intel will embrace that concept in time remains to be seen  Personally, I hope they don't; if Intel transforms into a chipless Fab company (like TSMC) everyone benefits 
I still think Samsung has the advantage long term because they have both the SOC and the memory products  As mentioned in the article, TSV's (Through Silicon Via's) are going to be quite a disruption  Today, people normally stack an LPDDR2 package on top of their SOC package (POP or Package On Package)  Within the LPDDR2 package, you could have a stack of DRAM die typically with wire bonding connecting the die within the package Once you more to TSV's, you can have a LOT more connections between the SOC and its DRAM's  While this is being standardized through JEDEC (http://www jedec org/category/technolog     a/3d-ics-0), Samsung has all the pieces in house to do whatever they want  You could see a 512 bit or higher bus from the SOC to the memory  The trick is that the memory and the SOC need to line up with each other when you stack them  This gives Samsung an inherent advantage This isn't just going to impact mobile either  Take a look at that JEDEC link  It also lists High Bandwidth Memory (HBM)  This is a 1024 bit bus that provides 128GBytes/s to 256GBytes/s of bandwidth to a stack of up to 8 DRAM's  Here is your processor that includes 8-16 cores and 4GBytes of really, really, fast DRAM    No DIMMs required  How many of them do you want in your server rack?If I was Intel or Apple, I would be thinking seriously about making some investments in Micron to guarantee they make some compelling DRAM's to integrate with their SOC's and processors    otherwise Samsung is going to blow them out of the water on bandwidth 
Great_Scott wrote:Intel had better decide that they are competing in this space "for real", or they are screwed  They've already let the Atom languish for five years, during which ARM has completely caught up in performance Just like Tim Cook said, if you don't cannibalize your own markets someone else will do it for you Whether Intel will embrace that concept in time remains to be seen  Personally, I hope they don't; if Intel transforms into a chipless Fab company (like TSMC) everyone benefits It's true that Atom has stood still for too long, but honestly it's pretty amazing how Atom is still competitive with current ARM chips  The Z2760 is even 32nm vs 28nm of the latest Krait and A15 chips But that's all changing with Atom moving to the tick tock schedule this year  It wouldn't even surprise me to see Apple move to Intel chips for IOS And I don't see how Intel moving to a chipless Fab company would help everyone  It certainly wouldn't help Intel 
Mabsark wrote:ggeezz wrote:Intel cannot abandon the phone/tablet market  Desktop/laptop sales are stagnating/decreasing and phones/tablets are on the rise  This trend is only going to increase going forward But you're right, they're going to have use their fabs that are a step or two behind the cutting the edge  But they're going to have to up their game in the tablet space to even be able to do that Actually, that trend will not simply keep increasing going forward  The reason desktop/laptop sales are stagnating/decreasing is due to the fact that most people already have one and therefore don't need to buy another one  The exact same thing will happen with tablets as well  Sales are increasing now because people without tablets are buying them  When most people already own a tablet, they won't be buying a new one every year and therefore sales will stagnate/decrease The PC market is saturated and in a couple of years, the tablet market will be saturated too  Basically, in order to increase sales in a saturated market, you need to increase the population growth or decrease the longevity of the product Yes and no  I'm not sure the tablet market will saturate in a "couple of years " It may be more like 5 years  But that's a quibble Here's the real issue  Right now Apple wants you to own an iPhone AND iPad AND Macbook AND iWatch AND Apple TV  Microsoft, OTOH, is making the Surface so that you could ditch your laptop and just use a Surface  Not everyone, but some people If 5 years from now, we're in a world where a significant number of people use a Surface-type device instead of a laptop, then the PC market is going to contract significantly  Maybe some of the tablet-like devices will use moderately expensive Intel chips, but some of them are going to use cheaper chips 
GravyGraphics wrote:I still think Samsung has the advantage long term because they have both the SOC and the memory products  As mentioned in the article, TSV's (Through Silicon Via's) are going to be quite a disruption  Today, people normally stack an LPDDR2 package on top of their SOC package (POP or Package On Package)  Within the LPDDR2 package, you could have a stack of DRAM die typically with wire bonding connecting the die within the package Once you more to TSV's, you can have a LOT more connections between the SOC and its DRAM's  While this is being standardized through JEDEC (http://www jedec org/category/technolog     a/3d-ics-0), Samsung has all the pieces in house to do whatever they want  You could see a 512 bit or higher bus from the SOC to the memory  The trick is that the memory and the SOC need to line up with each other when you stack them  This gives Samsung an inherent advantage This isn't just going to impact mobile either  Take a look at that JEDEC link  It also lists High Bandwidth Memory (HBM)  This is a 1024 bit bus that provides 128GBytes/s to 256GBytes/s of bandwidth to a stack of up to 8 DRAM's  Here is your processor that includes 8-16 cores and 4GBytes of really, really, fast DRAM    No DIMMs required  How many of them do you want in your server rack?If I was Intel or Apple, I would be thinking seriously about making some investments in Micron to guarantee they make some compelling DRAM's to integrate with their SOC's and processors    otherwise Samsung is going to blow them out of the water on bandwidth Why not AMD? Last I checked they still made memory   and processors/GPUs 
Context 3: Current address: Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom 
In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan  However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap  We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation  Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome  Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs  The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated  Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2  Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease 
Copyright: © 2018 De Vito 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 
Data Availability: All relevant data are within the paper and its Supporting Information files  RNAseq data are available in the ArrayExpress database under the accession number E-MTAB-6452 
Funding: This study was funded by the Ajinomoto Innovation Alliance Program, (AIAP; https://www ajinomoto com/en/rd/AIAP/index html#aiap) (to M V S and D G), which is a joint research initiative of Ajinomoto Co , Inc , Japan  One of the authors [M B ] is an employee of Ajinomoto Co , and his specific roles are articulated in the ‘author contributions’ section  The commercial funder provided support in the form of salary for author [M B ] and some of the necessary research materials (medium for cell culture), but did not have any additional role in the study design, data collection and analysis, or preparation of the manuscript, and the authors had unrestricted access to the data  Due to a confidentiality agreement, the commercial funder participated only in the decision to publish the data obtained during the study, without any restriction 
Competing interests: This study was funded by Ajinomoto Co , Inc , Japan and one of the authors [M B ] is an employee of this commercial funder  No other employment or consultancy relationships exist with the commercial funder, and no patents, products in development, or marketed products result from this study  The authors declare that no competing interests exist and that the commercial affiliation of one of the authors does not alter the adherence of authors to all PLOS ONE policies on sharing data and materials 
In animals, excessive, insufficient, or imbalanced nutrient availability is known to strongly impact on phenotype and health, both short and long-term, and across generations [1, 2]  In particular, studies in yeast, animal models and humans have shown that reduced food intake, reducing either overall calories, or only sugars, proteins, or even single amino acids (AA), such as Methionine (Met), may extend lifespan and healthspan, and reduce the risk of cancer and other age-related diseases [3–9]  In addition, fasting or specific AA deprivation have shown potential therapeutic applications, owing to their ability to directly reduce the growth of some tumor types [10, 11], sensitize cancer cells to chemo- or immunotherapy [12, 13], and allow efficient hematopoietic stem cell engraftment   However, little is known about the specific processes and molecular mechanisms mediating the roles of nutrient restriction in human health and longevity 
A properly balanced diet in metazoans contains optimal amounts of a subset of AA, which cannot be synthetized de novo and are therefore named essential amino acids (EAAs)  In humans these include Met, Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Phenylalanine (Phe), Threonine (Thr), Tryptophan (Trp), and Valine (Val), while a few others are considered as semi-essential, such as Glutamine (Gln) and Tyrosine (Tyr) [15, 16]  Consistently, EAA deprivation triggers a cell-autonomous adaptive response, characterized by extensive metabolic and gene expression modifications, implementing biosynthetic, catabolic, and plasma membrane transport processes, aimed at reconstituting the full AA complement [17, 18]  The best known and conserved pathways responding to AA deprivation are triggered by mechanistic Target of Rapamycin Complex 1 (mTORC1) and General amino acid Control Non-derepressible 2 (GCN2) protein kinases [15, 19, 20]  Activation of mTORC1 requires in particular the presence of Gln, Arg and Leu, but also Met , which activate the kinase through sensors mainly acting upstream of Rag GTPases at lysosomal membranes   In turn, mTORC1 promotes cell growth, proliferation and anabolism upon activation, and translational attenuation and autophagy upon inhibition [19, 20] 
By contrast, GCN2 is activated by deprivation of any individual EAA, by means of its histidyl-tRNA synthetase-related domain, which binds uncharged tRNAs accumulating during AA limitation [23, 24]  Upon activation, GCN2 phosphorylates and inhibits its only known downstream target, namely the eukaryotic Initiation Factor 2 α (eIF2α), thereby initiating the Integrated Stress Response (ISR)  This leads to attenuation of general translation, and induction of a transcriptional/translational program, aimed at increasing stress resistance and restoring cell homeostasis, by upregulating a specific subset of genes, including Activating Transcription Factor 4 (ATF4) and C/EBP-Homologous Protein (CHOP) [25–27]  Thus, inhibition of mTORC1 and activation of GCN2 by AA restriction cooperate to attenuate general translation at the initiation step, increase catabolism and turnover, and enhance stress resistance to promote adaptation   However, how these processes eventually induce protective mechanisms against the alterations associated with aging, which include pervasive epigenetic and transcriptional changes [28, 29], remains largely unknown 
We previously reported the unexpected observation that prolonged deprivation of either Tyr, or of both Methionine and Cysteine (Met/Cys), triggers the selective and reversible reactivation of exogenous transcriptional units, including plasmids, retroviral vectors and proviruses, integrated into the genome and transcriptionally repressed by defensive mechanisms against non-native DNA sequences [30, 31]  This phenomenon was observed both in HeLa epithelial and ACH-2 lymphocytic human cells, and was independent of the transgene or provirus (Ocular Albinism type 1, OA1; Green Fluorescent Protein, GFP; Lysosomal-Associated Membrane Protein 1, LAMP1; Human Immunodeficiency Virus-1, HIV-1), or of the exogenous promoter driving their transcription, either viral (cytomegalovirus, CMV; Long Terminal Repeat, LTR) or human (Phospho-Glycerate Kinase 1, PGK1; Elongation Factor-1α, EF-1α)   Furthermore, this transgene reactivation response was not reproduced by serum starvation, activation of p38, or pharmacological inhibitors of mTOR (PP242 or rapamycin), sirtuins and DNA methylation  By contrast, it was induced by pan histone deacetylase (HDAC) inhibitors, and by selective inhibitors of class II HDACs   Consistently, we found that the mechanism responsible involves epigenetic modifications at the transgene promoter, including reduced nucleosome occupancy and increased histone acetylation, and is mediated in part by reduced expression of a class II HDAC, namely HDAC4  
These findings indicate that AA deprivation induces a specific epigenetic and transcriptional response, affecting the expression of newly-integrated exogenous transgenes and proviruses, and suggesting that endogenous sequences sharing similar structural and functional features may represent a transcriptional target as well [30, 31]  In particular, transposable elements, such as LTR-retrotransposons (or endogenous retroviruses, ERVs), are genomic “parasites” anciently-integrated into the genome, and silenced by epigenetic mechanisms of mammalian cells against the spreading of mobile elements, eventually becoming "endogenized" during evolution [32, 33]  This raises the question of whether their expression is also sensitive to AA restriction  In addition, it remains unclear whether or not the transgene reactivation response is related to specific AA deprivations, and most importantly which is the AA sensing/signaling pathway involved, in particular whether the GCN2 kinase is implicated  Thus, here we used the reactivation of silenced transgenes in cultured cells, as a model to investigate a novel molecular pathway induced by imbalanced EAA starvation, implicated in the epigenetic/transcriptional regulation of exogenous non-native DNA sequences and possibly of other endogenous anciently-integrated genomic elements 
HeLa human epithelial carcinoma, HepG2 human hepatocellular carcinoma and C2C12 mouse skeletal muscle cells were maintained in DMEM containing glutaMAX (Invitrogen) and supplemented with 10% FBS (Sigma), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), at 37°C in a 5% CO2 humidified atmosphere  Cell lines carrying integrated and partially silenced transgenes were also maintained in 600–1000 μg/ml G418 
The C2C12 cell line was provided by ATCC  HeLa and HepG2 cells were obtained by Drs  F  Blasi and G  Tonon at San Raffaele Scientific Institute, Milan, Italy, respectively, and were authenticated by Short Tandem Repeat (STR) profiling, using the Cell ID System kit (Promega), according to the manufacturer’s instructions  Briefly, STR-based multiplex PCR was carried out in a final volume of 25 μL/reaction, including 5 μL Cell ID Enzyme Mix 5X, 2 5 μL Cell ID Primer Mix 10X and 3 ng of template DNA  The thermal cycling conditions were: 1 cycle at 96°C for 2 min, followed by 32 cycles at 94°C for 30 sec, 62°C for 90 sec, and 72°C for 90 sec, and 1 cycle at 60°C for 45 sec  The following STR loci were amplified: AMEL, CSF1PO, D13S317, D16S539, D21S11, D5S818, D7S820, TH01, TPOX, vWA  Fragment length analysis of STR-PCR products was performed by Eurofins Genomics, using standard procedures of capillary electrophoresis on the Applied Biosystems 3130 XL sequencing machine, and assessment of the STR profile was performed at the online STR matching analysis service provided at http://www dsmz de/fp/cgi-bin/str html 
Stable cell clones, expressing myc-tagged human OA1 (GPR143) or GFP transcripts, were generated using pcDNA3 1/OA1myc-His or pcDNA3 1/EGFP vectors   Briefly, HeLa, HepG2 and C2C12 cells were transfected using FuGENE 6 (Roche) and selected with 800, 1000, and 650 μg/ml of G418 (Sigma), respectively, which was maintained thereafter to avoid loss of plasmid integration  G418-resistant clones were isolated and analyzed for protein expression by epifluorescence and/or immunoblotting 
Full DMEM-based medium, carrying the entire AA complement, and media deprived of Met/Cys (both AAs), Met (only), Cys (only), Alanine (Ala), Thr, Gln, Val, Leu, Tyr, Trp, Lys and His were prepared using the Nutrition free DMEM (cat #09077–05, from Nacalai Tesque, Inc , Kyoto, Japan), by adding Glucose, NaHCO3, and either all 20 AAs (for full medium) or 18–19 AAs only (for deprivations of two-one AAs)  Single AAs, Glucose, and NaHCO3 were from Sigma  Further details and amounts utilized are indicated in S1 Table  All media were supplemented with 10% dialyzed FBS (Invitrogen), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and G418 as required  HBSS was from Invitrogen  Cells were seeded at 10–30% of confluency; cells to be starved for 48 h were plated 2–3 times more confluent compared to the control  The following day, cells were washed and cultured in the appropriate medium, with or without EAA, for 24–48 h 
L-Histidinol (HisOH), PP242, Integrated Stress Response Inhibitor (ISRIB), SP600125, Cycloheximide (CHX) were from Sigma; Salubrinal was from Tocris Bioscience; U0126 was from Promega  Drugs were used at the following final concentrations: HisOH at 4–16 mM; PP242 at 1–3 μM; ISRIB at 100 nM; SP600125 at 20 μM in HepG2 cells and 50 μM in HeLa cells; Cycloheximide (CHX) at 50 ug/ml in HepG2 cells and 100 ug/ml in HeLa cells; Salubrinal at 75 μM; U0126 at 50 μM  Vehicle was used as mock control  Treatments with drugs to be tested for their ability to inhibit transgene reactivation (ISRIB, SP600125 and U0126) were initiated 1h before the subsequent addition of L-Histidinol (ISRIB) or the subsequent depletion of Met/Cys (SP600125 and U0126) 
Total RNA was purified using the RNeasy Mini kit (Qiagen), according to manufacturer’s instructions  RNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific)  Equal amount (1 μg) of RNA from HeLa, HepG2 and C2C12 cells was reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) using oligo-dT as primers, and diluted to 5 ng/μl  The cDNA (2 μl) was amplified by real-time PCR using SYBR green Master Mix on a Light Cycler 480 (Roche), according to manufacturer’s instructions  The thermal cycling conditions were: 1 cycle at 95°C for 5 min, followed by 40–45 cycles at 95° for 20 sec, 56° for 20 sec and 72° for 20 sec  The sequences, efficiencies and annealing temperatures of the primers are provided in S2 Table  Data were analyzed with Microsoft Excel using the formula EtargetΔct target (control-sample) /EreferenceΔct reference (control-sample)   Reference genes for normalizations were ARPC2 (actin-related protein 2/3 complex, subunit 2) for HeLa and HepG2 cells; and Actb (actin beta) for C2C12 cells, unless otherwise indicated 
siRNA (Mission esiRNA, 200 ng/μL; Sigma) against ATF4 and GCN2 were designed against the targeted sequences NM_001675 and NM_001013703, respectively  Cells seeded in 6-well plates were transfected with 1 μg of siRNAs and 5 μL of Lipofectamine 2000 (Invitrogen), following manufacturer’s instructions, at day 1 post-plating for ATF4 and at day 1 and 2 post-plating for GCN2  At day 2 (ATF4) or 3 (GCN2) post-plating, cells were washed and cultured in medium in the absence or presence of HisOH 4 mM for 6 h  siRNAs against RLuc (Sigma), targeting Renilla Luciferase, were used as negative control  For CRISPR/Cas9 experiments, we used the “all-in-one Cas9-reporter” vector, expressing GFP (Sigma), which is characterized by a single vector format including the Cas9 protein expression cassette and gRNA (guide RNA)  GFP is co-expressed from the same mRNA as the Cas9 protein, enabling tracking of transfection efficiency and enrichment of transfected cells by fluorescence activated cell sorting (FACS)  The human U6 promoter drives gRNA expression, and the CMV promoter drives Cas9 and GFP expression  The oligonucleotide sequences for the three gRNAs targeting GCN2 exon 1 or 6 are listed in S2 Table  We transfected HeLa and HepG2 cells with these plasmids individually (one plasmid one guide) and sorted the GFP-positive, transfected cells by FACS  Screening GCN2-KO clones was performed by western blotting  In the case of HepG2-OA1 cells, two rounds of selection were necessary to obtain three GCN2-KO clones by using a guide RNA against exon 1  Compared to the original HepG2-OA1 cell line and to the clone resulting from the first round of selection (185#27), the selected clones E23, F22 and F27 showed a very low amount—if any—of residual GCN2 protein (see results) 
Genomic DNA of HeLa and HepG2 cells was purified using DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer’s instructions  DNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific)  PCR conditions for amplification of GCN2 exon 1 and 6 were as follows: 1 cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 40 sec, 56°C for 40 sec, and 72°C for 40 sec; and a final extension step of 5 min at 72°C  The primer sequences are provided in S2 Table 
For OA1, western immunoblotting was carried out as described   For GCN2, cells were lysed in RIPA buffer, boiled at 95°C for 5 min and resolved on a 7 5% polyacrylamide gel; immunoblotting was then performed following standard procedures  Primary Abs were as follows: anti-human OA1, previously developed by our group in rabbits ; anti-GCN2 (Cell Signaling, Cat  #3302) 
Statistical analyses were performed using Microsoft Excel for Mac (version 15 32, Microsoft) for Student’s t-test; or GraphPad Prism (version 5 0d for Mac, GraphPad Software, Inc ) for one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s multiple comparisons post-tests  T-test was used when only two means, typically sample versus control, were compared, as specified in the figure legends  One way ANOVA was used for multiple comparisons, followed by either a Dunnett’s (to compare every mean to a control mean), or a Tukey’s (to compare every mean with every other mean) post-test, by setting the significance level at 0 05 (95% confidence intervals)  Both tests compare the difference between means to the amount of scatter, quantified using information from all the groups  Specifically, Prism computes the Tukey-Kramer test, allowing unequal sample sizes  P values in Figures are generally referred to comparison between a sample and the control (full medium/mock), and are indicated as follows: *P<0 05, **P<0 01, ***P<0 001  Comparisons not involving the control are similarly indicated, by a horizontal line at the top of the graphs, encompassing the two samples under analysis  Additional details regarding the specific experiments are reported in the Figure Legends 
To examine the expression behavior of genomic repeats upon AA starvation, we performed a transcriptomic analysis taking advantage of an intramural sequencing facility  HeLa-OA1 cells were cultured in normal medium (for 6-30-120 hours) or in absence of Met/Cys (for 6-15-30-72-120 hours)  Total RNA was prepared using Trizol (Sigma) to preserve transcripts of both small and long sizes (from Alu, of about 0 3 kb, to Long Interspersed Nuclear Elements, LINEs, and ERVs, up to 6–8 kb long), DNase treated to avoid contamination of genomic DNA, and processed for NGS sequencing by Ovation RNA-Seq System V2 protocol and HiSeq 2000 apparatus  Raw sequence data (10–20 M reads/sample) were aligned to the human genome (build hg19) with SOAPSplice   Read counts over repeated regions, defined by RepeatMasker track from UCSC genome browser , were obtained using bedtools suite   Normalization factors and read dispersion (d) were estimated with edgeR , variation of abundance during time was analyzed using maSigPro package , fitting with a negative binomial distribution (Θ = 1/d, Q = 0 01), with a cutoff on stepwise regression fit r2 = 0 7  Read counts were transformed to RPKM for visualization purposes  The expression of the OA1 transgene and HDAC4, which are progressively up- and down-regulated during starvation, respectively , were used as internal controls 
For genomic repeat analysis, reads belonging to repetitive elements were classified according to RepeatMasker and assigned to repeat classes (total number in the genome = 21), families (total number in the genome = 56) and finally subfamilies (total number in the genome = 1396), each including a variable number of genomic loci (from a few hundred for endogenous retroviruses, up to several thousand for Alu)  Repeat subfamilies were then clustered according to their expression pattern in starved vs control cells, by maSigPro using default parameters, and repeats classes or families that are significantly enriched in each cluster, compared to all genomic repeats, were identified by applying a Fisher Exact test (using scipy stats, a statistical module of Python)  Alternatively, differentially expressed repeat subfamilies were identified by averaging three time points of starvation (15-30-72 h) and controls  Repeats significantly up- or downregulated (104 and 77, respectively) were selected based on a P value <0 05 (unpaired two-tailed Student’s t-test, assuming equal variance), and analyzed for their class enrichment by a Fisher Exact test as described above 
For gene set enrichment analysis of Met/Cys deprived vs control HeLa cells, differentially expressed genes were selected considering three time points of starvation (15-30-72 h) and controls, based on a P value <0 05 (unpaired two-tailed Student’s t-test, assuming equal variance) and a fold change >2  This led to a total of 2033 differentially expressed genes, 996 upregulated and 1037 downregulated  The enrichment analysis was performed separately for up and down regulated genes, or with all differentially expressed genes together (both), using the KEGG database  The analysis was performed with correction for the background of all expressed genes (about 13600 genes showing an average expression over 3 starvation and 3 control samples of at least 5 counts) and by using default parameters (adjusted P value and q-value cut-off of <0 05 and 0 2, respectively)  Differentially expressed genes were also selected considering all starvation time points, as with genomic repeats, by maSigPro using default parameters, and a fold change of at least 1 5, leading to similar enrichment results (not shown)  RNAseq gene expression data are available in the ArrayExpress database under the accession number E-MTAB-6452 
To provide proof-of-principle that AA starvation may affect the expression of transposable elements, we performed an RNAseq analysis of the previously described HeLa-OA1 cells, carrying an integrated and partially silenced OA1 transgene   Since the reactivation of the transgene by starvation is a progressive phenomenon , we performed a time-course experiment, where each time point represents one biological sample, rather than a biological triplicate of a single time point  To this aim, cells were cultured either in normal medium, or in absence of Met/Cys for different time points (6-15-30-72-120 hours), resulting in the progressive upregulation of the OA1 transgene during starvation (Fig 1A and 1B), consistent with previously published results   The expression of genomic repeats was determined according to RepeatMasker annotation and classification into classes, families, and subfamilies  Repeat species were then subjected to differential expression and enrichment analyses in starved vs control conditions  Out of 1396 annotated repeat subfamilies, 172 species displayed a differential expression profile during starvation 
Fig 1



ground_truth: O(t, L_{\parallel}; S_\Delta) = L_{\parallel}^{-\beta/[\nu(1+\Delta)]} \tilde f_O(t/L_{\parallel}^{z/(1+\Delta)}; S_\Delta).

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: How are smartphones and tablets different from a technical perspective?

Read the following text and answer briefly.

Context 3:  Exogenous transgene and endogenous retroviruses are upregulated in Met/Cys-deprived HeLa cells 
(A,B) Exogenous integrated transgene (OA1) mRNA abundance in HeLa-OA1 cells, cultured in Met/Cys-deprived medium for the indicated time points, and analyzed by RNAseq (A), or RT-qPCR (B), compared to full medium  Data represent RPKM (A), or mean ± SD of 2 technical replicates, expressed as fold change vs  control (full medium at 6 h = 1) (B)  (C) Clustering of 172 genomic repeat subfamilies, differentially expressed upon starvation, according to their expression profile  (D) Class distribution of repeat subfamilies belonging to differential expression clusters, compared to all genomic repeat subfamilies (first column)  Class DNA includes DNA transposons; SINE includes Alu; LINE includes L1 an L2; LTR includes endogenous retroviruses and solitary LTRs; Satellite includes centromeric acrosomal and telomeric satellites; Others includes SVA, simple repeats, snRNA, and tRNAs  LTR-retroelements are significantly enriched among repeats that are upregulated upon starvation, while LINEs are significantly enriched among repeats that are downregulated  *P<0 05, ***P<0 001 (Fisher exact test) 
As shown in Fig 1C, the clustering of differentially expressed repeats, according to their expression pattern, reveals profiles comparable to the behavior of the transgene in the same conditions, i e  upregulation upon starvation and no change in regular medium (Cluster 1 and 2)  In particular, Cluster 1 contains sequences that, similarly to the OA1 transgene, are progressively upregulated upon starvation (Fig 1A and 1C) , while Cluster 2 contains sequences that are upregulated at early time points  Interestingly, repeat families that are significantly enriched in these two clusters belong mostly to the group of LTR-retrotransposons, including ERV1, ERVK, ERVL, ERVL-MaLR and other LTR sequences (Fig 1D; S1A and S2A Figs)  By contrast, DNA transposons (such as TcMar-Tigger) and L1 non-LTR retrotransposons are enriched among repeats that are downregulated during starvation, particularly at late time points (Clusters 3 and 4) (Fig 1D; S1A and S2A Figs)  Consistent results were obtained by selecting significantly up- or downregulated genomic repeats (overall 181 species), based on their average expression out of three time points of starvation (15-30-72 h, when the transgene upregulation is more homogeneous) and controls, and on a P value <0 05 (S1B and S2B Figs)  These findings suggest that EAA starvation induces genome-wide effects involving repetitive elements, and that—among major repeat classes—it upregulates in particular the expression of ERVs 
In addition, to obtain a general overview of main gene pathways changing their expression together with the transgene during AA starvation, we performed gene expression and enrichment analyses of regular genes, by considering three time points of starvation (15-30-72 h) and controls  Differentially expressed genes were selected based on a P value <0 05 and a fold change between means of at least 2, and analyzed with the EnrichR tool   As shown in Fig 2 and S1 File, enrichment analyses against the KEGG and Reactome databases reveals a predominance of downregulated pathways, namely ribosome and translation, proteasome, AA metabolism, oxidative phosphorylation and other pathways related to mitochondrial functions, which are affected in Huntington, Alzheimer and Parkinson diseases (http://www genome jp/kegg/pathway html)  In particular, a large fraction of ribosomal protein mRNAs is downregulated upon Met/Cys starvation (Fig 2A and 2C; S1 File), consistent with the notion that their genes–despite being scattered throughout the genome—are coordinately expressed in a variety of conditions   This reduced expression may depend on multiple pathways that control ribosome biogenesis in response to external stimuli, including the downregulation of Myc activity , the downregulation of mTORC1 [42, 44], or possibly the activation of the ISR, as described in yeast   By contrast, upregulated genes show a significant enrichment for transcription and gene expression (Fig 2B)  Similar results were obtained by the Gene Ontology Biological Process (GO-BP) database (S1 File), overall indicating a general downregulation of translation and metabolism, and upregulation of transcription, during the time interval of Met/Cys starvation corresponding to the transgene upregulation 
Fig 2  Gene set enrichment analysis of Met/Cys-deprived HeLa cells 
Differentially expressed genes between three time points of starvation (15-30-72 h) and controls were selected based on a P value <0 05 and a fold change of at least 2, leading to a total of 996 upregulated, and 1037 downregulated genes  The enrichment analysis was performed separately for up and down regulated genes, using the EnrichR tool and the KEGG (A) and REACTOME (B, C) databases  Ranking is based on the combined score provided by EnrichR, and categories are displayed up to 20 items with an Adjusted P value <0 05  No significant categories were found with upregulated genes against the KEGG database  All data are shown in S1 File  The enrichment analysis using all differentially expressed genes together did not reveal any additional enriched process 
To characterize the pathway leading to the reactivation of silenced transgenes, we used HeLa-OA1 and HeLa-GFP cells, as described   In addition, to test cell types relevant for AA metabolism, such as liver and muscle, we generated clones of HepG2 human hepatoma and C2C12 mouse skeletal muscle cells, stably transfected with plasmids for OA1 and GFP transgenes, respectively (HepG2-OA1 and C2C12-GFP cells; endogenous OA1 is not expressed in any of these cell types)  In all cases, the integrated transgenes are under the control of the CMV promoter in the context of a pcDNA3 1 plasmid, are partially silenced, and can be efficiently upregulated by HDAC inhibitors (trichostatin A, TSA; ref   and S3A, S3B and S4A Figs), indicating that their expression is controlled at least in part by epigenetic mechanisms, as previously described  
To establish whether the reactivation response results from the shortage of specific AAs only, such as Met/Cys, or it is triggered by any AA deprivations, we cultured HeLa-OA1, HeLa-GFP, HepG2-OA1 and C2C12-GFP cells for 24–48 hours with a battery of media deprived of EAAs or semi-EAAs, including Met/Cys, Thr, Gln, Val, Leu, Tyr, Trp, Lys, and His  As negative controls, cells were cultured in full medium, carrying the entire AA complement, and in a medium deprived of Ala, a non-essential AA  The expression of the transgene transcript was then evaluated by RT-qPCR  As shown in Fig 3, and in S3C and S4B Figs, most EAA-deficiencies induced reactivation of the OA1 or GFP transgenes in all four cell lines, with the notable exception of Trp deprivation, which consistently resulted in no or minimal reactivation of the transgenes  Indeed, despite some variability, Met/Cys deficiency, but also Thr, Val, Tyr, and His deprivation always gave an efficient response, while Leu, Gln and Lys elicited evident responses in some cases, but not in others  Depletion of Phe gave results comparable to Tyr deprivation, however it significantly altered multiple reference genes used for normalization and therefore was eventually omitted from the analysis (not shown)  Finally, in the above experiments we used a combined Met/Cys deficiency, to avoid the potential sparing of Met by Cys  and for consistency with our previous studies   Nevertheless, the analysis of single Met or Cys starvation, both at the protein and transcript levels, revealed an exclusive role of Met deprivation in transgene reactivation, consistent with the notion that Cys is not an EAA (S3D and S3E Fig) 
Fig 3  EAA deprivation induces reactivation of silent transgenes in HeLa and HepG2 cells 
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in various AA-deprived media for 48 h and 24 h, respectively, compared to full medium  Mean ± SEM of 3 independent experiments  Data are expressed as fold change vs  control (full medium = 1)  *P<0 05, **P<0 01, ***P<0 001 (one way ANOVA, followed by Dunnett’s post-test vs  full medium) 
Collectively, these results indicate that transgene reactivation by EAA starvation is reproducible with most EAAs, shared by different cell types (epithelium, liver, and skeletal muscle), and conserved in different mammalian species (human, mouse) 
mTORC1 inhibition and GCN2 activation trigger the best-known signaling pathways responding to AA starvation   We previously showed that inhibition of mTORC1 is not sufficient to reproduce transgene reactivation in HeLa cells   By contrast, the involvement of GCN2 and the ISR, including the downstream effectors ATF4 and CHOP, has never been tested  In addition, this pathway has been typically assessed in transient assays, lasting for a few hours, which may not be comparable with the prolonged starvation conditions necessary to reactivate the transgene expression (at least 15–24 h)  Thus, we tested whether CHOP expression was upregulated upon incubation of HeLa-OA1, HepG2-OA1 and C2C12-GFP cells in media deprived of different EAAs for 24–48 h 
As shown in Fig 3 and S4B Fig, we found that CHOP expression is increased in all EAA-starvation conditions, but not in the absence of Ala, in all tested cell lines  Similar, yet less pronounced, results were obtained with ATF4, consistent with the notion that activation of this transcription factor is mainly mediated by translational upregulation (not shown) [15, 26]  However, the upregulation of CHOP does not parallel quantitatively that of the transgene, neither appears sufficient to induce it  In fact, CHOP is highly upregulated even upon Trp starvation, which consistently results in no or minimal reactivation of the transgenes (compare CHOP with OA1 or GFP expression; Fig 3 and S4B Fig)  Thus, while the ISR appears widely activated upon EAA starvation, the upregulation of its downstream effector CHOP only partly correlates with transgene reactivation and may not be sufficient to induce it 
The activation of the ISR upon AA starvation suggests that GCN2 may be involved in the transgene reactivation response  Therefore, we tested whether direct pharmacological activation of this kinase is sufficient to trigger the transgene reactivation similarly to starvation  In addition, we used pharmacological inhibitors of mTOR to corroborate previous negative results in HeLa cells  in the other cell lines under study  To this aim, HeLa-OA1 or GFP, HepG2-OA1 and C2C12-GFP cells were cultured in the presence of different concentrations of PP242 (mTOR inhibitor) or L-Histidinol (GCN2 activator, inhibiting tRNAHis charging by histidyl-tRNA synthetase), either alone or in combination for 24 h, compared to Met/Cys-deprived and full medium  As shown in Fig 4 and S5 Fig, while inhibition of mTORC1 consistently leads to minor or no effects, in agreement with previous findings , treatment with L-Histidinol results in efficient reactivation of the transgene in HepG2-OA1 and C2C12-GFP cells, but not in HeLa cells 
Fig 4  mTOR inhibition and GCN2 activation differently affect transgene expression in HeLa and HepG2 cells 
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in Met/Cys-deprived medium, or in the presence of PP242 (mTOR inhibitor; 1–3 μM) or L-Histidinol (HisOH, GCN2 activator; 4–16 mM), either alone or in combination for 24–48 h, compared to full medium  Mean ± SEM of 4 (A) or 3 (B) independent experiments  Data are expressed as fold change vs  control (full medium = 1)  *P<0 05, **P<0 01, ***P<0 001 (one way ANOVA, followed by Dunnett’s post-test vs  full medium)  PP-1 and PP-3, PP242 at 1 and 3 μM, respectively; HisOH-4 and HisOH-16, L-Histidinol at 4 and 16 mM, respectively 
Specifically, L-Histidinol is not effective in HeLa-OA1 and HeLa-GFP cells, either alone or in combination with PP242 (Fig 4A and S5A Fig), or by using different concentrations of the drug, with or without serum (not shown)  In these cells, L-Histidinol appears also unable to trigger the ISR, as indicated by lack of CHOP upregulation, possibly due to their different sensitivity to the drug  These findings are consistent with previous reports, describing the use of L-Histidinol in HeLa cells in conditions of low His concentration in the culture medium , which would resemble AA starvation in our system and therefore may not be applicable  Thus, even though the amount of the amino alcohol was adapted to exceed 20 to 80 times that of the amino acid, as described , HeLa cells may be resistant or able to compensate 
In contrast, in other cell types, L-Histidinol has been utilized in regular DMEM, to mimic the AA response triggered by DMEM lacking His [48, 49]  Consistently, in HepG2-OA1 cells, L-Histidinol is sufficient to elicit extremely high levels of transgene reactivation, and its combination with PP242 results in additive or even synergistic effects, possibly due to an indirect effect of mTOR inhibition on GCN2 activity (Fig 4B) [50, 51]  Similarly, C2C12-GFP cells efficiently reactivate the transgene upon treatment with L-Histidinol, but not PP242 (S5B Fig)  However, differently from HepG2-OA1 cells, simultaneous treatment of C2C12-GFP cells with L-Histidinol and PP242 does not lead to synergistic effects  Consistent with stimulation of the ISR, CHOP and to a minor extent ATF4 are upregulated by L-Histidinol in both cell lines, yet their expression levels show only an incomplete correlation with those of the transgene (Fig 4B, S5B Fig, and not shown) 
The finding that GCN2 activation by L-Histidinol is sufficient to reactivate the transgenes in both HepG2-OA1 and C2C12-GFP cells pointed to this kinase, and to the downstream ISR, as the pathway possibly involved in the EAA starvation response  Thus, we investigated whether the ISR is sufficient to trigger upregulation of the OA1 transgene in HepG2-OA1 cells by pharmacological means  As CHOP expression does not correspond quantitatively and is not sufficient to induce transgene reactivation, we tested the role of the core upstream event of the ISR, namely the phosphorylation of eIF2α , which can be induced by pharmacological treatments, independent of GCN2 (Fig 5A)  To this aim, we used Salubrinal, a specific phosphatase inhibitor that blocks both constitutive and ER stress-induced phosphatase complexes against eIF2α, thereby increasing its phosphorylation   We found that, while the ISR is activated upon Salubrinal treatment, as shown by increased CHOP expression, it does not induce OA1 transgene reactivation (Fig 5B) 
Fig 5  The ISR is neither sufficient nor necessary to induce transgene reactivation in HepG2 cells 
(A) Schematic representation of GCN2 activation by AA starvation, resulting in phosphorylation of eIF2a and initiation of the downstream ISR  In addition to GCN2, the ISR may be activated by other eIF2a kinases (PKR, HRI and PERK; not shown in the picture)  (B) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 24 h with Salubrinal (a drug that induces the ISR by inhibiting the dephosphorylation of eIF2α; 75 μM), compared to full medium  Mean ± range of two experiments  Data are expressed as fold change vs  control (DMEM = 1)  *P<0 05 (paired two-tailed Student’s t-test vs  control)  (C) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM), in the absence or presence of ISRIB (a drug that bypasses the phosphorylation of eIF2α, inhibiting triggering of the ISR; 100 nM)  Mean ± range of two experiments  Data are expressed as fold change vs  control (DMEM = 1)  **P<0 01, ***P<0 001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs  control, unless otherwise indicated)  (D) Relative transgene (OA1) and ATF4 mRNA abundance in HepG2-OA1 cells transfected with control (CTRL) or anti-ATF4 siRNAs, and incubated in the presence or absence of L-Histidinol (HisOH, GCN2 activator; 4 mM) for 6 h  Mean ± range of two experiments  Data are expressed as fold change vs  control (w/o HisOH = 1, top; control siRNA = 1, bottom)  *P<0 05 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs  control, unless otherwise indicated) 
To test whether the ISR is necessary to trigger the transgene response to L-Histidinol, we used the chemical compound ISRIB, which inhibits the activation of the ISR, even in the presence of phosphorylated eIF2α, likely by boosting the activity of the guanine-nucleotide exchange factor (GEF) for eIF2α, namely eIF2B [53, 54]  HepG2-OA1 cells were stimulated with L-Histidinol, either in the presence or absence of ISRIB  As shown in Fig 5C, while the expression of CHOP is inhibited by ISRIB, as expected, the reactivation of the OA1 transgene is not affected  In addition, knockdown of the closest eIF2α downstream effector ATF4 by siRNAs does not interfere with the reactivation of the OA1 transgene by L-Histidinol (Fig 5D)  Together, these data suggest that eIF2α phosphorylation and the downstream ISR pathway are neither sufficient nor necessary to induce transgene reactivation 
To definitively establish if GCN2 is necessary to trigger the transgene reactivation response to EAA starvation, we directly suppressed its expression by CRISPR/Cas9-mediated knock-out (KO)  We generated three independent GCN2-KO clones from the parental HeLa-OA1 cell line, by using three different guide RNAs, two against exon 1 (clones 183#11 and 185#5), and one against exon 6 (clone 239#1) of the GCN2 gene  Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone 183#11, and on both alleles of exon 6 in clone 239#1; by contrast, clone 185#5 showed multiple alleles in exon 1, consistent with the presence of two cell populations, and was not characterized further at the genomic level (S6 Fig)  None of these clones express GCN2 at the protein level, as shown by immunoblotting (Fig 6A)  To test the GCN2-KO cells for their ability to respond to EAA starvation, parental HeLa-OA1 cells and the three GCN2-KO clones were cultured in media deprived of Met/Cys or Thr (corresponding to the most effective treatments in this cell line; see Fig 3A) for 24–48 h and transgene expression was assessed by RT-qPCR  We found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, thus excluding that this kinase is necessary for the response to EAA starvation in HeLa-OA1 cells (Fig 6B and 6C) 
Fig 6  GCN2 knockout does not interfere with transgene reactivation in HeLa cells 
(A) Immunoblotting of protein extracts from the HeLa-OA1 parental cell line and GCN2-KO clones 183#11, 185#5 and 239#1, immunodecorated with anti-GCN2 antibody  Arrow, GCN2 specific band  Ponceau staining was used as loading control  (B, C) Relative transgene (OA1) mRNA abundance in HeLa-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or Thr (C) deprived medium for 24 h or 48 h, respectively, compared to full medium  Mean ± SD of 3 technical replicates from 1 experiment  Data are expressed as fold change vs  control (full medium = 1)  Since independent clones may display variable reactivation responses (e g  due to different levels of transgene expression in basal conditions), the results are not shown as means of the three clones, but as separate replicates 
Similarly, we generated GCN2-KO clones from the parental HepG2-OA1 cell line by the same strategy  By using a guide RNA against exon 1 of the GCN2 gene, we obtained three independent GCN2-KO clones, namely E23, F22 and F27  Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone F27 (S7 Fig) and all three clones showed a very low amount—if any—of residual GCN2 protein, compared to the original HepG2-OA1 cell line (Fig 7A)  To assess the ability of GCN2-KO cells to reactivate the transgene upon starvation, we cultured parental HepG2-OA1 cells and the three GCN2-KO clones in media deprived of Met/Cys or His (corresponding to the most effective treatments in this cell line; see Fig 3B) for 24 h, and evaluated the transgene expression by RT-qPCR  As shown in Fig 7B and 7C, we found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, as in HeLa cells  To further confirm this result, we knocked-down GCN2 by RNA interference (RNAi), and incubated the cells with or without L-Histidinol for 6 h  As shown in Fig 8, treatment of HepG2-OA1 cells with L-Histidinol results in efficient transgene reactivation, even upon significant GCN2 downregulation, both at the mRNA and protein levels  Taken together, these data strongly support the conclusion that GCN2 is not necessary for transgene reactivation in response to EAA starvation, either in HeLa or in HepG2 cells 
Fig 7  GCN2 knockout does not interfere with transgene reactivation in HepG2 cells 
(A) Immunoblotting of protein extracts from the HepG2-OA1 parental cell line and GCN2-KO clones 185#27, E23, F22, F27, immunodecorated with anti-GCN2 antibody  Clone 185#27 results from the first round of selection, and was used to generate clones E23, F22, F27  Arrow, GCN2 specific band  For GCN2 protein quantification, Ponceau staining was used as loading control and data are expressed as fold change vs  parental cell line (= 1)  (B, C) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or His (C) deprived medium for 24 h, compared to full medium  Mean ± SD of 3 technical replicates from 1 experiment 



ground_truth: Smartphones are more compact and power constrained.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: Is the ISR necessary for transgene reactivation?

Read the following text and answer briefly.

Context 4: 
Early tests revealed cooling problems, which prompted a radiator move from the firewall to a lower cowling location 
The first flight was almost a disaster, as test pilot Randy Smith lost power right after takeoff  He managed a 180 with a safe downwind landing with only minor nosewheel pant damage  The culprit proved to be a spark plug with too much reach, which was quickly remedied  Subsequent flights have shown water temp to be about 210 degrees, oil temp is 220-230, and airspeed is about 180 mph 
Shopping for the Partially Built KR 
This story starts about twenty years ago when I first started looking at the KR-2 as the plane I'd like to build  The only problem at that time was a lack of money, lack of knowledge, and a lack of job stability  I liked the design, except for the low ground clearance of the retractable gear and that a KR was going to be a tight fit for me to fly 
Over the past twenty years I've owned a number of planes, but still always wanted to build my own  I needed one that would fit me, my budget requirements, and have the speed and performance that I wanted  When "KITPLANES" published the article featuring Roy Marsh's new KR-2S, it was the first I had heard of any major modifications or improvements to the same old KR design  I believe that article and Roy Marsh's workmanship have probably been the greatest boon to Rand Robinson (RR) in the last twenty years  It certainly caught my eye! Here was the same design I had decided I wanted to build twenty years ago, with all of the improvements I wanted  It was sitting on fixed gear with some reasonable ground clearance  It had the capability to be built large enough to accommodate me  It has enough prefab parts available that it didn't have to be 100% scratch built if I decided to hurry the project along  And it had the speed I wanted  I knew that Roy's published speeds were probably not realistic expectations for the average KR, but after knocking around for the last three years in my Champ, anything over 90 mph seems pretty fast to me 
After purchasing the info kit and the sales video from Rand Robinson, the next step after deciding for sure to build this plane was to order the KR-2 plans and the KR-2S addendum  I finally got my plans and was putting together my first order to start the plane, when my partner in the Champ pointed out that there was a partially completed KR-2S for sale in Trade-a-plane  My initial answer was "No, I don't even want to look at it  I want to build my own from scratch " My partner insisted that for the advertised price and the fact that it wasn't too far away, I ought to at least give the guy a call and investigate it  "No, I don't think I want to buy someone else's problems," I persisted  That night I went home and crunched up some numbers on the calculator and finally came to the conclusion that for the sake of my budget for the next several years, I really should give this guy a call 
Three days later, I flew to his place about 400 miles away to take a look at his project  At this point I should probably mention that I consider myself to be fairly knowledgeable about airplane construction, although the vast majority of my experience is with tube and fabric  The rest of this article deals with what I looked for and more importantly what I missed and have had to repair in the last year since I purchased the project 
When we went to the seller's house, I found that the left wing was built using the Dan Diehl wing skins and the right wing skins were leaning against the wall inside the house  Also the canopy was in the house with the canopy covered with paper and tape  I wanted to inspect the fuselage first, so off we went to the shop 
There I found a fuselage sitting on it's gear painted in primer gray  The first step was to inspect the quality of workmanship of what could be seen as it sat  The interior of the fuselage looked as if it had been built with a great deal of care  The fit and finish of all of the interior wood was very nice  Even the gussets looked like they had been painstakingly perfectly fitted  The glass work on the turtle back also looked very precise and clean  It was evenly faired into the vertical and horizontal stabs  The tail also appeared to be well built with the exception of a depression directly over the front and rear spars in the horizontal stabs  He explained that when he moved recently, that he had shot the plane with gray primer to protect it from the weather since he wouldn't have ready access to a shop to put it in right away  It ended up sitting out in the hot south Texas summer sun for a few weeks before he got a shop rented to work in  That caused the glass (or possibly the foam inside the horizontal stab) to swell, except that it held onto the spar, so it was slightly ballooned in front of and behind the spars  His recommendation was to fill it back smooth with micro 
I also found a small linear crack in the lower left wing spar cap on the left wing stub  It appeared to be from over tightening the rear spar wing attach fitting bolts  His explanation was that the crack wasn't important because the rear spars only job is to keep the wings from folding back  I also noticed that the holes for attaching the outer wing to the wing stub were badly rounded out on the rear spar  He explained that the Diehl wing skins require the rear spar to be swept slightly more forward than the stock wings  This won't allow you to use the rear spar attach fittings from RR and that I would need to fabricate a new set of rear spar attach fittings 
I also found that the aileron bellcranks were not built or installed as per plans, but found that they looked professional  I couldn't check for function since the right bellcrank and sheeve wasn't installed, the left wing also wasn't installed, and the right wing didn't exist yet 
Next we pulled the inspection panels off of the fuselage and tail and looked at everything I could see with a good flashlight  I didn't find anything else that might be questionable about the fuselage except for a cracked elevator trim tab that was damaged when it fell off it's hanging place on the wall 
Next we spent some time going over his builders log and builders photo album  I still hadn't seen anything that would dissuade me from buying this project 
At this point it was starting to get late and my ride down needed to get airborne for the flight home  I needed to make a decision about whether I wanted this project or not, but I hadn't inspected the wings and canopy yet  I took a cursory look at the left wing and saw lots on micro built up on it and some bubbles in the leading edge, but nothing that looked seriously wrong to my amateur eye  The right wing was only a set of spars in the shop and the Diehl wing skins in the house, so there wasn't much to look at there  The canopy was wrapped in paper and tape, so there wasn't much to look at there either  I decided that even if there were serious problems in the wing that was built, I would be money ahead to go ahead and buy the project  For the advertised price, I could build a new set of wings and still be way ahead financially  We negotiated a final price, shook hands, took my ride to the airport, and started off in search of a U-haul to haul the project home 
Now, at this point, some of you are thinking about what I surely must have forgotten to inspect and why didn't I take a local A & P or EAA member along for the ride  First of all, I don't know any mechanics locally that have any experience with glass and our EAA chapter of which I am VP is woefully lacking in fiberglass knowledge  Secondly, as you will see, I missed plenty  Some by ignorance, some by just not looking close enough 
Now for a list of the problems that I found over the last year and a few of the fixes that I came up with 
I found that the lower set of rear spar attach fittings on the left rear spar were installed backwards with the longer spaced hole towards the fuselage  Since this is the same place that also had the cracked spar cap, it required a major change  Also in the same area he had drilled through the rear spar with a hole saw to create a place for the aileron cable to pass through and managed to cut out the second from the outside vertical brace in the spar  Then he chose to install the aileron bellcranks in front of the rear spar, and cut another hole through the rear spar for the aileron push rod  He also managed to cut out the outside vertical brace in the spar  Since the holes were already drilled through the spar, the choices were to either cut out that section of spar cap and scarf a new piece in, cut the whole rear spar carrythrough out of the fuselage including ruining the left lower wing skin, or do something else creative to reinforce the spar cap and install a custom built set of attach fittings 
I also found that after I built and installed the right side wing stub ribs and skin that the aileron bellcrank setup would not work as installed  The cable that crosses between the two bellcranks had a sharp uphill from the sheeve to the bellcrank in the last 12 inches on either side  This combined with the radius that the bellcranks turn caused the cross cable to pull up tight when the ailerons were pushed to either end of their travel, but allowed the cables to go very slack when the ailerons were centered  Also the Aileron pushrods needed to pass directly through the lower set of rear wing attach fittings to attach to the aileron  This whole rear spar and aileron bellcrank setup was going to either have to be redesigned or cut out and built to plans  The bottom line is that the problems I observed when I inspected this part were much more serious than expected when I had to fix it 
I decided that I had to remove the rear fittings from the left wing to be replaced with the new set that my neighborhood machinist was cutting out for me  When I put the wing on the work bench to start removing the rear fittings, I thought I had better take a closer look at the bubbles in the leading edge  I found that as I pushed on the leading edge, it delaminated between the glass lay-up on top and the upper and lower wing skin edges that were floxed together underneath  I concluded that that area had to come apart and took a belt sander to the leading edge  What I found was that the leading edge had been floxed together and glassed over, but the mold release had never been scrubbed off the leading edge of the wing  It peeled apart for rebuild quite easily 
When I got back to removing the rear spar attach fittings, I noticed that the woodwork inside the wing looked awfully dull  The reason was that the wing had been closed up without varnishing any of the woodwork  This was rectified with a small hole saw, a number of extensions and a modified undercoating sprayer 
I also found that the aluminum drain fitting in the bottom of the left wing tank had been glassed into place upside down  The tapered pipe threads were tapered the wrong way to install the draincock into the tank  Retapping the fitting the right direction seemed to be a good fix for that problem 
When I finally got around to attaching the wing to the fuselage, I found that the front spar attach fittings were badly misaligned  Although they could be forced into alignment, I didn't think I needed that kind of preload on the main spar fittings  This problem was fixed by calling on my local neighborhood machinist to build me an aligning fixture and reaming the attach holes to the next larger size and ordering the new sized bolts 
On the fuselage I found that although it had new Cleveland wheels and brakes on it, one of the brakes had a severe wobble to it  I must complement the manufacturers for taking care of that problem  One call to the Cleveland factory and they shipped me a new set of wheels and brakes even though the receipt for this set was over four years old and in the original builders name  Their only concern was that this set had never been placed in service yet 
I chose to sand the load of micro off the left wing to see what it was covering  When I got down to the glass, I found that there was no glass for the aft inch and a half of the underside of the wing in front of the aileron hinge  With the Diehl wing skins, you build the wings, then cut the ailerons out of trailing edge of the wing  He had mismeasured and cut too much material off the bottom side of the trailing edge in front of the aileron  It was filled by floxing a piece of spruce into the gap to fill the space between the back edge of the fiberglass and the aileron mount  I chose to wrap the trailing edge of that wing, and the other wing to match with a couple of lay-ups of glass 
When I sanded the primer off the aforementioned damaged trim tab, I found that the hinge was floxed to the leading edge of the foam insides of the tab, but not the glass  I also chose to wrap the front of the trim tab with a lay-up of glass 
I decided to pull the paper off the canopy and take a look at it before I'm ready to bolt it on and fly  The original builder had blown his own canopy and after some of the previous problems, I was beginning to have some concerns about not having looked it over closely enough  The canopy turned out to have been blow a little too large  It ended up with a little larger bubble for headroom, which I didn't object to  However, it had more headroom on the right side than the left  Yes, it was just a little bit lopsided  The main problem was that the canopy is stretched thin enough that it can be easily pushed in with one hand when the weather is warm   My fear was that this is just thin enough that it may decide to lay on my head or in my lap when flying on a warm day  It will have to be replaced 
I'm sure that many that are reading this could see several of the potential problems before I mentioned them, but some others may not have and I'm sure that there could have been many other problems that didn't but could have existed on this project  This is also not intended to be critical of the gentleman that started this project as many parts of it, especially the wood work are better than I could have done and much of his work is outstanding  I prefer to think that I'll end up with a better plane with his woodwork combined with my glasswork  This article is intended to feature some of the problems that you may run into in buying someone else's project 
The final question is, knowing what I have found over the past year, would I have still purchased this project  The answer is yes, but primarily because the price was right in that I am still money and work ahead of where I would be if I had started the project from scratch  There are a few things that I would have done differently, but nothing that I can't live with  Although I won't be able to say that I built it all from scratch, I have built and rebuild enough of the plane that I should have no problem qualifying under the 51% rule 
You can send comments directly to the author via e-mail at "jscott@LANL GOV" 
Here is an brief explanation of how I built my turtledecks  The jig was constructed from scrap plywood and a few 1x4s that I ripped into stringers  I made two temporary bulkheads from the plywood, one for each end  Remember the forward bulkhead needs to be shaped in a way that will closely match the aft end of your canopy frame  Make an aft bulkhead by placing a straight edge at the top of your forward bulkhead and the trailing edge of your horizontal stabilizer  This will give you an idea of how tall your aft bulkhead needs to be  As far as location, I placed my aft bulkhead just forward of the lower/front of my vertical fin  I constructed the jig on the fuselage, it is glued together with automotive bondo 
After the bulkheads were bondoed to the fuselage I used the stringers that I ripped from the 1x4s and bondoed them to the bulkheads  This gave me a male form to cover with thin plastic or posterboard  I stapled two layers of posterboard to the jig(thin plastic would work better)  The posterboard wraps down two inches onto the fuselage  After I was satisfied with the way it looked, I then covered the entire thing with duct tape (fiberglass will not stick to duct tape) On top of this I wetout one layer of tri-ply cloth (22oz) that I had left over from an earlier project, and one layer of 8oz  bid  Remember to mask off your fuselage so you don't get epoxy on it  If you are not familiar with composite lay-ups, you should plan on razor cutting your lay-ups 4 to 6 hours after wetout while the lay-up is still soft enough to cut with a razorblade 
After the lay-up cured (2 or 3 days) it was removed from the jig, and the jig was removed from the fuselage and discarded  (be careful, the bondo sticks very well to the spruce, you could splinter your wood during removal) I now have a fiberglass skin that tends to hold the shape of the jig but is still flexible enough to work with  I made two bulkheads out of 1/4 last-a-foam (AS&S) using the plywood formers from the jig as a guide  I covered these foam bulkheads with one 8oz layer of glass on each side, with a glass to glass edge on the bottom  After cure these bulkheads were bondoed into place (to the fuselage)and the fiberglass skin was pulled down tight and floxed to the bulkheads  When the flox cured the bondo joints were broken, again being careful not to harm the wood  The turtledeck was removed from the fuselage and 2 inch tapes added to the bulkheads inside and out 
At this point the turtledeck looked great and only weighed about 5lbs  but I noticed you could deform the skin by pushing hard on the outside  So I flipped the turtledeck over and from 1/4 inch last-a-foam, I cut two inch wide strips that would run the entire length, forward and aft inside the turtledeck  In effect these would act as composite stringers, I made enough of these two inch wide strips to make up three stringers  One down the center (sort of a backbone) and one on each side of the "backbone" half the distance to the edge of the turtledeck  I sanded the edge of the foam so that when covered with a layer of bid @ 45degrees there would be a nice transition from the turtledeck skin up onto the foam and then back onto the turtledeck I scuff sanded and glued the foam stringers in with micro  I covered the foam stringers with one layer of 8oz bid @ 45degrees 
You can also send me email at: mikemims@pacbell net if you have any questions or want to share your ideas 
KROnline is an online KR Newsletter devoted to sharing KR information with other builders and pilots in a timely manner  The first issue (September 96) is now available as a zipped MicroSoft Word file at http://members aol com/bshadr or as an html document at kronline9 html  If you'd like to submit articles or photos, email Randy Stein at BSHADR@aol com ------------------------------------------------------------ Don't bother to email Randy though  KROnline has been retired since the KR Newsletter has improved 



ground_truth: No, it is not necessary.