Biomimetic Mineralization of Large Enzymes Utilizing a Stable Zirconium-Based Metal-Organic Frameworks.

Enzymes are natural catalysts for a wide range of metabolic chemical transformations, including selective hydrolysis, oxidation, and phosphorylation. Herein, we demonstrate a strategy for the encapsulation of enzymes within a highly stable zirconium-based metal-organic framework. UiO-66-F4 was synthesized under mild conditions using an enzyme-compatible amino acid modulator, serine, at a modest temperature in an aqueous solution. Enzyme@UiO-66-F4 biocomposites were then formed by an in situ encapsulation route in which UiO-66-F4 grows around the enzymes and, consequently, provides protection for the enzymes. A range of enzymes, namely, lysozyme, horseradish peroxidase, and amano lipase, were successfully encapsulated within UiO-66-F4. We further demonstrate that the resulting biocomposites are stable under conditions that could denature many enzymes. Horseradish peroxidase encapsulated within UiO-66-F4 maintained its biological activity even after being treated with the proteolytic enzyme pepsin and heated at 60 °C. This strategy expands the toolbox of potential metal-organic frameworks with different topologies or functionalities that can be used as enzyme encapsulation hosts. We also demonstrate that this versatile process of in situ encapsulation of enzymes under mild conditions (i.e., submerged in water and at a modest temperature) can be generalized to encapsulate enzymes of various sizes within UiO-66-F4 while protecting them from harsh conditions (i.e., high temperatures, contact with denaturants or organic solvents).

Assessment of the occlusal outcomes in patients treated with orthognathic surgery and clear aligners.

OBJECTIVE: This retrospective two-centre study aimed to evaluate the occlusal outcomes in patients undergoing orthognathic surgery with clear aligners. METHODS: A retrospective chart review and occlusal outcomes for 15 patients (10 females and five males) with different types of dentofacial deformities in the anteroposterior, vertical and transverse dimensions, who underwent orthognathic surgery in conjunction with clear aligners were evaluated. Weighed Peer Assessment Rating (PAR) index scores of the pre-treatment and post-treatment digital models were used to assess initial complexity, final occlusal outcomes and degree of improvement with surgery and clear aligners. RESULTS: The mean post-treatment PAR score was 3.5 ± 2.54, which was a statistically significant improvement from the pre-treatment PAR score of 27.63 ± 12.09, an 87% improvement was achieved. All subcategories of the PAR index showed statistically significant improvement except for midline assessment component. CONCLUSIONS: Occlusal outcomes with aligners showed great improvement as indicated with the PAR index scores. Orthognathic surgical cases can be treated efficiently with aligners and future studies should compare occlusal outcomes between orthognathic surgical patients treated with clear aligners and those treated with fixed appliances.

Mechanochemical Enhancement of the Structural Stability of Pseudorotaxane Intermediates in the Synthesis of Rotaxanes.

Mechanochemical syntheses of rotaxanes have attracted considerable attention of late because of the superior reaction rates and higher yields associated with their production compared with analogous reactions carried out in solution. Previous investigators, however, have focused on the demonstration of the mechanochemical syntheses of rotaxanes per se, rather than on studying the solid-phase host-guest molecular interplay related to their rapid formation and high yields. In this investigation, we attribute the lower yields of rotaxanes prepared in solution to the limited concentration and a desolvation energy penalty that must be compensated for by host-guest interactions during complexation that precedes the templation leading to rotaxane formation. It follows that, if the desolvation energy can be removed and higher concentrations can be attained, even weak host-guest interactions can drive the complexation of host and guest molecules efficiently. In order to test this hypothesis, we chose two host-guest pairs of permethylated pillar[5]arene/1,6-diaminohexane and permethylated pillar[5]arene/2,2'-(ethylenedioxy)bis(ethylamine) for the simple reason that they exhibit extremely low binding constants (2.7 ± 0.4 M-1 when 1,6-diaminohexane is the guest and <0.1 M-1 when 2,2'-(ethylenedioxy)bis(ethylamine) is the guest in CDCl3; i.e., ostensibly no pseudorotaxane formation is observed). We argue that the amount of pseudorotaxanes formed in the solid state is responsive to mechanical treatments or otherwise and changes in temperature during stoppering reactions. Compared to the amount of pseudorotaxanes that can be obtained in solution, large quantities of pseudorotaxanes are formed in the solid state because of concentration and desolvation effects. This mechanochemical enhancement of pseudorotaxane formation is referred to as a self-correction in the current investigation. Rotaxanes based on permethylated pillar[5]arene/1,6-diaminohexane and permethylated pillar[5]arene/2,2'-(ethylenedioxy)bis(ethylamine) have been synthesized in much higher yields compared to those obtained in solution, aided and abetted by self-correction effects during mechanical treatments and heating at a mild temperature of 50 °C.

Redox-Active Phenanthrenequinone Triangles in Aqueous Rechargeable Zinc Batteries.

Aqueous rechargeable zinc batteries (ZBs) have received considerable attention recently for large-scale energy storage systems in terms of rate performance, cost, and safety. Nevertheless, these ZBs still remain a subject for investigation, as researchers search for cathode materials enabling high performance. Among the various candidate cathode materials for ZBs, quinone compounds stand out as candidates because of their high specific capacity, sustainability, and low cost. Quinone-based cathodes, however, suffer from the critical limitation of undergoing dissolution during battery cycling, leading to a deterioration in battery life. To address this problem, we have introduced a redox-active triangular phenanthrenequinone-based macrocycle (PQ-Δ) with a rigid geometry and layered superstructure. Notably, we have confirmed that Zn2+ ions, together with H2O molecules, can be inserted into the PQ-Δ organic cathode, and, as a consequence, the interfacial resistance between the cathode and electrolytes is decreased effectively. Density functional theory calculations have revealed that the low interfacial resistance can be attributed mainly to decreasing the desolvation energy penalty as a result of the insertion of hydrated Zn2+ ions in the PQ-Δ cathode. The combined effects of the insertion of hydrated Zn2+ ions and the robust triangular structure of PQ-Δ serve to achieve a large reversible capacity of 210 mAh g-1 at a high current density of 150 mA g-1, along with an excellent cycle-life, that is, 99.9% retention after 500 cycles. These findings suggest that the utilization of electron-active organic macrocycles, combined with the low interfacial resistance associated with the solvation of divalent carrier ions, is essential for the overall performance of divalent battery systems.

The emerging era of supramolecular polymeric binders in silicon anodes.

Silicon (Si) anode is among the most promising candidates for the next-generation high-capacity electrodes in Li-ion batteries owing to its unparalleled theoretical capacity (4200 mA h g-1 for Li4.4Si) that is approximately 10 times higher than that of commercialized graphitic anodes (372 mA h g-1 for LiC6). The battery community has witnessed substantial advances in research on new polymeric binders for silicon anodes mainly due to the shortcomings of conventional binders such as polyvinylidene difluoride (PVDF) to address problems caused by the massive volume change of Si (300%) upon (de)lithiation. Unlike conventional battery electrodes, polymeric binders have been shown to play an active role in silicon anodes to alleviate various capacity decay pathways. While the initial focus in binder research was primarily to maintain the electrode morphology, it has been recently shown that polymeric binders can in fact help to stabilize cracked Si microparticles along with the solid-electrolyte-interphase (SEI) layer, thus substantially improving the electrochemical performance. In this review article, we aim to provide an in-depth analysis and molecular-level design principles of polymeric binders for silicon anodes in terms of their chemical structure, superstructure, and supramolecular interactions to achieve good electrochemical performance. We further highlight that supramolecular chemistry offers practical tools to address challenging problems associated with emerging electrode materials in rechargeable batteries.

Energy Band-Gap Engineering of Conjugated Microporous Polymers via Acidity-Dependent in Situ Cyclization.

Conjugated microporous polymers (CMPs) offer a unique structure integrating π-conjugated backbone into a porous network for the simultaneous transport of charges and materials. However, tuning electronic properties of CMPs so far has been limited to an approach of varying the monomers, and the precious metal catalysts are inevitably needed for the C-C coupling reaction. Here, we present a powerful strategy to synthesize CMPs and precisely tune their optical band gap and surface area through metal-free in situ cyclization reaction controlled by the acid strength of acid catalysts. Notably, the optical band gap of CMPs showed a linear relationship with the p Ka of acid catalysts, which provides us with the ability to obtain the desired band gap between 2.07 and 3.35 eV, falling in the range of the visible solar spectrum. Moreover, CMPs exhibited excellent textural properties such as microporosity and high specific surface area.

Epithelial cell shape change of Drosophila as a biomonitoring model for the dose assessment of environmental radiation.

Inevitable exposure to ionizing radiation from natural and human-made sources has been increasing over time. After nuclear disasters, such as the Fukushima accident, the public concerns on health risk of radiation exposure because of radioactive contamination of the environment have increased. However, it is very difficult to assess the biological effects of exposure caused by environmental radiation. A reliable and rapid bioassay to monitor the physiological effects of radiation exposure is therefore needed. Here, we quantitatively analyzed the changes in cell shape in Drosophila epidermis after irradiation as a model for biomonitoring of radiation. Interestingly, the number of irregularly shaped epithelial cells was increased by irradiation in a dose-dependent manner. A dose-response curve constructed with the obtained data suggests that the measurement of the number of irregular shaped cell in the epidermis is useful for the assessment of radiation dose. In addition, a comparison of the variation in the different samples and the data scored by different observers showed that our evaluation for cellular morphology was highly reliable and accurate and would, therefore, have immense practical application. Overall, our study suggests that detection of morphological changes in the epithelial cells is one of the efficient ways to quantify the levels of exposure to radioactive radiation from the environment.

Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis.

The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 µm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-ß transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-ß-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-ß transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.

CYP2E1 regulates the development of radiation-induced pulmonary fibrosis via ER stress- and ROS-dependent mechanisms.

Radiation-induced pulmonary fibrosis (RIPF) is one of the most common side effects of lung cancer radiotherapy. This study was conducted to identify the molecular mechanism responsible for RIPF. We revealed that the transcriptional level of cytochrome P450 2E1 (CYP2E1) was elevated by examining expression profile analysis of RIPF mouse models. We also confirmed that CYP2E1 regulated levels of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in alveolar epithelial type II (AE2) cells and lung fibroblasts. Inhibition of CYP2E1 via its siRNA or inhibitor significantly attenuated epithelial-to-mesenchymal transition and apoptosis of AE2 cells, as well as myofibroblast formation induced by radiation. Finally, the effects of a CYP2E1 inhibitor on development of RIPF were evaluated by in vivo studies. Taken together, the results of the present study suggest that CYP2E1 is an important mediator of RIPF development that functions by increasing cellular ER stress and ROS levels.

Effects of Bisphosphonate Administration on Cleft Bone Graft in a Rat Model.

OBJECTIVE: Bone grafts in patients with cleft lip and palate can undergo a significant amount of resorption. The aim of this study was to investigate the effects of bisphosphonates (BPs) on the success of bone grafts in rats. DESIGN: Thirty-five female 15-week-old Fischer F344 Inbred rats were divided into the following experimental groups, each receiving bone grafts to repair an intraoral CSD: (1) Graft/saline: systemic administration of saline and (2) systemic administration of zoledronic acid immediately following surgery (graft/BP/T0), (3) 1 week postoperatively (graft/BP/T1), and (4) 3 weeks postoperatively (graft/BP/T2). As an additional control, the defect was left empty without bone graft. MAIN OUTCOME MEASURES: Microcomputed tomography and histologic analyses were performed in addition to evaluation of osteoclasts through tartrate-resistant acid phosphatase staining. RESULTS: Bone volume fraction (bone volume/tissue volume) for the delayed BP treatment groups (graft/BP/T1 = 45.4% ± 8.8%; graft/BP/T2 = 46.1% ± 12.4%) were significantly greater than that for the graft/saline group (31.0% ± 7.9%) and the graft/BP/T0 (27.6% ± 5.9%) 6 weeks postoperatively (P < .05). Hematoxylin and eosin staining confirmed an evident increase in bone volume and fusion of defect margins with existing palatal bone in the graft/BP/T1 and graft/BP/T2 groups. The graft/BP/T0 group showed the lowest bone volume with signs of acute inflammation. CONCLUSIONS: Delayed BP administration following cleft bone graft surgery led to significant increase in bone volume and integration compared with saline controls. However, BP injection immediately after the surgery did not enhance bone volume, and rather, may negatively affect bone graft incorporation.

Inhibition of hedgehog signalling attenuates UVB-induced skin photoageing.

The hedgehog (Hh) signalling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis and progression of a variety of aggressive human cancers including skin cancer. Despite its importance, little is known about its role in photoageing, a type of UV-induced skin lesions. In this study, we investigated the involvement of Hh signalling in the photoageing process as well as the use of an Hh-regulating alkaloid compound as a novel therapeutic drug to regulate photoageing in keratinocytes. We found that UVB induced Hh signalling by the expression of Hh ligands and Hh-mediated transcription factors, respectively. Moreover, UVB-induced Hh activation relied on mitogen-activated protein kinase (p38, ERK and JNK) activity and inflammatory responses (upregulation of COX-2, IL-1ß, IL-6 and TNF-α), resulting in premature senescence and photoageing in vitro and in vivo. Notably, a selected Hh inhibitor, evodiamine, mediated photoageing blockade in a mouse skin model. Taken together, our findings demonstrated that Hh signalling is associated with UVB-induced photoageing, while pharmacological inhibition of Hh signalling significantly reduced experimental photoageing, indicating its potential for use as a therapeutic target for this disease.

Dermal Toxicity of Flake-Like α-Alumina Pigments.

Aluminum is one of the most widely used nonferrous metals and an important industrial material, especially for automotive coatings. However, potential toxicity caused by aluminum in humans limits the used of this metal. α-alumina is the most stable form of aluminum in various phases. Although the results of studies evaluating the dermal toxicity of α-alumina remained unclear, this compound can still be used as a pigment in cosmetics for humans. In the current study, we further evaluated the dermal cytotoxic effects of α-alumina on human skin cells and an in vivo mouse model. We also measured the in vitro penetration profile of flake-like α-alumina in porcine skin and assessed the degree of cellular metabolic disorders. Our findings demonstrated that treatment with flake-like α-alumina did not significantly affect cell viability up to 24 h. This compound was found to have a non-penetration profile based on a Franz modified diffusion cell assay. In addition, flake-like α-alumina was not found to induce dermal inflammation as assessed by histology of epidermal architecture, hyperplasia, and the expression of Interleukin-1ß and Cyclooxygenase-2. Results of the cellular metabolic disorder assay indicated that flake-like α-alumina does not exert a direct effect on human skin cells. Taken together, our findings provided not only evidence that flake-like α-alumina may serve as a pearlescent pigment in cosmetics but also experimental basis utilizing α-alumina for human application. Our results also obviously provide new insight of the further toxicity study to aluminum based nanoparticles for skin.

Hyperbranched ß-cyclodextrin polymer as an effective multidimensional binder for silicon anodes in lithium rechargeable batteries.

Polymeric binders play an important role in electrochemical performance of high-capacity silicon (Si) anodes that usually suffer from severe capacity fading due to unparalleled volume change of Si during cycling. In an effort to find efficient polymeric binders that could mitigate such capacity fading, herein, we introduce polymerized ß-cyclodextrin (ß-CDp) binder for Si nanoparticle anodes. Unlike one-dimensional binders, hyperbranched network structure of ß-CDp presents multidimensional hydrogen-bonding interactions with Si particles and therefore offers robust contacts between both components. Even the Si nanoparticles that lost the original contacts with the binder during cycling recover within the multidimensional binder network, thus creating a self-healing effect. Utilizing these advantageous features, ß-CDp-based Si electrode shows markedly improved cycling performance compared to those of other well-known binder cases, especially when combined with linear polymers at an appropriate ratio to form hybrid binders.

Enhanced performance on polymeric light-emitting diode with non-metallic ion in the water soluble non-conjugated polymer.

We have fabricated a polymer light-emitting diode (PLED) from the conventional blue-emitting polymer, polyfluorene (PFO), by constructing a multilayer structure with non-metal ion containing water soluble non-conjugated polymer, polyurethane with F- ion (PU:F-), on the top of the PFO. The device with PU:F- layer shows a maximum luminance of 5294 cd/m2 at an applied voltage of 10 V while the one without PU:F- layer shows only 4439 cd/m2 at the same applied voltage. We propose the improvement of device performance with PU:F- layer was due to not only an effective hole blocking at the polymer-polymer interface but also increase of electric field strength with anode after electro-stactic repulsion between electrons from the cathod and anions from the water soluble polymer layer. We will discuss the effect of multilayer polymer structure in PLED in terms of current/voltage characteristics, luminance, and quantum efficiency related with the applied bias.

PIM1 kinase inhibitors induce radiosensitization in non-small cell lung cancer cells.

Radiotherapy plays a critical role in the treatment of non-small cell lung cancer (NSCLC). However, radioresistance is a major barrier against increasing the efficiency of radiotherapy for NSCLC. To understand the mechanisms underlying NSCLC radioresistance, we previously focused on the potential involvement of PIM1, PRAS40, FOXO3a, 14-3-3, and protein phosphatases. Among these proteins, PIM1 functioned as an oncogene and was found to act as a crucial mediator in radioresistant NSCLC cells. Therefore, we investigated the use of PIM1-specific inhibitors as novel therapeutic drugs to regulate radiosensitivity in NSCLC. After structure-based drug selection, SGI-1776, ETP-45299, and tryptanthrin were selected as candidates of PIM1 inhibitors that act as radiosensitizers. With irradiation, these drugs inhibited only PIM1 kinase activity without affecting PIM1 mRNA/protein levels or cellular localization. When PIM1 kinase activity was suppressed by these inhibitors, PRAS40 was not phosphorylated. Consequently, unphosphorylated PRAS40 did not form trimeric complexes with 14-3-3 and FOXO3a, leading to increased nuclear localization of FOXO3a. Nuclear FOXO3a promoted the expression of pro-apoptotic proteins such as Bim and FasL, resulting in a radiosensitizing effect on radioresistant NSCLC cells. Moreover, an in vivo xenograft mouse model confirmed this radiosensitizing effect induced by PIM1 inhibitors. In these model systems, tumor volume was significantly reduced by a combinational treatment with irradiation and PIM1 inhibitors compared to irradiation alone. Taken together, our findings provided evidence that PIM1-specific inhibitors, SGI-1776, ETP-45299, and tryptanthrin, can act as novel radiosensitizers to enhance the efficacy of radiotherapy by inhibiting irradiation-induced signaling pathway associated with radioresistance.

Neuroprotective effects of bornyl acetate on experimental autoimmune encephalomyelitis via anti-inflammatory effects and maintaining blood-brain-barrier integrity.

BACKGROUND: Bornyl acetate (BA), a chemical component of essential oil in the Pinus family, has yet to be actively studies in terms of its therapeutic effect on numerous diseases, including autoimmune diseases. PURPOSE: This study aimed to investigate the pharmacological effects and molecular mechanisms of BA on myelin oligodendrocyte glycoprotein (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) mice in an animal model of multiple sclerosis (MS), a representative autoimmune disease in central nervous system. METHODS: BA (100, 200, or 400 mg/kg) was orally treated to EAE mice once daily for 30 days after immunization for the behavioral test and for the 16th-18th days for the histopathological and molecular analyses, from the onset stage (8th day) of EAE symptoms. RESULTS: BA mitigated behavioral dysfunction (motor disability) and demyelination in the spinal cord that were associated with the down-regulation of representative pro-inflammatory cytokines (interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha), enzymes (cyclooxygenase-2 and inducible nitric oxide synthase), and chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1 alpha, and regulated on activation), and decreased infiltration of microglia (CD11b+/CD45+(low)) and macrophages (CD11b+/CD45+(high)). The anti-inflammatory effect of BA was related to the inhibition of mitogen-activated protein kinases and nuclear factor-kappa B pathways. BA also reduced the recruitment/infiltration rates of CD4+ T, Th1, and Th17 cells into the spinal cords of EAE mice, which was related to reduced blood-spinal cord barrier (BSCB) disruption. CONCLUSION: These findings strongly suggest that BA may alleviate EAE due to its anti-inflammatory and BSCB protective activities. This indicates that BA is a potential therapeutic agent for treating autoimmune demyelinating diseases including MS.

Modified polyvinyl alcohol layer with hydrophobic surface for the passivation of pentacene thin-film transistor.

We modified the surface of a polyvinyl alcohol (PVA) layer by self assembly monolayer technique using a fluorine substituted silane compound (1H,1H,2H,2H-perfluorooctyl-trichlorosilane: FTS) to protect a pentacene thin-film transistor (TFT) from O2 and H2O. Surface modified PVA showed very low surface energy with water contact angle of 106.2 degrees. Surface treatment of PVA layer on pentacene TFT device was done in toluene solvent and we did not observe any damage to the PVA layer or pentacene TFT devices during surface modification process. Pentacene TFT with surface modified PVA passivation layer exhibited very stable TFT operation with almost no field effect mobility drop or threshold voltage shift up to 400 hrs. The performance of unpassivated OTFTs exponentially degraded and almost failed in 290 hrs. We propose that modified PVA layer can be used as a good passivation layer for oxygen and water in OTFT.

Optimizing hemocompatibility of surfactant-coated silver nanoparticles in human erythrocytes.

Several recent biological science studies have been focused on nanotechnology and nanomaterials due to their potential use in biomedicine. Drug delivery systems are an example of biomedical applications utilizing nanoparticles. Silver nanoparticles (AgNPs) can be used for these drug delivery systems. However, the effects of cytotoxicity caused by AgNPs are not fully understood. Determining the optimal characteristics to facilitate the biocompatibility of AgNPs is an important subject for application. In the present study, human erythrocytes were used as an in vitro model to examine the size, dose, and coating surfactant-dependent cytotoxicity of AgNPs. Our results demonstrated that polyvinylpyrrolidone (PVP) was a more suitable surfactant than polyethylene glycol (PEG) for AgNPs capping. In addition, we determined the appropriate particular size and dosage of AgNPs to reduce human erythrocytes hemolysis. Membrane damages including hemolysis, potassium efflux, protein leakage, and alterations in cell shape and membrane fragility were minimized with 100-nm AgNP particles. This study provides novel insights into AgNPs cytotoxicity and a basis for utilizing AgNPs for diagnostic and therapeutic applications.

Gene engineering, purification, crystallization and preliminary X-ray diffraction of cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis membrane protein.

Cytochrome P450s (P450s) are the most versatile biological catalysts in plants; however, because the structure of the P450s has not been fully established, their broad substrate specificity has been limitedly discussed. p-coumarate-3-hydroxylase (C3H) is an essential enzyme for the biosynthesis of phenolic natural products in plants, but all attempts to express and purify C3H, have failed. In this research, we developed a bacterial expression of Arabidopsis C3H by combinational mutagenesis and purified C3H as a catalytically active form. The modified C3H could be purified in the absence of detergent, and crystallized in two forms (orthorhombic and trigonal space group) under different conditions. X-ray diffraction was processed to a 4.0 Å resolution (first type crystal) and a 3.8 Å resolution (second type crystal). Although the diffraction results of C3H(mod) crystals are not enough to determine crystallographic structure due to low resolution, the simplicity and rapidity of this technology are competitive advantages in comparison with other methods, and may contribute to structural analyses of other membrane proteins including P450s family.