1_intro.tex

\section{Introduction} % - Vini 
\subsection{Background}
Solution pH is tightly regulated in biological environments \cite{Casey_Orlowski_2010_Nat.Rev.Mol.CellBiol.}. 
In normal adult cells, intracellular pH$_{\rm i}$ is about 7.2 and extracellular pH$_{\rm e}$ is about 7.4 \cite{Webb_Barber_2011_Nat.Rev.Cancer};
however, in cancer cells, the pH gradient is reversed,
which promotes cancer progression, metabolic adaptation, and metastasis
\cite{Webb_Barber_2011_Nat.Rev.Cancer,White_Barber_2017_J.CellSci.}. 
Virtually all proteins depend on pH to maintain their structure and function
\cite{Casey_Orlowski_2010_Nat.Rev.Mol.CellBiol.,Roos_Boron_1981_Physiol.Rev.}.
% Protein folding is one example of a pH-dependent process, where the unfolded protein only can reach its native state in a narrow pH range. 
A small change in pH can perturb the functions of many enzymes.
For example, SARS coronavirus main proteases have the maximal
cleavage activity in a narrow pH range around 7.0 \cite{Tan_Hilgenfeld_2005_J.Mol.Biol.}. 
Below the optimum pH, the active site collapses
due to the protonation state change of a histidine \cite{Tan_Hilgenfeld_2005_J.Mol.Biol.,Verma_Shen_2020_J.Am.Chem.Soc.}. 
The function of human $\beta$-secretase 1 peaks at pH 4.5,
\cite{Shimizu_Nukina_2008_Mol.CellBiol.}; at low or high pH
the protonation state of the catalytic dyad changes, 
which results in the closure of the ``flap'' that lies above
the active site and blockage of 
substrate entrance \cite{Ellis_Shen_2015_J.Am.Chem.Soc.}.
Solution pH is also an important parameter in the creation of dynamic, stimuli-responsive materials \cite{Li_Payne_2019_Biofabrication}. 
For example, pH triggers the formation of hydrogel films
from the biopolymer chitosan
\cite{Morrow_Shen_2015_J.Am.Chem.Soc.}; 
addition of surfactants allows the chitosan film to be reconfigured via a second pH signal to achieve different mechanical properties \cite{Tsai_Shen_2018_Chem.Mater.}.

In a conventional molecular dynamics (MD) simulation,
solution pH is implicitly taken into account by
fixing the protonation states of titratable sites according to the experimental {\pka} values of
model compounds (or peptides) or 
traditional {\pka} calculations based on a static structure. 
Traditional {\pka} methods include empirical
approaches, e.g.,
PropKa \cite{Olsson_Jensen_2011_J.Chem.TheoryComput.},
Rosetta \cite{Kilambi_Gray_2012_Biophys.J.},
Poisson-Boltzmann solvers
MCCE \cite{Song_Gunner_2009_J.Comput.Chem.},
APBS/PDB2PQR \cite{Unni_Baker_2011_J.Comput.Chem.}, 
DelPhiPKa \cite{Wang_Alexov_2016_Bioinformatics},  
H++ \cite{Anandakrishnan_Onufriev_2012_NucleicAcidsRes.},
and other variants \cite{WarwickerJim_WarwickerJim_2011_Proteins}.
These tools are fast; however, 
the predicted protonation states are often incorrect
for deeply buried sites
\cite{Alexov_Word_2011_Proteins},
enzyme active sites \cite{Huang_Shen_2018_J.Phys.Chem.Lett.},
transmembrane proteins \cite{Huang_Shen_2016_Nat.Commun.},
and cysteines or lysines in general \cite{Liu_Shen_2019_J.Am.Chem.Soc.,Harris_Shen_2020_J.Chem.TheoryComput.,Liu_Shen_2022_J.Med.Chem.}.
Also, even assuming an accurate assignment of protonation states at the beginning of MD, the conformational changes sampled
by the simulation may modify the electrostatic environment around the titratable groups, changing their protonation states. 
To account for 
the direct coupling between conformational dynamics and protonation state changes and to offer more accurate \pka\ predictions, constant pH MD methods have been developed.

Over the past two decades, much progress has been made in the
development of constant pH MD methods.
In a discrete (also known as stochastic) constant pH MD (DpHMD), the MD trajectory is periodically
interrupted by the Metropolis Monte-Carlo (MC) sampling of 
protonation states \cite{Baptista_Soares_2002_J.Chem.Phys.,Mongan_McCammon_2004_J.Comput.Chem.,Swails_Roitberg_2014_J.Chem.TheoryComput.,Stern_Stern_2007_J.Chem.Phys.,Chen_Roux_2015_J.Chem.TheoryComput.}. 
Here, protonation and deprotonation of titratable groups is
sampled discretely, assuming one of the two states. 
Besides the advantage that only physical (protonated and deprotonated) states are sampled, the DpHMD method based on
the hybrid MD/MC scheme is conceptually simple
and practically straightforward to implement.
In contrast, the continuous constant pH MD (CpHMD) 
\cite{Lee_Brooks_2004_Proteins,Khandogin_Brooks_2005_Biophys.J.,Khandogin_Brooks_2006_Biochemistry,Wallace_Shen_2011_J.Chem.TheoryComput.,Wallace_Shen_2012_J.Chem.Phys.,Huang_Shen_2016_J.Chem.TheoryComput.}
treats the protonation states of ionizable sites using an auxiliary set of continuous titration coordinates based on an extended Hamiltonian $\lambda$-dynamics approach \cite{Kong_Brooks_1996_J.Chem.Phys.}. 
This method allows the system to escape local energy minima by transiently accessing the partially protonated states.
Although sampling the unphysial intermediate states is a major caveat, the CpHMD method offers significantly faster convergence 
for coupled residues, and importantly it can be readily extended to all-atom simulations.
The faster convergence arises from the fact that at every MD step the $\lambda$ values of 
all titratable sites are updated, whereas in a typical DpHMD implementation, the protonation state of 
a single residue is attempted at each MC step.

Development of an all-atom DpHMD algorithm based on the
hybrid MD/MC approach is challenging, as in explicit solvent a switch 
in protonation state leads to a large change in electrostatic energy, 
which results in a nearly complete rejection of the MC move
\cite{Chen_Roux_2015_J.Chem.TheoryComput.}.
This problem, which is due to the lack of overlap between explicit solvent
configurations for the protonated and deprotonated states
\cite{Chen_Roux_2015_J.Chem.TheoryComput.},
has been tackled by introducing a free energy calculation
\cite{Burgi_vanGunsteren_2002_Proteins}
or a short non-equilibrium MD (neMD)
\cite{Stern_Stern_2007_J.Chem.Phys.,Chen_Roux_2015_J.Chem.TheoryComput.,Radak_Roux_2017_J.Chem.TheoryComput.} 
(see later discussion).

In an implicit-solvent constant pH scheme, sampling of both conformational dynamics 
and protonation states is performed using a continuum
electrostatic model, typically a generalized Born (GB) model.
Implementations of this scheme in the CHARMM package
 \cite{Brooks_Karplus_2009_J.Comput.Chem.}
include the 
GBMV-\cite{Lee_Brooks_2003_J.Comput.Chem.} and 
GBSW- \cite{Im_Brooks_2003_J.Comput.Chem.} based CpHMD \cite{Lee_Brooks_2004_Proteins,Khandogin_Brooks_2005_Biophys.J.,Khandogin_Brooks_2006_Biochemistry} methods.
Implementations in the Amber package \cite{Case_Kollman_2018}
include the OBC-GB \cite{Onufriev_Bashford_2002_J.Comput.Chem.}
based DpHMD 
\cite{Mongan_McCammon_2004_J.Comput.Chem.}
and the GBNeck2-based CpHMD
\cite{Huang_Shen_2018_J.Chem.Inf.Model.,Harris_Shen_2019_J.Chem.Inf.Model.}
methods.

In a hybrid-solvent constant pH scheme, conformational sampling
is conducted with explicit solvent, while a continuum model
e.g., GB or Poisson-Boltzmann (PB), is used to sample protonation states,
i.e., calculating the solvation forces 
on the $\lambda$ particles for the CpHMD methods
or the energy changes due to protonation-state changes
for the DpHMD methods.
Implementations of this scheme include
the first DpHMD method based on PB/TIP3P 
\cite{Baptista_Soares_2002_J.Chem.Phys.}
for GROMACS \cite{Spoel_Berendsen_2005_J.Comput.Chem.},
the GBSW/TIP3P based CpHMD method \cite{Wallace_Shen_2011_J.Chem.TheoryComput.}
in CHARMM \cite{Brooks_Karplus_2009_J.Comput.Chem.},
and the OBC-GB/TIP3P based DpHMD method 
\cite{Swails_Roitberg_2014_J.Chem.TheoryComput.}
in Amber \cite{Case_Kollman_2018}.
Note, the GBSW/TIP3P based CpHMD method \cite{Wallace_Shen_2011_J.Chem.TheoryComput.} was extended to the treatment of transmembrane systems \cite{Huang_Shen_2016_Nat.Commun.} by incorporating a membrane GBSW model \cite{Im_Brooks_2003_Biophys.J.}.

An all-atom or fully explicit-solvent constant pH scheme
removes the dependence on a continuum model in that
it samples both conformational and protonation states in
explicit solvent.
An all-atom CpHMD method 
called CPHMD$^{\rm MS\lambda D}$ \cite{Goh_Brooks_2014_Proteins}
was implemented by the Brooks group in the BLOCK module of the CHARMM package
\cite{Brooks_Karplus_2009_J.Comput.Chem.}
as a part of the multi-site $\lambda$-dynamics (MS$\lambda$D) functionality \cite{Knight_Brooks_2011_J.Chem.TheoryComput.}.
Most recently, the Brooks group developed
a standalone GPU program called basic lambda dynamics engine (BLaDE)
\cite{Hayes_Brooks_2021_J.Chem.TheoryComput.},
which enables GPU-accelerated MS$\lambda$D and
constant pH simulations.
The first all-atom particle mesh Ewald (PME) CpHMD method 
was implemented by the Shen group in CHARMM
\cite{Brooks_Karplus_2009_J.Comput.Chem.},
as an extension to the GBSW and hybrid-solvent CpHMD 
methods in the PHMD module
\cite{Wallace_Shen_2012_J.Chem.Phys.,Chen_Shen_2013_Biophys.J.,Huang_Shen_2016_J.Chem.TheoryComput.}.
To compensate for the net charge fluctuation due to proton titration,
the Shen group introduced co-titrating ions \cite{Wallace_Shen_2012_J.Chem.Phys.}
or water \cite{Chen_Shen_2013_Biophys.J.}.
Most recently, the all-atom PME CpHMD method was implemented
in Amber (version Amber22 \cite{Case_Kollman_2022}) 
by the Shen group \cite{Harris_Shen_Submitted}.
A $\lambda$-dynamics based all-atom CpHMD implementation 
was also developed by the Grubm\"{u}ller group
\cite{Donnini_Grubmuller_2011_J.Chem.TheoryComput.,Donnini_Grubmuller_2016_J.Chem.TheoryComput.}
in the GROMACS package
\cite{Spoel_Berendsen_2005_J.Comput.Chem.}, 
although the method has not been validated for a large number of proteins yet. The proper treatment of tautomer states and the use of PME for $\lambda$ dynamics remain to be addressed.

To circumvent the aforementioned configuration overlap problem
in a DpHMD framework, B\"{u}rgi and van Gunsteren used thermodynamic integration (TI)
to calculate the titration free energy change for the MC move
\cite{Burgi_vanGunsteren_2002_Proteins};
however, TI calculations are computationally costly and do not readily converge.
Recently, an all-atom DpHMD method based on a hybrid neMD/MC scheme
has been implemented in NAMD \cite{Phillips_Schulten_2005_J.Comput.Chem.}
by the Roux group
\cite{Chen_Roux_2015_J.Chem.TheoryComput.,Radak_Roux_2017_J.Chem.TheoryComput.}.
This approach, which was originally proposed by Stern \cite{Stern_Stern_2007_J.Chem.Phys.},
has the flavor of both TI and $\lambda$ dynamics
methods, as it utilizes a ($\lambda$) coupling parameter in 
a neMD trajectory to gradually change the protonation state 
thus allowing solvent to adjust 
\cite{Chen_Roux_2015_J.Chem.TheoryComput.,Radak_Roux_2017_J.Chem.TheoryComput.}. 
Analogous to the titratable ion or water approach of the Shen group
\cite{Wallace_Shen_2012_J.Chem.Phys.,Chen_Shen_2013_Biophys.J.},
the hybrid neMD/MC implementation of the Roux group used the chemical transformation 
between a counterion and water to maintain charge neutrality of the system
\cite{Chen_Roux_2015_J.Chem.TheoryComput.}.
Interestingly, while the Roux group reported that charge neutrality 
acts as a constraint and decreases the acceptance ratio in the MC steps
\cite{Chen_Roux_2015_J.Chem.TheoryComput.,Radak_Roux_2017_J.Chem.TheoryComput.}, 
the Shen group observed a slow down in convergence of protonation state sampling \cite{Chen_Shen_2013_Biophys.J.,Huang_Shen_2016_J.Chem.TheoryComput.}.
Having reviewed the various constant pH methods,
the remainder of the tutorial will focus on the CpHMD implementations 
developed by the Shen group.

While accurate prediction of protonation states or {\pka} values is a goal, 
a major application of constant pH MD is to elucidate the mechanisms of 
pH-dependent or proton-coupled conformational dynamics. 
One can argue that the latter can also be studied by conventional 
fixed-protonation-state MD using simulations with different protonation states.
While this may well be true when the switch of one protonation state is involved, 
the problem becomes intractable when the number $N$ of titratable sites is large
because the number of possible protonation states increases as 2$^N$.
Furthermore,
the identity of the titratable site responsible for proton-coupled conformational changes is
often unclear, which makes the fixed-protonation-state approach unfeasible.
Importantly, by running fixed-protonation-state MD one cannot obtain the {\pka} value for the conformational transition \cite{Wallace_Shen_2012_J.Phys.Chem.Lett.,Huang_Shen_2016_Nat.Commun.,Yue_Shen_2017_J.Chem.TheoryComput.,Henderson_Shen_2020_Proc.Natl.Acad.Sci.USA}, 
which can be compared with experiment to validate the microscopic details revealed by simulation.

Table~\ref{Table:applications} summarizes the applications of the implicit-, hybrid-, and fully explicit-solvent CpHMD methods developed in the Shen group.
% Over the past decade, CpHMD simulations have been applied 
% to proteins in different environments \cite{Chen_Shen_2014_Mol.Simulat.,Radak_Roux_2017_J.Chem.TheoryComput.}. 
The implicit-solvent GBSW- and GBNeck2-CpHMD methods have been mainly applied to {\pka} predictions of proteins
\cite{Khandogin_Brooks_2006_Biochemistry,Wallace_Shen_2009_MethodsEnzymol.,Wallace_Shen_2011_Proteins,Harris_Shen_2019_J.Chem.Inf.Model.,Harris_Shen_2020_J.Chem.TheoryComput.},
while the hybrid-solvent and all-atom CpHMD methods have been additionally applied to elucidate the mechanisms of pH-dependent or proton-coupled conformational processes, e.g.,
protein unfolding
\cite{Yue_Shen_2018_Phys.Chem.Chem.Phys.},
structure-function relationships of proteases and kinases \cite{Ellis_Shen_2015_J.Am.Chem.Soc.,Tsai_Shen_2019_J.Am.Chem.Soc.,Ma_Shen_2021_J.Chem.Inf.Model.,Verma_Shen_2020_J.Am.Chem.Soc.,Henderson_Shen_2020_J.Chem.Phys.}, protein/ligand binding \cite{Ellis_Shen_2016_J.Phys.Chem.Lett.,Harris_Shen_2017_J.Phys.Chem.Lett.,Henderson_Shen_2018_J.Phys.Chem.Lett.}, 
conformational activation of transmembrane channels and transporters \cite{Chen_Shen_2016_J.Phys.Chem.Lett.,Yue_Shen_2017_J.Chem.TheoryComput.,Huang_Shen_2016_Nat.Commun.},
and materials \cite{Morrow_Shen_2015_J.Am.Chem.Soc.,Tsai_Shen_2018_Chem.Mater.}.
A recent application of GBNeck2-CpHMD method is the prediction of nucleophilic cysteine and lysine sites for targeted covalent drug design \cite{Liu_Shen_2019_J.Am.Chem.Soc.,Verma_Shen_2020_J.Am.Chem.Soc.,Henderson_Shen_2020_J.Chem.Phys.,Liu_Shen_2022_J.Med.Chem.,Liu_Shen_2022_RSCMed.Chem.}. 
In this tutorial, we will discuss GBNeck2-CpHMD in Amber \cite{Huang_Shen_2018_J.Phys.Chem.Lett.,Harris_Shen_2019_J.Chem.Inf.Model.},
hybrid-solvent  \cite{Wallace_Shen_2011_J.Chem.TheoryComput.},
and particle-mesh Ewald (PME) all-atom CpHMD 
\cite{Huang_Shen_2016_J.Chem.TheoryComput.}
in CHARMM \cite{Brooks_Karplus_2009_J.Comput.Chem.}. 
% The GB-based CpHMD methods
% have been validated for a large number of proteins and the latter has additionally revealed the pH-dependent or proton-coupled mechanisms of various soluble and transmembrane proteins.

\begin{table*}
\begin{center}
\begin{tabular}{p{1.2in}p{3in}ll}
System &  Topic  & Method & Reference\\ 
\hline
\multicolumn{2}{l}{\bf Soluble proteins} & \\
Benchmark proteins &  {\pka} calculations & GBSW 
& \cite{Khandogin_Brooks_2006_Biochemistry,Wallace_Shen_2009_MethodsEnzymol.}\\
SNase  & Blind {\pka} predictions for 87 engineered mutant proteins   & GBSW & \cite{Wallace_Shen_2011_Proteins}\\
Peptides and mini-proteins & pH-dependent folding mechanisms & GBSW & \cite{Khandogin_Brooks_2006_Proc.Natl.Acad.Sci.USA,Khandogin_Brooks_2007_Proc.Natl.Acad.Sci.USA,Khandogin_Brooks_2007_J.Am.Chem.Soc.}\\
Benchmark proteins & {\pka} calculations & Hybrid & \cite{Wallace_Shen_2011_J.Chem.TheoryComput.}\\
NTL9, BBL & Unfolded states and unfolding mechanism & Hybrid& \cite{Shen_Shen_2010_J.Am.Chem.Soc.,Yue_Shen_2018_Phys.Chem.Chem.Phys.}\\
SNase   & {\pka} calculation for a deeply buried lysine; proton-coupled opening of the site   & Hybrid & \cite{Shi_Shen_2012_Biophys.J.}\\
Spider silk protein & pH sensing residues for dimerization & Hybrid & \cite{Wallace_Shen_2012_J.Phys.Chem.Lett.}\\
BACE1, BACE2, cathepsin D, renin, plasmepsin D & Acid/base roles of the catalytic dyad; binding-induced protonation state change of the inhibitor; pH-dependent conformational dynamics;
proton-coupled dynamics of binding site water;
pH-dependent 
inhibitor binding free energy calculations & Hybrid & \cite{Ellis_Shen_2015_J.Am.Chem.Soc.,Ellis_Shen_2016_J.Phys.Chem.Lett.,Ellis_Shen_2017_J.Comput.Chem.,Harris_Shen_2017_J.Phys.Chem.Lett.,Henderson_Shen_2018_J.Phys.Chem.Lett.,Ma_Shen_2021_J.Chem.Inf.Model.,Henderson_Shen_2021_J.Chem.Inf.Model.}\\
c-Src Kinase & Proton-coupled conformational change of the DFG motif & Hybrid & \cite{Tsai_Shen_2019_J.Am.Chem.Soc.}\\
% BACE1, cathepsin D &  pH-dependent binding free energy calculations & Hybrid & \cite{Harris_Shen_2017_J.Phys.Chem.Lett.}\\
Benchmark proteins & {\pka} calculations for Asp/Glu/His &  All-atom & \cite{Huang_Shen_2016_J.Chem.TheoryComput.} \\
% Plasmepsin & Dissect the pH-dependent interaction of the ligand binding site, identify proton coupled allostery, elucidate the catalytic roles of the active site & Hybrid-Solvent \\
Various enzymes & Prediction of proton donor and nucleophile and physical determinants & Hybrid & \cite{Huang_Shen_2018_J.Phys.Chem.Lett.}\\
Benchmark proteins & {\pka} calculations for Asp/Glu/His/Cys &  GBNeck2 & \cite{Huang_Shen_2018_J.Chem.Inf.Model.,Harris_Shen_2019_J.Chem.Inf.Model.}\\
Various kinases &  Prediction and rationalization of reactive Lys and Cys  & GBNeck2 & \cite{Liu_Shen_2019_J.Am.Chem.Soc.,Liu_Shen_2022_J.Med.Chem.,Liu_Shen_2022_RSCMed.Chem.}\\
Coronavirus papain-like proteases  &   Protonation states of His/Cys; proton-coupled conformational dynamics  & GBNeck2 & \cite{Henderson_Shen_2020_J.Chem.Phys.}\\
Coronavirus main proteases  & Protonation states of His and Cys; proton-coupled conformational change of the binding site & GBNeck2 & \cite{Verma_Shen_2020_J.Am.Chem.Soc.}\\
\multicolumn{2}{l}{\bf Transmembrane proteins} & \\
Proton channel M2 & Proton-coupled channel opening/closing & Membrane-hybrid & \cite{Chen_Shen_2016_J.Phys.Chem.Lett.} \\
Sodium/proton antiporter NhaA   & Identification of proton binding residues; proton-coupled conformational changes & Membrane-hybrid & \cite{Huang_Shen_2016_Nat.Commun.,Henderson_Shen_2020_Proc.Natl.Acad.Sci.USA} \\ Efflux pump AcrB  &  Identification of proton binding residues; proton-coupled conformational transitions & Membrane-hybrid & \cite{Yue_Shen_2017_J.Chem.TheoryComput.}  \\
$\mu$-opioid receptor & {\pka} calculation & Membrane-hybrid & \cite{Vo_Ellis_2021_Nat.Commun.,Mahinthichaichan_Shen_2021_JACSAu}\\
\multicolumn{2}{l}{\bf Materials} & \\
Various surfactants & {\pka} calculation in micelle & GBSW and hybrid& \cite{Morrow_Shen_2011_J.Phys.Chem.B} \\
Fatty acid & {\pka} calculation in micelle and bilayer & All-atom& \cite{Morrow_Shen_2014_J.Chem.Phys.} \\
Fatty acid & pH-dependent micelle/bilayer formation & Hybrid& \cite{Morrow_Shen_2012_J.Chem.Phys.,Morrow_Shen_2013_Langmuir}\\
Peptide & pH-dependent unfolding of $\beta$-sheets; phase transition {\pka} & All-atom &  \cite{Cote_Shen_2014_J.Phys.Chem.C}\\
Polysaccharide chitosan & Glucosamine titration; pH-dependent dissociation of a model crystallite; phase transition {\pka}; pH-dependent interaction with a surfactant & All-atom & \cite{Morrow_Shen_2015_J.Am.Chem.Soc.,Tsai_Shen_2018_Chem.Mater.}\\
\end{tabular}
\end{center}
\caption{\textbf{Applications of the continuous constant pH MD (CpHMD) methods.}
GBSW-CpHMD \cite{Khandogin_Brooks_2005_Biophys.J.,Khandogin_Brooks_2006_Biochemistry},
hybrid-solvent \cite{Wallace_Shen_2011_J.Chem.TheoryComput.} and membrane-enabled hybrid-solvent \cite{Huang_Shen_2016_Nat.Commun.} CpHMD methods are implemented in CHARMM \cite{Brooks_Karplus_2009_J.Comput.Chem.}.
The GRF \cite{Wallace_Shen_2012_J.Chem.Phys.,Chen_Shen_2013_Biophys.J.}
and PME \cite{Huang_Shen_2016_J.Chem.TheoryComput.}  based all-atom 
CpHMD methods are also implemented in CHARMM \cite{Brooks_Karplus_2009_J.Comput.Chem.} and they can be used with co-titrating ions \cite{Wallace_Shen_2012_J.Chem.Phys.} or water \cite{Chen_Shen_2013_Biophys.J.}.
GBNeck2-CpHMD \cite{Huang_Shen_2018_J.Chem.Inf.Model.,Harris_Shen_2019_J.Chem.Inf.Model.} method is implemented in Amber18 \cite{Case_Kollman_2018}.
The pH replica-exchange protocol \cite{Wallace_Shen_2011_J.Chem.TheoryComput.}
was used for all applications except for those with GBSW-CpHMD where the temperature replica-exchange protocol \cite{Khandogin_Brooks_2006_Biochemistry} was used.
All implementations except for GBNeck2-CpHMD are for CPU computing.
}
\label{Table:applications}
\end{table*}