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 algorithm is engaged in the PLMS-PPIC method?

Read the following text and answer briefly.

Context 1: \section{Introduction}\label{S1}

The multiple access interferences (MAI) is the root of user
limitation in CDMA systems \cite{R1,R3}  The parallel least mean
square-partial parallel interference cancelation (PLMS-PPIC) method
is a multiuser detector for code division multiple access (CDMA)
receivers which reduces the effect of MAI in bit detection  In this
method and similar to its former versions like LMS-PPIC \cite{R5}
(see also \cite{RR5}), a weighted value of the MAI of other users is
subtracted before making the decision for a specific user in
different stages \cite{cohpaper}  In both of these methods, the
normalized least mean square (NLMS) algorithm is engaged
\cite{Haykin96}  The $m^{\rm th}$ element of the weight vector in
each stage is the true transmitted binary value of the $m^{\rm th}$
user divided by its hard estimate value from the previous stage  The
magnitude of all weight elements in all stages are equal to unity 
Unlike the LMS-PPIC, the PLMS-PPIC method tries to keep this
property in each iteration by using a set of NLMS algorithms with
different step-sizes instead of one NLMS algorithm used in LMS-PPIC 
In each iteration, the parameter estimate of the NLMS algorithm is
chosen whose element magnitudes of cancelation weight estimate have
the best match with unity  In PLMS-PPIC implementation it is assumed
that the receiver knows the phases of all user channels  However in
practice, these phases are not known and should be estimated  In
this paper we improve the PLMS-PPIC procedure \cite{cohpaper} in
such a way that when there is only a partial information of the
channel phases, this modified version simultaneously estimates the
phases and the cancelation weights  The partial information is the
quarter of each channel phase in $(0,2\pi)$ 

The rest of the paper is organized as follows: In section \ref{S4}
the modified version of PLMS-PPIC with capability of channel phase
estimation is introduced  In section \ref{S5} some simulation
examples illustrate the results of the proposed method  Finally the
paper is concluded in section \ref{S6} 

\section{Multistage Parallel Interference Cancelation: Modified PLMS-PPIC Method}\label{S4}

We assume $M$ users synchronously send their symbols
$\alpha_1,\alpha_2,\cdots,\alpha_M$ via a base-band CDMA
transmission system where $\alpha_m\in\{-1,1\}$  The $m^{th}$ user
has its own code $p_m( )$ of length $N$, where $p_m(n)\in \{-1,1\}$,
for all $n$  It means that for each symbol $N$ bits are transmitted
by each user and the processing gain is equal to $N$  At the
receiver we assume that perfect power control scheme is applied 
Without loss of generality, we also assume that the power gains of
all channels are equal to unity and users' channels do not change
during each symbol transmission (it can change from one symbol
transmission to the next one) and the channel phase $\phi_m$ of
$m^{th}$ user is unknown for all $m=1,2,\cdots,M$ (see
\cite{cohpaper} for coherent transmission)  According to the above
assumptions the received signal is
\begin{equation}
\label{e1} r(n)=\sum\limits_{m=1}^{M}\alpha_m
e^{j\phi_m}p_m(n)+v(n),~~~~n=1,2,\cdots,N,
\end{equation}
where $v(n)$ is the additive white Gaussian noise with zero mean and
variance $\sigma^2$  Multistage parallel interference cancelation
method uses $\alpha^{s-1}_1,\alpha^{s-1}_2,\cdots,\alpha^{s-1}_M$,
the bit estimates outputs of the previous stage, $s-1$, to estimate
the related MAI of each user  It then subtracts it from the received
signal $r(n)$ and makes a new decision on each user variable
individually to make a new variable set
$\alpha^{s}_1,\alpha^{s}_2,\cdots,\alpha^{s}_M$ for the current
stage $s$  Usually the variable set of the first stage (stage $0$)
is the output of a conventional detector  The output of the last
stage is considered as the final estimate of transmitted bits  In
the following we explain the structure of a modified version of the
PLMS-PIC method \cite{cohpaper} with simultaneous capability of
estimating the cancelation weights and the channel phases 

Assume $\alpha_m^{(s-1)}\in\{-1,1\}$ is a given estimate of
$\alpha_m$ from stage $s-1$  Define
\begin{equation}
\label{e6} w^s_{m}=\frac{\alpha_m}{\alpha_m^{(s-1)}}e^{j\phi_m} 
\end{equation}
From (\ref{e1}) and (\ref{e6}) we have
\begin{equation}
\label{e7} r(n)=\sum\limits_{m=1}^{M}w^s_m\alpha^{(s-1)}_m
p_m(n)+v(n) 
\end{equation}
Define
\begin{subequations}
\begin{eqnarray}
\label{e8} W^s&=&[w^s_{1},w^s_{2},\cdots,w^s_{M}]^T,\\
\label{e9}
\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!X^{s}(n)\!\!\!&=&\!\!\![\alpha^{(s-1)}_1p_1(n),\alpha^{(s-1)}_2p_2(n),\cdots,\alpha^{(s-1)}_Mp_M(n)]^T 
\end{eqnarray}
\end{subequations}
where $T$ stands for transposition  From equations (\ref{e7}),
(\ref{e8}) and (\ref{e9}), we have
\begin{equation}
\label{e10} r(n)=W^{s^T}X^{s}(n)+v(n) 
\end{equation}
Given the observations $\{r(n),X^{s}(n)\}^{N}_{n=1}$, in modified
PLMS-PPIC, like the PLMS-PPIC \cite{cohpaper}, a set of NLMS
adaptive algorithm are used to compute
\begin{equation}
\label{te1} W^{s}(N)=[w^{s}_1(N),w^{s}_2(N),\cdots,w^{s}_M(N)]^T,
\end{equation}
which is an estimate of $W^s$ after iteration $N$  To do so, from
(\ref{e6}), we have
\begin{equation}
\label{e13} |w^s_{m}|=1 ~~~m=1,2,\cdots,M,
\end{equation}
which is equivalent to
\begin{equation}
\label{e14} \sum\limits_{m=1}^{M}||w^s_{m}|-1|=0 
\end{equation}
We divide $\Psi=\left(0,1-\sqrt{\frac{M-1}{M}}\right]$, a sharp
range for $\mu$ (the step-size of the NLMS algorithm) given in
\cite{sg2005}, into $L$ subintervals and consider $L$ individual
step-sizes $\Theta=\{\mu_1,\mu_2,\cdots,\mu_L\}$, where
$\mu_1=\frac{1-\sqrt{\frac{M-1}{M}}}{L}, \mu_2=2\mu_1,\cdots$, and
$\mu_L=L\mu_1$  In each stage, $L$ individual NLMS algorithms are
executed ($\mu_l$ is the step-size of the $l^{th}$ algorithm)  In
stage $s$ and at iteration $n$, if
$W^{s}_k(n)=[w^s_{1,k},\cdots,w^s_{M,k}]^T$, the parameter estimate
of the $k^{\rm th}$ algorithm, minimizes our criteria, then it is
considered as the parameter estimate at time iteration $n$  In other
words if the next equation holds
\begin{equation}
\label{e17} W^s_k(n)=\arg\min\limits_{W^s_l(n)\in I_{W^s}
}\left\{\sum\limits_{m=1}^{M}||w^s_{m,l}(n)|-1|\right\},
\end{equation}
where $W^{s}_l(n)=W^{s}(n-1)+\mu_l \frac{X^s(n)}{\|X^s(n)\|^2}e(n),
~~~ l=1,2,\cdots,k,\cdots,L-1,L$ and
$I_{W^s}=\{W^s_1(n),\cdots,W^s_L(n)\}$, then we have
$W^s(n)=W^s_k(n)$, and therefore all other algorithms replace their
weight estimate by $W^{s}_k(n)$  At time instant $n=N$, this
procedure gives $W^s(N)$, the final estimate of $W^s$, as the true
parameter of stage $s$ 

Now consider $R=(0,2\pi)$ and divide it into four equal parts
$R_1=(0,\frac{\pi}{2})$, $R_2=(\frac{\pi}{2},\pi)$,
$R_3=(\pi,\frac{3\pi}{2})$ and $R_4=(\frac{3\pi}{2},2\pi)$  The
partial information of channel phases (given by the receiver) is in
a way that it shows each $\phi_m$ ($m=1,2,\cdots,M$) belongs to
which one of the four quarters $R_i,~i=1,2,3,4$  Assume
$W^{s}(N)=[w^{s}_1(N),w^{s}_2(N),\cdots,w^{s}_M(N)]^T$ is the weight
estimate of the modified algorithm PLMS-PPIC at time instant $N$ of
the stage $s$  From equation (\ref{e6}) we have
\begin{equation}
\label{tt3}
\phi_m=\angle({\frac{\alpha^{(s-1)}_m}{\alpha_m}w^s_m}) 
\end{equation}
We estimate $\phi_m$ by $\hat{\phi}^s_m$, where
\begin{equation}
\label{ee3}
\hat{\phi}^s_m=\angle{(\frac{\alpha^{(s-1)}_m}{\alpha_m}w^s_m(N))} 
\end{equation}
Because $\frac{\alpha^{(s-1)}_m}{\alpha_m}=1$ or $-1$, we have
\begin{eqnarray}
\hat{\phi}^s_m=\left\{\begin{array}{ll} \angle{w^s_m(N)} &
\mbox{if}~
\frac{\alpha^{(s-1)}_m}{\alpha_m}=1\\
\pm\pi+\angle{w^s_m(N)} & \mbox{if}~
\frac{\alpha^{(s-1)}_m}{\alpha_m}=-1\end{array}\right 
\end{eqnarray}
Hence $\hat{\phi}^s_m\in P^s=\{\angle{w^s_m(N)},
\angle{w^s_m(N)+\pi, \angle{w^s_m(N)}-\pi}\}$  If $w^s_m(N)$
sufficiently converges to its true value $w^s_m$, the same region
for $\hat{\phi}^s_m$ and $\phi_m$ is expected  In this case only one
of the three members of $P^s$ has the same region as $\phi_m$  For
example if $\phi_m \in (0,\frac{\pi}{2})$, then $\hat{\phi}^s_m \in
(0,\frac{\pi}{2})$ and therefore only $\angle{w^s_m(N)}$ or
$\angle{w^s_m(N)}+\pi$ or $\angle{w^s_m(N)}-\pi$ belongs to
$(0,\frac{\pi}{2})$  If, for example, $\angle{w^s_m(N)}+\pi$ is such
a member between all three members of $P^s$, it is the best
candidate for phase estimation  In other words,
\[\phi_m\approx\hat{\phi}^s_m=\angle{w^s_m(N)}+\pi \]
We admit that when there is a member of $P^s$ in the quarter of
$\phi_m$, then $w^s_m(N)$ converges  What would happen when non of
the members of $P^s$ has the same quarter as $\phi_m$? This
situation will happen when the absolute difference between $\angle
w^s_m(N)$ and $\phi_m$ is greater than $\pi$  It means that
$w^s_m(N)$ has not converged yet  In this case where we can not
count on $w^s_m(N)$, the expected value is the optimum choice for
the channel phase estimation, e g  if $\phi_m \in (0,\frac{\pi}{2})$
then $\frac{\pi}{4}$ is the estimation of the channel phase
$\phi_m$, or if $\phi_m \in (\frac{\pi}{2},\pi)$ then
$\frac{3\pi}{4}$ is the estimation of the channel phase $\phi_m$ 
The results of the above discussion are summarized in the next
equation
\begin{eqnarray}
\nonumber \hat{\phi}^s_m = \left\{\begin{array}{llll} \angle
{w^s_m(N)} & \mbox{if}~
\angle{w^s_m(N)}, \phi_m\in R_i,~~i=1,2,3,4\\
\angle{w^s_m(N)}+\pi & \mbox{if}~ \angle{w^s_m(N)}+\pi, \phi_m\in
R_i,~~i=1,2,3,4\\
\angle{w^n_m(N)}-\pi & \mbox{if}~ \angle{w^s_m(N)}-\pi, \phi_m\in
R_i,~~i=1,2,3,4\\
\frac{(i-1)\pi+i\pi}{4} & \mbox{if}~ \phi_m\in
R_i,~~\angle{w^s_m(N)},\angle
{w^s_m(N)}\pm\pi\notin R_i,~~i=1,2,3,4 \\
\end{array}\right 
\end{eqnarray}
Having an estimation of the channel phases, the rest of the proposed
method is given by estimating $\alpha^{s}_m$ as follows:
\begin{equation}
\label{tt4}
\alpha^{s}_m=\mbox{sign}\left\{\mbox{real}\left\{\sum\limits_{n=1}^{N}
q^s_m(n)e^{-j\hat{\phi}^s_m}p_m(n)\right\}\right\},
\end{equation}
where
\begin{equation} \label{tt5}
q^{s}_{m}(n)=r(n)-\sum\limits_{m^{'}=1,m^{'}\ne
m}^{M}w^{s}_{m^{'}}(N)\alpha^{(s-1)}_{m^{'}} p_{m^{'}}(n) 
\end{equation}
The inputs of the first stage $\{\alpha^{0}_m\}_{m=1}^M$ (needed for
computing $X^1(n)$) are given by
\begin{equation}
\label{qte5}
\alpha^{0}_m=\mbox{sign}\left\{\mbox{real}\left\{\sum\limits_{n=1}^{N}
r(n)e^{-j\hat{\phi}^0_m}p_m(n)\right\}\right\} 
\end{equation}
Assuming $\phi_m\in R_i$, then
\begin{equation}
\label{qqpp} \hat{\phi}^0_m =\frac{(i-1)\pi+i\pi}{4} 
\end{equation}
Table \ref{tab4} shows the structure of the modified PLMS-PPIC
method  It is to be notified that
\begin{itemize}
\item Equation (\ref{qte5}) shows the conventional bit detection
method when the receiver only knows the quarter of channel phase in
$(0,2\pi)$  \item With $L=1$ (i e  only one NLMS algorithm), the
modified PLMS-PPIC can be thought as a modified version of the
LMS-PPIC method 
\end{itemize}

In the following section some examples are given to illustrate the
effectiveness of the proposed method 

\section{Simulations}\label{S5}

In this section we have considered some simulation examples 
Examples \ref{ex2}-\ref{ex4} compare the conventional, the modified
LMS-PPIC and the modified PLMS-PPIC methods in three cases: balanced
channels, unbalanced channels and time varying channels  In all
examples, the receivers have only the quarter of each channel phase 
Example \ref{ex2} is given to compare the modified LMS-PPIC and the
PLMS-PPIC in the case of balanced channels 

\begin{example}{\it Balanced channels}:
\label{ex2}
\begin{table}
\caption{Channel phase estimate of the first user (example
\ref{ex2})} \label{tabex5} \centerline{{
\begin{tabular}{|c|c|c|c|c|}
\hline
\multirow{6}{*}{\rotatebox{90}{$\phi_m=\frac{3\pi}{8},M=15~~$}} & N(Iteration) & Stage Number& NLMS & PNLMS  \\
&&&&\\
\cline{2-5} & \multirow{2}{*}{64}& s = 2 &  $\hat{\phi}^s_m=\frac{3 24\pi}{8}$ & $\hat{\phi}^s_m=\frac{3 18\pi}{8}$ \\
\cline{3-5} & & s = 3 & $\hat{\phi}^s_m=\frac{3 24\pi}{8}$ & $\hat{\phi}^s_m=\frac{3 18\pi}{8}$ \\
\cline{2-5} & \multirow{2}{*}{256}& s = 2 &  $\hat{\phi}^s_m=\frac{2 85\pi}{8}$ & $\hat{\phi}^s_m=\frac{2 88\pi}{8}$ \\
\cline{3-5} & & s = 3 & $\hat{\phi}^s_m=\frac{2 85\pi}{8}$ & $\hat{\phi}^s_m=\frac{2 88\pi}{8}$ \\
\cline{2-5} \hline
\end{tabular} }}
\end{table}
Consider the system model (\ref{e7}) in which $M$ users
synchronously send their bits to the receiver through their
channels  It is assumed that each user's information consists of
codes of length $N$  It is also assumd that the signal to noise
ratio (SNR) is 0dB  In this example there is no power-unbalanced or
channel loss is assumed  The step-size of the NLMS algorithm in
modified LMS-PPIC method is $\mu=0 1(1-\sqrt{\frac{M-1}{M}})$ and
the set of step-sizes of the parallel NLMS algorithms in modified
PLMS-PPIC method are
$\Theta=\{0 01,0 05,0 1,0 2,\cdots,1\}(1-\sqrt{\frac{M-1}{M}})$,
i e  $\mu_1=0 01(1-\sqrt{\frac{M-1}{M}}),\cdots,
\mu_4=0 2(1-\sqrt{\frac{M-1}{M}}),\cdots,
\mu_{12}=(1-\sqrt{\frac{M-1}{M}})$  Figure~\ref{Figexp1NonCoh}
illustrates the bit error rate (BER) for the case of two stages and
for $N=64$ and $N=256$  Simulations also show that there is no
remarkable difference between results in two stage and three stage
scenarios  Table~\ref{tabex5} compares the average channel phase
estimate of the first user in each stage and over $10$ runs of
modified LMS-PPIC and PLMS-PPIC, when the the number of users is
$M=15$ 
\end{example}

Although LMS-PPIC and PLMS-PPIC, as well as their modified versions,
are structured based on the assumption of no near-far problem
(examples \ref{ex3} and \ref{ex4}), these methods and especially the
second one have remarkable performance in the cases of unbalanced
and/or time varying channels 

\begin{example}{\it Unbalanced channels}:
\label{ex3}
\begin{table}
\caption{Channel phase estimate of the first user (example
\ref{ex3})} \label{tabex6} \centerline{{
\begin{tabular}{|c|c|c|c|c|}
\hline
\multirow{6}{*}{\rotatebox{90}{$\phi_m=\frac{3\pi}{8},M=15~~$}} & N(Iteration) & Stage Number& NLMS & PNLMS  \\
&&&&\\
\cline{2-5} & \multirow{2}{*}{64}& s=2 &  $\hat{\phi}^s_m=\frac{2 45\pi}{8}$ & $\hat{\phi}^s_m=\frac{2 36\pi}{8}$ \\
\cline{3-5} & & s=3 & $\hat{\phi}^s_m=\frac{2 71\pi}{8}$ & $\hat{\phi}^s_m=\frac{2 80\pi}{8}$ \\
\cline{2-5} & \multirow{2}{*}{256}& s=2 &  $\hat{\phi}^s_m=\frac{3 09\pi}{8}$ & $\hat{\phi}^s_m=\frac{2 86\pi}{8}$ \\
\cline{3-5} & & s=3 & $\hat{\phi}^s_m=\frac{2 93\pi}{8}$ & $\hat{\phi}^s_m=\frac{3 01\pi}{8}$ \\
\cline{2-5} \hline
\end{tabular} }}
\end{table}
Consider example \ref{ex2} with power unbalanced and/or channel loss
in transmission system, i e  the true model at stage $s$ is
\begin{equation}
\label{ve7} r(n)=\sum\limits_{m=1}^{M}\beta_m
w^s_m\alpha^{(s-1)}_m c_m(n)+v(n),
\end{equation}
where $0<\beta_m\leq 1$ for all $1\leq m \leq M$  Both the LMS-PPIC
and the PLMS-PPIC methods assume the model (\ref{e7}), and their
estimations are based on observations $\{r(n),X^s(n)\}$, instead of
$\{r(n),\mathbf{G}X^s(n)\}$, where the channel gain matrix is
$\mathbf{G}=\mbox{diag}(\beta_1,\beta_2,\cdots,\beta_m)$  In this
case we repeat example \ref{ex2}  We randomly get each element of
$G$ from $[0,0 3]$  Figure~\ref{Figexp2NonCoh} illustrates the BER
versus the number of users  Table~\ref{tabex6} compares the channel
phase estimate of the first user in each stage and over $10$ runs of
modified LMS-PPIC and modified PLMS-PPIC for $M=15$ 
\end{example}

\begin{example}
\label{ex4} {\it Time varying channels}: Consider example \ref{ex2}
with time varying Rayleigh fading channels  In this case we assume
the maximum Doppler shift of $40$HZ, the three-tap
frequency-selective channel with delay vector of $\{2\times
10^{-6},2 5\times 10^{-6},3\times 10^{-6}\}$sec and gain vector of
$\{-5,-3,-10\}$dB  Figure~\ref{Figexp3NonCoh} shows the average BER
over all users versus $M$ and using two stages 
\end{example}


\section{Conclusion}\label{S6}

In this paper, parallel interference cancelation using adaptive
multistage structure and employing a set of NLMS algorithms with
different step-sizes is proposed, when just the quarter of the
channel phase of each user is known  In fact, the algorithm has been
proposed for coherent transmission with full information on channel
phases in \cite{cohpaper}  This paper is a modification on the
previously proposed algorithm  Simulation results show that the new
method has a remarkable performance for different scenarios
including Rayleigh fading channels even if the channel is
unbalanced 


Context 2: 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 
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ground_truth: The normalized least mean square (NLMS) algorithm.

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 2:  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 
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(f) BROADJAM AND ITS AFFILIATES, LICENSORS, SUPPLIERS, ADVERTISERS, SPONSORS AND AGENTS DO NOT WARRANT THAT YOUR USE OF THE SITE WILL BE UNINTERRUPTED, ERROR-FREE OR SECURE, THAT DEFECTS WILL BE CORRECTED, OR THAT THE SITE OR THE SERVER(S) ON WHICH THE SITE IS HOSTED ARE FREE OF VIRUSES OR OTHER HARMFUL COMPONENTS  YOU ACKNOWLEDGE THAT YOU ARE RESPONSIBLE FOR OBTAINING AND MAINTAINING ALL TELEPHONE, COMPUTER HARDWARE AND OTHER EQUIPMENT NEEDED TO ACCESS AND USE THE SITE, AND ALL CHARGES RELATED THERETO  YOU ASSUME ALL RESPONSIBILITY AND RISK FOR YOUR USE OF THE SITE AND ANY SERVICE AND YOUR RELIANCE THEREON  YOU UNDERSTAND AND AGREE THAT YOU DOWNLOAD OR OTHERWISE OBTAIN MATERIAL, INFORMATION OR DATA THROUGH THE USE OF THE SITE AT YOUR OWN DISCRETION AND RISK AND THAT YOU WILL BE SOLELY RESPONSIBLE FOR ANY DAMAGES TO YOUR COMPUTER SYSTEM OR LOSS OF DATA THAT RESULTS FROM THE DOWNLOAD OF SUCH MATERIAL, INFORMATION OR DATA 
(g) SOME STATES OR OTHER JURISDICTIONS DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES, SO THE ABOVE EXCLUSIONS MAY NOT APPLY TO YOU  YOU MAY ALSO HAVE OTHER RIGHTS THAT VARY FROM STATE TO STATE AND JURISDICTION TO JURISDICTION  PROVIDED, HOWEVER, THAT TO THE EXTENT PERMITTED BY APPLICABLE LAW YOU HEREBY WAIVE THE PROVISIONS OF ANY STATE LAW LIMITING OR PROHIBITING SUCH EXCLUSIONS 
(a) NEITHER BROADJAM NOR ANY OF OUR AFFILIATES, LICENSORS, SUPPLIERS, ADVERTISERS OR SPONSORS, NOR OUR OR THEIR DIRECTORS, OFFICERS, EMPLOYEES, CONSULTANTS, AGENTS OR OTHER REPRESENTATIVES (TOGETHER, FOR PURPOSES OF THIS SECTION, "BROADJAM"), ARE RESPONSIBLE OR LIABLE FOR ANY INDIRECT, INCIDENTAL, CONSEQUENTIAL, SPECIAL, EXEMPLARY, PUNITIVE OR OTHER DAMAGES (INCLUDING, WITHOUT LIMITATION, DAMAGES FOR LOSS OF BUSINESS, LOSS OF DATA OR LOST PROFITS), UNDER ANY CONTRACT, NEGLIGENCE, WARRANTY, STRICT LIABILITY OR OTHER THEORY ARISING OUT OF OR RELATING IN ANY WAY TO USE OR MISUSE OF OR RELIANCE ON THE SITE OR ANY BROADJAM SERVICE OR ANY LINKED SITE, EVEN IF BROADJAM HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES, AND IN NO EVENT SHALL BROADJAM'S TOTAL CUMULATIVE LIABILITY UNDER THIS AGREEMENT EXCEED THE TOTAL AMOUNT PAID BY YOU, IF ANY, TO ACCESS THE SITE  SUCH LIMITATION OF LIABILITY SHALL APPLY WHETHER THE DAMAGES ARISE FROM USE OR MISUSE OF AND/OR RELIANCE ON THE SITE OR ANY BROADJAM SERVICE, FROM INABILITY TO USE THE SITE OR ANY BROADJAM SERVICE, OR FROM THE INTERRUPTION, SUSPENSION, OR TERMINATION OF THE SITE OR ANY BROADJAM SERVICE (INCLUDING SUCH DAMAGES INCURRED BY THIRD PARTIES)  THIS LIMITATION SHALL ALSO APPLY WITH RESPECT TO DAMAGES INCURRED BY REASON OF OTHER SERVICES OR GOODS RECEIVED THROUGH OR ADVERTISED ON THE SITE OR RECEIVED THROUGH ANY LINKS PROVIDED AT, IN OR THROUGH THE SITE, AS WELL AS BY REASON OF ANY INFORMATION OR ADVICE RECEIVED THROUGH OR ADVERTISED ON THE SITE OR RECEIVED THROUGH ANY LINKS PROVIDED ON THE SITE OR ANY BROADJAM SERVICE  THIS LIMITATION SHALL ALSO APPLY, WITHOUT LIMITATION, TO THE COSTS OF PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES, LOST PROFITS, AND LOST DATA  SUCH LIMITATION SHALL FURTHER APPLY WITH RESPECT TO THE PERFORMANCE OR NONPERFORMANCE OF THE SITE OR ANY BROADJAM SERVICE OR ANY INFORMATION OR MERCHANDISE THAT APPEARS ON, OR IS LINKED OR RELATED IN ANY WAY TO, THE SITE OR ANY BROADJAM SERVICE  SUCH LIMITATION SHALL APPLY NOTWITHSTANDING ANY FAILURE OF ESSENTIAL PURPOSE OF ANY LIMITED REMEDY AND TO THE FULLEST EXTENT PERMITTED BY LAW 
(b) SOME STATES OR OTHER JURISDICTIONS DO NOT ALLOW THE EXCLUSION OR LIMITATION OF LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES, SO THE ABOVE LIMITATIONS AND EXCLUSIONS MAY NOT APPLY TO YOU  PROVIDED, HOWEVER, THAT TO THE EXTENT PERMITTED BY APPLICABLE LAW YOU HEREBY WAIVE THE PROVISIONS OF ANY STATE LAW LIMITING OR PROHIBITING SUCH EXCLUSIONS OR LIMITATIONS 
(c) WITHOUT LIMITING THE FOREGOING, UNDER NO CIRCUMSTANCES SHALL BROADJAM BE HELD LIABLE FOR ANY DELAY OR FAILURE IN PERFORMANCE RESULTING DIRECTLY OR INDIRECTLY FROM ACTS OF NATURE, FORCES, OR CAUSES BEYOND ITS REASONABLE CONTROL, INCLUDING, WITHOUT LIMITATION, INTERNET FAILURES, COMPUTER EQUIPMENT FAILURES, TELECOMMUNICATION EQUIPMENT FAILURES, OTHER EQUIPMENT FAILURES, ELECTRICAL POWER FAILURES, STRIKES, LABOR DISPUTES, RIOTS, INSURRECTIONS, CIVIL DISTURBANCES, SHORTAGES OF LABOR OR MATERIALS, FIRES, FLOODS, STORMS, EXPLOSIONS, ACTS OF GOD, EPIDEMIC, WAR, GOVERNMENTAL ACTIONS, ORDERS OF DOMESTIC OR FOREIGN COURTS OR TRIBUNALS, NON-PERFORMANCE OF THIRD PARTIES, OR LOSS OF OR FLUCTUATIONS IN HEAT, LIGHT, OR AIR CONDITIONING 
(a) All content included on this Site, including but not limited to text, graphics, logos, button icons, images, data compilations, code and source code, multimedia content, including but not limited to images, illustrations, audio and video clips, html and other mark up languages, and all scripts within the Site or associated therewith, are the property of Broadjam or its content suppliers and is protected by United States and international copyright laws with All Rights Reserved  The compilation of all content on this Site is the exclusive property of Broadjam and is protected by United States and international copyright laws with All Rights Reserved  All software used on this site is the property of Broadjam or its software suppliers and is protected by United States and international copyright laws with All Rights Reserved 
(b) "Broadjam," "Broadjam Top 10," "Metajam", "broadjam com", "Musicians of Broadjam," Mini MoB, PRIMO MoB and other trademarks, service marks, logos, labels, product names and service names appearing on the Site (collectively, the "Marks") are owned or licensed by Broadjam  Marks not owned by Broadjam or its subsidiaries are the property of their respective owners, who may or may not be affiliated with, connected to, or sponsored by Broadjam 
(c) You agree not to copy, display or otherwise use any Marks without Broadjam's prior written permission  The Marks may never be used in any manner likely to cause confusion, disparage or dilute the Marks and/or in connection with any product or service that is not authorized or sponsored by Broadjam 
(a) We make no representation that products or services available on or through the Site or any Service are appropriate or available for use in other locations other than the United States  Those who choose to access the Site or any Service from other locations do so on their own initiative and at their own risk, and are responsible for compliance with local laws, if and to the extent local laws are applicable  By accessing the Site or using any Services you are consenting to have your personal data transferred to and processed in the United States 
(b) Products, including software, made available through the Site or any Service are further subject to United States export controls  You agree to comply with all applicable laws regarding the transmission of technical data exported from the United States or the country in which you reside  No such products may be downloaded or otherwise exported or re-exported (i) into (or to a national or resident of) any country to which the U S  has embargoed goods; or (ii) to anyone on the U S  Treasury Department's list of Specially Designated Nationals or the U S  Commerce Department's Table of Deny Orders  By downloading any product available through the Site or any Service, you represent and warrant that you are not located in, under the control of, or a national or resident of any such country or on any such list  We reserve the right to limit the availability of the Site and/or any Service or product described thereon to any person, geographic area or jurisdiction, at any time and in our sole discretion, and to limit the quantities of any such Service or product that we provide 
Broadjam may also provide access to certain services (including, without limitation and by way of example only: advertising, promotion, and submission processing services for contests, radio play, publishing, placement and licensing opportunities) that are supplied by others ("Third Party Services")  YOU EXPRESSLY ACKNOWLEDGE THAT BROADJAM BEARS NO RESPONSIBILITY FOR THIRD PARTY SERVICES; BROADJAM EXPRESSLY DISCLAIMS ANY/ALL LIABILITY FOR THIRD PARTY SERVICES; AND BROADJAM MAKES NO WARRANTY, REPRESENTATION OR GUARANTEE TO YOU REGARDING ANY ASPECT OF THIRD PARTY SERVICES  ANY CLAIM YOU MAY HAVE REGARDING ANY THIRD PARTY SERVICE MUST BE PURSUED DIRECTLY AND EXCLUSIVELY WITH THE INDIVIDUAL OR GROUP, WHETHER OR NOT ORGANIZED AS A LEGAL ENTITY (THE "THIRD PARTY PROVIDER"), THAT SUPPLIED THE THIRD PARTY SERVICE  BROADJAM IS NOT A PARTY TO ANY RULES, CONTRACTS OR OTHER AGREEMENTS BETWEEN YOU AND ANY THIRD PARTY PROVIDER, AND YOU EXPRESSLY AGREE NOT TO JOIN OR ATTEMPT TO JOIN BROADJAM AS A PARTY IN ANY DISPUTE BETWEEN YOU AND ANY THIRD PARTY PROVIDER 
Upon receipt of your written request, Broadjam will remove any of your Materials from the Site within a reasonable period of time  Broadjam's licenses to use such Materials will continue for any copies of such Materials that may have been disseminated in any format or media prior to the actual removal of such Materials from the Site 
You agree that, at any time, Broadjam may revise, change or modify any terms and conditions of this Agreement and/or any aspect of any Service, without notice to you  You can review the most current version of this Agreement at any time at: http://www broadjam com  When using any Service, you and Broadjam shall also be subject to any guidelines, Policies or rules applicable to such Service which may be posted on the Site from time to time  All such guidelines, Policies or rules are hereby incorporated by reference into this Agreement and you agree to their terms  Any such revisions, changes or modifications shall be binding and effective immediately upon posting of same to the Site 
(a) Your rights under this Agreement are not assignable and any attempt by your creditors to obtain an interest in your rights under this Agreement, whether by attachment, levy, garnishment or otherwise, renders this Agreement voidable at Broadjam's option 
(b) This Agreement is binding onthe Parties and their respective heirs, legatees, executors, successors and assigns  Except for Policies and other agreements incorporated by reference herein, this Agreement is the entire agreement between the Parties and supersedes all prior written or oral agreements between the Parties relating to the subject matter hereof  If any portion of this Agreement is found to be void or unenforceable, the remaining portion shall be enforceable with the invalid portion removed, giving all reasonable construction to permit the essential purposes of the Agreement to be achieved  The Parties' various rights and remedies hereunder shall be construed to be cumulative 
(c) This Agreement shall be deemed to have been made in the State of Wisconsin, and it shall be governed by the substantive laws of the State of Wisconsin without regard to any applicable conflict of laws provisions  The Parties submit to jurisdiction in the state and federal courts sitting in Dane County, Wisconsin, and you hereby waive any jurisdictional, venue or inconvenient forum objections  Provided, however, that if we are sued or joined in an action in any other court or forum in respect of any matter which may give rise to a claim by us hereunder, you consent to the jurisdiction of such court or forum over any such claim  Nothing in this paragraph or Agreement constitutes our consent to the assertion of personal jurisdiction over Broadjam otherwise than in Wisconsin 
(d) Nothing contained in this Agreement shall be construed to require the commission of any act contrary to law  Nothing in this Agreement shall be construed or deemed to create any partnership, agency, joint venture, employment or franchise relationship between the Parties 
(e) Each Party hereto agrees to execute all further and additional documents as may be necessary or desirable to effectuate and carry out the provisions of this Agreement 
(f) Captions and headings used in this Agreement are for purposes of convenience only and shall not be deemed to limit, affect the scope, meaning or intent of this Agreement, nor shall they otherwise be given any legal effect 
(g) No breach of this Agreement by Broadjam shall be deemed material unless the Party alleging such breach shall have given Broadjam written notice of such breach, and Broadjam shall fail to cure such breach within thirty (30) days after its receipt of such notice 
(h) All notices required to be sent to Broadjam under this Agreement shall be in writing and shall be sent by certified mail, return receipt requested, postage paid, or by overnight delivery service, to Broadjam Inc , 211 S  Paterson St  Ste  360 Madison, WI 53703 Attention: Legal (or such other address or addresses as may be designated by Broadjam herein) 
(i) All duties, liabilities, obligations, warranties, representations, covenants, authorizations, agreements and restrictions undertaken by and/or imposed upon you in connection with this Agreement shall be deemed to apply jointly and severally to all members collectively and each member individually of any group at any time comprising the Artist whose recordings or other Materials you post, upload or otherwise make available to Broadjam  You affirmatively represent that you have the authority to bind all such individuals to the terms and conditions of this Agreement 
(j) You agree that regardless of any statute or law to the contrary, any claim or cause of action against Broadjam, arising out of or related to use of the Site or any Service, must be filed within one (1) year after such claim or cause of action arose or be forever barred 
Sacramento, California 95834, or by telephone at (800) 952-5210 
available by contacting Broadjam at the above address, Attention: Customer Service 
(m) This Agreement has no intended third party beneficiaries 
(a) This Article II applies to any Person (hereinafter a "Subscriber") who subscribes to any member subscription service offered by Broadjam, including but not limited to, by way of example, Mini MoB or PRIMO MoB (hereinafter a "Subscription Service")



ground_truth: No, it is not necessary.

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 are the restrictions on the use of Broadjam's servers?

Read the following text and answer briefly.

Context 3:  For purposes of this Agreement all Subscribers are also Users as defined herein 
(b) You agree to provide true, accurate, current and complete information about yourself as prompted by the subscription registration processes (such information being your "Account Information")  You further agree that, in providing such Account Information, you will not knowingly omit or misrepresent any material facts or information and that you will promptly enter corrected or updated Account Information, or otherwise advise us promptly in writing of any such changes or updates  You further consent and authorize us to verify your Account Information as required for your use of and access to the Site and any Service, as applicable 
(c) As a Subscriber, you will receive a unique username and password in connection with your account (collectively referred to herein as your "Username")  You agree that you will not allow another person to use your Username to access and use the Site or any Service under any circumstances  You are solely and entirely responsible for maintaining the confidentiality of your Username and for any charges, damages, liabilities or losses incurred or suffered as a result of your failure to do so  Broadjam is not liable for any harm caused by or related to the theft of your Username, your disclosure of your Username, or your authorization to allow another person to access and use the Site or any Service using your Username  Furthermore, you are solely and entirely responsible for any and all activities that occur under your account, including, but not limited to, any charges incurred relating to the Site or any Service  You agree to immediately notify us of any unauthorized use of your account or any other breach of security known to you  You acknowledge that the complete privacy of your data transmitted while using the Site or any Service cannot be guaranteed 
The term of any Subscription Service shall commence when the Subscriber initiates payment for such Subscription Service or, if the Subscription Service is complimentary, when the Subscriber registers for such Subscription Service  All Subscription Services will extend for an initial period of oneyear (the "Term") and, unless terminated as provided herein, shall renew automatically for successive one-year periods  During the Term, the Subscriber shall be afforded the full use and benefit of the applicable Subscription Service as described on the Site (the "Service Benefits"), which Service Benefits may be revised by Broadjam from time to time without notice to the Subscriber  Due to technical considerations, certain Service Benefits may not be available to the Subscriber immediately upon commencement of the Term, but shall be provided to the Subscriber as soon as commercially reasonable  Please direct any questions about Subscription Services or Service Benefits to Broadjam by email at: customerservice@broadjam com or by US mail at: Broadjam Inc , 100 S  Baldwin St  Ste  #204, Madison, WI 53703, Attn: Customer Service 
(b) maintain and update such information as needed to keep it current, complete and accurate 
Subscriber acknowledges that Broadjam relies and will rely upon the accuracy of such information as supplied by Subscriber 
(a) Termination by Subscriber  Subscriber may terminate any Subscription Service at any time by providing Broadjam with written notice pursuant to this Agreement  Written notice will be followed by a confirmation request from Broadjam Customer Service  Confirmation is required to implement termination  Such termination will be effective after the paid period  In the case of termination by the Subscriber, the period that is already paid for will not be reimbursed  The Subscription Service will remain active until the end of the paid period 
(a) As consideration for a Subscription Service, Subscriber agrees to pay Broadjam all applicable subscription fees as posted on the Site at the time Subscriber applies for the Subscription Service  All subscription fees are due immediately pursuant to the payment option Subscriber chooses, and are non-refundable except as otherwise provided herein  Broadjam may exercise all available remedies to collect fees due and owing for any Subscription Service 
(b) Broadjam may, at its sole discretion and for any Subscription Service, offer Subscriber the option to pay Subscriber's annual subscription fee in monthly installments (a "Payment Plan")  If Subscriber elects a Payment Plan, Subscriber agrees to provide Broadjam with a valid credit card number, which Broadjam will charge on a monthly basis for twelve (12) consecutive months, in an amount each month equal to 1/12th of the subscription fee for the Subscription Service, plus a finance charge, until the Subscription Service is terminated pursuant to this Agreement  By providing credit card billing information, Subscriber shall be authorizing Broadjam to charge that credit card until termination of the Subscription Service  Broadjam shall have the right immediately to discontinue Subscriber's Service Benefits if Broadjam does not receive payment when due 
In order to change any of Subscriber's account information, Subscriber must use the User Name and the Password that Subscriber selected when Subscriber registered as a Broadjam User  In no event will Broadjam be liable for any unauthorized use or misuse of Subscriber's User Name and Password 
Subscriber agrees that Subscriber's failure to abide by any provision of this Agreement or any Broadjam operating rule or policy, Subscriber's willful provision of inaccurate or unreliable information as part of the application process, Subscriber's failure to update Subscriber's information to keep it current, complete or accurate, and/or Subscriber's failure to respond to inquiries from Broadjam concerning the accuracy of Subscriber's account information shall be considered a material breach of this Agreement  If within ten (10) calendar days after Broadjam provides notice (in any form and via any method of delivery) to Subscriber of such material breach, Subscriber fails to provide evidence, reasonably satisfactory to Broadjam, that Subscriber has not breached its obligations under this Agreement, Broadjam may terminate all Services, Subscription and otherwise, without further notice to Subscriber 
This Article III applies to any Person (hereinafter a "Hosting Subscriber") who subscribes to any web hosting subscription service offered by Broadjam, including but not limited to, by way of example, PRIMO MoB (hereinafter a "Hosting Service")  For purposes of this Agreement all Hosting Subscribers are also Subscribers and Users as defined herein 
Hosting Subscriber's Website will not be used in connection with any illegal activity 
(b) Hosting Subscriber is responsible for ensuring that there is no excessive overloading on Broadjam's DNS or servers  Broadjam prohibits the use of software or scripts run on its servers that cause the server to load beyond a reasonable level, as determined by Broadjam  Hosting Subscriber agrees that Broadjam reserves the right to remove Hosting Subscriber's Website temporarily or permanently from its hosting servers if Hosting Subscriber's Website threatens the stability of Broadjam's network 
(c) Hosting Subscriber may not use Broadjam's servers or Hosting Subscriber's Website as a source, intermediary, reply to address, or destination address for mail bombs, Internet packet flooding, packet corruption, denial of service, or any other abusive activities  Server hacking or other perpetration of security breaches is strictly prohibited and Broadjam reserves the right to remove websites that contain information about hacking or links to such information  Use of Hosting Subscriber's Website as an anonymous gateway is prohibited 
engage in any other activity deemed by Broadjam to be in conflict with the spirit or intent of this Agreement or any Broadjam policy 
Subject to the terms and conditions of this Agreement, Broadjam shall attempt to provide Hosting Services for twenty-four (24) hours a day, seven (7) days a week throughout the term of Hosting Subscriber's subscription  Hosting Subscriber agrees that from time to time the Hosting Service may be inaccessible or inoperable for any reason, including, without limitation, equipment malfunctions; periodic maintenance procedures or repairs which Broadjam may undertake from time to time; or causes beyond the control of Broadjam or which are not reasonably foreseeable by Broadjam, including, without limitation, interruption or failure of telecommunication or digital transmission links, hostile network attacks, network congestion or other failures  Hosting Subscriber agrees that Broadjam makes no representation or assurance that Hosting Services will be available on a continuous or uninterrupted basis 
At all times, Hosting Subscriber shall bear full risk of loss and damage to Hosting Subscriber's Website and all of Hosting Subscriber's Website content  Hosting Subscriber is solely responsible for maintaining the confidentiality of Hosting Subscriber's Password and account information  Hosting Subscriber agrees that Hosting Subscriber is solely responsible for all acts, omissions and use under and charges incurred with Hosting Subscriber's account or password or any of Hosting Subscriber's Website content  Hosting Subscriber shall be solely responsible for undertaking measures to: (i) prevent any loss or damage to Hosting Subscriber's Website content; (ii) maintain independent archival and backup copies of Hosting Subscriber's Website content; (iii) ensure the security, confidentiality and integrity of all of Hosting Subscriber's Website content transmitted through or stored on Broadjam servers; and (iv) ensure the confidentiality of Hosting Subscriber's password  Broadjam's servers and Hosting Services are not an archive and Broadjam shall have no liability to Hosting Subscriber or any other person for loss, damage or destruction of any of Hosting Subscriber's content  If Hosting Subscriber's password is lost, stolen or otherwise compromised, Hosting Subscriber shall promptly notify Broadjam, whereupon Broadjam shall suspend access to Hosting Subscriber's Website by use of such password and issue a replacement password to Hosting Subscriber or Hosting Subscriber's authorized representative  Broadjam will not be liable for any loss that Hosting Subscriber may incur as a result of someone else using Hosting Subscriber's password or account, either with or without Hosting Subscriber's knowledge  However, Hosting Subscriber could be held liable for losses incurred by Broadjam or another party due to someone else using Hosting Subscriber's account or password 
(a) Broadjam does not tolerate the transmission of spam  We monitor all traffic to and from our Web servers for indications of spamming and maintain a spam abuse compliant center to register allegations of spam abuse  Customers suspected to be using Broadjam products and services for the purposeof sending spam are fully investigated  Once Broadjam determines there is a problem with spam, Broadjam will take the appropriate action to resolve the situation  Our spam abuse compliant center can be reached by email at hosting@broadjam com 
(c) Broadjam will not allow its servers or services to be used for the purposes of spam as described above  In order to use our products and services, Hosting Subscriber shall abide by all applicable laws and regulations, including but not limited to the Can-Spam Act of 2003 and the Telephone Consumer Protection Act, as well as Broadjam's no-spam policies  Commercial advertising and/or bulk emails or faxes may only be sent to recipients who have already "opted-in" to receive messages from the sender specifically  They must include a legitimate return address and reply-to address, the sender's physical address, and an opt-out method in the footer of the email or fax  Upon request by Broadjam, conclusive proof of optin may be required for an email address or fax number 
(d) If Broadjam determines that Hosting Services are being used in association with spam, Broadjam will re-direct, suspend, or cancel such Hosting Service for a period of no less than 2 days  The Hosting Subscriber will be required to respond by email to Broadjam stating that Hosting Subscriber will cease to send spam and/or have spam sent on their behalf  Broadjam will require a non-refundable reactivation fee to be paid before Hosting Subscriber's Website, email boxes and/or other Hosting Services are reactivated  In the event Broadjam determines the abuse has not stopped after services have been restored the first time, Broadjam may terminate all Services associated with the Hosting Subscriber 
This Article IV applies to all Users 
Fees and prices appearing on the Site are based on United States dollars  Payments for any Service or purchase made on or through the Site shall be made to Broadjam in United States dollars, except as provided in Section 4 05 herein 
You agree to pay for all fees and charges incurred under your Broadjam account or Username  If you have configured the account associated with your Username (your "Account") to pay for Services or purchases with a credit or debit card or similar form of payment (a "Card" payment method), you authorize any and all charges and fees incurred under your Account to be billed from time to time to your Card account  Regardless of the method of payment, it is your sole responsibility to advise Broadjam of any billing problems or discrepancies within thirty (30) days after such discrepancies or problems become known to you  Your Card issuer agreement governs the use of your designated Card account in connection with any fee, purchase or Service; you must refer exclusively to such issuer agreement, and not this Agreement, to determine your rights and liabilities as a Cardholder  If you submit a payment that results in Broadjam being charged non-sufficient funds, chargeback fees, or other similar fees, you agree to reimburse all such fees 
Monthly Billing Subscriptions  No refunds will be issued for monthly billing subscriptions  If monthly billing is selected and is not cancelled by the end of the monthly period (30 days from the sign up date), your Card will be billed at the beginning of the next 30 day period  In order to avoid additional charges to your Card, you must contact Broadjam Customer Service by email (customerservice@broadjam com) at least 5 days before your next billing period, to cancel your Subscription Service  Your email should include the following: registered name on the account, registered email address on the account, and the service to be cancelled  Notice will be followed by a confirmation request from Broadjam Customer Service  Confirmation is required to implement cancellation 
(a) Merchants who elect to be paid in Purchase Credits ("PCs") for sales at Broadjam, Buyers who choose to purchase PCs and Users who otherwise obtain PCs (collectively, "Holders" of PCs) shall hold PCs subject to the provisions of this Section 4 05 as well as all rules and policies posted on the Site relating to PCs 
(b) PCS ARE NONRETURNABLE AND NONREFUNDABLE 
(c) PCs do not have an expiration date  However, if there exists rules defined by the laws of your state that require Broadjam to terminate your right to use PCs if you have not used them within a specified number of years  Under those laws, Broadjam will attempt to contact you before terminating your right to use PCs 
(e) Holders shall have no right to demand cash or any other thing of value in exchange for PCs, except as provided in Section 4 05 (d) 
(f) Interest shall not accrue on PCs 
(a) Buyers who choose to purchase the Primo MoB membership which includes complimentary Weekly Submission Credits ("WSCs") for the term of the membership purchased for use towards Music Licensing Opportunities services and shall hold WSCs subject to the provisions of this Section 4 06 as well as all rules and policies posted on the Site relating to WSCs 
(b) WSCs ARE NONRETURNABLE AND NONREFUNDABLE 
(c) One WSC is available for use each week for the duration of the membership purchased  One WSC is available each week starting Sunday at 12:00 am midnight CST  If unused, each WSC will expire on the following Sunday at 11:59 pm CST 
ii  wholly controlled by Broadjam 
(f) Holders shall have no right to demand cash or any other thing of value in exchange for WSCs, except as provided in Section 4 06 (e) 
(g) Interest shall not accrue on WSCs 
(a) Buyers who choose to purchase the Film/TV membership which includes complimentary Monthly Submission Credits ("MSCs") for the term of the membership purchased for use towards Music Licensing Opportunities services and shall hold MSCs subject to the provisions of this Section 4 07 as well as all rules and policies posted on the Site relating to MSCs 
(b) MSCs ARE NONRETURNABLE AND NONREFUNDABLE 
(c) One MSC is available for use each month for the duration of the membership purchased  One MSC is available each month starting the first day of the month at 12:00 am midnight CST  If unused, each WSC will expire on the last day of the month at 11:59 pm CST 
(f) Holders shall have no right to demand cash or any other thing of value in exchange for MSCs, except as provided in Section 4 07 (e) 
(g) Interest shall not accrue on MSCs 
Checks issued by Broadjam to any User, for any purpose, are VOID after 180 days from the date of issue  Users who fail to cash Broadjam-issued checks within such 180-day period will be charged a $2 00 fee for re-depositing funds from the stale check to the User's account  Users requesting replacement checks will be charged an additional $5 00 fee for issuance of the replacement check 
The following shall apply if you purchase Broadjam's Deliveries services 
Refunds will not be issued for Broadjam Deliveries services  If you experience a technical problem related to Broadjam Deliveries services, Broadjam will take steps in accordance with Section 1 10 to ensure your transaction is completed successfully  Broadjam may at its sole discretion convey complimentary services to you in the event of a verified technical problem 
The following shall apply if you purchase Broadjam's Music Software services 
Refunds will not be issued for Music Software services  If you experience a technical problem related to Broadjam Music Software services, Broadjam will take steps in accordance with Section 1 10 to ensure your transaction is completed successfully  Broadjam may at its sole discretion convey complimentary services to you in the event of a verified technical problem 



ground_truth: No excessive overloading and no use for illegal activity.