A chiral porous organic polymer COP-1 used as stationary phase for HPLC enantioseparation under normal-phase and reversed-phase conditions.

A spherical chiral porous organic polymer (POPs) COP-1 is synthesized by the Friedel-Crafts alkylation reaction of Boc-3-(4-biphenyl)-L-alanine (BBLA) and 4,4'-bis(chloromethyl)-1,1'-biphenyl (BCMBP), which was used as a novel chiral stationary phase (CSPs) for mixed-mode high-performance liquid chromatography (HPLC) enantioseparation. The racemic compounds were resolved in normal-phase liquid chromatography (NPLC) using n-hexane/isopropanol as mobile phase and reversed-phase liquid chromatography (RPLC) using methanol/water as mobile phase. The COP-1-packed column exhibited excellent separation performance toward various racemic compounds including alcohols, amines, ketones, esters, epoxy compounds, organic acids, and amino acids in NPLC and RPLC modes. The effects of analyte mass and column temperature on the separation efficiency of racemic compounds were investigated. In addition, the chiral resolution ability of the COP-1-packed column not only can be complementary in RPLC/NPLC modes but also exhibit a good chiral recognition complementarity with Chiralpak AD-H column and chiral porous organic cage (POC) NC1-R column. The relative standard deviations (RSD) (n = 5) of the retention time, resolution value, and peak area by repeated separation of 1-(4-chiorophenyl)ethanol are all below 3.0%. The COP-1 column shows high column efficiency (e.g., 17,320 plates m-1 for 1-(4-chlorophenyl)ethanol on COP-1 column in NPLC), high enantioselectivity, and good reproducibility toward various racemates. This work demonstrates that chiral POPs microspheres are promising chiral materials for HPLC enantioseparation.

Rapid determination of lignosulfonate depolymerization products by advanced polymer chromatography.

Depolymerized lignin products are very complex mixtures. Based on a depolymerization solution of commercially available sodium lignosulfonate under mild conditions, a fast and efficient method for the separation and direct characterization of the degree and efficiency of the acid-catalyzed depolymerization of lignin was developed in this study. Using an ultraviolet detector, the depolymerized lignosulfonate products were well separated and characterized according to the relative molar mass distribution on an advanced polymer chromatographic system with three ethylene-bridged hybrid columns having small pore sizes (45 Å) in series and tetrahydrofuran as the mobile phase. The developed advanced polymer chromatography method enabled the detection of low-molecular-weight lignin degradation products (Mn  = 260-1100 Da) with high peak resolutions in less than 7.2 min. Furthermore, preliminary advanced polymer chromatography studies to determine the influence of reaction temperature on the depolymerized products indicated that the depolymerized aromatics fell in several molecular weight ranges with an extremely low dispersity. This new approach can be used for the rapid analysis of lignin depolymerization products.

Percutaneous vertebroplasty performed with an 18-gauge needle for treatment of metastatic severe compression fracture of the cervical vertebral body.

Percutaneous vertebroplasty (PVP) is considered technically difficult in patients with severe vertebral body compression, especially in the cervical spine. In this study, PVP was successfully performed with the use of an 18-gauge angiographic needle in four patients with severe cervical compression fractures from metastatic disease. There were no complications, and relief of pain was immediate in all patients. This technique may be an alternative treatment for intractable pain secondary to severe cervical compression fractures.

Effect of glycine powder air-polishing as an adjunct in the treatment of peri-implant mucositis: a pilot clinical trial.

BACKGROUND: Glycine powder air-polishing (GPAP) has the potential to effectively erase biofilms and may improve the treatment efficacy of peri-implant mucositis. This pilot clinical trial evaluated the effect of GPAP as an adjunct in treating peri-implant mucositis. MATERIALS AND METHODS: Twenty-four subjects having at least one implant with peri-implant mucositis were randomly assigned to test (12 subjects with 17 implants) and control (12 subjects with 16 implants) groups. Following baseline assessment, all subjects received oral hygiene instruction and non-surgical debridement. In the test group, the sites with probing depth (PD) ≥4 mm were additionally treated by GPAP for 5 sec. Clinical parameters were measured at 1-week, 1-month, and 3-month recall visits. RESULTS: At the 3-month visit, the mean reductions in PD at site level were 0.93 ± 0.93 mm and 0.91 ± 0.98 mm in the test and control groups, respectively (P < 0.05), and no significant difference existed between two groups. Mean bleeding score was also significantly reduced in both groups after the intervention. No complications or discomfort were reported during the study. CONCLUSIONS: This pilot clinical trial suggests that non-surgical mechanical debridement may effectively control peri-implant mucositis, and adjunctive GPAP treatment seems to have a limited beneficial effect as compared with mechanical debridement alone. However, further clinical trials with a large sample size are needed to confirm this preliminary observation.

A uniform quantitative enzyme-linked immunosorbent assay for Coxsackievirus A16 antigen in vaccine.

Coxsackievirus A16 (CV-A16), one of major etiological agents of hand, foot and mouth disease (HFMD), causes outbreaks of the disease in young children all over the world. In order to promote the prevention and control of HFMD, the research and development of CV-A16 vaccine have been carried out in China. However, due to lacking of a recognized CV-A16 antigen detection method, the evaluation and quality control (QC) of vaccine effectiveness are greatly limited. In this study, we established a quantitative enzyme-linked immunosorbent assay (Q-ELISA) to determine the antigen concentration in CV-A16 vaccines that can be applied in manufacturing in China. A neutralizing antibody 16E1 was used as a capture antibody that can bind to various CV-A16 antigens of different subgenotypes, and an antiserum from CV-A16-immunized rabbit conjugated by HRP was suitable for detecting and quantifying CV-A16 antigens. The Q-ELISA was validated for specificity, linearity, accuracy, precision and robustness by using the CV-A16 antigen national standard (NS). Furthermore, we utilized the Q-ELISA to quantify antigen contents of vaccine bulks from six manufacturers and other intermediate products from one manufacturer. The results indicated that the Q-ELISA can satisfy the requirements of QC for all manufacturers involved.

Structure of SF-1 bound by different phospholipids: evidence for regulatory ligands.

Despite the fact that many nuclear receptors are ligand dependent, the existence of obligate regulatory ligands is debated for some receptors, including steroidogenic factor 1 (SF-1). Although fortuitously bound bacterial phospholipids were discovered in the structures of the SF-1 ligand-binding domain (LBD), these lipids might serve merely as structural ligands. Thus, we examined whether exogenously added phospholipids would exchange for these bacterial lipids and bind to SF-1. Here, we report the first crystal structure of the SF-1 LBD bound by the exchanged phosphatidylcholine. Although the bound phosphatidylcholine phospholipid mimics the conformation of bound bacterial phosphoplipids, two surface loops, L2-3 and L11-12, surrounding the entrance to the pocket vary significantly between different SF-1 LBD structures. Based on this observation, we hypothesized that a bound ligand might control the conformations of loops L2-3 and L11-12, and that conserved residues in these dynamic loops could influence ligand binding and the receptor function. Consistent with this hypothesis, impaired phospholipid exchange and diminished transcriptional activity were observed for loop L11-12 SF-1 mutants and for the loop L2-3 human mutant R255L. The endocrine disease associated with this L2-3 mutation coupled with our cellular and biochemical data suggest that critical residues at the mouth of the ligand-binding pocket have evolved for efficient binding of phospholipid ligands and for achieving optimal SF-1 activity.

[Evaluation of the in vitro quantitative measurement of II degree furcation involvements in mandibular molars by cone-beam computed tomography].

OBJECTIVE: To evaluate the accuracy of measurement of II degree furcation involvements in molars of dry mandibles by cone-beam computed tomography (CBCT). METHODS: Twenty molars with II degree furcation involvements in dry mandibles were examined directly and measured by CBCT. Eight parameters were selected to describe the exact appearance of each II degree furcation involvements, including vertical defect dimensions, horizontal defect dimensions and furcation entrance dimensions. The results were compared with the corresponding data obtained by probing and periapical radiograph. RESULTS: All furcation involvements could be correctly classified by CBCT. For 5 of 8 parameters, no significant difference was found between the data obtained by CBCT and probing measurements (P>0.05). The distances from furcation entrance to alveolar crest, to bottom of bone pocket, and to the deepest site of horizontal bone defect measured by CBCT were less than those probed directly (P<0.05), but the differences were less than 0.5 mm (0.21, 0.24, 0.35 mm, respectively). The localization of furcation entrance may cause the differences. Two out of 20 furcation involvements could not be detected on periapical radiographs, and only 2 of 8 parameters could be measured on periapical radiographs. CONCLUSION: CBCT could provide precise and detailed 3D images of II degree furcation involvements in vitro.

[Association of Fc gamma receptors IIIA gene polymorphisms with the susceptibility to periodontitis in Chinese patients].

OBJECTIVE: Fc gamma receptors IIIA (Fc gamma RIIIA) mediates phagocytosis by macrophages, and cytokine production by NK cells and lymphocytes. The Fc gamma R IIIA-158V/F polymorphism may play a role in the pathogenesis of periodontitis. This study was to detect Fc gamma R IIIA-158V/F genotypes in Chinese patients with different forms of periodontitis. METHODS: Thirty aggressive periodontitis (AgP) patients (aged from 9 to 40, 13 males and 17 females), 131 chronic periodontitis (CP) patients (aged from 22 to 63, 76 males and 55 females), and 47 healthy subjects (H) (aged from 22 to 75, 21 males and 26 females) consented to participate in this study. Swabs were taken from each subject by ten strokes on the buccal mucosa. DNA was isolated from each swab by Chelex-100 methods. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)was performed to determine the Fc gamma R IIIA-158 genotypes in each groups of subject. RESULTS: The allele frequency was in Hardy-Weinberg equilibrium in our cohort for the V-158F polymorphism in Fc gamma R IIIA gene (P>0.05). The detected frequency of 158F/F in AgP patients was significantly higher than that in CP patients (16.7% vs 0.8%, P<0.05), and also higher than that in H subjects (16.7% vs 4.3%) but was not statistically significant. No significant difference in distribution of the Fc gamma R IIIA-158V/F genotype was found between CP and H groups (P>0.05). CONCLUSION: The distribution of Fc gamma R IIIA-158F/F genotype might have effects on the phenotypes of periodontitis.

Correlation of MDR1 gene polymorphism with propofol combined with remifentanil anesthesia in pediatric tonsillectomy.

The motive of this study was to investigate the interaction between polymorphisms in the MDR1 gene and anesthetic effects following pediatric tonsillectomy. In total, 240 children undergoing tonsillectomy with preoperative propofol-remifentanil anesthesia were selected. Genomic DNA was extracted from the peripheral blood of children after operation, and the MDR1 gene polymorphisms of 2677 G>T/A, 1236 C>T and 3435 C>T were detected by direct sequencing. We tested mean arterial pressure, diastolic blood pressure, systolic blood pressure, and heart rate at several time-points: T0 (5 mins after the repose), T1 (0 min after tracheal intubation), T2 (5 mins after the tracheal intubation), T3 (0 min after the tonsillectomy), T4 (0 min after removal of the mouth-gag) and T5 (5 min after the extubation). The visual analog scale, the face, legs, activity, cry, and consolability pain assessment, and the Ramsay sedation score were recorded after the patients regained consciousness. Adverse reactions were also recorded. The time of induction, respiration recovery, eye-opening, and extubation of children with the CC genotype were found to be shorter compared to the CT + TT genotype of MDR1 1236C > T (all P <.05). The mean arterial pressure, diastolic blood pressure, systolic blood pressure, and heart rate were significantly reduced at T5 in children with the CC genotype (all P <.05). The visual analog scale at 1, 2, 4, and 8 hours post-operation, and the Ramsay sedation score at 5, 10, and 30 min after the extubation were decreased, while the face, legs, activity, cry, and consolability pain assessment score increased (all P <0.05). There was no statistically significant difference in the adverse reaction of MDR1 mutations (P> 0.05). It could be concluded that anesthetic effect following pediatric tonsillectomy in patients with the MDR1 1236C > T CC genotype was stronger than in those carrying the CT + TT genotype.

[Construction of 3D complex of porous beta-tricalcium phosphate/collagen scaffolds and dog periodontal ligament cells].

OBJECTIVE: To investigate the construction of 3D complex of porous beta-tricalcium phosphate/collagen scaffolds (beta-TCP/col) and dog periodontal ligament cells (PDLCs). METHODS: Dog PDLCs were isolated, cultured and identified. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of beta-TCP/col on the proliferation of PDLCs. The cells were seeded onto porous beta-TCP/col scaffolds. The cellular capability of adhesion and growth on porous beta-TCP/col surface was investigated visually by scanning electron microscopy (SEM). RESULTS: The cytotoxicity assay indicated that there was no significant difference between beta-TCP/col and the control during the 7 days (P>0.05). SEM showed cells successfully adhered to porous beta-TCP/col scaffolds and spread extensively. Matrix secretions were found on the cell surface. CONCLUSION: Porous beta-tricalcium phosphate/collagen scaffolds were of good biocompatibility to the dog periodontal ligament cells, and were potential ideal candidates for periodontal tissue engineering.

Teeth under High Occlusal Force may Reflect Occlusal Trauma-associated Periodontal Conditions in Subjects with Untreated Chronic Periodontitis.

OBJECTIVE: To determine the association of high occlusal force (HOF) with the signs of occlusal trauma and periodontal conditions in periodontitis patients, and elaborate the relevant clinical implications. METHODS: Periodontal parameters and signs of occlusal trauma were recorded for 807 teeth in 30 subjects with untreated chronic periodontitis. The T-scan II occlusal analysis system determined the HOF during maximum intercuspation, lateral excursion and protrusive excursion. The correlation of HOF with periodontal parameters and signs of occlusal trauma was analysed. RESULTS: Overall, the teeth with HOF existed mainly in molars and presented with deeper probing depth (PD) and higher frequency of bleeding on probing (BOP) than those without HOF. The fixed-effect analysis showed that HOF was positively correlated with PD and BOP (P < 0.05) in posterior teeth; widened periodontal ligament space on radiographs in upper (r = 0.179, P < 0.01) and lower posterior teeth (r = 0.205; P < 0.05); as well as functional mobility in upper posterior teeth (r = 0.168; P < 0.05). CONCLUSION: This study suggests that the posterior teeth with HOF in subjects with chronic periodontitis may reflect occlusal trauma-associated periodontal conditions that could probably increase the risk of further periodontal destruction. These findings may improve the clinical assessment of occlusal trauma and related periodontal conditions for better patient management and treatment outcomes.

Mutation analysis of the small heat shock protein 27 gene in chinese patients with Charcot-Marie-Tooth disease.

BACKGROUND: Charcot-Marie-Tooth (CMT) disease, the most common hereditary peripheral neuropathy, is highly clinically and genetically heterogeneous, and mutations in at least 18 genes have been identified. Recently, mutations in small heat shock protein 27 (Hsp27) were reported to cause CMT disease type 2F and distal hereditary motor neuropathy. OBJECTIVE: To investigate the frequency and phenotypic features of an Hsp27 mutation in Chinese patients with CMT disease. DESIGN: DNA samples from 114 unrelated patients with CMT disease were screened for mutations in Hsp27 by polymerase chain reaction and direct sequencing. A cosegregated study was performed using the MbiI restriction endonuclease, and 50 healthy control subjects were analyzed. Haplotype analysis was performed using 5 short tandem repeat markers to analyze whether the families with the same mutation probably had a common ancestor. RESULTS: One missense mutation, C379T, was detected in 4 autosomal dominant families with CMT disease type 2, and haplotype analysis indicated that the 4 families probably had a common founder. The frequency of the Hsp27 mutation is 0.9% (1/111) in Chinese patients with CMT disease in our study, and the phenotypes were characterized by later onset (age, 35-60 years) and mild sensory impairments. Electrophysiological findings showed moderately to severely slowed nerve conduction velocities in lower limb nerves but normal or mildly reduced velocities in upper limb nerves. CONCLUSIONS: To our knowledge, this is the first report of an Hsp27 mutation in the People's Republic of China. The C379T mutation in Hsp27 also causes CMT disease type 2, except for distal hereditary motor neuropathy, and the phenotypes are distinct from the family with CMT disease type 2F described previously. A mutation of Hsp27 may be uncommon in Chinese patients with CMT disease.

[Mutation analysis of small heat shock protein 27 gene in Chinese patients with Charcot-Marie-Tooth disease].

OBJECTIVE: To investigate the features of small heat shock protein 27 (HSP27) gene mutation in Chinese patients with Charcot-Marie-Tooth disease (CMT). METHODS: DNA samples from 114 CMT probands were screened for mutations in HSP27 gene by polymerase chain reaction and direct sequencing, and haplotype analysis was further carried out on the mutation detected families. RESULTS: One missense mutation C379T was detected in 4 autosomal dominant CMT2 families. Haplotype analysis indicated that the 4 families probably had a common ancestor. CONCLUSION: To the authors' knowledge, this is the first report of HSP27 gene mutation in Chinese patients with CMT, but it may be not common(0.90%). The C379T mutation in HSP27 gene also causes CMT2 except for distal hereditary motor neuropathy, thus providing further evidence that even the same mutation in the same gene may lead to distinct phenotypes.

[Detection of duplications or deletions of the PMP22 gene using real-time quantitative PCR].

OBJECTIVE: To detect the duplication or deletion of peripheral myelin protein 22(PMP22) gene in Chinese patients with Charcot-Marie-Tooth disease(CMT) or hereditary neuropathy with liability to pressure palsies(HNPP) using real-time quantitative polymerase chain reaction. METHODS: Duplications or deletions of PMP22 gene were detected in 113 CMT cases, 4 HNPP cases and 50 normal controls by using real-time quantitative PCR. RESULTS: Thirty-six of 113 CMT cases had the PMP22 duplication, 4 HNPP cases had the PMP22 deletion. No duplication or deletion was found in 50 normal controls. CONCLUSION: The PMP22 duplication rate in Chinese patients with CMT is 31.9%(36/113). PMP22 deletion is the common cause of HNPP.

Mutation analysis of small heat-shock protein 22 gene in Chinese patients with Charcot-Marie-Tooth disease.

OBJECTIVE: To study the characteristics of the mutation of small heat-shock protein 22 (HSP22) gene in Chinese patients with Charcot-Marie-Tooth (CMT) disease. METHODS: A CMT2L proband with 423(G--> T) mutation in HSP22 gene had been studied and reported by the present authors. In this study, mutation analysis of HSP22 gene was performed using polymerase chain reaction and DNA direct sequencing in 114 CMT probands. RESULTS: In the 114 CMT probands, a 582(C--> T)(T194T)samesense mutation was found in two unrelated families. CONCLUSION: The rate of HSP22 gene mutation in Chinese patients with CMT is as low as 0.87%(1/115).

[Comparative study of the effects of removing plaque by two toothbrushing methods].

OBJECTIVE: To compare the effects of removing plaque by two toothbrushing methods. METHODS: Forty subjects were chosen to take part in the randomized, examiner-blind, crossover study. RESULTS: Modified Bass method has better effect of removing plaque, especially that at the central margin than Vertical method (P<0.05). CONCLUSION: Modified Bass method needs to be recommended.

Mutation screening of Cx32 in Han Chinese patients with Charcot-Marie-Tooth disease.

OBJECTIVE: To investigate the Cx32 mutation features and the clinical manifestations of Chinese patients with Charcot-Marie-Tooth disease(CMT). METHODS: Twenty-four of 65 unrelated CMT patients were selected for Cx32 mutation screening after the exclusion of the CMT1A 1.5 Mb duplication and male-to-male transmission. The motor and sensory nerve conduction studies were performed in all probands and most of their affected family members to establish the clinical CMT1 ,CMT2 or CMT intermediate diagnosis. The presence of mutations in the coding region of Cx32 was detected by single-strand conformation polymorphism analysis combined with direct sequencing. RESULTS: We found 7 different point mutations in the coding region of Cx32 in a total of 7 families. All the patients were mildly to moderately affected with a clinical CMT1 or CMT intermediate diagnosis. The mutation Arg15Gln was inherited with X-linked recessive trait in family 1 involved in our study. The Arg75Trp mutation was detected in a family with X-linked dominant CMT and autosomal recessive nonsydromic hearing loss. The clinical phenotype of the Thr188Ala mutation was firstly reported. CONCLUSION: Seven different Cx32 point mutations were detected and the percentage of Chinese CMT families with Cx32 mutation is about 10% in our study. The inheritance model of CMT secondary to Cx32 mutation could be X-linked dominant, X-linked recessive or sporadic. Male patients are usually more severely affected than females with slower nerve conduction velocities. Cx32 mutation screening should be firstly performed in those CMT families without male-to-male transmission and CMT1A duplication.

[The characteristics of gene mutations in Chinese patients with Charcot-Marie-Tooth disease].

OBJECTIVE: To study the characteristics of gene mutations in Chinese patients with Charcot-Marie-Tooth disease (CMT). METHODS: Real-time quantitative PCR, PCR-SSCP, and/or direct sequencing were used to analyze the mutation of the pathogenic genes PMP22, MPZ, CX32, EGR2, GDAP1, NEFL, HSP22 and HSP27 in 113 probands of CMT families, 45 of which had family history, from different provinces in China. The whole family members of the subjects with abnormal electrophoretic bands and 50 normal controls underwent the same examination. RESULTS: Thirty-six cases of PMP22 duplication, 7 cases of CX32 mutation, 1 case of HSP22 mutation, 1 case of HSP27 mutation, 1 case of MPZ mutation, and 1 case of GDAP1 mutation were found in the 113 CMT probands. No point mutation was found in PMP22, EGR2 and NEFL genes. CONCLUSION: Among the Chinese CMT patients 31.9% are caused by PMP22 duplication, 6.2% by CX32, and 0.9% by HSP22, HSP27, MPZ and GDAP1. Point mutations of PMP22, EGR2 and NEFL are rare.

[The contrast study between single and double balloon bilateral dilatation of kyphoplasty].

OBJECTIVE: To contrast single and double balloon-inflated kyphoplasty for vertebral compression fractures (VCFs) and evaluate its clinical efficacy. METHODS: From May 2000 to May 2004, 90 consecutive procedures were performed in 58 patients who suffered from painful vertebral compression fractures, transferring tumour and angioma. Ninety vertebrae were inflated while 62 as A group were double balloon and 28 as B group were single balloon, fracture reduction and bone cement augmentation. Preoperative and postoperative symptom levels, variables, complications were recorded and the vertebral height and Cobb angle were measured and analyzed. RESULTS: All patients' pain was alleviated or disappeared without syndrome, and the vertebral height and Cobb angle of both groups were improved. The average recovery rate was 72.6% (22.9% approximately 100%), Cobb angle from 17.9 degrees (3.1 degrees approximately 31.6 degrees ) were corrected to 9.6 degrees (0.6 degrees approximately 28.2 degrees ), the average angle was 8.7 degrees (0.3 degrees approximately 27.2 degrees ), and the contrast between preoperative and postoperative showed obvious differences (P <0.001). The average recovery rate of A group was 77.6% (55.3% approximately 100%), B group was 64.3% (22.9% approximately 100%). The average postoperative Cobb angle of A group was 9.9 degrees (0.3 degrees approximately 27.2 degrees ), B group was 8.6 degrees (0.6 degrees approximately 19.8 degrees ) (P >0.05). CONCLUSIONS: As a promising minimally invasive surgery, balloon kyphoplasty can provide early relief of pain and improve the function as well as spinal alignment in treatment of painful compression fracture owing to recovering the vertebral height and Cobb angle of the vertebral body. Single balloon-inflated kyphoplasty can improve VCFs as double balloon.

[Longitudinal genetic effect on incisor position in cephalograms of female twins from six to twelve years old].

PURPOSE: A better understanding of the relative effect of genes and environment on craniofacial parameters should improve the possibilities and limitations of the orthodontic treatment and treatment planning. This is especially important for early treatment started in the mixed dentitions that is performed to enhance the dental and skeletal development before the eruption of the permanent dentition. METHODS: The sample used in this study consisted of lateral cephalograms of eighty-nine pairs of female twins in Beijing. With a mixed longitudinal method, the effective twins were 183 pairs (MZ 110, DZ 73). With the statistical methods of twins, the genetic and environmental effects were analyzed on the position of incisors in female twins from six to twelve years old. SPSS13.0 software package was used for independent-sample t test. RESULTS: Statistical comparisons revealed significant environmental effects on 6 positions of incisors (P<0.05), whilst family genetic effects on angle LI-MP were significant (P<0.05). CONCLUSION: Although the relatively low inherited proportions of dental variations could be found, genetic effects on incisor position should be carefully treated. Supported in part by a Grants-in-Aid for Scientific Research (International Scientific Exchange, 1994-1996) from the Japanese Ministry of Education, Culture, Science & Technology.