Stability and cavitation of nanobubble: Insights from large-scale atomistic molecular dynamics simulations.

We perform large-scale atomistic simulations of a system containing 12 × 106 atoms, comprising an oxygen gas-filled bubble immersed in water, to understand the stability and cavitation induced by ultrasound. First, we propose a method to construct a bubble/water system. For a given bubble radius, the pressure inside the bubble is estimated using the Young-Laplace equation. Then, this pressure is used as a reference for a constant temperature, constant pressure simulation of an oxygen system, enabling us to extract a sphere of oxygen gas and place it into a cavity within an equilibrated water box. This ensures that the Young-Laplace equation is satisfied and the bubble is stable in water. Second, this stable bubble is used for ultrasound-induced cavitation simulations. We demonstrate that under weak ultrasound excitation, the bubble undergoes stable cavitation, revealing various fluid velocity patterns, including the first-order velocity field and microstreaming. These fluid patterns emerge around the bubble on a nanometer scale within a few nanoseconds, a phenomenon challenging to observe experimentally. With stronger ultrasound intensities, the bubble expands significantly and then collapses violently. The gas core of the collapsed bubble, measuring 3-4 nm, exhibits starfish shapes with temperatures around 1500 K and pressures around 6000 bar. The simulation results are compared with those from Rayleigh-Plesset equation modeling, showing good agreement. Our simulations provide insights into the stability and cavitation of nanosized bubbles.

Deciphering the free energy landscapes of SARS-CoV-2 wild type and Omicron variant interacting with human ACE2.

The binding of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein to the host cell receptor angiotensin-converting enzyme 2 (ACE2) is the first step in human viral infection. Therefore, understanding the mechanism of interaction between RBD and ACE2 at the molecular level is critical for the prevention of COVID-19, as more variants of concern, such as Omicron, appear. Recently, atomic force microscopy has been applied to characterize the free energy landscape of the RBD-ACE2 complex, including estimation of the distance between the transition state and the bound state, xu. Here, using a coarse-grained model and replica-exchange umbrella sampling, we studied the free energy landscape of both the wild type and Omicron subvariants BA.1 and XBB.1.5 interacting with ACE2. In agreement with experiment, we find that the wild type and Omicron subvariants have similar xu values, but Omicron binds ACE2 more strongly than the wild type, having a lower dissociation constant KD.

Interaction of SARS-CoV-2 with host cells and antibodies: experiment and simulation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the devastating global COVID-19 pandemic announced by WHO in March 2020. Through unprecedented scientific effort, several vaccines, drugs and antibodies have been developed, saving millions of lives, but the fight against COVID-19 continues as immune escape variants of concern such as Delta and Omicron emerge. To develop more effective treatments and to elucidate the side effects caused by vaccines and therapeutic agents, a deeper understanding of the molecular interactions of SARS-CoV-2 with them and human cells is required. With special interest in computational approaches, we will focus on the structure of SARS-CoV-2 and the interaction of its spike protein with human angiotensin-converting enzyme-2 (ACE2) as a prime entry point of the virus into host cells. In addition, other possible viral receptors will be considered. The fusion of viral and human membranes and the interaction of the spike protein with antibodies and nanobodies will be discussed, as well as the effect of SARS-CoV-2 on protein synthesis in host cells.

A Practical Guide to All-Atom and Coarse-Grained Molecular Dynamics Simulations Using Amber and Gromacs: A Case Study of Disulfide-Bond Impact on the Intrinsically Disordered Amyloid Beta.

Intrinsically disordered proteins (IDPs) pose challenges to conventional experimental techniques due to their large-scale conformational fluctuations and transient structural elements. This work presents computational methods for studying IDPs at various resolutions using the Amber and Gromacs packages with both all-atom (Amber ff19SB with the OPC water model) and coarse-grained (Martini 3 and SIRAH) approaches. The effectiveness of these methodologies is demonstrated by examining the monomeric form of amyloid-ß (Aß42), an IDP, with and without disulfide bonds at different resolutions. Our results clearly show that the addition of a disulfide bond decreases the ß-content of Aß42; however, it increases the tendency of the monomeric Aß42 to form fibril-like conformations, explaining the various aggregation rates observed in experiments. Moreover, analysis of the monomeric Aß42 compactness, secondary structure content, and comparison between calculated and experimental chemical shifts demonstrates that all three methods provide a reasonable choice to study IDPs; however, coarse-grained approaches may lack some atomistic details, such as secondary structure recognition, due to the simplifications used. In general, this study not only explains the role of disulfide bonds in Aß42 but also provides a step-by-step protocol for setting up, conducting, and analyzing molecular dynamics (MD) simulations, which is adaptable for studying other biomacromolecules, including folded and disordered proteins and peptides.

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aß, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

Amyloid-ß Tetramers and Divalent Cations at the Membrane/Water Interface: Simple Models Support a Functional Role.

Charge polarization at the membrane interface is a fundamental process in biology. Despite the lower concentration compared to the abundant monovalent ions, the relative abundance of divalent cations (Ca2+, Mg2+, Zn2+, Fe2+, Cu2+) in particular spaces, such as the neuron synapse, raised many questions on the possible effects of free multivalent ions and of the required protection of membranes by the eventual defects caused by the free forms of the cations. In this work, we first applied a recent realistic model of divalent cations to a well-investigated model of a polar lipid bilayer, di-myristoyl phosphatidyl choline (DMPC). The full atomistic model allows a fairly good description of changes in the hydration of charged and polar groups upon the association of cations to lipid atoms. The lipid-bound configurations were analyzed in detail. In parallel, amyloid-ß 1-42 (Aß42) peptides assembled into tetramers were modeled at the surface of the same bilayer. Two of the protein tetramers' models were loaded with four Cu2+ ions, the latter bound as in DMPC-free Aß42 oligomers. The two Cu-bound models differ in the binding topology: one with each Cu ion binding each of the monomers in the tetramer; one with pairs of Cu ions linking two monomers into dimers, forming tetramers as dimers of dimers. The models here described provide hints on the possible role of Cu ions in synaptic plasticity and of Aß42 oligomers in storing the same ions away from lipids. The release of structurally disordered peptides in the synapse can be a mechanism to recover ion homeostasis and lipid membranes from changes in the divalent cation concentration.

Elastic moduli of normal and cancer cell membranes revealed by molecular dynamics simulations.

Recent studies indicate that there are mechanical differences between normal cells and cancer cells. Because the cell membrane takes part in a variety of vital processes, we test the hypothesis of whether or not two fundamental alterations in the cell membrane, i.e., the overexpression of phosphatidylserine lipids in the outer leaflet and a reduction in cholesterol concentration, could cause the softening in cancer cells. Adopting ten models of normal and cancer cell membranes, we carry out 1 µs all-atom molecular dynamics simulations to compare the structural properties and elasticity properties of two membrane types. We find that the overexpression of the phosphatidylserine lipids in the outer leaflet does not significantly alter the area per lipid, the membrane thickness, the lipid order parameters and the elasticity moduli of the cancer membranes. However, a reduction in the cholesterol concentration leads to clear changes in those quantities, especially decreases in the bending, tilt and twist moduli. This implies that the reduction of cholesterol concentration in the cancer membranes could contribute to the softening of cancer cells.

Protein aggregation rate depends on mechanical stability of fibrillar structure.

The formation of the fibrillar structure of amyloid proteins/peptides is believed to be associated with neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Since the rate of aggregation can influence neurotoxicity, finding the key factors that control this rate is of paramount importance. It was recently found that the rate of protein aggregation is related to the mechanical stability of the fibrillar structure such that the higher the mechanical stability, the faster the fibril is formed. However, this conclusion was supported by a limited dataset. In this work, we expand the previous study to a larger dataset, including the wild type of Aß42 peptide and its 20 mutants, the aggregation rate of which was measured experimentally. By using all-atom steered molecular dynamics (SMD) simulations, we can assess the mechanical stability of the fibril structure, which is characterized by the rupture force, pulling work, and unbinding free energy barrier. Our result confirms that mechanical stability is indeed related to the aggregation rate. Since the estimation of the aggregation rate using all-atom simulations is almost forbidden by the current computational capabilities, our result is useful for predicting it based on information obtained from fast SMD simulations for fibrils.

Molecular dynamics simulation of cancer cell membrane perforated by shockwave induced bubble collapse.

It has been widely accepted that cancer cells are softer than their normal counterparts. This motivates us to propose, as a proof-of-concept, a method for the efficient delivery of therapeutic agents into cancer cells, while normal cells are less affected. The basic idea of this method is to use a water jet generated by the collapse of the bubble under shockwaves to perforate pores in the cell membrane. Given a combination of shockwave and bubble parameters, the cancer membrane is more susceptible to bending, stretching, and perforating than the normal membrane because the bending modulus of the cancer cell membrane is smaller than that of the normal cell membrane. Therefore, the therapeutic agent delivery into cancer cells is easier than in normal cells. Adopting two well-studied models of the normal and cancer membranes, we perform shockwave induced bubble collapse molecular dynamics simulations to investigate the difference in the response of two membranes over a range of shockwave impulse 15-30 mPa s and bubble diameter 4-10 nm. The simulation shows that the presence of bubbles is essential for generating a water jet, which is required for perforation; otherwise, pores are not formed. Given a set of shockwave impulse and bubble parameters, the pore area in the cancer membrane is always larger than that in the normal membrane. However, a too strong shockwave and/or too large bubble results in too fast disruption of membranes, and pore areas are similar between two membrane types. The pore closure time in the cancer membrane is slower than that in the normal membrane. The implications of our results for applications in real cells are discussed in some details. Our simulation may be useful for encouraging future experimental work on novel approaches for cancer treatment.

Ribosome Elongation Kinetics of Consecutively Charged Residues Are Coupled to Electrostatic Force.

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multiscale simulations, that positively charged residues generate large forces that move the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but move the A- and P-sites closer together. These conformational changes, respectively, increase and decrease the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation and provide a framework for interpreting experimental results on translation speed.

Molecular Mechanism of Ultrasound-Induced Structural Defects in Liposomes: A Nonequilibrium Molecular Dynamics Simulation Study.

The use of ultrasound in combination with liposomes is a promising approach to improve drug delivery. To achieve an optimal drug release rate, it is important to understand how ultrasound induces pathways on the liposome surface where drugs can be released from the liposome. To this end, we carry out large-scale ultrasound-induced molecular dynamics simulations for three single lipid component liposomes formed from the commonly used phospholipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), or phosphatidylcholine (POPC). The results show that ultrasound induces the detachment of two leaflets of the DOPC surface, suggesting that the drug release pathway may be through the low lipid packing areas on the stretched surface. In contrast, ultrasound induces pore formation on the surface of DPPC and DOPC, where drugs could escape from the liposomes. While the leaflet detachment and transient pore formation are the mechanisms of DOPC and DPPC, respectively, in both liquid-ordered and liquid-disordered phases, the leaflet detachment mechanism is switched to the transient pore formation mechanism on going from the liquid-ordered phase to the liquid-disordered phase in the POPC liposome. By adding 30% mol cholesterol, the leaflet detachment mechanism is observed in all liposomes. We found that the molecular origin that determines a mechanism is the competition between the intraleaflet and interleaflet interacting energy of lipids. The connection to experimental and theoretical modeling is discussed in some detail.

Zinc binding promotes greater hydrophobicity in Alzheimer's Aß42 peptide than copper binding: Molecular dynamics and solvation thermodynamics studies.

The aggregation of Aß42 peptides is considered as one of the main causes for the development of Alzheimer's disease. In this context, Zn2+ and Cu2+ play a significant role in regulating the aggregation mechanism, due to changes in the structural and the solvation free energy of Aß42. In practice, experimental studies are not able to determine the latter properties, since the Aß42-Zn2+ and Aß42-Cu2+ peptide complexes are intrinsically disordered, exhibiting rapid conformational changes in the aqueous environment. Here, we investigate atomic structural variations and the solvation thermodynamics of Aß42, Aß42-Cu2+ , and Aß42-Zn2+ systems in explicit solvent (water) by using quantum chemical structures as templates for a metal binding site and combining extensive all-atom molecular dynamics (MD) simulations with a thorough solvation thermodynamic analysis. Our results show that the zinc and copper coordination results in a significant decrease of the solvation free energy in the C-terminal region (Met35-Val40), which in turn leads to a higher structural disorder. In contrast, the ß-sheet formation at the same C-terminal region indicates a higher solvation free energy in the case of Aß42. The solvation free energy of Aß42 increases upon Zn2+ binding, due to the higher tendency of forming the ß-sheet structure at the Leu17-Ala42 residues, in contrast to the case of binding with Cu2+ . Finally, we find the hydrophobicity of Aß42-Zn2+ in water is greater than in the case of Aß42-Cu2+ .

Electrostatic Interactions Govern Extreme Nascent Protein Ejection Times from Ribosomes and Can Delay Ribosome Recycling.

The ejection of nascent proteins out of the ribosome exit tunnel, after their covalent bond to transfer-RNA has been broken, has not been experimentally studied due to challenges in sample preparation. Here, we investigate this process using a combination of multiscale modeling, ribosome profiling, and gene ontology analyses. Simulating the ejection of a representative set of 122 E. coli proteins we find a greater than 1000-fold variation in ejection times. Nascent proteins enriched in negatively charged residues near their C-terminus eject the fastest, while nascent chains enriched in positively charged residues tend to eject much more slowly. More work is required to pull slowly ejecting proteins out of the exit tunnel than quickly ejecting proteins, according to all-atom simulations. An energetic decomposition reveals, for slowly ejecting proteins, that this is due to the strong attractive electrostatic interactions between the nascent chain and the negatively charged ribosomal-RNA lining the exit tunnel, and for quickly ejecting proteins, it is due to their repulsive electrostatic interactions with the exit tunnel. Ribosome profiling data from E. coli reveals that the presence of slowly ejecting sequences correlates with ribosomes spending more time at stop codons, indicating that the ejection process might delay ribosome recycling. Proteins that have the highest positive charge density at their C-terminus are overwhelmingly ribosomal proteins, suggesting the possibility that this sequence feature may aid in the cotranslational assembly of ribosomes by delaying the release of nascent ribosomal proteins into the cytosol. Thus, nascent chain ejection times from the ribosome can vary greatly between proteins due to differential electrostatic interactions, can influence ribosome recycling, and could be particularly relevant to the synthesis and cotranslational behavior of some proteins.

Fast Estimation of the Blood-Brain Barrier Permeability by Pulling a Ligand through a Lipid Membrane.

The blood-brain barrier (BBB) is a physical barrier that regulates the homeostasis of the neural microenvironment. A relative estimate of the BBB permeability, which is important for drug design, may be experimentally provided by the logBB (the blood-brain concentration ratio) and the logPS (permeability-surface-area product), while many computational methods aim to identify key properties that correlate well with these quantities. Although currently existing computational methods (e.g., quantitative structure activity relation) have made a significant contribution in screening various compounds that could potentially translocate through the BBB, they are unable to provide a physical explanation of the underlying processes and they can often be computationally demanding. Here, we use steered molecular dynamics simulation to estimate the BBB permeability of various compounds on the basis of simple lipid-membrane models by computing the nonequilibrium work, Wneq, produced by pulling the compounds through the membrane. We found that the values of Wneq correlate remarkably well with logBB and logPS for a range of compounds and different membrane types and pulling speeds, independently of the choice of force field. Moreover, our results provide insight into the role of hydrogen bonds, the energetic barriers, and the forces exerted on the ligands during their pulling. Our method is computationally easy to implement and fast. Therefore, we anticipate that it could provide a reliable prescreening tool for estimating the relative permeability of the BBB to various substances.

Electric dipole image forces in three-layer systems: The classical electrostatic model.

General exact analytical expressions have been derived for the image force energy Wi(Z, φ) of a point dipole in a classical three-layer system composed of dispersionless media with arbitrary constant dielectric permittivities εi. Here, i = 1-3 is the layer number, and Z and φ are the dipole coordinate and orientation angle, respectively. It was found that the long-range asymptotics Wi(Z→∞,φ) in both covers (i = 1, 3) are reached unexpectedly far from the interlayer (i = 2). Another specific feature of the solution consists in that the interference of the fields created by polarization charges emerging at both interfaces leads to the appearance of a constant contribution inside the interlayer with a non-standard dependence on the dipole orientation angle φ. It was shown that by changing the dielectric constants of the structure components, one can realize two peculiar regimes of the Wi(Z, φ) behavior in the covers; namely, there arises either a potential barrier preventing adsorption or a well far from the interface, both being of a totally electrostatic origin, i.e., without involving the Pauli exchange repulsion, which is taken into account in the conventional theories of physical adsorption. The results obtained provide a fresh insight into the physics of adsorption in physical electronics, chemical physics, and electrochemistry.

Heat-induced degradation of fibrils: Exponential vs logistic kinetics.

The degradation of fibrils under the influence of thermal fluctuations was studied experimentally by various groups around the world. In the first set of experiments, it was shown that the decay of fibril content, which can be measured by the ThT fluorescence assay, obeys a bi-exponential function. In the second series of experiments, it was demonstrated that when the monomers separated from the aggregate are not recyclable, the time dependence of the number of monomers belonging to the dominant cluster is described by a single-exponential function if the fraction of bound chains becomes less than a certain threshold. Note that the time dependence of the fraction of bound chains can be measured by tryptophan fluorescence. To understand these interesting experimental results, we developed a phenomenological theory and performed molecular simulation. According to our theory and simulations using the lattice and all-atom models, the time dependence of bound chains is described by a logistic function, which slowly decreases at short time scales but becomes a single exponential function at large time scales. The results, obtained by using lattice and all-atom simulations, ascertained that the time dependence of the fibril content can be described by a bi-exponential function that decays faster than the logistic function on short time scales. We have uncovered the molecular mechanism for the distinction between the logistic and bi-exponential behavior. Since the dissociation of the chain from the fibrils requires the breaking of a greater number of inter-chain contacts as compared to the breaking of the beta sheet structure, the decrease in the number of connected chains is slower than the fibril content. Therefore, the time dependence of the aggregate size is logistic, while the two-exponential behavior is preserved for the content of fibrils. Our results are in agreement with the results obtained in both sets of experiments.

Molecular mechanism of ultrasound interaction with a blood brain barrier model.

The brain is strictly protected by the blood brain barrier preventing the crossing of therapeutics to treat brain diseases. The high and low intensity focused ultrasound methods have been used to temporarily open the blood brain barrier, facilitating the transport of drugs. The methods are very promising because the opening is transient, localized, and noninvasive. However, the molecular mechanism of the opening is unknown, and this limits the development and application of these methods. With this in mind, we carry out a molecular dynamics simulation study to understand the interaction of ultrasound with the cell membrane and the tight junction. Our minimal blood brain barrier model is composed of two lipid bilayers, mimicking two portions of neighboring cells, connected together by a tight junction formed by a pair of two cis-dimers of the claudin-5 protein. Using an experimental ultrasound frequency of 50 MHz, simulations show that at low intensities, ultrasound does not impact the structure of the cell membranes and tight junction, implying that the direct interaction of ultrasound with the blood brain barrier is not responsible for the experimentally observed opening. At high intensities, the ultrasound pulls the monolayers of individual cell membrane lipid bilayers apart, creating air compartments inside the bilayers. This reduces the free energy barrier for the translocation of drugs across the lipid bilayer and enhances drug permeability. At very high intensities, the two monolayers are largely separated, resulting in cell damage and implying that the blood brain barrier is primarily opened at the experimentally observed damaged areas.

Computational models explain how copper binding to amyloid-ß peptide oligomers enhances oxidative pathways.

Amyloid-ß (Aß) peptides are intrinsically disordered peptides and their aggregation is the major hallmark of Alzheimer's disease (AD) development. The interactions between copper ions and Aß peptides create catalysts that activate the production of reactive oxygen species in the synaptic region, a reactivity that is strongly related to AD onset. Recent experimental work [Gu et al., Sci. Rep., 2018, 8(1), 16190] confirmed that the oxidative reactivity of Cu-Aß catalyzes the formation of Tyr-Tyr crosslinks in peptide dimers. This work provides a structural basis to these observations, describing structures of Cu-Aß dimers that enhance the propagation of the oxidative pathways activated around the Cu center. Among these, the formation of Tyr-Tyr crosslinks becomes more likely when previous crosslinks involve Cu forming bridges between different peptides. Peptides are, therefore, easily assembled into dimers and tetramers, the latter being dimers of dimers. The size of such dimers and tetramers fits with ion mobility mass spectrometry results [Sitkiewicz et al., J. Mol. Biol., 2014, 426(15), 2871].

Nonequilibrium atomistic molecular dynamics simulation of tubular nanomotor propelled by bubble propulsion.

We develop a molecular nanoscaled model for tubular motors propelled by bubble propulsion. The motor is modeled by a carbon nanotube, and the bubble is represented by a particle interacting with water by a time-dependent potential. Effects of liquid viscosity, fuel concentration, geometry, and size of the tube on the performance of the motor are effectively encoded into two parameters: time scales of the bubble expansion and bubble formation. Our results are qualitatively consistent with experimental data of much larger motors. Simulations suggest that (i) the displacement of the tube is optimized if two time scales are as short as possible, (ii) the compromise between the performance and fuel consumption is achieved if the bubble formation time is shorter than the velocity correlation time of the tube, (iii) the motor efficiency is higher with slow expansion, short formation of the bubble than fast growth but long formation time, and (iv) the tube is propelled by strong forces on the order of mN, reaching high speeds up to ∼60 m/s. Our simulation may be useful for refining and encouraging future experimental work on nanomotors having the size of a few nanometers. The tiny size and high speed motors could have great potential applications in real life.

Interaction mechanism between the focused ultrasound and lipid membrane at the molecular level.

Focused ultrasound (FUS) has a wide range of medical applications. Nowadays, the diagnostic and therapeutic ultrasound procedures are routinely used; effects of ultrasound on biological systems at the molecular level are, however, not fully understood. Experimental results on the interaction of the cell membrane, a simplest but important system component, with ultrasound are controversial. Molecular dynamics (MD) simulations could provide valuable insights, but there is no single study on the mechanism of the FUS induced structural changes in cell membranes. With this in mind, we develop a simple method to include FUS into a standard MD simulation. Adopting the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid membrane as a representative model described by the MARTINI coarse-grained force field, and using experimental values of the ultrasound frequency and intensity, we show that the heat and bubble cavitation are not the primary direct mechanisms that cause structural changes in the membrane. The spatial pressure gradients between the focused and free regions and between the parallel and perpendicular directions to the membrane are the origin of the mechanism. These gradients force lipids to move out of the focused region, forming a lipid flow along the membrane diagonal. Lipids in the free region move in the opposite direction due to the conservation of the total momentum. These opposite motions create wrinkles along the membrane diagonal at low FUS intensities and tear up the membrane at high FUS intensities. Once the membrane is torn up, it is not easy to reform. The implication of our findings in the FUS-induced drug delivery is discussed in some detail.