text:
\section*{Introduction}
Single-molecule experimental techniques are widely used to understand the kinetics and energetics involved into biological phenomena. Among them, because of its simplicity and amenability to parallelization, nanopore force spectroscopy is an increasingly used technique for ultra-sensitive detection of macromolecules. The method consists in applying an electric potential difference to guide a charged molecule through a nanopore (artificial or biological) inserted in a solid or lipid membrane, in presence of a salt solution~\cite{Kasianowicz1996, Dudko2010, Meller2000, Oukhaled2007, Oukhaled2008}. A macromolecule going through the pore, partially blocking it, creates a variation in the electrical current according to the nature of the molecule and the pore characteristics: size, conformation, and structure. Ionic current measurements are a probe for the detection. Amongst the various biological nanopore, $\alpha$-hemolysin is the most widely employed in nanopore analytics. It has been commonly used as a nanopore for DNA or RNA translocation~\cite{Kasianowicz1996,Meller2000}, DNA sequencing or biosensing~\cite{Deamer2016}, DNA unzipping of individual hairpins~\cite{Mathe2004,Mathe2005,Muzard2010}, and the study of protein translocation~\cite{Oukhaled2007}.

The understanding of the physical mechanisms governing the translocation of ions or molecules through a nanopore is not only helpful to reveal relevant biological phenomena but also for the design of new devices based on such nanopores. The microscopic details of these processes cannot be deduced straightforwardly from the experiments. In this sense, an increasing effort to provide theoretical and computational approaches in order to understand such processes is being done~\cite{Modi2012}. In general, these approaches require a choice between the amount of computational resources and the microscopic level of accuracy. Among continuum approaches, the Poisson–Nernst–Planck theory (PNP)~\cite{Roux2004, Noskov2004, Cervera2005, Constantin2007, Cozmuta2004, Birlea2012} is useful to study ion transport at a relatively small computational cost, although not very accurate for narrow pores. At the same level, Brownian or Langevin dynamics methods (BD)~\cite{DeBiase2014, DeBiase2016, Millar2008, Noskov2004} can be an alternative. In this case, the main challenge is the representation of the protein-continuum solvent interface.

In All-Atom Molecular Dynamics (AA-MD) simulations, the individual motion of ions, water, protein and membrane atoms are fully computed based on a classical atomic force field. This approach will be limited by the classical force field approximation used, but AA-MD simulations are undoubtedly excellent to provide microscopic details of biological translocation processes "at short times". The huge computational cost is the main disadvantage of this method and therefore, there are several ways to accelerate the transport of molecules through the $\alpha$-hemolysin pore: using very high electric potentials (1~V compared to hundreds of mV for experiments)~\cite{Aksimentiev2004, Aksimentiev2005, Mathe2005} or forcing the transport of molecules through the pore using velocity-pulling methods~\cite{DiMarino2015}. Although some results of these AA-MD are in accordance with experiments, long time phenomena cannot be observed in such simulations, generally limited to a few hundred nanoseconds. The high-potential/pulling methods can also induce artifacts in the results.

Coarse-Grained (CG) force fields, representing several atoms by one site, for lipid membrane and biomolecules, are a very promising alternative to All-Atoms models, as they enable simulations of time-lengths closer to experiments. In this sense, our article focuses on MD simulations where the AA representation is replaced by an "atomistic" CG force field, in our case the MARTINI force field. The CG MARTINI force field~\cite{Marrink2007}, originally developed for lipids, is widely used to simulate membrane systems for time-lengths up to hundreds of microseconds~\cite{Ingolfsson2014,Pinot2014,VanEerden2015}. Furthermore, a polarizable CG water model is needed to correctly describe the electrostatic behavior of electrolytes.  Among the several CG polarizable solvent models available~\cite{Ha-Duong2009,Wu2010,Yesylevskyy2010}, the Polarizable Water (PW) model~\cite{Yesylevskyy2010} has been parametrized to be used together with the standard MARTINI force field. Whereas classical AA simulations of ionic current through $\alpha$-hemolysin have been done, to our knowledge, such a study using a CG model has not been yet performed.

In this work, our MARTINI CG MD simulations for determining ion currents through $\alpha$-hemolysin are detailed and discussed. We begin by studying the electrostatic behavior of solvent, ions and membrane using this force field. The ionic conductivity of CG MARTINI ions in PW solution as a function of concentration is discussed. Secondly, we study the electrostatic behavior of a solvated DPPC (dipalmitoylphosphatidylcholine) lipid bilayer with excess ions in solution. We will confirm that applying an electric field in a direction perpendicular to the membrane plan is accurate to represent the experimentally applied potential difference. Finally, we will study the current through the protein nanopore at various electric potentials on microseconds-long MD.

\section*{Results}

All our MD simulations were performed using the Gromacs software package~\cite{VanDerSpoel2005} and the MARTINI 2.2 Coarse-Grained (CG) model for ions, lipids~\cite{Marrink2007}, proteins~\cite{Monticelli2008,Jong2013} and the PW (Polarizable Water) model~\cite{Yesylevskyy2010} for water. The systems were simulated either in the NVT or the NPT ensemble at a temperature of $T=320$~K, where PW water presents a density of 1020 kg/$m^3$~\cite{Yesylevskyy2010}. MARTINI CG ions consist in particles of the same size and mass as a PW particle, carrying positive or negative charge. Although they are called "Na$^+$" and "Cl$^-$" in the force field parameters files, they do not represent a realistic sodium or chlorine ion \cite{Marrink2004}. Since the MARTINI salt is perfectly symmetrical, we considered that it represents the KCl salt instead of NaCl. Methodological details of the simulations can be found in the Methods.The details of the ionic molar concentration setting for NVT simulations is discussed in the Supplementary information.

\subsection*{Ionic conductivity}

In first place, we focused on the ionic conductivity of MARTINI ions in bulk PW water. The correct interplay between ions and water force fields is of critical importance to simulate conduction processes. In the case of MARTINI PW water, bulk properties such as density and dielectric constant have been optimized to construct the PW force field, while for ions the equilibrium bulk correlation function ION-PW water has been benchmarked to determine ions parameters~\cite{Yesylevskyy2010}. We would like to address the ability of the combination of these ion and water force fields to yield reasonable results when used to simulate transport processes.

Our simulations consisted on a cubic box containing PW water particles, cations (K$^{+}$) and anions (Cl$^-$) with concentrations from 0.1~M to 2~M (see Methods for details). For each concentration, three different conditions were simulated: no external electric field, and electric fields of 0.02~V/nm and 0.04~V/nm in the $z$ direction.

\begin{figure}[htb]
\centering
\includegraphics[width=0.5\linewidth]{KCLconducVSmolar.jpg}
\caption{Ionic conductivity of KCl as a function of molarity. Experimental results (in red) and all-atom simulations using the CHARMM (blue) and AMBER (green) force field performed by Pezeshki et al~\cite{Pezeshki2009} for KCl at 320~K, and CG results (black) computed with our CG MARTINI MD simulations.}
\label{fig:KClconduc}
\end{figure}
To calculate the ionic conductivity $\kappa$ of CG ions as a function of concentration, we computed the average total ionic current $<I>$ in the $z$ direction as follows: the $z$-component of the velocity of the center of mass of all K$^{+}$ ions, $v^{+}_z$, and the one of all Cl$^-$ ions, $v^{-}_z$, were calculated every 500~ps, and the instantaneous current in the $z$ direction was then computed using the formula:

\begin{eqnarray}
I(t) = \frac{1}{L_z} N_{ions} e (v^{+}_z-v^{-}_z) ,
\end{eqnarray}
where $N_{ions}$ is the total number of ions of each category, and $e$ is the elementary charge. The cumulative sum of the current over the length of the simulation (1~$\mu$s) was then calculated and fitted with a linear regression, the resulting slope of this fit yielding the average current $<I>$. The conductivity $\kappa$ is defined by $\kappa=\frac{<I>}{E A}$, where $A$ is the cross-sectional area, which is here: $A=L_xL_y$ ($L_x$ and $L_y$ are the boxes dimensions in $x$ and $y$ directions). Since two distinct electric fields were applied in the simulations at each concentration, we computed the average of the two calculated conductivities.

The concentration dependence of the conductivity computed at a fixed temperature of 320~K is shown on Fig~\ref{fig:KClconduc}, as well as the experimental results and AA simulations using the CHARMM and AMBER force field performed by Pezeshki \textit{et al}~\cite{Pezeshki2009,Lu2013} for KCl (numerical values from the figure of ref.~\cite{Pezeshki2009} were kindly provided by Ulrich Kleinekath\"ofer). The CG conductivity increases with the ionic concentration for concentrations below 1.15~M and is slightly lower than the reference values. For concentrations above this value, the conductivity decreases with the concentration. This effect can be explained by the size of the CG particles: at concentrations larger than 1.15~M, two opposite CG ions are separated by very few CG water molecules, and therefore positive ions going in one direction are disturbed by negative ions going into the opposite direction. Using AA models, a saturation of the conductivity was also observed above 1~M, compared to experiments, but there is no decrease. A saturation effect is also observed experimentally for conductivity in the bulk for certain types of ions with a large hydration shell~\cite{Bhattacharya2011}.

The CG ionic conductivities values with PW at 320~K are in very good agreement with experimental and all-atom theoretical data for concentrations of KCl below 1.15~M. This result is quite different from ~\cite{Vogele2015} where the authors, using a Nernst-Einstein method, show that conductivity of CG MARTINI ions is close to experiments only if an effective concentration is used.

\subsection*{Charge distribution and electric potential of a DPPC bilayer}

The electric potential across lipid membranes is essential for various biological processes in the cell, such as signaling, ion transport through membrane channels, or translocation of molecules through pores. \emph{In vivo}, such potential difference across the membrane is caused by ionic concentration gradients.

\begin{figure}[tbh]
\centering
\includegraphics[width=0.7\linewidth]{denscharNACL_noEz.jpg}
\caption{(a): Total linear charge density along the $z$ axis around a CG DPPC bilayer with (0.4~M in red and 0.8~M in green) and without ions (in black), and its decomposition: (b): DPPC charge density, (c): solvent, (d): ions. (e-f): Particle density for K$^{+}$ (e) and Cl$^-$ (f) ions.}
\label{fig:densNACL}
\end{figure}

In nanopore force spectroscopy, this difference is established and monitored using two electrodes placed across the membrane~\cite{Kasianowicz1996}, while there are several options to simulate the trans-membrane potential \emph{in silico}. In this work, a uniform electric field $E$ in the $z$ direction perpendicular to the membrane surface is applied to every charged atom of the system~\cite{Roux2008}. This  method has been previously used by Aksimentiev and Schulten~\cite{Aksimentiev2005} on the $\alpha$-hemolysin pore, and successfully applied to various biological problems~\cite{Pezeshki2009,Modi2012, Bockmann2008, Mathe2005}. An extensive MD study on several membrane systems showed that this method accurately represents an electric potential difference of $\Delta V=EL_z$, where $L_z$ is the system size in the $z$ direction, independently of the membrane thickness~\cite{Gumbart2012}.

In the original paper on the PW model~\cite{Yesylevskyy2010}, the electric potential around a DPPC membrane was computed without any ions and external electric field. Therefore, a study of the electrostatic properties of a system composed of MARTINI \{DPPC+PW+IONS\} under an external electric field is needed to assure the validity of the model for describing the ionic transport.
After equilibration of a system composed of DPPC lipids, PW water molecules and ions (see Methods), the MD production was performed in the NPT ensemble for two microseconds at 320~K and 1~bar. Several ionic concentrations were simulated: pure water, 0.2~M, 0.4~M and 0.8~M. Moreover, at each concentration, three different electric fields were applied: no external field, 0.01~V/nm  and 0.02~V/nm in the $z$ direction. These electric fields correspond to a theoretical transmembrane potential difference of 0~mV, 160~mV and 320~mV, respectively. It should be noted that, in the NPT ensemble, the box dimension in the $z$ direction increased at the beginning of the dynamics, and the $L_z$ length was around 16~nm on average during all the membrane MD (from 16.24~nm without ions to 16.41~nm at 0.8~M).

\begin{figure}[htb]
\centering
\includegraphics[width=0.8\linewidth]{potFigAll.jpg}
\caption{(a-c): Total electric potential along the $z$ axis of a CG DPPC membrane (black) and its decomposition, without ions (a), with 0.8~M ionic concentration (b), with no external electric field, and in the presence of an external electric field of 0.02~V/nm (c). (d): Total electrostatic potentials with no electric field (black), electric fields of 0.01~V/nm (red) and 0.02~V/nm (green) without ions (solid line) and with 0.4~M and 0.8~M concentrations (dashed lines).}
\label{fig:potNACL}
\end{figure}
The averaged charge density, on the last 500~ns of the 2~$\mu$s-simulation along the $z$ direction for each species, DPPC, water and ions, was computed for each simulation. Fig.~\ref{fig:densNACL}a shows the total linear charge density and its decomposition (b-d), without an electric field, as a function of the $z$ coordinate, without ions and at 0.4~M and 0.8~M concentrations. The total charge density is globally similar to previous AA simulations~\cite{Rodriguez2011}, showing four peaks from either side of the membrane. The decomposition of the charge density is also consistent with the AA one, so that water and ions, when present, rearrange in reaction of the membrane charge density. The DPPC charge density (Fig.~\ref{fig:densNACL}b) is slightly different when ions are present in the system, showing that the lipids rearrange a little in the presence of salt. The solvent charge density is very different with and without the presence of ions (Fig.~\ref{fig:densNACL}c), since the dipolar moments of water molecules react to the ionic charge distribution. Fig.~\ref{fig:densNACL}e-f show the particle density for each ions species (K$^{+}$ (e) and Cl$^-$ (f)). From these figures, we can infer that there is a layer of cations close to the negative charges of the lipid heads, and an overlay of anions close to the positive beads of the lipid heads. This ionic structure on the membrane surface is also consistent with AA simulations.

The electrostatic potential along the $z$ direction for each was then calculated by the double numerical integration of the charge density, according to~\cite{Bockmann2003,Lee2008}. In the absence of an external electric field, the system, centered on the bilayer, is theoretically symmetrical in the transformation ($x$,$y$,$z$) to ($x$,$y$,$-z$). Therefore, for the systems without electric field, the charge density $\rho(z)$ was symmetrized before calculation of the potential. Fig.~\ref{fig:potNACL}a shows the decomposition of the electric potential without ions. When no ions are present, the PW particles are oriented to compensate the electric potential created by the lipids. However, the total electric potential inside the membrane is negative, contrary to AA~\cite{Rodriguez2011} for which the total electric potential is slightly positive inside the membrane. The PW solvent electric potential does not compensate enough the potential created by the DPPC membrane, as it was previously shown in the original PW paper~\cite{Yesylevskyy2010}.

On Fig.~\ref{fig:potNACL}b, the decomposition of the electric potential at 0.8~M ionic concentration is shown. The total electric potential inside the membrane is less negative than without ions. The potential created by the membrane increases with the ion concentration, as in AA simulations~\cite{Rodriguez2011}, proving that the bilayer is sensitive to the ions presence. When ions are present, the solvent potential is much lower and the rearrangement of ions is compensating the lipids potential. This effect is consistent with the AA study by Rodriguez and Garcia~\cite{Rodriguez2011}, that showed that in the presence of ions, water plays a significantly smaller contribution to the total electric potential. Therefore, although the total electric potential inside the membrane is negative instead of positive, CG ions and water adopt the same behavior around a lipid membrane as an AA force-field.

Fig.~\ref{fig:potNACL}c shows the same decomposition for simulations with an external electric field. The total potential thus also contains the applied external potential (in dashed line). The figure shows that the ionic polarization potential is strongly altered by the external potential. This is not the case for other species whose charge density distribution is much less modified by the external field. On Fig.~\ref{fig:potNACL}d, the total electrostatic potentials of all simulations are represented. In the bulk, the reaction polarization potential is opposed to the external imposed one, and the resulting total electric potential difference is therefore only effective over the membrane. This result is consistent with previous AA-MD~\cite{Gumbart2012} studies which attest that the electric potential does not depend on the thickness of the membrane. The authors showed that the reaction potential of the solution, composed of solvent and ions, to the external field, combined to the external electric field, results in an electric potential difference across the membrane only, with a zero-potential difference within the two bulk regions, proving that using the relation $\Delta V=EL_z$ is accurate. Our results show that this assumption is also accurate with the MARTINI CG model.

We showed that the MARTINI force field with the PW solvent reproduces accurately the ionic conductivity in the bulk and the electrostatics properties around a lipid membrane in the presence of an external electric field. We can therefore now try to simulate ionic transport through a nanopore using this methodology.

\subsection*{Ionic current through the nanopore}

\begin{figure}[htb]
	\centering
    \includegraphics[width=0.4\linewidth]{image_memb_courb1.jpg}
    \caption{\label{memb_courb} Representation of simulation setup. Water has been removed for clearness purposes.
    Section of the pore (\emph{orange}) along the Z axis. The DPPC membrane (\emph{blue}) is curved. K$^+$ (\emph{green}) and Cl$^-$ (\emph{magenta}) ions are going through the pore channel.}
    \end{figure}

In this section, we present the results of the simulations for the system composed by the $\alpha$-hemolysin (PDB entry: 7AHL~\cite{Song1996}) inserted in a DPPC bilayer and surrounded by PW water, ions and counter-ions in the presence of an external electric field. A value of 1,1~M$\pm~0.1$~M ionic concentration was measured at the bulk region. Further details of these procedures and the parameters used can be found in Methods.

The initial condition for the ensemble $\alpha$-hemolysin suspended in a lipid bilayer membrane was taken from final state of Prof. Sansom simulations~\cite{Scott2008}, where the lipid bilayer grows spontaneously around the protein. This is the reason for the observed membrane curvature in the pore vicinity in our system (see Figure~\ref{memb_courb}). MD simulations of $\alpha$-hemolysin in DMPC lipid membrane using MARTINI force field were recently studied~\cite{Desikan2017} and showed a considerable distortion of the protein stem. In our simulations, the ElNeDyn network\cite{Periole2009} was applied inside and between the different protomers, and, as a result, the protein stem remains stable during the whole simulation.

After equilibration of the system, production simulations were carried out in the NPT ensemble ($T=320$~K and $P=1$~atm), with an external electric field applied perpendicular to the lipid layer, ranging from $-0.04$~V/nm to $+0.04$~V/nm. The potential bias $\Delta V$ value for a given electric field is calculated using an average $L_z=18.5$~nm, $\Delta V = L_z E_z $. This $L_z$ value varies less than $5~\%$ during the simulation time. All simulations, including the no-field simulation, start from the same initial condition (see Methods). The complete list of simulations performed with their corresponding electric fields and simulation times are shown in Table~\ref{TableSimPore}.

To provide reasonable statistics, we performed simulations of 2~$\mu$s and 3~$\mu$s, depending on the applied external electric field. We do not present data for an electric field $E_z=-0.04$~V/nm, since the membrane broke early during the simulation. This is also the case in experiments where the allowed potential differences are $|\Delta V| < 350~mV$.

The instant current was calculated computing the number of cations and anions ($n_{c}(t)$ and $n_{a}(t)$) crossing a $xy$ plane perpendicular to the $z$ axis, with $z$ abscissa equal to the position of the center of mass of the groups in the \textit{trans} end of the pore, between $t$ and $t+\Delta t$ ($\Delta t = 100~ps$) (see Supplementary Information). The sign represents the direction of the velocity in the $z$ axis. These values are normally 0 or 1, so we define the number of ions passing through the plane in the interval $[0,t]$ as $ N_{c,a}(t) =\int_0^t n_{c,a}(t') dt' $, and the total charge going through the pore, which is the cumulative function of the current, is : $Q(t)= e \left( N_{c}(t)-N_{a}(t) \right) $.  The instant current is defined then as the slope of the function $Q(t)$ : $I(t) = \frac{dQ(t)}{dt} $.
\FloatBarrier
\begin{table}[htb]
\centering
\caption{Simulation parameters and number of ion passages during simulations. Electric field  $E_z$, computed potential bias $\Delta V$, total simulation time $t_s$, number of cations and anions traversing during the total simulation time $N_c(t_s)$ , $N_a(t_s)$  and total charge traversing during the simulation time $Q(t_s)$.}

\label{table}
\begin{tabular}{|r|ccccc|ccccc|}

 \hline
\bfseries  &\multicolumn{5}{|c|}{\textbf{Positive Bias}} & \multicolumn{5}{c|}{\textbf{Negative Bias}} \\
\hline
   $E_z$ (V/nm) & $\Delta V (mV)$ & $t_s (\mu s)$ & $N_ {c}(t_s) $
& $N_{a}(t_s) $ & $Q_{ts}  (e)$ &
$\Delta V (mV)$ & $t_s (\mu s)$ & $N_ {c}(t_s) $
& $N_{a}(t_s) $ & $Q_{ts}  (e)$

\\
  \hline
$\pm$ 0.00& 0 & 2 & -4 & 6 & -10 &  &  & &  &  \\

$\pm$ 0.005 & 92 & 3 & 45 & -108 & 150 & -92 & 3 & -21 & 103 & -124 \\

$\pm$ 0.01 & 185 & 3 & 85 & -180 & 265 & -185 & 3 & -33 & 209 & -242 \\

$\pm$  0.015 & 277 & 3 & 118 & -212 & 330 & -277 & 3 & -31 & 269 & -300 \\

 $\pm$ 0.02 & 370 & 2 & 144 & -228 &  372& -370 & 2 & -28  & 218 &- 246 \\

 $\pm$ 0.025 & 462 & 2 & 80 & -130 & 210 & -463 & 2 & -26 & 178 & -204 \\

 $\pm$ 0.03 & 555 & 2 & 109 & -113 & 222 &-555 & 2 & -32 & 194 & -226 \\

$\pm$ 0.04 & 740 & 2 & 241 & 229 & 470 & -740 & X & X & X & X \\

\hline

\end{tabular}
\label{TableSimPore}
\end{table}

The number of ions crossing the pore during the whole simulation time, $N_{a}(t_s)$ and $N_c(t_s)$ are represented in Table~\ref{TableSimPore}, together with the total charge variation during the whole simulation $Q(t_s)$ in \emph{e} units. These values are in the limit of "reasonable" statistics although, as we will see below, they evolve with time. The rectification and selectivity of the pore can be also deduced from this global values.

The instant cumulative current $Q(t)$ is represented in Fig.~\ref{fig:currentCumul} for potentials under (side a)) and over (side b)) 300~mV. Since the instant current $I(t)$ is the slope of this function at each time $t$, the linear increase of the cumulative current with time would indicate a stationary current. This seems to be the case for potential bias under 300~mV, although it should be pointed out that the stationary current is established only after some hundreds of nanoseconds, especially in the case of positive potential bias. The fact that the slope of the function increases with increasing potential is the reflect of a linear-response regime.

\begin{figure}[htb]
	\centering
   \includegraphics[width=0.9\linewidth]{CumulCurrentTot_SuperNew3.jpg}
	\caption{Cumulative current as a function of time, for potentials (a) under or (b) over 300~mV computed during $3~\mu s$ or $2~\mu s$, respectively. Fig. a) : $\pm$ 92~mV (green),  $\pm$ 185~mV (red) and  $\pm$ 277~mV (blue). Fig. b) : $\pm$ 370~mV (green),  $\pm$ 462~mV (red), $\pm$ 555~mV (blue) and  $\pm$ 740~mV (violet). }
    \label{fig:currentCumul}
\end{figure}

This is not the case for potentials over 300~mV, as shown in Fig.~\ref{fig:currentCumul} b). The cumulative currents are not proportional to the potential bias at the end of the simulation, implying that the simulated systems are not in the linear response regime. Moreover, we cannot be confident that the stationary state has been reached at the end of some simulations as the slope of the cumulative current is still varying.

Fig.~\ref{fig:CurrentIV} shows the evolution in time of the $I-V$ curve of the system averaged at a fix number of crossings ($N= 40$ events). For every simulation, an initial time $t_{ini}$ is defined as $Q(t_{ini})= 5~e$, which is the time when five charges have already crossed the pore. From this origin $t_{ini}$, we computed the total time $t_{end}$ such as $\Delta Q=Q(t_{end})-Q(t_{ini})= 40~e$. In this interval, the current $\Delta t=t_{end}-t_{ini}$, is estimated as $\bar{ I} = \frac{\Delta Q}{\Delta t}$. The error bars were calculated with the standard deviation $\sigma=\frac1N
\sum_{i=1..N} | \Delta Q_i-I \,\Delta t_i| $ so that
$\delta I = \frac{2\,\sigma}{\Delta t}$.

In order to compare our results to experimental values, we have defined a reference current in the linear regime, as $
 I^{ref} = R^{ref} \Delta V  $,
where $R^{ref}= 1~G\Omega$ is a typical resistance observed experimentally for these systems~\cite{Bhattacharya2011}. For negative electric fields, $I^{ref}$ value is multiplied by 0.7 to take into account the observed rectification of $30~\%$ in experimental setups. The black continuous line observed in the figure is the function $I^{ref}/10$. Thus, in our simulations, we observe a $Q_s$ value ten times smaller than the expected one. This is a consequence of the CG model: the resistance of the pore increases since the mobility of the ions inside the channel is highly reduced because of the larger size of PW water molecules. This ratio increases for higher potential differences where the linear regime is not valid anymore.

The measured current is clearly evolving during our simulations. In spite of this, a linear response regime can be established for potential biases below $\sim 350$~mV.  In this regime, the final current appears slightly smaller than the one measured at the beginning of the simulation and, as mentioned, it is one order of magnitude smaller than the experimental one. For potentials above this value, a limiting current is observed. We find some asymmetry between positive and negative biases: while, for negative biases, the limiting current seems to converge to a well defined value, for the positive ones, this value appears much more noisy.

\begin{figure}[htb]
	\centering
    \includegraphics[width=0.7\linewidth]{CurrentSuperNew2.jpg}
    \caption{Average current during different passages windows as a function of potential bias. Note the difference between the begining of the simulation (5-45 crossings) and the end of the simulation (195-235 crossings). The black solid line corresponds to $I^{ref}/10$.}
    \label{fig:CurrentIV}
\end{figure}

The origin of the non-linear ohmic behavior of the current-voltage curve has been explained as due to the crowding or depletion regions at the \textit{trans} and \textit{cis} ends of the pore in presence of an applied external potential~\cite{Tagliazucchi2015}. Both limiting and over-limiting currents in nanopores have been discussed in van Oeffelen \textit{et al}~\cite{vanOeffelen2015}. In our simulations, we observe a limiting current at a quite low voltage $\sim 350$~mV. This value is probably higher in AA simulations, although it is difficult to observe as the simulated time is not enough to get to the steady state~\cite{Aksimentiev2005}. In Ref.~\cite{Liu2013}, the authors compare linear-response theory with AA-MD simulations to determine the departure from the linear regime for two pore models, one of them being a modified $\alpha$-hemolysin pore. Their conclusion, and also ours as regard of our results, is that $\alpha$-hemolysin has a small linear response range and that currents at low voltages cannot be safely extrapolated from the values obtained at higher potential biases. The limiting current cannot be experimentally observed since, as mentioned, the membrane breaks over $\sim 300$~mV .

It is not clear from our results if an over-limiting current appears over $+700$~mV~\cite{vanOeffelen2015}. This could be the case but it cannot be excluded that the stationary state has not been reached, that the effective protein charge (protein plus decorating ions) evolved, or that the periodic boundary conditions are inducing such phenomenon.

As observed, the average current cannot be accurately computed before reaching the stationary state and therefore,  an interesting issue is which simulation time is needed to get to this regime. In van Oeffelen et al.~\cite{vanOeffelen2015}, it is pointed out that, due to slow co-diffusion of ions, the time for the charge-current to decay to a stationary state value is determined by the diffusion of particles. This time is estimated as a typical diffusion time $\tau=L^2/D$, where $L$ is a typical distance and $D = 2 (D_{+}\, D_{-})/(D_{+}+D_{-})$ is an averaged diffusion coefficient for cations and anions. In our simulations, this time is estimated to $\sim 140~ns$, where we have used $L$ the length of the system occupied by the solvent, $D_{+}=2.54~10^-9~m^2/s$ and $D_{-}=2.45~10^-9~m^2/s$ measured during our conductivity calculations. We have computed the evolution of the number of ions inside the stem, the region of the pore going from the trans side to 4~nm above (see Supplementary Information).We observed that the time to get to a stationary number of ions goes from $\sim$ 100 to 300~ns, being shorter for negative voltage biases. The asymmetry for positive and negative biases is in agreement with AA simulations, as well as the number of ions in the stem computed in previous AA and PNP studies~\cite{Noskov2004,Cozmuta2003}.

The time to get to a stationary regime is longer than this estimation according to our simulations. The relatively quick convergence of the number of ions inside the pore is not a guarantee for the stationary state, where anions and cations currents, or conversely charge-current and particle-current, should be, in average, time independent. This is not the case for all our simulations. We observe the convergence of the current for voltage biases under $+350$~mV as well as for negative higher biases. Positive high biases would require longer simulations to get confident results. We can conclude that a 1-1.5$\mu$s simulation should be enough to get a stationary state in the linear regime.

A final pertinent question about CG-MD simulations of such complex systems is if the pore dynamics play a determining role on the current. If this was not the case, simulation time could be saved by tabulating the pore and membrane potential. To test this possibility, we have performed CG-MD simulations where the backbone of the $\alpha$-hemolysin was constrained to a fixed configuration. The external electric fields imposed were $\pm~0.01$~V/nm, $\pm~0.02$~V/nm and $\pm~0.03$~V/nm. The measured current in this case is much smaller than the corresponding one for the flexible protein and we found clogging periods during the simulation, going up to 200 ns. The current saturates for small potential biases going to a value close to zero (see the Supplementary Information). This result proves that the flexibility of the protein is essential to simulate the translocation process.

\section*{Discussion}

The use of MARTINI force field to simulate the transport of ions through the $\alpha$-hemolysin in a DPPC bilayer is studied for the first time, to our knowledge, in this article.

In first place, we have investigated the transport properties of the original Martini hydrated ions as a function of concentration. This leads us to the problem of comparing CG results to AA or experimental ones. Since the ratio ion/water valid for AA systems is no longer a suitable parameter to set a molar concentration for the CG simulations, we have decided to use the number of ions per volume as concentration. We are aware that this definition may not represent exactly the same concentration in NVT and NPT ensembles, and definitely cannot be directly compared to AA results. In spite of this incertitude, we can conclude that MARTINI ions in PW water present a conductivity that could represent a KCl solution below 1.1~M concentration.

Secondly, we have investigated the properties of the DPPC+IONS+PW system in the absence and under different applied electric fields. It is already known that the MARTINI force field leads to a unrealistic positive potential inside the membrane in the absence of an applied electric field~\cite{Yesylevskyy2010}. In the presence of an external field, we found that the expected potential differences through the membrane are recovered, such as in previous AA simulations~\cite{Gumbart2012}. We have also pointed out the role of the polarization of water to describe the correct electrostatics, proving that it is necessary to use a polarizable model of water.

Finally, we have studied the whole system $\alpha$-hemolysin+DPPC+PW+IONS under different applied external fields focusing on the ionic transport through the pore. Principally due to the size of MARTINI PW water, the pore presents a smaller conductance than the experimental system. This deviation seems to be systematic for low applied external fields. The qualitative behavior of the system nevertheless appears to be correct. Our $I-V$ curves show a small range $\pm~350$~mV where the linear response regime is established. For higher potential biases, a saturation of the current is observed. These limits cannot be compared to experiments, for practical reasons, or to AA-MD simulations, since the simulated time is not enough as to get to a stationary state. We have also performed simulations to conclude that the flexibility of the protein channel plays an important role.

PNP solutions have provided to be extremely useful to describe the stationary state of ionic currents in many systems, but a precise description of this particular system would require excluded volume effects, water-ion correlations, hydrodynamics, finite system size correlations and the possibility to take into account the flexibility of the protein. On the other side, AA-MD simulations provide good current statistics at reasonable simulation times for high voltages but a) reaching the steady state seems to take a long time, if it is possible due to the finite system size, and b) the system is at a non-linear regime. Although the non-linearity will appear at higher potentials in AA simulations than in CG simulations, in Ref.~\cite{Liu2013}, they estimate this limit to be below $500$~mV in a AA-MD study of a modified $\alpha$-hemolysin, interpolating low voltage values from high voltage ones can be somehow risky. At low voltages, direct sampling of net ion fluxes with AA simulations would require very long simulation times to acquire sufficient statistics.

Our conclusion, as regard to our results, is that CG-MD simulations could result a good alternative to study the details, such as mutation effects, of translocation through protein nanopores, saving some of the simulation time required for the equivalent AA simulations for these kind of problems.

\section*{Methods}

\subsection*{Simulation details}
We performed Molecular Dynamics (MD) using the Gromacs software package~\cite{VanDerSpoel2005} and the MARTINI 2.2 CG model for ions, lipids~\cite{Marrink2007}, proteins~\cite{Monticelli2008,Jong2013} and the PW (Polarizable Water) model~\cite{Yesylevskyy2010} for water. Conductivity simulations systems contained only water and ions at different concentrations, whereas membrane simulations systems contained a DPPC bilayer, water, and ions at different concentrations. In the nanopore simulations, the protein nanopore was inserted in the lipid membrane.\\

A timestep of 20~fs was used for all MD simulations, which is the recommended timestep for CG MD simulations using MARTINI force field. Periodic boundary conditions in the three dimensions were applied. All MD were performed using the PME method for electrostatic interactions, with a 2~\AA\ spacing for the Fourier grid and a direct space cut-off radius of 13~\AA. A relative dielectric constant of $\epsilon_r=2.5$ was used for PW as recommended\cite{Yesylevskyy2010}. Van der Waals interactions were shifted at 13~\AA. A Nos\'e-Hoover thermostat was used to maintain the temperature at 320~K.

\subsubsection*{Conductivity simulations}

For conductivity simulations, the system was originally built as a cubic box of 15.8~nm$^3$, containing 32,756 PW water particles. The system was minimized and slightly equilibrated (2~ns at NVT) before the introduction of ions at different concentrations. The \emph{genion} tool of Gromacs was used to replace random water particles by positive (the MARTINI Na$^+$ ion) and negative (Cl$^-$) ions in the same quantity, in order to keep the system neutral. The system containing water and ions was then again minimized and equilibrated with a short MD (10~ns) in the NVT ensemble, using the Berendsen thermostat. Then, a one-microsecond-long MD was performed in the NVT ensemble at 320~K, using the Nos\'e-Hoover thermostat, with a characteristic time interval $\tau_T=1$~ps for both groups (ions and water).
For each concentration, ranging from 0.1~M to 2~M, three different simulations were performed: one with no external electric field, one with an electric field of 0.02~V/nm in the $z$ direction, and one with an electric field of 0.04~V/nm in the same direction.

\subsubsection*{Membrane simulations}
As for the membrane simulations, a box containing 128 DPPC CG lipids organized in a bilayer was downloaded from the MARTINI website~\cite{MARTINIlipids}. 4,232 water molecules (PW) were added around the membrane in a box of originally $6.7 \times 6.1 \times 12.0$~nm$^3$, the $z$ axis oriented perpendicularly to the membrane plan. The system was minimized and shortly equilibrated before addition of ions at different concentrations using the same procedure as the conductivity simulations. Simulations without ions were also performed using the same methodology. After another minimization of the system, a NVT MD simulation was performed for 2~ns using the Berendsen thermostat, followed by 120~ns of NPT MD using the Berendsen thermostat and Berendsen semi-isotropic barostat at 1 bar. The MD production was performed for two microseconds using the Nos\'e-Hoover thermostat at 320~K (with a characteristic time interval $\tau_T=1$~ps for each group) and the Parrinello-Rahman barostat to keep the pressure at 1 bar (with a characteristic time interval of $\tau_P=5$~ps and a compressibility of $4.5\times 10^{-5}$~bar$^{-1}$). It should be noted that, in the NPT ensemble, the box dimension in the $z$ direction increased at the beginning of the dynamics, and the $L_z$ length was around 16~nm on average during all the membrane MD. In addition to simulations without ions, three different KCl concentrations were simulated: 0.2~M (60 ions of each species), 0.4~M (124 ions) and 0.8~M (248 ions). For each concentration, three different simulations were performed: one with no external electric field, one with an electric field of 0.01~V/nm in the $z$ direction, and one with an electric field of 0.02~V/nm. The averaged charge density was calculated on the last 500~ns of the 2~$\mu$s-simulation, using the \emph{g\_potential} tool of Gromacs, decomposing the system into 100 slices in the $z$ direction.

\subsubsection*{Nanopore simulations}

The crystallographic structure of $\alpha$-hemolysin was taken from the PDB (entry 7AHL~\cite{Song1996}). The coordinates of the missing atoms were added using the \emph{pdb2pqr} software and the crystallographic water molecules were remove.
The protein was reduced in coarse grains using the \emph{martinize.py} script. The ElNeDyn MARTINI force field was applied on the protein~\cite{Periole2009}. It is composed of an elastic network model that maintains the secondary structures during the dynamics using springs (500~kJ.mol$^{-1}$.nm$^{-2}$ elastic bond strength and a 0.9~nm cutoff).
After a short energy minimization, the resulting CG protein structure was inserted in a DPPC bilayer using the following procedure: the PDB structure of a CG $\alpha$-hemolysin inserted in a DPPC bilayer of 756 lipid molecules obtained by Prof. Sansom \cite{Scott2008} was downloaded from CGDB (coarse grain database, a database of inserted membrane proteins inside DPPC bilayers~\cite{CGDBSamson}. Since the protein CG model used for CGDB is different from the MARTINI one, a least-squared fit of the backbone of our CG protein on the CGDB one was performed, then the initial protein from CGDB was deleted to keep only our MARTINI CG protein inserted in the DPPC bilayer. The size of the simulation box was initially set at 15.85 x 15.85 x 20~nm. The system was solvated with 28,758 standard W MARTINI CG water molecules using the \emph{genbox} Gromacs command. Another minimization was carried out followed by two equilibration steps: 1~ns in the NVT ensemble (using a 10~fs timestep) followed by 5~ns in the NPT ensemble (using a 20~fs timestep).\\

The ionic concentration was set around 1~M, corresponding to 2054 ions of each species (calculated according to the number of initial water molecules and using the ions/PW molecules ratio from conductivity simulations concentrations).
Under normal conditions, the nanopore carries a global charge of $+7e$. Therefore, in order to neutralize the system, 2050 K$^+$ ions and 2057 Cl$^-$ ions were added to the system.
A short dynamics of 5~ns was then performed at NPT. After transformation of MARTINI W water into polarizable PW water, a short minimization was carried out, followed by two additional equilibration dynamics of 1 ns at NVT and 10~ns at NPT, both using position restraints on the protein backbone (with a 20~fs integration timestep). A last dynamics of 1 ns at NPT without position restraints was performed using the PME method for electrostatic interactions. The resulting system was used as initial state for all the simulations of the pore as well as the dynamics with the pore under position restraints on the protein backbone.

Several electric fields were applied in the $z$ direction of the system, perpendicular to the membrane plane, both positive and negative: no electric field, +/-0.005~V/nm, +/-0.01~V/nm, +/-0.015~V/nm, +/-0.02~V/nm, +/-0.025~V/nm, +/-0.03~V/nm and +/-0.04~V/nm. Molecular dynamics were carried out for 2 to 3~$\mu$s (+/-0.005~V/nm, +/-0.01~V/nm, +/-0.015~V/nm). See Table~\ref{table} for further details.

\section*{Supplementary Information}
\beginsupplement

In the original MARTINI article, a CG ion is presumed to contain a first hydration shell composed by six water molecules. This number is commonly used to set the ratio between PW molecules and ions for a given molar concentration, although for concentration setting purposes, the 4 to 1 standard MARTINI mapping seems to be a better choice. In our NPT simulations, we have computed this ratio together with the molar concentration in the bulk regions. In our NVT simulations, we will consider that one CG ion represents an all-atom ion with three water molecules.

\begin{figure}[htb]
	\centering
	\includegraphics[width=0.7\linewidth]{comp-density1.jpg}
	\caption{Bulk molar concentration vs. PW/Ion ratio. Circles correspond to measured concentrations in our NPT simulations and triangles correspond to fixed concentrations in our NVT simulations. The dashed line is the theoretical value of molar concentration assuming one CG ion represents an ion plus three AA water molecules. The solid line is the same theoretical value, when a CG ion represents a ion plus six AA water molecules. }
    \label{comp-dens}

\end{figure}

The instant current was calculated computing the number of cations and anions crossing a $xy$ plane perpendicular to the $z$ axis, with $z$ abscissa equal to the position of the center of mass of the groups in the \textit{trans} end of the pore, colored in blue in the figure.

\begin{figure}[htb]
	\centering
	\includegraphics[width=0.3\linewidth]{alpha_compt_cut.jpg}
	\caption{Partial section of $\alpha$-hemolysin. The group in the \emph{trans} side of the pore (\emph{cyan}), composed of Asp127, Asp128, Thr129, Gly130 and Lys131, is used to define the plan position for the current calculations. The evolution of the number of ions was computed inside the restricted cylinder of 4~nm in height and width (\emph{mauve}) surrounding the stem.}
    \label{bott_group}
\end{figure}

\begin{figure}[htb]
	\centering
    \includegraphics[width=0.5\linewidth]{countinpore_ch_4.jpg}
    \caption{Number of ions in the stem region of the pore along time for different external electric fields applied on the system.}
    \label{fig:countinpore}
\end{figure}

We have defined the "stem" as a region inside the pore going from the $z$ abscissa defined as the "trans" to 4~nm above. It is colored in mauve in the figure.

The evolution of the number of ions inside the stem for different positive and negative biases is represented in \ref{fig:countinpore}. The asymmetry between positive and negative bias can be observed. We have estimated to 1~$\mu$s the time to get to a stationary state.

During the simulations where the protein was constrained to a fix position, the number of ions crossing the membrane is not enough as to compute statistics in the way used for the flexible protein.
In order to compute the current, a different approach was used.

A simple model to describe the current evolution from the initial condition to the stationary state is

\begin{equation}
I(t)=I_{\infty}+\left( I_0 - I_{\infty} \right) e^{- t/\tau}
\end{equation}

So that the total cumulative current is
\begin{equation}
Q(t) = I_{\infty} t -\tau \left( I_0 - I_{\infty} \right)\left(  e^{- t/\tau}
- 1 \right )
\label{fitequation}
\end{equation}

We have fitted the parameters of eq.[\ref{fitequation}] with the computed values of $Q(t)$ for times each $\Delta t = 15$~ns. While $I_0$ and $\tau$ depend on the initial condition, it can be expected, close enough to the steady state, that $I_{\infty}$ is the asymptotic value of the current. To test the model, $I_{\infty}$ has been computed for the flexible protein and compared to the averaged current between $190$ and $230$ crossings. As shown in figure \ref{fig:IV-CH-PR} the results are quite similar between these two methods. It can be observed that the current saturates very quickly going to a value close to zero. We believe that since the flexible-non-fixed protein vibrates and travels along the membrane, it transmits energy to the particles inside the pore and prevents clogging. This is not the case for a rigid fixed protein. The dynamics of the protein seems then essential to simulate the translocation process.

\begin{figure}[htb]
	\centering
    \includegraphics[width=0.6\linewidth]{IV-temps-CH-PR.jpg}
    \caption{IV curve for flexible protein and rigid fixed protein. Position restrained protein and time statistics (circles), flexible protein and time statistics (diamonds) and flexible protein with crossing statistics (triangles). The black solid line is the reference experimental current, as defined in the main text, divided by a factor of 10.}
    \label{fig:IV-CH-PR}
\end{figure}

\end{document}

redpajama_set_name: RedPajamaArXiv


Prior to the deployment phase of Operation Redback VI, MMEA crew were provided vessel search training funded through DIBPs Regional Skills Development Programme and delivered by the Australian Border Force College.
As of June 30, the company had 348 hotels across MMEA, with a total of 52,185 rooms.
Euler Hermes' future growth is closely linked to the group's ability to seize opportunities arising from changes in the global economy, including in the GCC countries," said Michele Pignotti, head of the company's MMEA region.
1999), a partir de la EAS, proponen la MMEA reduciendo el numero de items a 28.
An MMEA official told Kyodo News that there was no major oil spillage.
MMEA proves to be more effective in finding multiple optima of the considered test functions.
Malaysia's MMEA to Acquire Air Assets," DefenseNews, 5 February 2007, p.
Building maritime security in Southeast Asia: outsiders not welcome?
Owing to the poor impact resistance and color changes (possibly due to degradation) it was decided to add 10 wt% of the MMEA copolymer as a processing aid.
Flint's blue-collar origins are key to understanding the need for the program, says MMEA president Doug Gould, a design team member on full-time release from his teaching assignment.
MMEA Deputy director-general (Operations), Rear Admiral (Maritime) Datuk Che Hassan Jusoh, said the aircraft had started water scooping 10 times and water bombing 10 times under 4 hours of assignment on the initial day.
According to MMEA enforcement chief Captain Anor Jaafar, only three crew members were initially found on the boat.
We could see more security solutions designed to protect IoT," notes Ravi Patil, Technical Director, MMEA, Trend Micro.


World Sport Group, Asia's leading sports marketing, media and event Management Company has appointed Ten Sports, India's largest sports network to broadcast the 2011 South Asian Football Federation (SAFF) Championship on Ten Action+.
All 15 matches of this 7th edition of the SAFF Championship which will be played at Jawaharlal Nehru Stadium New Delhi from 2nd December to 11th December 2011 will be telecast live on Ten Action+. Eight countries from the region: India, Bangladesh, Maldives, Pakistan, Nepal, Sri Lanka, Bhutan and Afghanistan will participate in this prestigious tournament.
Atul Pande, CEO-Ten Sports said, "We are delighted to broadcast the 2011 SAFF Championship. The championship will not only help in the development of football in the region but also in fostering goodwill among neighboring nations. The telecast of this prestigious tournament reaffirms our commitment to grow our football and sports broadcasting business, and consolidate our position as the leading sports broadcaster."
Harish Krishnamachar, Senior Vice President and Country Head, India, World Sport Group said, "World Sport Group has worked on seven editions of the SAFF Championship and we are happy to be producing this event in India. Together, our capability and the football pedigree of Ten Sports will help us deliver a world class event".
Drawn in Group A alongside Sri Lanka, Afghanistan and Bhutan, five-time winners and defending champions India will kick-off their campaign in the SAFF Championship with a match against Afghanistan on December 3. Pakistan are in Group B along with runners-up Maldives, Bangladesh and Nepal. The top two teams from each group will advance to the semifinals, which will be held on December 9th, two days before the summit clash.


text:
Robin Driver
Kim Kardashian West announces "elevated" rebrand for KKW Beauty
American reality star Kim Kardashian West has taken to social media to announce that KKW Beauty, the cosmetics brand she founded in 2017, will be going offline in August in order to undergo a comprehensive rebrand.
The KKW Beauty website will be shutting down on August 1 - Instagram: @kkwbeauty
Posting the news on Twitter and Instagram, the 40-year-old celebrity and entrepreneur explained, "on August 1 at midnight we will be shutting down the kkwbeauty.com site so that we can come back to you under a completely new brand with new formulas that are more modern, innovative and packaged in an elevated and sustainable new look."
"I'm excited to continue to develop and expand my product range and for you to finally be able to experience it the way that I have always envisioned," Kardashian West continued. "In addition, my team is hard at work to improve the customer shopping experience where you will be able to purchase my beauty and cosmetic offerings in all categories from one single website."
Kardashian West did not provide a prevision for a return date for the brand, but did say, "I promise we won't be gone for too long."
The rebrand of the business, which was valued at $1 billion by New York-based beauty conglomerate Coty, Inc. when it acquired a 20% stake in the company in 2020, comes on the heels of a similar announcement concerning an imminent relaunch of fellow Kardashian-Jenner clan member Kylie Jenner's Kylie Cosmetics brand.
Coty, which is also the majority owner of the brand founded by Jenner in 2014, said on July 1 that the new vision for Kylie Cosmetics will focus on "improved clean and vegan formulas." Furthermore, the rebrand will involve the launch of a new website on July 15, which Coty says will sell products from the Kylie Skin skincare line as well.
The abrupt announcement of the two rebrands in quick succession has led to speculation about the reasons behind the apparent rush to relaunch the businesses, with some theorizing that the decisions could be related to legal issues.
In June of last year, Forbes reported that Seed Beauty, the company which manufactures both KKW Beauty and Kylie Cosmetics products, had brought a lawsuit against Coty, alleging that "Kylie Cosmetics knowingly disclosed Seed's trade secrets — confidential intellectual property" to the beauty conglomerate. According to Forbes, Seed Beauty had also brought a civil lawsuit against KKW Beauty to ensure that the brand did not share its trade secrets.
It certainly wouldn't be the first time that Kardashian West has been pressured into a rebrand. The reality star's Kimono shapewear label was renamed as "Skims" shortly after launch in 2019, following accusations of cultural appropriation.
In any case, this initial controversy does not appear to have had much of a negative impact on the business moving forward. Skims, which was valued at $1.6 billion by The New York Times in April of this year, recently announced that it will be supplying Team USA with undergarments, nightwear and loungewear at the Tokyo Olympics.
Visa profit beats estimates on resilient consumer spending

redpajama_set_name: RedPajamaCommonCrawl


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Last week's column on smartphone battery life brought some follow-up questions about location services.
Applications and websites have different reasons for wanting to your knowing your location, based on the features they offer.
Apps like Google's Waze, which gives driving directions with real-time traffic information, need to know your location to help you navigate.
Individual websites may ask for your location as well. When I place online orders from Torchy's Tacos, they ask to access my location to help me find the closest restaurant.
In the location services settings on my iPhone, which is in the Privacy settings, I have three choices for granting permission to my location data.
I can choose to let an app always know my location, only access my location when the app is in use or never know my location.
In the location services settings, you'll see a list of all the apps you have installed that can use your location data. Beside each app, you'll see its status.
When you load and launch an app for the first time, it will ask for your permission to access your location. Many times, you might be in a hurry and click OK without realizing how much privacy you may be giving up.
Therefore, it's a good idea to periodically check your location services list to make sure you realize how each app is using your location.
Most of my apps are set to access my location only when I'm using them, but I do let a few apps know where I am all the time.
For example, I use the app MyRadar to keep track of the weather. By letting the app know my location, I get alerts when severe weather is headed my way based on my phone's location.
Whenever I remember to check the list, I usually find an app or two that I don't think need my location data. I just found a restaurant app that was set to always know my location. There's no need for it to always know where I am, so I changed the setting.
Android users can find their location settings in the Connections page in their Settings app.
Google's support page on location data is here: https://support.google.com/accounts/answer/3467281?hl=en.
To read more about Apple's location settings, go to https://support.apple.com/en-us/HT203033.


If you still haven't read how and what exactly led to the demise of the MintPal crypto currency exchange, one of the big ones for altcoin trading until a few months ago, then you might want to check out the website called MintPal Justice. The website is the official effort of the former partners of Alex Green aka Ryan Kennedy that have purchased the crypto currency exchange earlier this year to bring him to justice. On that website Michael and Ferdous tell the whole story from their point of view and how they became partners with Ryan Kennedy, or "Alex Green" – the CEO of MooPay Ltd for the acquisition of MintPal from its previous owners. It seems that Ryan Kennedy not only cheated and lied to the users of the MintPal exchange, but also his partners and employees in the end getting away with thousands of Bitcoins that are not his own. The website MintPal Justice also takes donations with the goal to cover the legal fees of the effort to bring Ryan Kennedy to justice. So far the effort has generated over 22 BTC in donations out of the goal of 75 total required (roughly $25000 USD).
Moopay LTD, the company behind Moolah and the MintPal exchange, has officially announced that it will be filing for bankruptcy and dissolution following the administration process; and ceasing operations. The company ensures that all funds held in consumer accounts are safe – consumer funds are essentially held in the equivalent of a client trust account and are fully separate. People should be able to withdraw them via the normal route up until the end of the month. Any businesses or entities with funds held in any other fashion with the company will be contacted over the next month as a part of the bankruptcy and creditor process.
As a result of the company closing doors and filing for bankruptcy, MintPal is currently down and will no longer be managed by Moolah who are apparently do not own the exchange, but were only responsible for management. There will be a new management team who will be responsible for making a number of stability changes, UI improvements and bug fixes as well as handling the resolution of issues surrounding missing balances. An update with more information should follow on mintpal.com shortly, with details of further timeframes according to the announcement made. It is a fact however that MintPal needs to earn back the trust of the thousands of users it had and that is getting harder to achieve as each ay passes with things not getting much better than they were the day before.


Poodle skirt loving, 50's Jessica!
Jessica will be available in the store Friday along with the other 2 stamps we preview this week.
Here is my sample made with the new Jessica stamp. I sorta giggle when I see a poodle skirt. I had started a new job and saw this flyer for the company Halloween Party. I was so thrilled, I made myself a poodle skirt and even had vintage accessories from my mom to finish the 50s look. I went to work.. no one had a costume on! Whatever!
Enough rambling.. Jessica is colored with Copic Markers to match the colors of my very own poodle skirt. ;) The card was created with dies from Jaded Blossom.


text:
Pacers add help to backcourt by signing Darren Collison
BY AP • July 7, 2017
INDIANAPOLIS (AP) The Indiana Pacers have signed free agent Darren Collison.
Terms of the deal were not released Friday amid reports it is a two-year contract worth $20 million.
The point guard returns to a team he played for from 2010-12. Collison averaged 11.9 points and 5.0 in 139 games during his first stint with the Pacers.
Since being taken with 21st overall pick in the 2009 NBA draft, Collison has career averages of 12.7 points and 4.8 assists.
He joins a backcourt that will be led by Victor Oladipo and Lance Stephenson The Pacers are rebuilding after trading four-time All-Star Paul George to Oklahoma City and losing point guard Jeff Teague in free agency.
Indiana also waived forward Rakeem Christmas.
More AP NBA: www.apnews.com/tag/NBAbasketball
NBA odds: Championship futures for every team after Kevin Durant's injury
Lakers coach Frank Vogel doesn't deserve to be fired
NBA Power Rankings: Suns, Warriors flying high at midseason
Cade Cunningham, Evan Mobley highlight NBA's standout rookies so far this season
Ben Simmons, Philadelphia 76ers are dancing toward an inevitable breakup


I am writing to enquire about the following item which I saw on the Mary Cooke Antiques website: "An exceptionally fine George III cast Bsket Weave Pocket Snuff Box made in London in 1810 by John Reilly."
An exceptionally fine George III cast Bsket Weave Pocket Snuff Box made in London in 1810 by John Reilly.
This exceptional example is palely gilded and is unusually cast. The box has a slightly rounded form and is decorated all over with basket weave designs. The basket weave has niello in the design to highlight the patterning. The interior displays fine original gilding and this example is very well marked. This is almost certainly the finest basket weave snuff box we have offered over the years and it is in excellent condition. Cast pocket Snuff Boxes are rare and this is certainly the heaviest we have offered. A very fine box !


text:
What: All Issues : Government Checks on Corporate Power : Requiring publicly traded companies to allow shareholders to cast non-binding votes on compensation packages for top executives beginning in 2009 (H.R. 1257)/Rep. Pete Sessions (R-Texas) amendment to prohibit shareholders' votes from being counted if they spent money trying to influence the votes of others without disclosure (2007 house Roll Call 236)
Requiring publicly traded companies to allow shareholders to cast non-binding votes on compensation packages for top executives beginning in 2009 (H.R. 1257)/Rep. Pete Sessions (R-Texas) amendment to prohibit shareholders' votes from being counted if they spent money trying to influence the votes of others without disclosure
This vote was on an amendment to a bill requiring publicly traded companies to allow shareholders a say in the compensation packages of top executives. Proposed by Rep. Pete Sessions (R-Texas), the amendment would have prohibited a shareholder's vote from being counted if the shareholder spent more than a minimal amount of money trying to influence the votes of others and did not fully disclose those activities to the Securities and Exchange Commission (SEC).
The legislation Sessions was seeking to amend would require that corporations conduct annual nonbinding advisory votes on the compensation of their executives beginning in 2009. The impetus for the bill was a growing concern among lawmakers as well as many economists that the widening discrepancy between pay for executives and working- and middle-class Americans was harmful for both the economy and social mobility.
Sessions described his amendment as a way to provide "sunshine and transparency for shareholders so that there is full disclosure about who is financing efforts to influence their vote on this new congressionally mandated, nonbinding shareholder resolution."
He said the purpose was not to impede the ability of organizations to influence the shareholder votes on executive pay but simply to provide everyone with access to information about who is spending money to influence that vote. The amendment would prompt the SEC to set a minimum spending threshold that would trigger the disclosure requirement.
"If an individual wants to spend more than this de minimis amount and not disclose their identity to shareholders, they are still perfectly able to do so," Sessions continued, saying that their votes simply would not count.
Rep. George Miller (D-Calif.) said the amendment was designed to close down public discourse and conversations between shareholders about executive pay. Miller said Session's amendment would effectively silence individuals who donate money to a campaign aimed at swaying non-binding votes on executive pay.
"So what you are really doing here is, you are trying to chill the speech and freeze the speech by putting them and holding them responsible for the disclosure that they may not have any control over," Miller said. "They may know they just don't like that executive compensation or they want a discussion of it. They don't necessarily know the activities engaged in to influence the vote."
After Miller's lengthy discourse opposing the amendment, Rep. Barney Frank (D-Mass.) responded with a two-sentence observation about Republicans' definition of democracy, which, he said, "has recently frequently included throwing votes away."
To which Miller responded: "You mean those 13,000 in Florida that are missing?" referring the disputed results from the 2000 election.
Democratic opposition to Sessions' amendment was nearly unanimous. All but two Democrats voted against it, and all Republicans present but three voted for it, not enough to muster a majority. Thus, on a party-line vote of 177 to 222, the House rejected an amendment prohibiting shareholders' votes from being counted if they spent money trying to influence the votes of others without disclosure, and legislation requiring publicly traded companies to allow shareholders to cast non-binding votes on compensation packages proceeded without the provision.
GOVERNMENT CHECKS ON CORPORATE POWER — General


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Liverpool Philh...
Liverpool Philharmonic announces 5th Christopher Brooks Composition Prize for young composers in association with The Rushworth Foundation
Friday 7 June 2019
The Christopher Brooks Composition Prize provides a unique opportunity for a young North West composer to develop their talent and access mentoring and guidance from leading artists and music industry professionals, culminating in writing a new work for performance by Royal Liverpool Philharmonic Orchestra musicians which will be premiered in autumn 2020.
Now in its fifth year, the prize represents Liverpool Philharmonic's continuing commitment to the commissioning and performance of new music and supporting home-grown talent.
The Christopher Brooks Composition Prize is open to any composer aged 18-30 living, studying or working in the counties of Merseyside, Cheshire, Cumbria, Lancashire, and Greater Manchester.
The prize-winning composer will write a new work for an ensemble from the Royal Liverpool Philharmonic Orchestra which will be performed in concert during the 2020/21 concert season.
A cash prize of £1,000 has been made possible through the support, for a fifth year, of the Lancashire Sinfonietta Legacy Fund and The Rushworth Foundation. The voice for music writers Ivors Academy will also provide a year's complimentary membership for the winning composer.
The prize-winner will also be supported with a bespoke programme of workshops, masterclasses and mentoring sessions from resident and visiting musicians, conductors, composers, performers and other industry professionals associated with Liverpool Philharmonic. They will develop their composition and teaching practice through access to Liverpool Philharmonic's programme for children and young people, including Liverpool Philharmonic Youth Company, Youth Orchestra and Choirs, higher education partnerships and the El-Sistema inspired In Harmony Liverpool.
Carmel Smickersgill was the 2018 winner of the Christopher Brooks Composition Prize, and her new work, Charcoal, will be premiered in concert by Ensemble 10/10, Liverpool Philharmonic's contemporary music group, on Wednesday 6 November at Liverpool's St. George's Hall Concert Room. Another previous prize-winner, Grace-Evangeline Mason, will have her new work Mahler's Letters premiered on 11 June 2020 by the Royal Liverpool Philharmonic Choir.
Entries will be judged by a panel of leading composers, Royal Liverpool Philharmonic Orchestra musicians and senior management, and music industry professionals. This year's panel includes:
Peter Garden (Executive Director, Performance and Learning, Liverpool Philharmonic)
Andrew Cornall (Liverpool Philharmonic Artistic Consultant, Record Producer and former Vice President of A&R at EMI Classics)
Sally Groves (former Creative Director of Schott Music Publishers)
Stephen Pratt (Emeritus Professor of Music, Liverpool Hope University, Emeritus Professor, Gresham College, London)
Hilary Browning (Associate Principal cellist with Royal Liverpool Philharmonic Orchestra)
Fiona Sinclair, (Chief Executive Leeds International Piano Competition)
Peter Garden, Liverpool Philharmonic's Executive Director, Performance & Learning said:
Liverpool Philharmonic is always looking for ways to create exciting new opportunities for emerging creative talent from Liverpool and the North West region. This fifth Christopher Brooks Competition Prize builds on our extensive work supporting composition students in the City's universities, along with the many local, national and international artists we commission to write new work for the Royal Liverpool Philharmonic Orchestra and associated ensembles.
Jonathan Rushworth of The Rushworth Foundation said:
The Rushworth name has been synonymous with music in Liverpool and the North West for well over a century. We are keen to preserve the legacy and heritage of the family's support for music and the arts in Liverpool and are delighted to support this second competition to encourage composition by young people.
Bethan Morgan Williams (previous participant) said:
The Christopher Brooks Composition Prize provides a young composer with a unique opportunity to absorb them self in the day to day life of the Royal Liverpool Philharmonic. The orchestra is the most friendly and welcoming organisation, with everyone I've come into contact with wanting to do anything and everything they can to help me on my way. I have been able to meet with and pick the brains of many of the players, and develop relationships with musicians and non-musicians alike. I even had the opportunity to meet childhood hero Andrew Manze!
Application Deadline is Friday 5 July at 5pm. Full details about to how to apply at: www.liverpoolphil.com/christopher-brooks-composition-prize
The late Christopher Brooks was a founder of the Lancashire Sinfonietta (now sadly disbanded) and gave an immense amount of support and encouragement to young musicians during his lifetime. The Rushworth Foundation, founded by Jonathan Rushworth whose family were founders of Liverpool's famous music retail and pipe organ building business, Rushworth & Dreaper, continues a 175-year tradition of support for the musical life of the city of Liverpool.
Photograph: Vasily Petrenko, Chief Conductor of the Royal Liverpool Philharmonic Orchestra and Carmel Smickersgill winner of the 2018 Christopher Brooks Composition Prize.
Media enquiries and further information:
Millicent Jones, Executive Director, Audiences and Development, Liverpool Philharmonic
Tel: 0151 210 2915 / Millicent.jones@liverpoolphil.com
Royal Liverpool Philharmonic Welcomes New Members to Board of Directors
Vanessa Bakewell and Angela Bellingham have been appointed to the Board of Directors of the Royal Li...
Dan Carden MP launches In Harmony Liverpool partnership with All Saints Catholic Primary School, Anfield
Dan Carden MP launches In Harmony Liverpool partnership with All Saints Catholic Primary School, Anf...
Liverpool Philharmonic Commemorates the 100th Anniversary of the end of World War One
Liverpool Cathedral performance of Britten's War Requiem (10 Nov) is centrepiece of Liverpool Philha...
Jessica Cottis conducts the Royal Liverpool Philharmonic Orchestra in music by three of France's Finest Historical Female Musical Voices
A generous gift from the Association of British Orchestra's Sirens fund sees Jessica Cottis conduct...
Liverpool Philharmonic Announces Winner of Christopher Brooks Composition Prize 2018
Liverpool Philharmonic is delighted to announce 22-year-old Carmel Snickersgill as the 2018 winner o...
And In The End…A Celebration of Abbey Road and Le
The award-winning Royal Liverpool Philharmonic Orchestra and the nation's premier Beatle group The B...
Thu 19 & Sun 22 September 2019 Various Times Liverpool Philharmonic Hall
Dani Howard Coalescence (Liverpool Philharmonic commission/World premiere), Rachmaninov Piano Concerto No.2, Berlioz Symphonie Fantastique conducted by Vasily Petrenko and pianist Nobuyuki Tsujii .
Symphonie Fantastique
Wednesday 4 September 2019 7:30pm Liverpool Philharmonic Hall
Fry divides his gripping tales of love and war, debauchery and revenge into three full-length shows as Gods takes us from primordial chaos to the creation of the dazzling Greek pantheon.
Mythos: A Trilogy by Stephen Fry - Gods
Thu 26 - Fri 27 September 2019 7:30pm Liverpool Philharmonic Hall
Following on from their hugely successful tour in 2017/18, the award-winning Royal Liverpool Philharmonic Orchestra and the nation's premier Beatles group, The Bootleg Beatles, join forces once again to deliver a Beatles experience like no other.
And In The End
Last Few Tickets
Thursday 17 October 2019 7:30pm Liverpool Philharmonic Hall
UK pop icon Will Young has released his brand new single 'All The Songs' - the first track to be heard from his long awaited seventh album Lexicon.
Friday 29 November 2019 7:30pm Liverpool Philharmonic Hall
The Stylistics are known instantly for their many recognisable hits such as the Grammy nominated 'You Make Me Feel Brand New'.
Saturday 16 November 2019 7:30pm Liverpool Philharmonic Hall
Multi-platinum, award-winning singer-songwriter Emeli Sandé MBE announces her U.K tour, ahead of the release of her much anticipated third album Real Life..


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Round 10 – Carlton v Hawthorn: There is merit in struggle
May 26, 2021 by John Butler Leave a Comment
Few stats sum up Carlton's 21st century struggles more abjectly than the fact the Blues last beat the Hawks at the MCG on the day Sam Walsh was born. Playing them anywhere, we'd won one of the last fifteen. So I couldn't care less how ugly you thought this win looked. Any win over Hawthorn is a good win.
This game had more turnovers than a tumble dryer, but you never doubted both sides were committed. Having provided North their first win last week, Hawthorn were seeking redemption. As for Carlton, we just couldn't afford to lose. A loss to this battling Hawthorn side would have mocked respectable (but unsuccessful) efforts against better opponents.
We began the game on our terms. Perhaps mindful of that aforementioned birthday stat, Sam Walsh had brought his own footy and was everywhere early. We should have led by more, but our forward entries were wasteful. Harry opted to stand and grapple with opponent Kyle Hartigan, which suited Hartigan just fine. Both of Hawthorn's first term goals were a product of kicking turnovers from Liam Jones. For all our efforts we only led by 10 points at the break.
We stretched that to 23 points by midway through the 2nd term, but then squandered the advantage with a poor last 10 minutes before half time. All day we had the Hawks' tall forwards under control, but struggled to defend when the ball hit the deck. Inevitably, Luke Breust crumbed a couple. Eddie replied with one of his own. Then we contrived to cough up 2 goals in the final 30 seconds of play. Admittedly, Mitch Lewis' goal was a flukey tumbling bouncer that cleared two contests before dribbling through. But David Teague had grounds to be grumpy at the long break, as we only had a 3 point lead to show for a half we appeared to largely control.
At this point, memories of last year's clash with the Hawks hardly eased anxieties. On that occasion, we'd jumped out to a 5 goal advantage, only to see Hawthorn swing the momentum, eventually slicing us up for our biggest defeat of 2020. Hawthorn had squeaked their 2 previous wins this season by just hanging in until their chance presented. It felt like we had offered just such an opportunity.
That concern persisted in a 3rd term where Hawthorn began to play on their terms. Winning first possession, they now had the majority of play. They only led briefly, but we couldn't draw clear. This is precisely the kind of scenario where Carlton has struggled in recent years. This time, it was encouraging that as the momentum shifted the scoreboard didn't. This was partly due to Hawthorn fluffing some chances, but we also had a steadier feel about us. Zac Williams unsurprisingly looked much more comfortable at half back, and he formed an effective rebounding duo with Adam Saad. Moved to a wing to accommodate Williams, Sam Docherty has added substance to our midfield group in the last two games. We look less desperately dependent on Crippa and Walshy. This time we prevailed to add three red-time goals ourselves, to lead at the final break by 19 points.
Breust opened the final term by scrounging his 3rd goal, then proceedings settled into a long, strenuous arm-wrestle. I remember being overtaken by an unfamiliar calmness at this point. It felt like we had Hawthorn's measure, but just couldn't quite find the killer blow. It turned out that the fatal blow came courtesy of an otherwise inconspicuous Harry, who managed to land a set shot from well outside 50 at the 21 minute mark. It was the last goal of the game.
There's no doubt that calmness I felt was a reflection on the lack of real threat the Hawks presented. They tried hard, but they look a shadow of what we remember Hawthorn sides to be. They are missing a few key players, but so are we. I think it's only Clarkson's reputation, and the memory of those earlier sides, that currently keeps the media hounds bluffed. Jeff Kennett seems to have supreme confidence in their ability to rebuild, but his comments have a decided late period Jack Elliot "we don't do rebuilds at Carlton" feel about them to these Navy Blue ears. We'll see how that all works out for them.
Tribunal matters have dominated the post-game discussion. Kyle Hartigan's suspension looks straight forward. Only the fact the condemning video came via a crowd source adds novelty.
Then there's Lachie Plowman's two week suspension. Anyone who has ever read any football history knows that tribunals in their various forms have baffled for well over a century, so I am rarely shocked by any decision they might produce. But the direction of the Tribunal chairman to the panel that the collision between Plowman and Jaeger O'Meara should not be considered a marking contest is genuinely bewildering to me. I doubt anyone who saw the incident live would have formed that conclusion at the time. This leaves me wondering about the reliance on technology for the process of laying charges, and whether that reliance can strip incidents of their game context, turning them into something other than what they actually were. I certainly couldn't relate the incident I saw to the way that incident was subsequently discussed.
No Bluebagger walked away from this game thinking we'd turned any corners. But the fact we could arrest an opponent's momentum marks a small step in the direction we need to take. We are far from settled personnel or position-wise. We didn't play anywhere near the best football we have produced in other games this season, yet we defeated an opponent that has consistently confounded us.
With the AFL, media and fans seemingly obsessed with how the game looks, a good old-fashioned slog feels generally undervalued. So I will conclude as I began, any win over Hawthorn is a good win.
CARLTON 4.1 8.3 12.4 13.8 (86)
HAWTHORN 2.3 7.6 8.9 9.9 (63)
Carlton: Owies 2, Betts 2, McKay 2, Silvagni, De Koning, Williams, Walsh, Pittonet, Gibbons, Murphy
Hawthorn: Breust 3, Morrison 2, Koschitzke, Lewis, Howe, Moore
Carlton: Walsh, Docherty, Williams, Saad, Cripps, Weitering
Hawthorn: Mitchell, Worpel, Scrimshaw, Wingard, Breust, Hartigan
For more from John, click HERE.
The Tigers (Covid) Almanac 2020 will be published in 2021. It will have all the usual features – a game by game account of the Tigers season – and will also include some of the best Almanac writing from the Covid winter. Pre-order HERE
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About John Butler
John Butler has fled the World's Most Liveable Car Park and now breathes the rarefied air of the Ballarat Plateau. For his sins, he has passed his 40th year as a Carlton member.


My apologies for any cross-posting.
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