Structured Water Molecules on Membrane Proteins Resolved by Atomic Force Microscopy.

Water structuring on the outer surface of protein molecules called the hydration shell is essential as well as the internal water structures for higher-order structuring of protein molecules and their biological activities in vivo. We now show the molecular-scale hydration structure measurements of native purple membrane patches composed of proton pump proteins by a noninvasive three-dimensional force mapping technique based on frequency modulation atomic force microscopy. We successfully resolved the ordered water molecules localized near the proton uptake channels on the cytoplasmic side of the individual bacteriorhodopsin proteins in the purple membrane. We demonstrate that the three-dimensional force mapping can be widely applicable for molecular-scale investigations of the solid-liquid interfaces of various soft nanomaterials.

Molecular-Resolution Imaging of Interfacial Solvation of Electrolytes for Lithium-Ion Batteries by Frequency Modulation Atomic Force Microscopy.

Solvation structures formed by ions and solvent molecules at solid/electrolyte interfaces affect the energy storage performance of electrochemical devices, such as lithium-ion batteries. In this study, the molecular-scale solvation structures of an electrolyte, a solution of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) in propylene carbonate (PC) at the electrolyte-mica interface, were measured using frequency-modulation atomic force microscopy (FM-AFM). The spacing of the characteristic force oscillation in the force versus distance curves increased with increasing ion concentration, suggesting an increase in the effective size of molecules at the interface. Molecular dynamics simulations showed that the effective size of molecular assemblies, namely, solvated ions formed at the interface, increased with increasing ion concentrations, which was consistent with the experimental results. Knowledge of molecular-scale structures of solid/electrolyte interfaces obtained by a combination of FM-AFM and molecular dynamics simulations is important in the design of electrolytes for future energy devices and in improving their properties.

Surface charge density measurement of a single protein molecule with a controlled orientation by AFM.

The spatial distribution of functional groups causes a charge distribution that often has a close relationship with its biofunctions. To understand them of the protein molecules, measurements of the charge distribution under physiological conditions are desired. Atomic force microscopy (AFM) has been utilized to measure the surface charge density by measuring the electric double layer (EDL) force caused by the overlap of the EDLs on the surfaces of the AFM tip and the biomolecule. Here, we demonstrated the surface charge density measurement of a single streptavidin (SA) protein molecule by the three-dimensional force mapping method based on frequency modulation AFM (FM-AFM). The SA has a strong affinity to biotin because of the electrostatic interactions between the molecules. Therefore, the surface charge density measurements of the biotin-binding sites and other surface areas of the molecule have been anticipated. However, the surface charge density of the surfaces other than the biotin-binding side has never been measured. We demonstrate the surface charge density measurement of the top surface of the single SA molecule, which is perpendicular to the biotin-binding sides, with a controlled orientation using DNA origami as a template by FM-AFM in an electrolyte solution. The surface charge density of the top surface of the SA molecule was estimated by fitting the experimental force curves to the Derjaguin-Landau-Verwey-Overbeck theory. We found that the surface charge density of the top surface of the SA molecule is comparable to those reported earlier for the biotin-binding sides of the molecule. We expect that, by using the DNA origami technology, one can control the orientation of a biomolecule attached to the substrate and measure the surface charge density of the specific surface areas of the biomolecule to obtain information that will help us to understand the relationship between their structures and functions.

Low-Background Tip-Enhanced Raman Spectroscopy Enabled by a Plasmon Thin-Film Waveguide Probe.

Tip-enhanced Raman spectroscopy (TERS) is a nano-optical approach to extract spatially resolved chemical information with nanometer precision. However, in the case of direct-illumination TERS, which is often employed in commercial TERS instruments, strong fluorescence or far-field Raman signals from the illuminated areas may be excited as a background. They may overwhelm the near-field TERS signal and dramatically decrease the near-field to far-field signal contrast of TERS spectra. It is still challenging for TERS to study the surface of fluorescent materials or a bulk sample that cannot be placed on an Au/Ag substrate. In this study, we developed an indirect-illumination TERS probe that allows a laser to be focused on a flat interface of a thin-film waveguide located far away from the region generating the TERS signal. Surface plasmon polaritons are generated stably on the waveguide and eventually accumulated at the tip apex, thereby producing a spatially and energetically confined hotspot to ensure stable and high-resolution TERS measurements with a low background. With this thin-film waveguide probe, TERS spectra with obvious contrast from a diamond plate can be acquired. Furthermore, the TERS technique based on this probe exhibits excellent TERS signal stability, a long lifetime, and good spatial resolution. This technique is expected to have commercial potential and enable further popularization and development of TERS technology as a powerful analytical method.

TS-1 add-on therapy in Japanese patients with triple-negative breast cancer after neoadjuvant or adjuvant chemotherapy: a feasibility study.

Purpose We examined the feasibility, efficacy, and safety of TS-1 add-on therapy (TAT) in Japanese patients with triple-negative breast caner (TNBC). Methods TAT (TS-1, 80 mg/m2/day, BID, PO), consisting of the 21-day cycles of 14-day consecutive administration followed by 7-day drug holiday, was conducted for 365 days. The median follow-up was 75.2 months (range, 7.3-103.3 months). The primary endpoint was the feasibility of TAT. The secondary endpoints included relapse-free survival (RFS), overall survival (OS), and safety. Results 63 Japanese patients with TNBC (median age, 52.5 years; range, 23.7-68.6 years) were examined. Among them, 34 (54.0%) were postmenopausal, 54 (93.7%) had TNBC of common histological type, 51 (81.0%) had T1 to 3 tumors, 63 (100%) had undergone standardized surgery, and 44 (69.8%) and 19 (30.2%) had undergone neoadjuvant chemotherapy and adjuvant chemotherapy, respectively. The 365-day cumulative rate of TS-1 administration was 68.3% (95% confidence interval, 55.3-79.4), being comparable to 65.8% previously reported for gastric cancer. The 5-year RFS rates were 52.3% and 84.2% in the neoadjuvant and adjuvant chemotherapy groups, respectively, and the 5-year OS rates were 68.0% and 89.5%, respectively. The most common adverse events (AEs) were leucocyte count decreased (50.8%), total bilirubin decreased (44.4%), and pigmentation (42.9%). AEs were manageable clinically, and any grade 4 AEs did not develop. Conclusions The 365-day cumulative rate of TS-1 administration in TNBC patients was comparable to that in gastric cancer patients despite previous chemotherapy with anthracyclines and/or taxanes. TAT was feasible for TNBC patients after standard primary therapy.

Eribulin, trastuzumab, and pertuzumab as first-line therapy for patients with HER2-positive metastatic breast cancer: a phase II, multicenter, collaborative, open-label, single-arm clinical trial.

Purpose To examine the efficacy and safety of triple therapy with eribulin, trastuzumab, and pertuzumab in patients with HER2-positive metastatic breast cancer (MBC) who never received any prior therapy in the first-line metastatic/advanced setting. Methods Eribulin 1.4 mg/m2 (days 1 and 8), trastuzumab 8 mg/kg over 90 min and 6 mg/kg over 30 min, and pertuzumab 840 mg/body over 60 min and 420 mg/body over 30 min were administered intravenously in 21-day cycles. Results 25 women (median age, 57 years [range, 41-75 years]) received a median of 10 cycles (range, 0-34 cycles); 24 had performance status (PS) 0, 1 PS 1, 8 stage IV breast cancer, and 17 recurrence. Lung and liver metastases occurred in 9 and 9 patients, respectively. Median time to treatment failure with eribulin was 9.1 months (95% confidence interval [CI], 4.3-13.9 months), and median progression-free survival was 23.1 months (95% CI, 14.4-31.8 months). The overall response rate (complete response [CR] + partial response [PR]) was 80.0% (95% CI, 59.3-93.2%), and the clinical benefit rate (CR + PR + stable disease ≥24 weeks) was 84.0% (95% CI, 63.9-95.5%). The most common treatment-emergent adverse events (TEAEs) were alopecia (92.0%), fatigue (68.0%), and sensory peripheral neuropathy (60.0%). Grade 3/4 TEAEs occurred in 11 patients (44.0%). The only grade 4 TEAE was neutrophil count decreased (16.0%). Neither grade 4 peripheral neuropathy nor febrile neutropenia occurred. Conclusions ETP therapy showed acceptable efficacy and safety and is a potential first-line therapy for patients with HER2-positive MBC.

Correction to: Eribulin, trastuzumab, and pertuzumab as first-line therapy for patients with HER2-positive metastatic breast cancer: a phase II, multicenter, collaborative, open-label, single-arm clinical trial.

The authors would like to note the replacement of Fig. 2b, for which Fig. 2a was placed erringly, with appropriate Fig. 2b.

Atomic-Level Viscosity Distribution in the Hydration Layer.

The viscosity of solvation structures is crucial for the development of energy-efficient biofunctional and electrochemical devices. Elucidating their subnanoscale distributions can cause the formation of a sustainable energy society. Here, we visualize the site-specific three-dimensional damping distribution on a CaCO_{3} surface composed of binary ion species using ultra-low-noise frequency modulation atomic force microscopy. With the support from molecular dynamics simulation, we found a strikingly large damping at the calcium sites, which demonstrates the capability of this methodology to visualize atomic-scale viscosity in the hydration layers. Our finding will expedite the evolutions of various functional devices.

Rhombic-Shaped Nanostructures and Mechanical Properties of 2D DNA Origami Constructed with Different Crossover/Nick Designs.

DNA origami methods enable the fabrication of various nanostructures and nanodevices, but their effective use depends on an understanding of their structural and mechanical properties and the effects of basic structural features. Frequency-modulation atomic force microscopy is introduced to directly characterize, in aqueous solution, the crossover regions of sets of 2D DNA origami based on different crossover/nick designs. Rhombic-shaped nanostructures formed under the influence of flexible crossovers placed between DNA helices are observed in DNA origami incorporating crossovers every 3, 4, or 6 DNA turns. The bending rigidity of crossovers is determined to be only one-third of that of the DNA helix, based on interhelical electrostatic forces reported elsewhere, and the measured pitches of the 3-turn crossover design rhombic-shaped nanostructures undergoing negligible bending. To evaluate the robustness of their structural integrity, they are intentionally and simultaneously stressed using force-controlled atomic force microscopy. DNA crossovers are verified to have a stabilizing effect on the structural robustness, while the nicks have an opposite effect. The structural and mechanical properties of DNA origami and the effects of crossovers and nicks revealed in this paper can provide information essential for the design of versatile DNA origami structures that exhibit specified and desirable properties.

Investigation of Local Hydration Structures of Alkanethiol Self-Assembled Monolayers with Different Molecular Structures by FM-AFM.

Hydration structures play crucial roles in a wide variety of chemical and biological phenomena. However, the key factors that determine a hydration structure remain an open question. Most recent studies have focused on the electrostatic interactions between the surface charges and dipoles of water molecules, which are determined by the atomic/ionic species of the outermost solid surface, as the dominating factor. The number of studies on the correlation between the hydration structure and the atomic-scale surface corrugation has been limited. In this study, we investigated the hydration structures of alkanethiol self-assembled monolayers terminated with a hydroxyl group using frequency-modulated atomic force microscopy. We observed two molecular structures, namely, the (√3 × âˆš3) R30° structure and the c(4 × 2) superlattice structure, and found that their hydration structures are different mainly because of the slight differences in their molecular arrangements. This result suggests that a slight difference in the molecular/atomic arrangements as well as the atomic/ionic species in the outermost solid surface strongly influences the local hydration structures.

Atomic-Scale 3D Local Hydration Structures Influenced by Water-Restricting Dimensions.

Hydration structures at solid-liquid interfaces mediate between the atomic-level surface structures and macroscopic functionalities in various physical, chemical, and biological processes. Atomic-scale local hydration measurements have been enabled by ultralow noise three-dimensional (3D) frequency-modulation atomic force microscopy. However, for their application to complicated surface structures, e.g., biomolecular devices, understanding the relationship between the hydration and surface structures is necessary. Herein, we present a systematic study based on the concept of the structural dimensionality, which is crucial in various scientific fields. We performed 3D measurements and molecular dynamics simulations with silicate surfaces that allow for 0, 1, and 2 degrees of freedom to water molecules. Consequently, we found that the 3D hydration structures reflect the structural dimensions and the hydration contrasts decrease with increasing dimension due to the enlarged water self-diffusion coefficient and increased embedded hydration layers. Our results provide guidelines for the analysis of complicated hydration structures, which will be exploited in extensive fields.

Flexible DNA Path in the MCM Double Hexamer Loaded on DNA.

The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.

Phase II clinical study of eribulin monotherapy in Japanese patients with metastatic breast cancer who had well-defined taxane resistance.

No clinical evidence on the efficacy and safety of eribulin monotherapy has been obtained by a prospective clinical study in patients with metastatic breast cancer (MBC) who had well-defined taxane resistance. The present Phase II, multicenter, single-arm, open-label study aimed to obtain the evidence. Japanese female patients, aged 33-74 years who had the metastasis of taxane-resistant and histopathologically confirmed breast cancer, received eribulin mesylate 1.4 mg/m(2) (equivalent to eribulin 1.23 mg/m(2) [expressed as free base]) as a 2- to 5-min intravenous infusion on days 1 and 8 of each 21-day cycle. The primary endpoint was the clinical benefit rate (CBR) [complete response (CR), partial response (PR), and long-term stable disease (LSD) ≥24 weeks]. A total of 51 patients underwent chemotherapy cycles (median 4; range 1-42 cycles). The CBR was 39.2 % (CR 2.0 %; PR 23.5 %; and LSD 13.7 %), and the rate of progressive disease was 49.0 %. The median progression-free survival and the median overall survival were 3.6 months [95 % confidence interval (CI) 2.6-4.6 months] and 11.7 months (95 % CI 9.2-14.2 months), respectively. Grade 3 or greater adverse events were leukopenia (23.5 %), neutropenia (35.3 %), anemia (5.9 %), and febrile neutropenia (7.8 %). The incidences of grade 3 and 4 peripheral sensory neuropathy were 2.0 and 0 %, respectively. Eribulin showed a clinically manageable tolerability profile by dose adjustments or symptomatic treatment. Eribulin was effective and well tolerated in heavily pretreated patients with MBC who had well-defined taxane resistance, thus providing a potential therapeutic option in the clinical settings.

Visualization of subsurface nanoparticles in a polymer matrix using resonance tracking atomic force acoustic microscopy and contact resonance spectroscopy.

A visualization technique of subsurface features with a nanometer-scale spatial resolution is strongly demanded. Some research groups have demonstrated the visualization of subsurface features using various techniques based on atomic force microscopy. However, the imaging mechanisms have not yet been fully understood. In this study, we demonstrated the visualization of subsurface Au nanoparticles buried in a polymer matrix 900 nm from the surface using two techniques; i.e., resonance tracking atomic force acoustic microscopy and contact resonance spectroscopy. It was clarified that the subsurface features were visualized by the two techniques as the area with a higher contact resonance frequency and a higher Q-factor than those in the surrounding area, which suggests that the visualization is realized by the variation of the contact stiffness and damping of the polymer matrix due to the existence of the buried nanoparticles.

Immunoactive two-dimensional self-assembly of monoclonal antibodies in aqueous solution revealed by atomic force microscopy.

The conformational flexibility of antibodies in solution directly affects their immune function. Namely, the flexible hinge regions of immunoglobulin G (IgG) antibodies are essential in epitope-specific antigen recognition and biological effector function. The antibody structure, which is strongly related to its functions, has been partially revealed by electron microscopy and X-ray crystallography, but only under non-physiological conditions. Here we observed monoclonal IgG antibodies in aqueous solution by high-resolution frequency modulation atomic force microscopy (FM-AFM). We found that monoclonal antibodies self-assemble into hexamers, which form two-dimensional crystals in aqueous solution. Furthermore, by directly observing antibody-antigen interactions using FM-AFM, we revealed that IgG molecules in the crystal retain immunoactivity. As the self-assembled monolayer crystal of antibodies retains immunoactivity at a neutral pH and is functionally stable at a wide range of pH and temperature, the antibody crystal is applicable to new biotechnological platforms for biosensors or bioassays.

Atom-resolved analysis of an ionic KBr(001) crystal surface covered with a thin water layer by frequency modulation atomic force microscopy.

An ionic KBr(001) crystal surface covered with a thin water layer was observed with a frequency modulation atomic force microscope (FM-AFM) with atomic resolution. By immersing only the tip apex of the AFM cantilever in the thin water layer, the Q-factor of the cantilever in probing the solid-liquid interface can be maintained as high as that of FM-AFM operation in air, leading to improvement of the minimum detection of a differential force determined by the noise. Two types of images with atom-resolved contrast were observed, possibly owing to the different types of ions (K(+) or Br(-)) adsorbed on the tip apex that incorporated into the hydration layers on the tip and on the sample surface. The force-distance characteristics at the solid-water interface were analyzed by taking spatial variation maps of the resonant frequency shift of the AFM cantilever with the high Q-factor. The oscillatory frequency shift-distance curves exhibited atomic site dependence. The roles of hydration and the ions on the tip and on the sample surface in the measurements were discussed.

Molecular-scale quantitative charge density measurement of biological molecule by frequency modulation atomic force microscopy in aqueous solutions.

Surface charge distributions on biological molecules in aqueous solutions are essential for the interactions between biomolecules, such as DNA condensation, antibody-antigen interactions, and enzyme reactions. There has been a significant demand for a molecular-scale charge density measurement technique for better understanding such interactions. In this paper, we present the local electric double layer (EDL) force measurements on DNA molecules in aqueous solutions using frequency modulation atomic force microscopy (FM-AFM) with a three-dimensional force mapping technique. The EDL forces measured in a 100 mM KCl solution well agreed with the theoretical EDL forces calculated using reasonable parameters, suggesting that FM-AFM can be used for molecular-scale quantitative charge density measurements on biological molecules especially in a highly concentrated electrolyte.

Molecular-scale investigations of structures and surface charge distribution of surfactant aggregates by three-dimensional force mapping.

Surface charges on nanoscale structures in liquids, such as biomolecules and nano-micelles, play an essentially important role in their structural stability as well as their chemical activities. These structures interact with each other through electric double layers (EDLs) formed by the counter ions in electrolyte solution. Although static-mode atomic force microscopy (AFM) including colloidal-probe AFM is a powerful technique for surface charge density measurements and EDL analysis on a submicron scale in liquids, precise surface charge density analysis with single-nanometer resolution has not been made because of its limitation of the resolution and the detection sensitivity. Here we demonstrate molecular-scale surface charge measurements of self-assembled micellar structures, molecular hemicylinders of sodium dodecyl sulfate (SDS), by three-dimensional (3D) force mapping based on frequency modulation AFM. The SDS hemicylindrical structures with a diameter of 4.8 nm on a graphite surface were clearly imaged. We have succeeded in visualizing 3D EDL forces on the SDS hemicylinder surfaces and obtaining the molecular-scale charge density for the first time. The results showed that the surface charge on the trench regions between the hemicylinders was much smaller than that on the hemicylinder tops. The method can be applied to a wide variety of local charge distribution studies, such as spatial charge variation on a single protein molecule.

Diffusion tensor imaging analysis of optic radiation using readout-segmented echo-planar imaging.

PURPOSE: To investigate the diffusion tensor imaging parameters of the optic radiation and surrounding structures using the high-resolution readout-segmented diffusion tensor imaging method. MATERIALS AND METHODS: Coronal readout-segmented diffusion tensor images were acquired in 15 healthy volunteers. On three slices of each image, eigenvalue 1, fractional anisotropy, radial diffusivity, apparent diffusion coefficient, and signal intensity on T2-weighted images were measured in the lateral inferior longitudinal fasciculus, external and internal layers of the optic radiation, and the tapetum within regions of interest delineated by two independent observers. Profile curve analysis of regions of interest across the optic radiation and surrounding structures was performed for a representative typical case. RESULTS: Significant differences in fractional anisotropy, radial diffusivity and apparent diffusion coefficient were observed between external and internal layers of the optic radiation, while there was no significant difference in eigenvalue 1. In fractional anisotropy maps, two low signal bands were observed between the inferior longitudinal fasciculus, the optic radiation and the tapetum. Profile curve analysis showed a minimum on the fractional anisotropy and eigenvalue 1 images and a maximum in the radial diffusivity image. CONCLUSION: Readout-segmented diffusion tensor imaging revealed significant differences in the diffusion tensor imaging parameters between internal and external layers of the optic radiation.

Monotonic damping in nanoscopic hydration experiments.

We present the first accurate damping profiles acquired in atomic-resolution hydration force spectroscopy, revealing a monotonic damping profile at the nanoscopic tip apex. Atomic resolution is confirmed by the observed inhomogeneity caused by random substitution of Si by Al on the mica surface, and two distinct damping regimes are identified. Damping only appears above our detection limit of 1 nNs/m once the tip interacts with the second hydration layer, and an onset of strong damping above 100 nNs/m appears upon direct interaction with the adsorbed layer and first hydration layer. These results are compared to various simulations to interpret the damping signals and determine the tip-sample distance.