Dewetting transition assisted clearance of (NFGAILS) amyloid fibrils from cellmembranes by grapheneJiajia Liu, Zaixing Yang, Haotian Li, Zonglin Gu, Jose Antonio Garate, and Ruhong Zhou Citation: The Journal of Chemical Physics 141, 22D520 (2014); doi: 10.1063/1.4901113 View online: http://dx.doi.org/10.1063/1.4901113 View Table of Contents: http://scitation.aip.org/content/aip/journal/jcp/141/22?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Sticky water surfaces: Helix–coil transitions suppressed in a cell-penetrating peptide at the air-water interface J. Chem. Phys. 141, 22D517 (2014); 10.1063/1.4898711 Adhesive characteristics of low dimensional carbon nanomaterial on actin Appl. Phys. Lett. 104, 023702 (2014); 10.1063/1.4862200 Relationship between disease-specific structures of amyloid fibrils and their mechanical properties Appl. Phys. Lett. 102, 011914 (2013); 10.1063/1.4774296 Effect of membrane charge density on the protein corona of cationic liposomes: Interplay between cationiccharge and surface area Appl. Phys. Lett. 99, 033702 (2011); 10.1063/1.3615055 Effect of trehalose on amyloid β (29–40)-membrane interaction J. Chem. Phys. 131, 085101 (2009); 10.1063/1.3193726
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THE JOURNAL OF CHEMICAL PHYSICS 141, 22D520 (2014)
Dewetting transition assisted clearance of (NFGAILS) amyloid fibrilsfrom cell membranes by graphene
Jiajia Liu,1,a) Zaixing Yang,1,a) Haotian Li,2 Zonglin Gu,1 Jose Antonio Garate,3
and Ruhong Zhou1,3,4,b)
1Institute of Quantitative Biology and Medicine, SRMP and RAD-X, Collaborative InnovationCenter of Radiation Medicine of Jiangsu Higher Education Institutions, and Jiangsu ProvincialKey Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China2Bio-X Lab, Department of Physics, Zhejiang University, Hangzhou 310027, China3IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598, USA4Department of Chemistry, Columbia University, New York, New York 10027, USA
(Received 14 September 2014; accepted 24 October 2014; published online 10 November 2014)
Clearance of partially ordered oligomers and monomers deposited on cell membrane surfacesis believed to be an effective route to alleviate many potential protein conformational diseases(PCDs). With large-scale all-atom molecular dynamics simulations, here we show that graphenenanosheets can easily and quickly win a competitive adsorption of human islet amyloid polypep-tides (hIAPP22-28) NFGAILS and associated fibrils against cell membrane, due to graphene’s uniquetwo-dimensional, highly hydrophobic surface with its all-sp2 hybrid structure. A nanoscale dewet-ting transition was observed at the interfacial region between the fibril (originally deposited on themembrane) and the graphene nanosheet, which significantly assisted the adsorption of fibrils ontographene from the membrane. The π–π stacking interaction between Phe23 and graphene played acrucial role, providing the driving force for the adsorption at the graphene surface. This study ren-ders new insight towards the importance of water during the interactions between amyloid peptides,the phospholipidic membrane, and graphene, which might shed some light on future developmentsof graphene-based nanomedicine for preventing/curing PCDs like type II diabetes mellitus. © 2014AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4901113]
INTRODUCTION
Aggregation of misfolded proteins or peptides is a ma-jor risk factor for many fatal protein-conformational-diseases(PCDs),1–4 ranging from type II diabetes,5, 6 Alzheimer’s dis-ease (AD)7, 8 to prion disease.9 There is growing evidencesupporting the idea that rather than mature fibrils, the small,partially ordered, oligomers (with relatively lower β-sheetscontent) formed during the early aggregation stage, alongwith the aggregation process itself, are more likely to be themain toxic agents for cells.10–19 The molecular underpinningsof this cytotoxicity have been extensively studied in the lit-erature. Despite some remaining disputes and controversies,three most common mechanisms have been proposed: (i) thepartially ordered oligomers directly penetrate and disrupt thestructure of membrane which induces cell death;20 (ii) thepartially ordered oligomers can form ions or small-moleculemembrane channels altering ion concentration balances be-tween extracellular and intracellular environments, causingpremature cell apoptosis;21–25 (iii) the growth of rigid amy-loid fibrils on a flexible membrane results in a change inthe membrane curvature, largely disrupting the membranestructure and causing membrane breaches.10, 17, 19, 26, 27 It isnoteworthy that an essential prerequisite for the appearance
a)J. Liu and Z. Yang contributed equally to this work.b)Author to whom correspondence should be addressed. Electronic mail:
of small partially ordered oligomers (concurrently actingas amyloid nucleation seeds) is that the concentrations ofmisfolded monomeric proteins or peptides exceed the crit-ical threshold value.1 Furthermore, the formation of amy-loid nucleation seeds can trigger the subsequent large scaleamyloidogenesis.1 Therefore, from a design point of view,clearing the small partially ordered oligomers and monomersdeposited on the surface of membranes stands for an impor-tant strategy for curing and preventing PCDs.
Numerous efforts have been devoted to the generationof therapeutics based on these findings, and as such, the cur-rent primary therapeutic strategies include the suppression ofmonomer fibrillization through direct disruption of peptide-peptide interactions and dispersion of amyloid fibrillar ag-gregations through direct attacking. In addition, modulatingand interrupting the interaction between the cell membraneand peptide oligomers represents an alternative and impor-tant approach for the development of anti-PCD therapies.28–30
Despite these great efforts, current commonly used anti-PCDs agents, such as small drug molecules,31, 32 polymers,33
peptides,34, 35 and metal oxides36 show only a moderate sup-pressive effect on the monomer fibrillization and a weak dis-assembling capability on the mature amyloid aggregations. Itis thus an urgent task to explore and design novel approaches,including nanomaterial-based therapies, for PCDs.
Meanwhile, the recent years have witnessed an enormousadvance in nanomaterials science and their applications inbiomedical fields. In particular, carbon-based nanomaterials
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such as graphene and its derivatives have received great at-tention in this emerging field due to their unique structural,mechanical, and electronic properties.19, 20 Graphene’s bio-persistence plus its smooth and contiguous topography playa distinct role in tumor progression and induced carcinogen-esis by foreign bodies.23, 24 Moreover, graphene sheets areideal candidates for drug delivery systems due to their tremen-dous specific surface area, which allows high-density bio-functionalization.37–39 Additionally, in the recent years, sev-eral works have pointed towards clear antibacterial activitiesof graphene and graphene oxide (GO).40–44 It is thus of greatinterest to thoroughly assess the potential usage of graphenein the development of anti-PCDs remedies. It is appealingto note that there already exist some studies on the interac-tion between graphene derivatives and amyloid fibrils.45–48
Among those, a protective effect of GO sheets due to the inhi-bition of Aβ fibrillization was associated with adsorption pro-cesses due to the vast surface area of GO sheets.45 Moreover,Qu et al.50 has described that amyloid deposits, via infrared(IR) laser, are thermal-ablated in the presence of thioflavinmodified GO-sheets. Interestingly, these effects were shownto work not only in buffer solution studies, but also in micecerebrospinal fluid. Furthermore, versatile and novel bio-nanomaterials based on a combination of graphene and lac-toglobulin amyloid fibrils have been reported by Mezzengaand co-workers.49 Nonetheless, to the best of our knowl-edge, no study has systematically inspected the potential us-age of graphene as a removing agent of small partially or-dered oligomers and monomers adsorbed on the surface ofcell membranes, and the role of water during that process.
NFGAILS peptide is the core fibrillization segment ofhuman islet amyloid polypeptide (hIAPP22-28), and is directlyinvolved in type II diabetes mellitus.5, 6 Many experimentsand simulations have demonstrated the similarities betweenthe NFGAILS aggregation cytotoxicity in pancreatic β-isletcell with respect to its full length peptide.1, 40–43 Therefore, tofill in the blank and answer the question stated above, we havestudied the dynamic process and underlying molecular mech-anism of the adsorption of preformed NFGAILS amyloidaggregates and monomers off the membrane by a graphenesheet, highlighting the water’s role in this process. In detail,extensive all-atom molecular dynamics (MD) simulations ofthe competitive adsorption of NFGAILS peptides and fibrilsbetween a graphene sheet and a phospholipidic membranewere carried out. Our findings unraveled the important roleof water in the interactions among amyloid peptides, mem-brane, and graphene, which might pave the way towards thefuture development of graphene-based nanomedicine for pre-venting/curing type II diabetes mellitus or and other PCDs.
MODELS AND METHODS
Extensive all-atom MD simulations were performed inorder to evaluate graphene’s adsorption capacity of amy-loid fibrils deposited over a phospholipidic membrane. Inorder to mimic different types of amyloid related cytotox-icity, two model systems of deposited fibrils were prepared(see Figs. 1): (i) the “fibril+membrane” system: a small NF-GAILS fibril, consisting of two stable, flat, antiparallel β-
FIG. 1. The initial (left column) and final (right column) configurations ofone representative trajectory for the simulation of (a) “fibril+membrane”(control), (b) “graphene+fibril+membrane,” (c) “peptides+membrane”(control), and (d) “graphene+peptides+membrane” systems, respectively.The NFGAILS fibril is shown in gray as a cartoon representation with itssurface colored based on residue type (hydrophobic in white and hydrophilicin green). The graphene nano-sheet is depicted with yellow sticks. For clarity,water molecules are not shown.
sheets, each containing four anti-parallel hIAPP22-28 (Ace-N22-F23-G24-A25-I26-L27-S28-NH2) peptides) was placed onthe surface of the membrane (see Fig. 1(a)); (ii) the“peptides+membrane” system: eight separated NFGAILSpeptides were randomly placed on the surface of the samemembrane (Fig. 1(c)). After an equilibration protocol (seeparagraph below), a graphene nanosheet was introduced andlocated over the membrane with its surface aligned par-allel to the membrane surface, generating two new sys-tems designated: (iii) graphene+fibril+membrane and (iv)graphene+peptides+membrane (see Figs. 1(b) and 1(d), re-spectively). Thus, a total of four different idealized modelsystems were employed to study the competitive adsorptionof peptides/fibrils between graphene and cell membrane.
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The NFGAILS fibril (and monomeric peptide) struc-ture was obtained from the Protein Data Bank (PDB code:2KIB).51 The coordinates of the graphene nanosheets weregenerated using the nanotube builder plugin of the VMDsoftware,52 with a size of 4.92×4.92 nm2. A palmitoy-loleoylphosphatidylethanolamine (POPE) lipid bilayer com-prising 264 lipids was adopted. The POPE membrane wasfirst solvated in an 8×8×16 nm3 water box, with a con-tinuous membrane in the x-y plane. The resulting solvatedPOPE system was subjected to a minimization protocol for10 000 steps employing the conjugate gradient method, fol-lowed by 50 ns of consecutive NVT and NPT relaxation runs.Following, the NFGAILS fibril/peptides were introduced andplaced adjacent to the lipid surface, with overlapping watermolecules removed. The complex systems were subsequentlyre-equilibrated with an energy minimization and a NPT equi-libration protocol. Finally, a graphene nanosheet was intro-duced in order to prepare the other two graphene-containingmodel systems, with the overlapping water molecules re-moved. For the “graphene+fibril+membrane” system, theinitial distance between the center of mass (COM) of thefibril and the graphene nanosheet was set in the order of1.7 nm, with the closest distance between any heavy atomsof fibril and graphene larger than 1.0 nm. Regarding the“graphene+peptides+membrane” system, the initial averagedistance between peptides and graphene was around 2.0 nm.Position restraints using harmonic potentials were applied tothe graphene nanosheet and the peptides atoms along the equi-libration process.
MD simulations were performed using the Gromacs-4.6.6 package.53 VMD was used for trajectory visualizationand analysis.52 The simple point charge (SPC) model54 wasadopted for the water molecules, the OPLS-AA force field forthe protein, and the Berger force field for the POPE lipids.55
This force field combination has proven to be reasonable formembrane-protein simulations.24, 56, 57 The carbon atoms ofgraphene were modeled as uncharged Lennard-Jones parti-cles with a cross-section σ cc = 0.34 nm and a depth of thepotential well εcc = 0.3598 kJ mol−1. Carbon-carbon bondlengths and bond angles were maintained by harmonic poten-tials with spring constants of 392 460 kJ mol−1 nm−2 and 527kJ mol−1 rad−2, respectively. A constant temperature (310 K)was maintained by using a v-rescale thermostat.58 The pres-
sure was kept constant via a semi-isotropic coupling scheme59
in which the lateral (Pl) and perpendicular (Pz) pressures arecoupled independently at 1 atm. Periodic boundary conditionsare applied in all directions. The PME method60 is used totreat long-range electrostatic interactions, whereas the vdWinteraction is treated with a cutoff distance of 1.2 nm. All so-lute bonds were constrained to their equilibrium values em-ploying the LINCS algorithm.61 The constrained water ge-ometry was solved employing the SETTLE62 algorithm. Dur-ing productions runs, the graphene atoms positions were re-strained via harmonic potentials. A time step of 2.0 fs wasused, and coordinates were collected every 2 ps. All modelsystems were subjected to five independent runs, each lasting20 ns. The total aggregate simulation time accounted for morethan 0.4 μs.
RESULTS AND DISCUSSION
Clearance of membrane-adhesive amyloid fibrilsby graphene
The partially ordered amyloid oligomers were shown toaccumulate on cell membrane surfaces (membrane-adhesive)of various tissues. These accumulated oligomers and (short)fibrils can directly penetrate and disrupt the structure of themembrane, with some even forming ion or small moleculeschannels, thus disrupting the osmotic balance between the ex-tracellular and intracellular reservoirs. These factors are be-lieved to be the main cause of premature cell apoptosis. OurMD simulation results supports this notion, indicating thatmembrane-adsorbed NFGAILS peptides or small β-sheet fib-rils form a stable complex where spontaneous detaching isvery unlikely. As shown in Fig. 1(a), at the end of simula-tion (t = 20 ns), the NFGAILS fibril is still tightly adhered tothe membrane. Furthermore, in all five independent runs, theadsorption and structure of NFGAILS β-sheets were alwaysmaintained.
Under the presence of graphene (Fig. 1(b)), NFGAILSfibrils originally tightly adsorbed onto the membrane, rapidlyswitched to adsorb onto the graphene surface, in all the fiveindependent runs. The switching process is very fast with anaverage time of 14.43 ± 2.47 ns for the fibril to be fully ad-sorbed on the graphene. In Fig. 2, the adsorption/desorption
FIG. 2. The distance between the COM of fibril with graphene (black curve), and with membrane surface (red curve) as function of simulation time for threerepresentative runs of the “graphene+ fibril+membrane” system.
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FIG. 3. Representative trajectory snapshots exemplifying the water dynamics during the adsorption process of the NFGAILS fibril onto the graphene nanosheet.These important intermediate states are depicted from the top view (top panel) and side view (bottom panel), respectively. The NFGAILS fibril is shown as acartoon representation in gray, graphene in yellow, and water in red (oxygen) and white (hydrogen). For clarity, only water molecules that are in the interfaceregion between the graphene and fibril and near the fibril are shown. This trajectory corresponds to the first representative one in Fig. 2.
process as function of time, is projected along the fibril’sCOM distance with respect to the membrane (red curve) andthe graphene (black curve). As expected, the desorption pro-cess of the fibril from membrane and adsorption process ofthe fibril onto the graphene are inversely correlated. The lat-ter implies that both membrane and graphene are intendedto drag NFGAILS fibrils closer to their respective surfaces,but the favorable graphene-fibril interaction rapidly over-comes the attractive forces ejected by the membrane (morebelow).
To further illustrate the molecular picture of NFGAILSfibrils’ adsorption onto graphene, some important intermedi-ate states during this process have been carefully examined.As shown in Fig. 2, the initial distance between the COMof the fibril and graphene is about 1.7 nm, and the inter-facial region in-between the graphene and the membrane isfully hydrated with about 92 water molecules (see the firstimage of Fig. 3 at t = 0 ns). Surprisingly, only after 2.5 ns,when the distance between the COM of fibril and graphenestill remains very far (∼1.56 nm), there are only 43 watermolecules left. Moreover, near the center of the interfacial re-gion, an apparent drying zone emerged. As time progresses,at t = 6.5 ns, the distance between the COM of fibril andgraphene sharply decreases to ∼1.24 nm, accompanied by theexpelling of nearly all water molecules from the interfacialregion (only 9 water molecules left; see Fig. 3), despite theaforementioned region is still large enough to accommodatemore water molecules. From 6.5 ns till 12 ns, the distance be-tween the COM of the fibril and graphene further collapsesto 1.09 nm, while the number of water molecules stays rela-tively constant. The previous analysis, strongly suggests thata fascinating nanoscale dewetting phenomenon occurred dur-ing the desorption/adsorption process, which will provide astrong drying-induced attraction between the fibril and thegraphene, and thus assist the clearance of NFGAILS fibrilsfrom the membrane surface, by adsorbing onto the graphenenanosheet.
With the aim of quantitatively characterizing this fas-cinating dewetting phenomenon, a scatter plot of the num-ber of water molecules at the interface between fibril andgraphene versus the distance between the COM of the fibriland graphene is presented in Fig. 4(a), with three representa-tive trajectories shown (corresponding to those of Fig. 2). Inall cases, a sharp decrease in the number of water moleculesbefore the reduction in the distance between the COM of fibriland graphene is observed (a reversed “L-shape” – typical fornanoscale dewetting transition63–65), though the exact timesfor each pace were slightly different. Overall, the water de-pletion process was very fast. On average it takes only 6.32± 2.02 ns for complete or near-complete water depletion. Onthe other hand, the graphene-fibril adsorption process takesmuch longer and the average absorption time is up to 14.43± 2.47 ns. These results confirmed that a robust nanoscaledewetting transition indeed happened in the interface regionbetween graphene and the NFGAILS fibril, which provideda strong drying-induced attraction63–65 between the fibril andgraphene and thus assisted in the clearance of NFGAILS amy-loid fibrils from the surface of the membrane.
A simple approach is also used to estimate the interfa-cial gap volume, which is defined as the number of watermolecules needed to fully resolvate that region divided by thebulk water number density. Knowing this volume with time,we can then estimate the water density in this gap as a func-tion of time by counting the number of molecules inside thegap divided by this irregular shaped volume. The resultingwater density along with the interface distance is shown inFig. 4(b). It is clear a similar trend in the interfacial water den-sity emerges as well, indicating a nanoscale dewetting transi-tion has occurred in this case, which helped providing a strongattraction force in assisting the clearance of NFGAILS fibrils.
Therefore, a general pathway for the adsorption of NF-GAILS fibrils onto graphene can be drawn: due to the stronghydrophobic interactions between the graphene and fibril, wa-ter molecules originally at the interface region were quickly
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FIG. 4. The number of water molecules at the interface between the fibril and graphene versus the distance between the COM of the fibril and graphene surface(a), and water density at the interface between the fibril and graphene (b). Each data point is colored according to its time sequence. The three trajectoriescorrespond to those shown in Fig. 2.
expelled, resulting in a depletion layer, which helped push-ing the NFGAILS fibril closer to the surface of graphene dueto the strong drying-induced attraction forces. This nanoscaledewetting transition was previously found to be crucial inmany important biological processes by providing large driv-ing forces, such as folding of various globular proteins,63, 64, 66
collapse of multi-domain proteins,67 self-assembly of amy-loidogenic peptide β-sheet protofilaments, ligand-receptorbinding,68–71 as well as ion-channel gating.72
Clearance of membrane-adhesive NFGAILSmonomers by graphene
Once the concentration of misfolded monomeric proteinsor peptides exceeds the critical threshold value, a large-scaleaggregation would be triggered, which has proven to be di-rectly related to cytotoxicity. In consequence, clearing themonomers accumulated on the surface of membrane couldeffectively inhibit the aggregation process, and in turn re-markably reduce cytotoxicity effects. As shown in Fig. 1(c),monomers that accumulated on the surface of membranedid not spontaneously desorb from membrane surface, eventhough these monomers can self-assemble into relative largeroligomers. These oligomers are also toxic to the cell. Oncea graphene nanosheet was introduced on the top of pep-tides (see Fig. 1(d)), in a similar fashion to the above fibrilcase, three out of eight peptides quickly (<∼17 ns) departedfrom the membrane and adsorbed onto the graphene’s sur-face (see Figs. 1(d) and 5(a)). Interestingly, the adsorptionof monomers onto the graphene surface was accomplishedat in a single-step mode, a process that was accompaniedby several spatial adjustments of the peptides with respectto graphene. As shown for one of the representative cases inFig. 5(a), initially, the N-terminal (which contains the aro-
matic residue Phe23) was further away from the graphenerelative to the C-terminal. At the first 5 ns, the distance be-tween the COM of the peptide became even larger (see thefirst graph in Fig. 5(a)). This was mainly due to the spatialadjustment of the peptide: the strong π–π stacking interac-tion between Phe23 and graphene caused the Phe23 to rotateand gradually face its carbon ring towards the graphene sur-face. This rotation also drove a full-body rotation of the pep-tide, which induced the N-terminal to approach the graphene,while the C-terminal moved away. After 10 ns, the distancebetween the peptide and graphene underwent a dramatic de-crease, at t = 12 ns the edge of Phe23 started to touch thesurface of graphene (the third graph in Fig. 5(b)). During theinterval between 12 and 13 ns, the carbon ring of Phe23 be-came fully paved on the surface of graphene and the π–π
interaction between Phe23 and graphene were significantlystrengthened. Meanwhile, the other residues of the peptidewere also fully adsorbed onto the graphene surface (see thefourth graph in Fig. 5(b)). After 13 ns, the distance betweenpeptide and graphene remained constant until the end of thesimulation. From the results mentioned above, it was clearthat the π–π stacking interaction also played a significant rolein the adsorption process of amyloid peptides onto the surfaceof graphene.
Many previous experimental and theoretical studies haveemphasized on the important role of π–π stacking interac-tions in the adsorption of carbon-based nanomaterials andproteins/peptides.73–83 Our recent work,81 which combinedquantum mechanical-based dispersion corrected DFTB-Dmethod and classical molecular mechanics-based MD simu-lations, has demonstrated that classical force-fields can prop-erly model the energetic strength of π–π stacking interactionbetween aromatic amino acid analogues and graphene derivematerials like carbon nanotubes.
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FIG. 5. (a) The time evolution of the distance between COM of four different peptides and the graphene surface (black curve) and the membrane surface (redcurve). (b) Some representative snapshots at various time points extracted from the first figure in the top panel in order to show the importance of Phe23 in theadsorption process of a monomer onto the surface of graphene.
CONCLUSION
In this paper, we used all-atom molecule dynamics simu-lation to investigate the competitive adsorption of NFGAILSpeptides and fibrils between graphene and the cell membrane.Four different idealized model systems were investigated,including (i) “fibril+membrane” and “peptides+membrane”systems that mimic the cytotoxicity of amyloid aggregates;(ii) graphene-included “graphene+fibril+membrane” and“graphene+peptides+membrane” systems in order to illus-trate the competitive binding with fibrils/peptides and the pro-tective role of graphene by purging these amyloid aggregates.
It was found that graphene could win a competitive ad-sorption of fibrils and peptides against a phospholipidic mem-brane due to its unusual two-dimensional, highly hydropho-bic surface with all sp2 aromatic rings. As a result, thepeptide aggregation process and associated cytotoxicity ofamyloid fibrils can be significantly mitigated by the presenceof graphene nanosheets. Interestingly, a nanoscale dewettingtransition was observed at the interfacial region between theNFGAILS fibril and graphene, which provides a strong driv-ing force for the adsorption of the fibril onto graphene andthus assists the clearance of these peptide aggregates. Fur-thermore, the π–π stacking interaction between Phe23 andgraphene also plays a significant role in the adsorption ofmonomers by graphene.
Our current findings provide an atomistic description ofthe nature of the interactions among amyloid fibrils/peptides,graphene, and cell membranes, enlightening the way towardsfuture developments of graphene-based nanomedicine forPDC related diseases.
ACKNOWLEDGMENTS
We thank Bruce Berne, Cuicui Ge, Xuanyu Meng, Xi-angfeng Sheng, and Bo Zhou for helpful discussions. This
work was partially supported by the National Natural ScienceFoundation of China (NNSFC) under Grant Nos. 11374221,21320102003, and 11404233. R.Z. acknowledges the sup-port from IBM Blue Gene Science Program, a project fundedby the Priority Academic Program Development of JiangsuHigher Education Institutions (PAPD).
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