Feasibility study of a LED light irradiation device for the treatment of chronic neck with shoulder muscle pain/stiffness.

Neck with shoulder muscle stiffness/pain is a common disorder. Commonly used physical therapy, pharmacotherapy, acupuncture, and moxibustion only temporarily alleviate the disorder in most cases, thus the disorder often recurs. Low power laser therapy is often used for neck and shoulder stiffness/pain and has been effective in clinical trials. In this study, we evaluated the safety and effectiveness of a newly developed self-care device for disorders including neck with shoulder muscle stiffness/pain. The device incorporates light-emitting diodes (LEDs), which are safer than lasers, as its light source. Ten adults with neck with shoulder muscle stiffness/pain were subject to LED irradiation (wavelength 780 nm ± 15 nm, output 750 mW, power density 3.8 W/cm2, energy density 5.7×102 J/cm2) for 3 minutes on the affected shoulder at a standard acupuncture point (GB21, Jianjing). Immediately after irradiation, the subjective symptoms of the neck with shoulder muscle stiffness and pain evaluated by a visual analog scale were improved from 58.3 mm ± 18.7 mm to 45.5 mm ± 21.5 mm and from 45.8 mm ± 23.3 mm to 39.4 mm ± 21.8 mm, respectively. The symptoms further improved after 15 minutes of irradiation. The skin temperature at the irradiated point increased from 34.3°C ± 1.1°C to 41.0°C ± 0.7°C. The increase in skin temperature was observed within approximately 5 cm of the irradiated area. There was no effect on the heart rate variability, a measure of the autonomic nervous system; however, the baroreflex sensitivity was slightly increased. No irradiation-related adverse skin events were observed. Our LED irradiation device was found to be safe, and it improved the subjective symptoms of muscle stiff neck with shoulders.

Quantitative and kinetic single-molecule analysis of DNA unwinding by Escherichia coli UvrD helicase.

Helicases are nucleic acid-unwinding enzymes involved in the maintenance of genome integrity. Helicases share several "helicase motifs" that are highly conserved amino acid sequences and are classified into six superfamilies (SFs). The helicase SFs are further grouped into two classes based on their functional units. One class that includes SFs 3-6 functions as a hexamer that can form a ring around DNA. Another class that includes SFs 1 and 2 functions in a non-hexameric form. The high homology in the primary and tertiary structures among SF1 helicases suggests that SF1 helicases have a common underlying mechanism. However, two opposing models for the functional unit, monomer and dimer models, have been proposed to explain DNA unwinding by SF1 helicases. This paper briefly describes the classification of helicase SFs and discusses the structural homology and the two opposing non-hexameric helicase models of SF1 helicases by focusing on Escherichia coli SF1 helicase UvrD, which plays a significant role in both nucleotide-excision repair and methyl-directed mismatch repair. This paper reviews past and recent studies on UvrD, including the author's single-molecule direct visualization of wild-type UvrD and a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C), the latter of which was used in genetic and biochemical assays that supported the monomer model. The visualization revealed that multiple UvrDΔ40C molecules jointly unwind DNA, presumably in an oligomeric form, similar to wild-type UvrD. Therefore, single-molecule direct visualization of nucleic acid-binding proteins can provide quantitative and kinetic information to reveal their fundamental mechanisms.

Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD.

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed "helicase motifs". Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

DNA-Unwinding Dynamics of Escherichia coli UvrD Lacking the C-Terminal 40 Amino Acids.

The E. coli UvrD protein is a nonhexameric DNA helicase that belongs to superfamily I and plays a crucial role in both nucleotide excision repair and methyl-directed mismatch repair. Previous data suggested that wild-type UvrD has optimal activity in its oligomeric form. However, crystal structures of the UvrD-DNA complex were only resolved for monomeric UvrD, using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). However, biochemical findings performed using UvrDΔ40C indicated that this mutant failed to dimerize, although its DNA-unwinding activity was comparable to that of wild-type UvrD. Although the C-terminus plays essential roles in nucleic acid binding for many proteins with helicase and dimerization activities, the exact function of the C-terminus is poorly understood. Thus, to understand the function of the C-terminal amino acids of UvrD, we performed single-molecule direct visualization. Photobleaching of dye-labeled UvrDΔ40C molecules revealed that two or three UvrDΔ40C molecules could bind simultaneously to an 18-bp double-stranded DNA with a 20-nucleotide, 3' single-stranded DNA tail in the absence of ATP. Simultaneous visualization of association/dissociation of the mutant with/from DNA and the DNA-unwinding dynamics of the mutant in the presence of ATP demonstrated that, as with wild-type UvrD, two or three UvrDΔ40C molecules were primarily responsible for DNA unwinding. The determined association/dissociation rate constants for the second bound monomer were ∼2.5-fold larger than that of wild-type UvrD. The involvement of multiple UvrDΔ40C molecules in DNA unwinding was also observed under a physiological salt concentration (200 mM NaCl). These results suggest that multiple UvrDΔ40C molecules, which may form an oligomer, play an active role in DNA unwinding in vivo and that deleting the C-terminal 40 residues altered the interaction of the second UvrD monomer with DNA without affecting the interaction with the first bound UvrD monomer.

Fluorescence microscopy for visualizing single-molecule protein dynamics.

BACKGROUND: Single-molecule fluorescence imaging (smFI) has evolved into a valuable method used in biophysical and biochemical studies as it can observe the real-time behavior of individual protein molecules, enabling understanding of their detailed dynamic features. smFI is also closely related to other state-of-the-art microscopic methods, optics, and nanomaterials in that smFI and these technologies have developed synergistically. SCOPE OF REVIEW: This paper provides an overview of the recently developed single-molecule fluorescence microscopy methods, focusing on critical techniques employed in higher-precision measurements in vitro and fluorescent nanodiamond, an emerging promising fluorophore that will improve single-molecule fluorescence microscopy. MAJOR CONCLUSIONS: smFI will continue to improve regarding the photostability of fluorophores and will develop via combination with other techniques based on nanofabrication, single-molecule manipulation, and so on. GENERAL SIGNIFICANCE: Quantitative, high-resolution single-molecule studies will help establish an understanding of protein dynamics and complex biomolecular systems.

A gold nano-electrode for single ion channel recordings.

The artificial bilayer single channel recording technique is commonly used to observe the detailed physiological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency. Here, we developed a novel artificial bilayer single channel recording technique in which solubilized ion channel proteins immobilized on a gold nano-electrode are directly incorporated into a lipid bilayer at the same time as the bilayer is formed at the tip of it on coming in contact with an aqueous-oil interface. Using this technique, we measured the single channel currents of several types of channels including KcsA, MthK, hBK and P2X4. This technique requires only one action to simultaneously form the bilayers and reconstitute the channels into the membranes. This simplicity greatly increases the measurement efficiency and allows the technique to potentially be combined with high-throughput screening devices.

A Simple Method for Ion Channel Recordings Using Fine Gold Electrode.

The artificial bilayer single-channel recording technique is commonly used to observe detailed pharmacological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency. Here, we develop a novel artificial bilayer single-channel recording technique in which bilayers are made and channels are reconstituted into the membranes by contacting a gold electrode to the lipid-solution interface. Using this technique, we measured the single-channel currents of two channel-forming peptides, gramicidin and alamethicin, and a channel-forming protein, α-hemolysin. This technique requires only one action, allowing the technique to potentially be combined with high-throughput screening devices.

Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation.

The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday junction DNA complex formation.

Optically Detected Magnetic Resonance of Nanodiamonds In Vivo; Implementation of Selective Imaging and Fast Sampling.

Single-molecule fluorescence measurements of biological samples frequently suffer from background autofluorescence originating from fluorescent materials pre-existing in living samples, and from unstable photo-physical properties of fluorescent labeling molecules. In this study, we first describe our method of selective imaging of nanodiamonds containing nitrogen-vacancy centers, promising fluorescent color centers, by a combination of optically detected magnetic resonance. The resultant images exhibit perfect elimination of extraneous fluorescence in real-time microscope observations. As the practical example applied to an in vivo system, we measured the resonance spectrum of nanodiamonds introduced into the intestine of Caenorhabditis elegans in the clear background and compared the spectral profile over time. The observed evolution strongly suggests that the rotation of the nanodiamond was detected. We also report our recent progress in the development of a spectrometer equipped with an avalanche photo-diode for fast sampling of photons, which can be used while observing the selective image of a field of view in a real-time manner. This apparatus is suitable for exploring dynamics through the measurement of fluctuation in fluorescence intensity caused by a rotating nanodiamond.

Construction and characterization of Cy3- or Cy5-conjugated hairpin pyrrole-imidazole polyamides binding to DNA in the nucleosome.

Sequence-specific DNA-binding modules, N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamides have been recently conjugated with fluorophores, and some of these conjugates could be used for the detection of specific DNA sequences. In this study, we synthesized two Py-Im polyamides 1 and 2, which interact with the 145-bp nucleosome positioning sequence 601. We conjugated the cyanine dye Cy3 or Cy5 with 1 or 2. In the absence of target DNA, the fluorescent conjugate of a Py-Im polyamide had lower fluorescence intensity compared with Cy3 or Cy5 alone. In the presence of either the target DNA or the nucleosome, the fluorescence intensity of the conjugates increased. Furthermore, we observed a Förster resonance energy transfer between the Cy3-Py-Im polyamide and the Cy5-Py-Im polyamide on the nucleosome. These results open up the possibilities that fluorescent conjugates of Py-Im polyamides can be used for characterization of the dynamic interactions within protein-DNA complexes.

Single-molecule imaging of the oligomer formation of the nonhexameric Escherichia coli UvrD helicase.

Superfamily I helicases are nonhexameric helicases responsible for the unwinding of nucleic acids. However, whether they unwind DNA in the form of monomers or oligomers remains a controversy. In this study, we addressed this question using direct single-molecule fluorescence visualization of Escherichia coli UvrD, a superfamily I DNA helicase. We performed a photobleaching-step analysis of dye-labeled helicases and determined that the helicase is bound to 18-basepair (bp) double-stranded DNA (dsDNA) with a 3' single-stranded DNA (ssDNA) tail (12, 20, or 40 nt) in a dimeric or trimeric form in the absence of ATP. We also discovered through simultaneous visualization of association/dissociation of the helicase with/from DNA and the DNA unwinding dynamics of the helicase in the presence of ATP that these dimeric and trimeric forms are responsible for the unwinding of DNA. We can therefore propose a new kinetic scheme for the helicase-DNA interaction in which not only a dimeric helicase but also a trimeric helicase can unwind DNA. This is, to our knowledge, the first direct single-molecule nonhexameric helicase quantification study, and it strongly supports a model in which an oligomer is the active form of the helicase, which carries important implications for the DNA unwinding mechanism of all superfamily I helicases.

Challenges to the clinical placement and evaluation of adhesively-bonded, cervical composite restorations.

OBJECTIVES: The incidence of non-carious cervical lesions (NCCLs) has been increasing. The clinical performance of resin composites in NCCLS was previously unsatisfactory due to their non-retentive forms and margins lying on dentin. In order to address this problem, a lot of effort has been put into developing new dentin adhesives and restorative techniques. This article discusses these challenges and the criteria used for evaluating clinical performance as they relate to clinical studies, especially long-term clinical trials. Polymerization contraction, thermal changes and occlusal forces generate debonding stresses at adhesive interfaces. METHODS: In laboratory studies, we have investigated how these stresses can be relieved by various restorative techniques and how bond strength and durability can be enhanced. Lesion forms, restorative techniques, adhesives (adhesive strategies, bond strengths, bond durability, and the relationship between enamel and dentin bond strengths) were found to have a complex relationship with microleakage. With regard to some restorative techniques, only several short-term clinical studies were available. RESULTS: Although in laboratory tests marginal sealing improved with a low-viscosity resin liner, an enamel bevel or prior enamel etching with phosphoric acid, clinical studies failed to detect significant effects associated with these techniques. Long-term clinical trials demonstrated that adhesive bonds continuously degraded in various ways, regardless of the adhesion strategy used. SIGNIFICANCE: Early loss of restoration may no longer be the main clinical problem when reliable adhesives are properly used. Marginal discoloration increased over time and may become a more prominent reason for repair or replacement. Reliable and standardized criteria for the clinical evaluation of marginal discoloration should be established as soon as possible and they should be based on evidence and a policy of minimal intervention.

Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants.

N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py-Im polyamides 1-5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides. The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py-Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides. This fundamental knowledge of the binding of Py-Im polyamides to DNA will facilitate the design of hairpin Py-Im polyamides as synthetic DNA-binding modules.

Real-time background-free selective imaging of fluorescent nanodiamonds in vivo.

Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence.

Three-year clinical evaluation of a flowable and a hybrid resin composite in non-carious cervical lesions.

OBJECTIVES: This randomized controlled clinical trial evaluated the 3-year clinical performance of a hybrid (Clearfil AP-X; AP) and a flowable (Clearfil Flow FX; FX) resin composite in 98 non-carious cervical lesions. METHODS: Twenty-two patients, 11 males and 11 females (mean age: 61.9 years, range: 29-78 years) regularly visiting the Nagasaki University Hospital, participated in the study. Each patient received both materials randomly. All restorations (48 restorations for AP and 50 restorations for FX) were placed in conjunction with an all-in-one system (Clearfil S(3) Bond) by one dentist. The restorations were blindly evaluated by two examiners at baseline, 6 months, 1, 2 and 3 years using modified USPHS criteria. The data were statistically analyzed using the Cochran's Q-test and Fisher's exact test. RESULTS: All the patients were examined at each recall. However, five restorations could not be evaluated at 3-year recall as two teeth had been extracted and three restorations had been lost. The only minor problem was the integrity of the enamel margin. The incidence and extent of marginal staining increased with time, but it was still superficial. Marginal staining occurred adjacent to 11 restorations for AP and 12 restorations for FX after 3 years. Neither lesion size nor depth had influence on marginal staining adjacent to each type of resin composite. There were no significant differences in the clinical performances between AP and FX for each variable. CONCLUSIONS: Under the protocol used in this study, both types of resin composite in conjunction with S(3) Bond demonstrated an acceptable clinical performance up to 3 years.

Two-year clinical evaluation of one-step self-etch systems in non-carious cervical lesions.

OBJECTIVES: This randomized controlled clinical trial evaluated the 2-year clinical performance of S3 Bond (S3) and G-Bond (GB) in 108 non-carious cervical lesions. METHODS: Twenty-three patients, 12 male and 11 female (mean age: 61.8 years, range: 30-79 years) regularly visiting the Nagasaki University Hospital of Medicine and Dentistry, participated in the study. Each patient received both materials randomly. All restorations (53 restorations for S3 and 55 restorations for GB) were placed by one dentist. The restorations were blindly evaluated by two examiners at baseline, 6 months, 1 and 2 years using modified USPHS criteria. The data were statistically analyzed using the Cochran Q test and Fisher's exact test. RESULTS: One restoration of each material was lost during 2 years. The only minor clinical problem was the integrity of the enamel margin. Slight marginal staining occurred adjacent to 11 restorations of both S3 and GB. There was no significant difference in the clinical performance between S3 and GB for each variable. CONCLUSIONS: Under the protocol used in this study, S3 and GB have demonstrated an acceptable clinical performance up to 2 years.

Dynamic polymorphism of actin as activation mechanism for cell motility.

Actin filament dynamics are crucial in cell motility. Actin filaments, and their bundles, networks, and gels assemble and disassemble spontaneously according to thermodynamic rules. These dynamically changing structures of actin are harnessed for some of its functions in cells. The actin systems respond to external signals, forces, or environments by biasing the fluctuation of actin assembly structures. In this study, dynamic conformation of actin molecules was studied by monitoring conformational dynamics of actin molecules at the single molecule level in real time. Actin conformation spontaneously fluctuates between multiple conformational states. Regarding myosin motility, the dynamic equilibrium of actin conformation was interpreted as between states that activates and inhibits the motility. The binding of myosin to actin filaments activates myosin motility by shifting the conformational fluctuation of actin towards the state that activates the motility. Thus, the activation mechanism based on thermal fluctuation is suggested at molecular level as well as at cellular level.

Effect of a caries-detecting solution on the tensile bond strength of four dentin adhesive systems.

This study investigated the effect of a caries-detecting solution on the tensile bond strength (TBS) to sound bovine dentin--which was either rinsed thoroughly of or contaminated with the caries-detecting solution. Caries Detector (1.0% acid red in propylene glycol) was applied on flat dentin surfaces for 10 seconds, rinsed, and dried with syringe air. In another group, Caries Detector was not rinsed but air-dried. Then, the surfaces were treated with one of the following adhesive systems: Clearfil Protect Bond, Clearfil SE Bond, One-Up Bond F, or Single Bond. Furthermore, an ingredient of Caries Detector, either 1.0% acid red aqueous solution or propylene glycol, was applied to evaluate the effect of each component. In the control groups, Caries Detector was not applied to the dentin surfaces. Finally, a resin composite was light-cured and the TBS measured. Fractured specimens and treated dentin surfaces were observed by SEM. Caries Detector did not reduce the tensile bond strength of any adhesive system (p>0.05) when rinsed thoroughly. On the other hand, when dentin surface was contaminated with Caries Detector, TBS decreased significantly with Clearfil SE Bond and Single Bond. As for the ingredients of Caries Detector, the effect of acid red on TBS was not significant, but that of propylene glycol was significant.

Dynamic polymorphism of Ras observed by single molecule FRET is the basis for molecular recognition.

Ras regulates signal transduction pathway function by dynamically interacting with various effectors. To understand the basis for Ras function, its conformational dynamics were measured in the absence and presence of effectors using single molecule fluorescence resonance energy transfer (FRET) between probes located on the Switch II region and GTP. The time trajectories of FRET efficiency from GTP-bound Ras showed that this conformation spontaneously varies among multiple states. Among them, a low FRET state was identified as an inactive state. The transition involving the inactive conformational state occurred in the time range of seconds. In contrast, fluctuation occurring most probably between multiple active high FRET conformational states lasted approximately 30 ms but converged to a specific conformational state upon binding to an effector. Thus, Ras conformation spontaneously fluctuates to readily interact with various effectors.

Dynamic polymorphism of single actin molecules in the actin filament.

Actin filament dynamics are critical in cell motility. The structure of actin filament changes spontaneously and can also be regulated by actin-binding proteins, allowing actin to readily function in response to external stimuli. The interaction with the motor protein myosin changes the dynamic nature of actin filaments. However, the molecular bases for the dynamic processes of actin filaments are not well understood. Here, we observed the dynamics of rabbit skeletal-muscle actin conformation by monitoring individual molecules in the actin filaments using single-molecule fluorescence resonance energy transfer (FRET) imaging with total internal reflection fluorescence microscopy (TIRFM). The time trajectories of FRET show that actin switches between low- and high-FRET efficiency states on a timescale of seconds. If actin filaments are chemically cross-linked, a state that inhibits myosin motility, the equilibrium shifts to the low-FRET conformation, whereas when the actin filament is interacting with myosin, the high-FRET conformation is favored. This dynamic equilibrium suggests that actin can switch between active and inactive conformations in response to external signals.