TDI-like multi-slit hyperspectral imaging for enhanced throughput via the Kalman filter.

The time-delay integration (TDI) technique is increasingly used to improve the signal-to-noise ratio (SNR) of remote sensing and imaging by exposing the scene multiple times. Inspired by the principle of TDI, we propose a TDI-like pushbroom multi-slit hyperspectral imaging (MSHSI) approach. In our system, multiple slits are used to significantly improve the throughput of the system, thereby enhancing the sensitivity and SNR through multiple exposures of the same scene during pushbroom scan. Meanwhile, a linear dynamic model for the pushbroom MSHSI is established, where the Kalman filter (KF) is employed to reconstruct the time-varying overlapped spectral images on a single conventional image sensor. Further, we designed and fabricated a customized optical system that can operate in both multi-slit and single slit modes to experimentally verify the feasibility of the proposed method. Experimental results indicate that the developed system improved SNR by a factor of about 7 compared to that of the single slit mode, while demonstrating excellent resolution in both spatial and spectral dimensions.

Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition.

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

Automation of AMOEBA polarizable force field for small molecules: Poltype 2.

A next-generation protocol (Poltype 2) has been developed which automatically generates AMOEBA polarizable force field parameters for small molecules. Both features and computational efficiency have been drastically improved. Notable advances include improved database transferability using SMILES, robust torsion fitting, non-aromatic ring torsion parameterization, coupled torsion-torsion parameterization, Van der Waals parameter refinement using ab initio dimer data and an intelligent fragmentation scheme that produces parameters with dramatically reduced ab initio computational cost. Additional improvements include better local frame assignment for atomic multipoles, automated formal charge assignment, Zwitterion detection, smart memory resource defaults, parallelized fragment job submission, incorporation of Psi4 quantum package, ab initio error handling, ionization state enumeration, hydration free energy prediction and binding free energy prediction. For validation, we have applied Poltype 2 to ~1000 FDA approved drug molecules from DrugBank. The ab initio molecular dipole moments and electrostatic potential values were compared with Poltype 2 derived AMOEBA counterparts. Parameters were further substantiated by calculating hydration free energy (HFE) on 40 small organic molecules and were compared with experimental data, resulting in an RMSE error of 0.59 kcal/mol. The torsion database has expanded to include 3543 fragments derived from FDA approved drugs. Poltype 2 provides a convenient utility for applications including binding free energy prediction for computational drug discovery. Further improvement will focus on automated parameter refinement by experimental liquid properties, expansion of the Van der Waals parameter database and automated parametrization of modified bio-fragments such as amino and nucleic acids.

Hydration of divalent lanthanides, Sm2+ and Eu2+ : A molecular dynamics study with polarizable AMOEBA force field.

The chemistry of divalent lanthanides, Ln2+ , is a growing sub-field of heavy element chemistry owing to new synthetic approaches. However, some theoretical aspects of these unusual cations are currently underdeveloped, especially as they relate to their dynamic properties in solution. In this work, we address the hydration of two of the classical Ln2+ cations, Sm2+ and Eu2+ , using atomic multipole optimized energetic for biomolecular applications (AMOEBA) force fields. These cations have not been parameterized to date with AMOEBA, and few studies are available because of their instability with respect to oxidation in aqueous media. Coordination numbers (CN's) of 8.2 and 8.1 respectively for Sm2+ and Eu2+ , and 8.8 for both Sm3+ and Eu3+ have been obtained and are in good agreement with the few available AIMD and X-ray absorption fine structures studies. The decreased CN of Ln2+ compared with Ln3+ arises from progressive water exchange events that indicates the gradual stabilization of 8-coordinate structures with respect to 9-coordinate geometries. Moreover, the effects of the chloride counter anions on the coordination of Ln2+ cations have been studied at different chloride concentrations in this work. Lastly, water exchange times of Ln2+ cations have been calculated to provide a comprehensive understanding of the behavior of Eu2+ and Sm2+ in aqueous chloride media.

Multi-echo balanced SSFP with a sequential phase-encoding order for functional MR imaging at 7T.

PURPOSE: To develop a 2D multi-echo passband balanced SSFP (bSSFP) sequence using an echo-train readout with a sequential phase-encoding order (sequential multi-echo bSSFP), and evaluate its performance in fast functional brain imaging at 7 T. METHODS: As images of sequential multi-echo bSSFP exhibit multiple ghosts due to periodic k-space modulations, a GRAPPA-based reconstruction method was proposed to eliminate ghosting artifacts. MRI experiments were performed to compare the image quality of multi-echo bSSFP and conventional single-echo bSSFP. Submillimeter-resolution fMRI using a checkerboard visual stimulus was conducted to compare the activation characteristics of multi-echo bSSFP, conventional single-echo bSSFP and standard gradient-echo EPI (GE-EPI). RESULTS: A higher mean structural similarity index was found between images of single-echo bSSFP and multi-echo bSSFP with a shorter echo train length (ETL). Multi-echo bSSFP (ETL = 3) showed higher temporal SNR (tSNR) values than GRAPPA-accelerated single-echo bSSFP (R = 2). In submillimeter-resolution fMRI experiments, multi-echo bSSFP (ETL = 3) approached the imaging speed of GRAPPA-accelerated single-echo bSSFP (R = 2), but without tSNR penalty and reduced activation due to acceleration. The median t-value and the number of significantly activated voxels were comparable between GE-EPI and multi-echo bSSFP (ETL = 3) that provides virtually distortion-free functional images and inherits the activation patterns of conventional bSSFP. CONCLUSION: Sequential multi-echo bSSFP (ETL = 3) is suitable for fast fMRI with submillimeter in-plane resolution, and offers an option to accelerate bSSFP imaging without tSNR penalty like parallel imaging.

Atomic Polarizabilities for Interactive Dipole Induction Models.

Thole-style mutual induction models for molecular polarization have been adopted by several popular polarizable force fields (FFs) for their simplicity and transferability. The atomic polarizability parameters of these models are typically derived by fitting to ab initio or/and experimental molecular polarizabilities. In this work, we improve upon Thole polarizability parameters by employing both high-level quantum mechanics molecular polarizabilities and electrostatic potential (ESP) responses on three-dimensional grids. Our results indicate that the two approaches to derive atomic polarizability parameters are both effective, while the ESP approaches can also capture the polarization for the atoms with lone pair electrons. The resulting polarizability parameters have been validated on a set of over 7200 molecules covering the most common elements found in organic molecules (C, H, O, N, P, S, F, Cl, Br, and I). These parameters have also been tested on the experimentally measured molecular polarizabilities of 422 molecules. The final set of parameters derived in this work show notable improvement over the current AMOEBA set. The result is a highly transferable, expanded set of atomic polarizabilities defined by the local chemical environment in the form of SMARTS patterns. These parameters can be used directly in molecular mechanics polarizable potential energy functions such as AMOEBA, AMOEBA+, and other Thole-style models.

An Efficient Approach to Large-Scale Ab Initio Conformational Energy Profiles of Small Molecules.

Accurate conformational energetics of molecules are of great significance to understand maby chemical properties. They are also fundamental for high-quality parameterization of force fields. Traditionally, accurate conformational profiles are obtained with density functional theory (DFT) methods. However, obtaining a reliable energy profile can be time-consuming when the molecular sizes are relatively large or when there are many molecules of interest. Furthermore, incorporation of data-driven deep learning methods into force field development has great requirements for high-quality geometry and energy data. To this end, we compared several possible alternatives to the traditional DFT methods for conformational scans, including the semi-empirical method GFN2-xTB and the neural network potential ANI-2x. It was found that a sequential protocol of geometry optimization with the semi-empirical method and single-point energy calculation with high-level DFT methods can provide satisfactory conformational energy profiles hundreds of times faster in terms of optimization.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.

Advanced Electrostatic Model for Monovalent Ions Based on Ab Initio Energy Decomposition.

Ions play important roles in the structures and functions of biomolecules. In biomolecular simulations, ions either directly interact with biomolecules or provide an ionic environment that influences electrostatic interactions of solutes. The AMOEBA+ water model has demonstrated significant advancement of the classical force field for describing molecular interactions due to its improvements on the functional forms to account for essential physics. This work expands the applicability of the AMOEBA+ model toward alkali metal (Li, Na, K, Rb, and Cs) and halogen (F, Cl, Br, and I) ions. Various quantum chemical data on ion-ion and ion-water interactions, experimental ion hydration free energies, and lattice energies of salt crystals are used in the parametrization. The final parameters are verified with other properties outside of the parametrization data, including lattice energies of additional salt crystals and ionic activity coefficients in solution. The new model captures a wide range of ion properties from the gas phase to solution phase and crystals. More importantly, AMOEBA+ provides energy components that are consistent with ab initio energy decomposition. Thus, we expect AMOEBA+ to be more general, transferable, and valuable for the interpretation of intermolecular forces in efficient classical simulations.

Many-body effect determines the selectivity for Ca2+ and Mg2+ in proteins.

Calcium ion is a versatile messenger in many cell-signaling processes. To achieve their functions, calcium-binding proteins selectively bind Ca2+ against a background of competing ions such as Mg2+ The high specificity of calcium-binding proteins has been intriguing since Mg2+ has a higher charge density than Ca2+ and is expected to bind more tightly to the carboxylate groups in calcium-binding pockets. Here, we showed that the specificity for Ca2+ is dictated by the many-body polarization effect, which is an energetic cost arising from the dense packing of multiple residues around the metal ion. Since polarization has stronger distance dependence compared with permanent electrostatics, the cost associated with the smaller Mg2+ is much higher than that with Ca2+ and outweighs the electrostatic attraction favorable for Mg2+ With the AMOEBA (atomic multipole optimized energetics for biomolecular simulation) polarizable force field, our simulations captured the relative binding free energy between Ca2+ and Mg2+ for proteins with various types of binding pockets and explained the nonmonotonic size dependence of the binding free energy in EF-hand proteins. Without electronic polarization, the smaller ions are always favored over larger ions and the relative binding free energy is roughly proportional to the net charge of the pocket. The many-body effect depends on both the number and the arrangement of charged residues. Fine-tuning of the ion selectivity could be achieved by combining the many-body effect and geometric constraint.

Three-site and five-site fixed-charge water models compatible with AMOEBA force field.

In a typical biomolecular simulation using Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field, the vast majority molecules in the simulation box consist of water, and these water molecules consume the most CPU power due to the explicit mutual induction effect. To improve the computational efficiency, we here develop two new nonpolarizable water models (with flexible bonds and fixed charges) that are compatible with AMOEBA solute: the 3-site AW3C and 5-site AW5C. To derive the force-field parameters for AW3C and AW5C, we fit to six experimental liquid thermodynamic properties: liquid density, enthalpy of vaporization, dielectric constant, isobaric heat capacity, isothermal compressibility and thermal expansion coefficient, at a broad range of temperatures from 261.15 to 353.15 K under 1.0 atm pressure. We further validate our AW3C and AW5C water models by showing that they can well reproduce the radial distribution function g(r), self-diffusion constant D, and hydration free energy from the AMOEBA03 water model and the experimental observations. Furthermore, we show that our AW3C and AW5C water models can greatly accelerate (>5 times) the bulk water as well as biomolecular simulations when compared to AMOEBA water. Specifically, we demonstrate that the applications of AW3C and AW5C water models to simulate a DNA duplex lead to a threefold acceleration, and in the meanwhile well maintain the structural properties as the fully polarizable AMOEBA water. We expect that our AW3C and AW5C water models hold great promise to be widely applied to simulate complex bio-molecules using the AMOEBA force field.

Accurate description of molecular dipole surface with charge flux implemented for molecular mechanics.

The molecular dipole moment is strongly coupled to molecular geometry among different phases, conformational states, intermolecular interaction energy, and vibrational spectroscopy. Our previous inclusion of geometry dependent charge flux into the atomic multipole-based polarizable AMOEBA+ force field has shown significant improvement of water properties from gaseous to condensed phases [C. Liu et al., J. Phys. Chem. Lett. 11(2), 419-426 (2020)]. In this work, the parameterization of the CF model for a broad range of organic and biomolecular fragments is presented. Atom types are automatically assigned by matching the predefined SMARTS patterns. Comparing to the current AMOEBA+ model without the CF component, it is shown that the AMOEBA+ (CF) model improves the description of molecular dipole moments for the molecules we studied over both equilibrium and distorted geometries. For the equilibrium-geometry structures, AMOEBA+ (CF) reduces the mean square error (MSE) from 6.806 × 10-1 (without CF) to 4.249 × 10-4 D2. For non-equilibrium structures, the MSE is reduced from 5.766 × 10-1 (without CF) to 2.237 × 10-3 D2. Finally, the transferability of the CF model and parameters were validated on two sets of molecules: one includes molecules in the training set but with different geometries, and the other one involves new molecules outside of the training set. A similar improvement on dipole surfaces was obtained on the validation sets. The CF algorithms and parameters derived in this work are general and can be implemented into any existing molecular mechanical force fields.

Hyperandrogen enhances apoptosis of human ovarian granulosa cells via up-regulation and demethylation of PDCD4.

Apoptosis of granulosa cells (GCs) induced by hyperandrogen plays a key role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the mechanism of androgen-induced apoptosis of GCs has not been clarified to date. Recent studies have reported that PDCD4 expression is higher in PCOS patients and might be a key factor in PCOS progression. In this study, we aimed to investigate the role of PDCD4 in regulating apoptosis of human GCs and whether hyperandrogen regulate PDCD4 expression through DNA methylation. Overexpression of PDCD4 in human ovarian granulosa cell line KGN cells promoted cells apoptosis. Meanwhile, expression of caspase-3 and caspase-9 were significantly elevated. High concentration of testosterone treatment resulted in up-regulation of PDCD4 and a significant increase of apoptosis in KGN cells. In addition, knockdown of PDCD4 in KGN cells treated with high concentration of testosterone abolished the hyperandrogen-induced apoptosis. Furthermore, high concentration of testosterone down-regulated DNMT1, DNMT3A and DNMT3B expression and the methylation level in the promoter region of PDCD4 was decreased. In conclusion, PDCD4 can promote apoptosis of human ovarian GCs. The mechanism of hyperandrogen-induced apoptosis may be mediated by PDCD4. Furthermore, the up-regulation of PDCD4 induced by hyperandrogen may through demethylation of its promoter regions.

Organic Semiconducting Alloys with Tunable Energy Levels.

Continuous band structure tuning, e.g., doping with different atoms, is one of the most important features of inorganic semiconductors. However, this can hardly be realized in organic semicondutors. Here, we report the first example of fine-tuning organic semiconductor band structures by alloying structurally similar derivatives into one single phase. By incorporating halogen atoms on different positions of the backbone, BDOPV derivatives with complementary intramolecular or intermolecular charge distributions were obtained. To maximize the Coloumbic attractive interactions and minimize repulsive interactions, they form antiparallel cofacial stacking in monocomponent or in alloy single crystals, resulting in efficient π orbital overlap. Benefiting from self-assembly induced solid state "olefin metathesis" reaction, it was observed, for the first time, that three BDOPV derivatives cocrystallized in one single crystal. Molecules with different energy levels serve like the dopants in inorganic semiconductors. Consequently, as the total number of halogen atoms increased, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) levels of the alloy single crystals decreased monotonously in the range from -5.94 to -6.96 eV and -4.19 to -4.48 eV, respectively.

A physically grounded damped dispersion model with particle mesh Ewald summation.

Accurate modeling of dispersion is critical to the goal of predictive biomolecular simulations. To achieve this accuracy, a model must be able to correctly capture both the short-range and asymptotic behavior of dispersion interactions. We present here a damped dispersion model based on the overlap of charge densities that correctly captures both regimes. The overlap damped dispersion model represents a classical physical interpretation of dispersion: the interaction between the instantaneous induced dipoles of two distinct charge distributions. This model is shown to be an excellent fit with symmetry adapted perturbation theory dispersion energy calculations, yielding an RMS error on the S101x7 database of 0.5 kcal/mol. Moreover, the damping function used in this model is wholly derived and parameterized from the electrostatic dipole-dipole interaction, making it not only physically grounded but transferable as well.

Molecular dynamics simulation, ab initio calculation, and size-selected anion photoelectron spectroscopy study of initial hydration processes of calcium chloride.

To understand the initial hydration processes of CaCl2, we performed molecular simulations employing the force field based on the theory of electronic continuum correction with rescaling. Integrated tempering sampling molecular dynamics were combined with ab initio calculations to overcome the sampling challenge in cluster structure search and refinement. The calculated vertical detachment energies of CaCl2(H2O)n- (n = 0-8) were compared with the values obtained from photoelectron spectra, and consistency was found between the experiment and computation. Separation of the Cl-Ca ion pair is investigated in CaCl2(H2O)n- anions, where the first Ca-Cl ionic bond required 4 water molecules, and both Ca-Cl bonds are broken when the number of water molecules is larger than 7. For neutral CaCl2(H2O)n clusters, breaking of the first Ca-Cl bond starts at n = 5, and 8 water molecules are not enough to separate the two ion pairs. Comparing with the observations on magnesium chloride, it shows that separating one ion pair in CaCl2(H2O)n requires fewer water molecules than those for MgCl2(H2O)n. Coincidentally, the solubility of calcium chloride is higher than that of magnesium chloride in bulk solutions.

Initial hydration processes of magnesium chloride: size-selected anion photoelectron spectroscopy and ab initio calculations.

To understand the initial hydration processes of MgCl2, we measured photoelectron spectra of MgCl2(H2O)n- (n = 0-6) and conducted ab initio calculations on MgCl2(H2O)n- and their neutral counterparts up to n = 7. A dramatic drop in the vertical detachment energy (VDE) was observed upon addition of the first water molecule to bare MgCl2-. This large variation in VDE can be associated with the charge-transfer-to-solvent (CTTS) effect occurring in the MgCl2(H2O)n- clusters, as hydration induces transfer of the excess electron of MgCl2- to the water molecules. Investigation of the separation of Cl--Mg2+ ion pair shows that, in MgCl2(H2O)n- anions, breaking of the first Mg-Cl bond occurs at n = 4, while breaking of the second Mg-Cl bond takes place at n = 6. For neutral MgCl2(H2O)n clusters, breaking of the first Mg-Cl bond starts at n = 7.

Study of interactions between metal ions and protein model compounds by energy decomposition analyses and the AMOEBA force field.

The interactions between metal ions and proteins are ubiquitous in biology. The selective binding of metal ions has a variety of regulatory functions. Therefore, there is a need to understand the mechanism of protein-ion binding. The interactions involving metal ions are complicated in nature, where short-range charge-penetration, charge transfer, polarization, and many-body effects all contribute significantly, and a quantitative description of all these interactions is lacking. In addition, it is unclear how well current polarizable force fields can capture these energy terms and whether these polarization models are good enough to describe the many-body effects. In this work, two energy decomposition methods, absolutely localized molecular orbitals and symmetry-adapted perturbation theory, were utilized to study the interactions between Mg2+/Ca2+ and model compounds for amino acids. Comparison of individual interaction components revealed that while there are significant charge-penetration and charge-transfer effects in Ca complexes, these effects can be captured by the van der Waals (vdW) term in the AMOEBA force field. The electrostatic interaction in Mg complexes is well described by AMOEBA since the charge penetration is small, but the distance-dependent polarization energy is problematic. Many-body effects were shown to be important for protein-ion binding. In the absence of many-body effects, highly charged binding pockets will be over-stabilized, and the pockets will always favor Mg and thus lose selectivity. Therefore, many-body effects must be incorporated in the force field in order to predict the structure and energetics of metalloproteins. Also, the many-body effects of charge transfer in Ca complexes were found to be non-negligible. The absorption of charge-transfer energy into the additive vdW term was a main source of error for the AMOEBA many-body interaction energies.

Screening and Functional Analyses of Nilaparvata lugens Salivary Proteome.

Most phloem-feeding insects secrete gelling and watery saliva during the feeding process. However, the functions of salivary proteins are poorly understood. In this study, our purpose was to reveal the components and functions of saliva in a rice sap-sucking insect pest, Nilaparvata lugens. The accomplishment of the whole genome and transcriptome sequencing in N. lugens would be helpful for elucidating the gene information and expression specificity of the salivary proteins. In this study, we have, for the first time, identified the abundant protein components from gelling and watery saliva in a monophagous sap-sucking insect species through shotgun proteomic detection combined with the genomic and transcriptomic analysis. Eight unknown secreted proteins were limited to N. lugens, indicating species-specific saliva components. A group of annexin-like proteins first identified in the secreted saliva displayed different domain structure and expression specificity with typical insect annexins. Nineteen genes encoding five annexin-like proteins, six salivaps (salivary glands-specific proteins with unknown function), seven putative enzymes, and a mucin-like protein showed salivary gland-specific expression pattern, suggesting their importance in the physiological mechanisms of salivary gland and saliva in this insect species. RNA interference revealed that salivap-3 is a key protein factor in forming the salivary sheath, while annexin-like5 and carbonic anhydrase are indispensable for N. lugens survival. These novel findings will greatly help to clarify the detailed functions of salivary proteins in the physiological process of N. lugens and elucidate the interaction mechanisms between N. lugens and the rice plant, which could provide important targets for the future management of rice pests.

From thermodynamics to kinetics: enhanced sampling of rare events.

Despite great advances in molecular dynamics simulations, there remain large gaps between the simulations and experimental observations in terms of the time and length scales that can be approached. Developing fast and accurate algorithms and methods is of ultimate importance to bridge these gaps. In this Account, we briefly summarize recent efforts in such directions. In particular, we focus on integrated tempering sampling. The efficiency of this sampling method has been demonstrated by applications to a range of chemical and biological problems: protein folding, molecular cluster structure searches, and chemical reactions. The combination of integrated tempering sampling and a trajectory sampling method allows the calculation of rate constants and reaction pathways without predefined collective coordinates.