Unraveling Polymorphism and Twisting in Near-Perfect Protein Crystals.

Crystals ideally have well-formed shapes and periodic arrangements of constituent components, such as atoms and molecules. Twisting, an unconventional crystal morphology, presents itself as a puzzling and natural phenomenon. The coexistence of a continuous twisting structure and crystalline order poses a paradox. Numerous mechanisms to explain twisting have been proposed, and the elucidation of the underlying causes of spontaneous nonlong-range translational order twisting in crystals has been desired. Here, we demonstrate twisting and perfect crystals controlled by the crystal polymorphs of macromolecular crystals. We establish that the presence of either a perfectly periodic crystalline arrangement or twisting is linked to anisotropic interactions arising from salt bridges among protein molecules. Employing the dynamical theory of X-ray diffraction, we discern that twisting serves as an imperfection that cannot be attributed to conventional crystal defects within crystals. These insights suggest the origin of crystal twisting and methods for controlling crystal perfection.

Unique Mechanical Properties of Gel-Incorporating Protein Crystals.

Crystalline materials that are grown in gel media exhibit reinforced mechanical characteristics. Studies on the mechanical properties of protein crystals are limited in numbers because of the difficulty in growing high-quality large crystals. This study shows the demonstration of the unique macroscopic mechanical properties by compression tests of large protein crystals grown in both solution and agarose gel. Particularly, the gel-incorporating protein crystals exhibit larger elastic limits and a higher fracture stress compared with the native protein crystals without gel. Conversely, the change in the Young's modulus corresponding to if the crystals incorporate the gel network is negligible. This suggests that gel networks affect only the fracture phenomenon. Thus, reinforced mechanical characteristics that cannot be obtained by the gel or the protein crystal alone can be developed. By combining the gel media and protein crystals, the gel-incorporating protein crystals show the potential to toughen without sacrificing other mechanical properties.

Diffusion Coefficient of Intracrystalline Water in Intrinsic Hen Egg-White Lysozyme Crystals Determined by Confocal Raman Spectroscopy.

Protein crystals composed of protein molecules are expected as a novel porous material. They have high porosity, and the knowledge of the diffusion of intracrystalline water is important. In this study, the diffusion coefficient of intracrystalline water in intrinsic hen egg-white lysozyme (HEWL) crystals was determined by a method that combines confocal Raman spectroscopy and air convection with controlled relative humidity. Similar to common porous materials, the drying process of the protein crystals includes three periods: constant-rate drying, falling-rate drying, and equilibrium state. During the falling-rate drying period, the drying rate depends on the diffusion of intracrystalline water in the protein crystal. The gradient of the water content was measured using confocal Raman spectroscopy. The diffusion coefficient of the intrinsic HEWL crystals was determined as 3.1 × 10-7 cm2/s with a water content of 36.3 vol %. The estimated diffusion coefficients of the intrinsic HEWL crystals without cross-linking were in close agreement with those of the cross-linked protein crystals. This study is timely as the knowledge of the intrinsic diffusion coefficient is useful not only for understanding the mechanism of hydration of proteins but also in practical applications such as porous materials, drug binding, and cryoprotectant soaks.

Existence of twisting in dislocation-free protein single crystals.

The growth of high-quality protein crystals is a prerequisite for the structure analysis of proteins by X-ray diffraction. However, dislocation-free perfect crystals such as silicon and diamond have been so far limited to only two kinds of protein crystals, such as glucose isomerase and ferritin crystals. It is expected that many other high-quality or dislocation-free protein crystals still exhibit some imperfection. The clarification of the cause of imperfection is essential for the improvement of crystallinity. Here, we explore twisting as a cause of the imperfection in high-quality protein crystals of hen egg-white lysozyme crystals with polymorphisms (different crystal forms) by digital X-ray topography with synchrotron radiation. The magnitude of the observed twisting is 10−6 to 10−5°/µm which is more than two orders smaller than 10−3 to 104°/µm in other twisted crystals owing to technique limitations with optical and electron microscopy. Twisting is clearly observed in small crystals or in the initial stage of crystal growth. It is uniformly relaxed with crystal growth and becomes smaller in larger crystals. Twisting is one of main residual defects in high-quality crystals and determines the crystal perfection. Furthermore, it is presumed that the handedness of twisting can be ascribed to the anisotropic interaction of chiral protein molecules associated with asymmetric units in the crystal forms. This mechanism of twisting may correspond to the geometric frustration proposed as a primary mechanism of twisting in molecular crystals. Our finding provides insights for the understanding of growth mechanism and the growth control of high-quality crystals.

Radiation-induced defects in protein crystals observed by X-ray topography.

The characterization of crystal defects induced by irradiation, such as X-rays, charged particles and neutrons, is important for understanding radiation damage and the associated generation of defects. Radiation damage to protein crystals has been measured using various methods. Until now, these methods have focused on decreased diffraction intensity, volume expansion of unit cells and specific damage to side chains. Here, the direct observation of specific crystal defects, such as dislocations, induced by X-ray irradiation of protein crystals at room temperature is reported. Dislocations are induced even by low absorbed doses of X-ray irradiation. This study revealed that for the same total absorbed dose, the formation of defects appears to critically depend on the dose rate. The relationship between dislocation energy and dose energy was analyzed based on dislocation theory associated with elasticity theory for crystalline materials. This demonstration of the crystal defects induced by X-ray irradiation could help to understand the underlying mechanisms of X-ray-induced radiation damage.

Control of strain in subgrains of protein crystals by the introduction of grown-in dislocations.

It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structure of protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals even though the overall crystal quality is rather high, as shown by clear equal-thickness fringes in X-ray topography. Thus, a large stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals to radiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in the crystal. This suggests that the introduction of dislocations in protein crystals can be effective in enhancing the crystal quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased, leading to the collection of X-ray diffraction data sets with high diffractivity.

Evaluation of crystal quality of thin protein crystals based on the dynamical theory of X-ray diffraction.

Knowledge of X-ray diffraction in macromolecular crystals is important for not only structural analysis of proteins but also diffraction physics. Dynamical diffraction provides evidence of perfect crystals. Until now, clear dynamical diffraction in protein crystals has only been observed in glucose isomerase crystals. We wondered whether there were other protein crystals with high quality that exhibit dynamical diffraction. Here we report the observation of dynamical diffraction in thin ferritin crystals by rocking-curve measurement and imaging techniques such as X-ray topography. It is generally known that in the case of thin crystals it is difficult to distinguish whether dynamical diffraction occurs from only rocking-curve profiles. Therefore, our results clarified that dynamical diffraction occurs in thin protein crystals because fringe contrasts similar to Pendellösung fringes were clearly observed in the X-ray topographic images. For macromolecular crystallography, it is hard to obtain large crystals because they are difficult to crystallize. For thin crystals, dynamical diffraction can be demonstrated by analysis of the equal-thickness fringes observed by X-ray topography.

Operando spectroscopic analyses for the ammonia absorption process of sodium borohydride.

The ammonia absorption process of sodium borohydride for ammonia storage has been studied by using the operando NMR and FT-IR measurements under various ammonia pressures. As a result, the characteristic variation in the chemical states of both materials due to the liquefaction has been clarified.

Analysis of oscillatory rocking curve by dynamical diffraction in protein crystals.

High-quality protein crystals meant for structural analysis by X-ray diffraction have been grown by various methods. The observation of dynamical diffraction in protein crystals is an interesting topic because dynamical diffraction generally occurs in perfect crystals such as Si crystals. However, to our knowledge, there is no report yet on protein crystals showing clear dynamical diffraction. We wonder whether the perfection of protein crystals might still be low compared with that of high-quality Si crystals. Here, we present observations of the oscillatory profile of rocking curves for protein crystals such as glucose isomerase crystals. The oscillatory profiles are in good agreement with those predicted by the dynamical theory of diffraction. We demonstrate that dynamical diffraction occurs even in protein crystals. This suggests the possibility of the use of dynamical diffraction for the determination of the structure and charge density of proteins.

Electrochemical microdevice for on-site determination of rice freshness.

An electrochemical microdevice was fabricated for on-site rapid freshness determination of rice. The activity of peroxidase was used as an indicator for freshness. With hydrogen peroxide and hydroquinone as substrates, the enzymatic reaction of peroxidase produced benzoquinone, which was subsequently reduced on a gold working electrode electrochemically. The progress of the enzymatic reaction could clearly be observed in the change of cyclic voltammograms. Distinct differences were observed in the current change between fresh rice and aged rice, reflecting the remaining activity of peroxide. The current also depended on the time at which rice grains were milled. The result agreed well with those obtained by the conventional fluorometric and guaiacol methods. Comparison of results obtained with different numbers of grains showed that even the measurement with a single grain provided fairly consistent data. Furthermore, the existence of grains of different freshness could be identified using a device with multiple sensing sites.

Autonomous microfluidic control by chemically actuated micropumps and its application to chemical analyses.

Autonomous control of microfluidic transport was realized through the use of chemically actuated diaphragm micropumps connected to a network of controlling flow channels. A hydrogen peroxide (H(2)O(2)) solution was transported in the controlling flow channel by capillary action. Upon the solution's arrival at the lower compartment of a micropump filled with manganese dioxide (MnO(2)) powder, a volume change that accompanied the production of oxygen caused by the catalytic decomposition of H(2)O(2) induced inflation of the diaphragm. This in turn caused the movement of a solution in another network of flow channels formed in the upper layer. Micropumps that only exert pressure were also fabricated. By positioning the micropumps at appropriate locations in conjunction with additional flow-delaying components, the ejection of solutions from the reservoir of each micropump could be initiated at coordinated times. Furthermore, the solutions could be transported by the application of pressure from other micropumps. In other words, the information for switching from one micropump to another could be described on the chip in the form of a network of flow channels. This autonomous processing of solutions was demonstrated for enzymatic analyses of H(2)O(2), glucose, and lactate.

Channel-switching crystal with guest stress drive.

By the addition of a "leverage" mechanism in a host component of a metal-organic crystal, a channel-switching property was developed in a porous single-crystal adsorbent driven by incorporated guest gas stress, which may provide new single-crystal devices with the active controllability of anisotropic guest diffusivity.

Complete removal of HIV-1 RNA and proviral DNA from semen by the swim-up method: assisted reproduction technique using spermatozoa free from HIV-1.

BACKGROUND: Use of antiretroviral drugs has reduced the mortality rate for HIV infection and many HIV-discordant couples wish to have children. It is possible for an HIV-infected man to father children without risk of HIV transmission if HIV-free spermatozoa can be obtained from his semen. METHODS: An improved swim-up method was used to collect HIV-free spermatozoa from the semen of HIV-positive males. Diluted semen was layered over a Percoll solution with a continuous density gradient of 30-98%, and then centrifuged. The bottom layer was collected by cutting the end from the tube and the sperm suspension was collected using the swim-up method. Spermatozoa were tested by nested polymerase chain reaction (PCR) for HIV-1 RNA and DNA, with a detection limit of one copy. Spermatozoa were used for assisted reproduction in 43 couples. RESULTS: HIV-1 RNA and proviral DNA were not detected by nested-PCR assay in all 73 of the collected spermatozoa samples from 52 patients. The HIV-1-negative sperm was used for in vitro fertilization in 12 couples and for intracytoplasmic sperm injection in 31 couples. No detection of HIV-1 RNA or proviral DNA in the culture medium of the fertilized eggs was confirmed again before embryo transfer. Of the 43 female partners, 20 conceived and 27 babies were born. HIV antibodies, HIV RNA and proviral DNA were negative in all of the females and babies. CONCLUSIONS: HIV-negative spermatozoa could be obtained from semen of HIV-positive men. The method involves no risk of HIV transmission to female partners and their children.

Electron transfer mediator micro-biosensor fabrication by organic plasma process.

We propose a new strategy for constructing a mediator-type biosensor as a Bio-MicroElectroMechanical Systems (BioMEMS) application. A vinylferrocene plasma-polymerized film (PPF) was deposited directly onto the surface of an electrode under dry conditions. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly crosslinked network structure. This technique, capable of polymeric deposition of any kind of monomer, can also serve the purpose of anti-fouling coating, or layer-to-layer interface creation. With a subsequent plasma process, additional polymeric layer of hydrophilic acetonitrile was superimposed onto the existing vinylferrocene-PPF surface to offer crucial features such that the wettability could be adjusted for a better electron transfer, and amino functional groups could be attached to immobilize a large amount of enzyme. Based upon this scheme, the device fabrication could be designed in a manner that the whole procedure was made up of dry wafer-handling processes, which is compatible with mass production. A prototype device was fabricated to have an array of needle-shaped amperometric micro-biosensors. The resultant thin polymer layer carried a large number of the mediator molecules, accomplishing a lower overpotential (+410 mV) and a rapid response time (<5s). Stressing the advantages of the plasma polymerization process together with some additional features accomplished in our device fabrication, we would discuss new possibilities in the field of BioMEMS.

Valence transition of YbInCu4 observed in hard X-ray photoemission spectra.

Yb 3d and valence-band photoemission spectra of the first-order valence-transition compound YbInCu4 have been measured with hard x ray at an excitation energy of 5.95 keV. Abrupt changes are clearly observed in both spectra around the transition temperature T(V)=42 K, in comparison with ultraviolet and soft x-ray photoemission (VUV-PES and SX-PES) spectra. From the Yb 3d spectra, the Yb valence has been estimated to be approximately 2.90 from 220 down to 50 K and approximately 2.74 at 30-10 K. We propose that Yb 3d hard x-ray photoemission spectroscopy is a very powerful method to estimate the valence of Yb with high accuracy. On the other hand, the Yb2+ 4f-derived peaks in the valence-band spectra exhibit a remarkable enhancement below T(V). The shape of the valence-band spectra is different from those of the VUV-PES and SX-PES spectra above T(V), reflecting the In 5s and 5p contributions.

Electrochemical protein chip with arrayed immunosensors with antibodies immobilized in a plasma-polymerized film.

An electrochemical protein chip was microfabricated. A thin-film three-electrode system, including an array of 36 platinum working electrodes, a set of thin-film Ag/AgCl electrodes, and platinum auxiliary electrodes, was integrated on a glass substrate. Capture antibodies were immobilized in a 4.5-nm-thick double layer of a hexamethyldisiloxane plasma-polymerized film. Because of their highly cross-linked network structure, the capture antibodies could be firmly immobilized. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross talk between neighboring sites was negligible. The sandwich immunoassay was conducted on a single chip using model proteins, alpha-1-fetoprotein and beta2-microglobulin. A distinct current increase following the oxidation of hydrogen peroxide produced by the enzymatic reaction of glucose oxidase was observed, which indicates that the capture proteins could actually bind the target proteins. Two kinds of protein were detected independently on multiple sites with respective capture antibodies.

Psychosocial factors associated with quality of life in Japanese patients with human immunodeficiency virus infection.

The purpose of this study was to identify psychosocial factors, including coping style, that are associated with quality of life (QOL) in Japanese patients with human immunodeficiency virus (HIV) infection and to clarify the difference in coping style between patients with hemophilia and patients with sexually transmitted infection. We administered the World Health Organization QOL-26 questionnaire, the Mental Adjustment Toward Cancer scale, and the Social Support scale to 50 patients with HIV infection. Regarding QOL scores, Fighting Spirit was the positive coping style; Helplessness/Hopelessness and Anxious Preoccupation were negative coping styles. Psychological QOL scores in patients with hemophilia were lower than those in patients with sexually transmitted infection. Patients with hemophilia had a significantly weaker Fighting Spirit than patients with sexually transmitted infection.

Model tests on seismic performance of retaining walls

This paper is a preliminary report of ongoing studies for the purpose of developing a rational design method to evaluate stability for several types of conventional retaining walls during an earthquake. Since the seismic behavior of retaining walls is not yet completely understood, a series of tilting and shaking table tests on small scale model are performed. The observed failure pattern and critical condition are compared with the predicted ones that are calculated based on pseudo-static approach. The results show that they are qualitatively consistent. However, the angle of failure planes observed for shaking table tests is steeper than what is predicted. It may suggest that the seismic behavior assumed in the prediction is not fully correct (AU)