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Molecular tests are the mainstay of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, their accessibility can be limited by the long examination time and inability to evaluate multiple samples at once. This study evaluated the analytical performance of the newly developed rapid molecular assays GENECUBE® HQ SARS-CoV-2 and GENECUBE® FLU A/B. This prospective study was conducted between 14 December 2020 and 9 January 2021 at a polymerase chain reaction (PCR) center. Samples were collected from the nasopharynx with flocked swabs. Molecular tests were performed with the GENECUBE® system and reference reverse transcription (RT)-PCR, and the results of the two assays were compared. Among 1065 samples, 81 (7.6%) were positive for SARS-CoV-2 on the reference RT-PCR. Three showed discordance between GENECUBE® HQ SARS-CoV-2 and the reference RT-PCR; the total, positive, and negative samples of concordance for the two assays were 99.7%, 100%, and 99.7%, respectively. All discordant cases were positive with GENECUBE® HQ SARS-CoV-2 and negative with the reference RT-PCR. SARS-CoV-2 was detected in all three samples using another molecular assay for SARS-CoV-2. For GENECUBE® FLU A/B, the total, positive, and negative samples of concordance for the two assays were 99.5%, 100%, and 99.1%. The GENECUBE® HQ SARS-CoV-2 and GENECUBE® FLU A/B demonstrated sufficient analytical performance to detect SARS-CoV-2 and influenza virus A/B.

Introduction. Resistance against macrolide antibiotics in Mycoplasma pneumoniae is becoming non-negligible in terms of both appropriate therapy and diagnostic stewardship. Molecular methods have attractive features for the identification of Mycoplasma pneumoniae as well as its resistance-associated mutations of 23S ribosomal RNA (rRNA). Hypothesis/Gap Statement. The automated molecular diagnostic sytem can identify macrolide-resistant M. pneumoniae . Aim. To assess the performance of an automated molecular diagnostic system, GENECUBE Mycoplasma, in the detection of macrolide resistance-associated mutations. Methodology. To evaluate whether the system can distinguish mutant from wild-type 23S rRNA, synthetic oligonucleotides mimicking known mutations (high-level macrolide resistance, mutation in positions 2063 and 2064; low-level macrolide resistance, mutation in position 2067) were assayed. To evaluate clinical oropharyngeal samples, purified nucleic acids were obtained from M. pneumoniae -positive samples by using the GENECUBE system from nine hospitals. After confirmation by re-evaluation of M. pneumoniae positivity, Sanger-based sequencing of 23S rRNA and mutant typing using GENECUBE Mycoplasma were performed. Results. The system reproducibly identified all synthetic oligonucleotides associated with high-level macrolide resistance. Detection errors were only observed for A2067G (in 2 of the 10 measurements). The point mutation in 23S rRNA was detected in 67 (26.9 %) of 249 confirmed M. pneumoniae -positive clinical samples. The mutations at positions 2063, 2064 and 2617 were observed in 65 (97.0 %), 2 (3.0 %) and 0 (0.0 %) of the 67 samples, respectively. The mutations at positions 2063 and 2064 were A2063G and A2064G, respectively. The results from mutant typing using GENECUBE Mycoplasma were in full agreement with the results from sequence-based typing. Conclusion. GENECUBE Mycoplasma is a reliable test for the identification of clinically significant macrolide-resistant M. pneumoniae .

BackgroundMolecular tests are the mainstay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, their accessibility can be limited by the long examination time and inability to evaluate multiple samples at once. This study evaluated the analytical performance of the newly developed rapid molecular assays GENECUBE® HQ SARS-CoV-2 and the GENECUBE® FLU A/B.MethodThis prospective study was conducted between December 14, 2020, and January 9, 2021, at a polymerase chain reaction (PCR) center. Samples were collected from the nasopharynx with flocked swabs. Molecular tests were performed with the GENECUBE® system and reference reverse transcription (RT)-PCR, and the results of the two assays were compared.ResultAmong 1065 samples, 81 (7.6%) were positive for SARS-CoV-2 on the reference RT-PCR. Three showed discordance between GENECUBE® HQ SARS-CoV-2 and the reference RT-PCR; the total, positive and negative samples of concordance for the two assays were 99.7%, 100%, and 99.7%, respectively. All discordant cases were positive for GENECUBE® HQ SARS-CoV-2 and negative for the reference RT-PCR. SARS-CoV-2 was detected from all three samples by another molecular assay for SARS-CoV-2. For the GENECUBE® FLU A/B, the total, positive and negative samples of concordance for the two assays were 99.5%, 100%, and 99.1%.ConclusionThe GENECUBE® HQ SARS-CoV-2 and GENECUBE® FLU A/B demonstrated sufficient analytical performance to detect SARS-CoV-2 and influenza virus A/B.Key pointsWe prospectively evaluated the analytical performance of the newly developed rapid molecular assays GENECUBE® HQ SARS-CoV-2 and the GENECUBE® FLU A/B. The two assays showed >99% concordance rate compared with a reference PCR, which indicated their sufficient analytical performance to detect SARS-CoV-2 and influenza virus A/B.

Recently, a new rapid assay for the detection of tcdB gene of Clostridioides difficile was developed using the GENECUBE. The assay can directly detect the tcdB gene from stool samples without a purification in approximately 35 minutes with a few minutes of preparation process. We performed a prospective comparative study of the performance of the assay at eight institutions in Japan. Fresh residual stool samples (Bristol stool scale ≥5) were used and comparisons were performed with the BD MAX Cdiff assay and toxigenic cultures. For the evaluation of 383 stool samples compared with the BD MAX Cdiff assay, the sensitivity, and specificity of the two assays was 99.0% (379/383), 98.1% (52/53), 99.1% (327/330), respectively. In the comparison with toxigenic culture, the total, sensitivity, and specificity were 96.6% (370/383), 85.0% (51/60), and 98.8% (319/323), respectively. The current investigation indicated the GENECUBE Clostridioides difficile assay has equivalent performance with the BD MAX Cdiff assay for the detection of tcdB gene of C. difficile.

The present study aimed to develop a practical method for preparing nanosuspension formulations of poorly water-soluble compounds for enhancing oral absorption in toxicology studies in the discovery stage. To obtain a suitable nanosuspension formulation for the intended purpose, formulations were optimized with a focus on the following characteristics: i) containing a high drug concentration, ii) consisting of commonly used excipient types in proper quantities for toxicology studies, iii) having long-term stability, and iv) having versatility for use with diverse compounds. Test compounds were milled with various excipients by wet media milling methods using a mixer mill (10 mg/batch) and a rotation/revolution mixer (0.5 g/batch). As a result, 100 mg/mL nanosuspensions of all 11 test compounds could be prepared with an optimized dispersing agent, 0.5% hydroxypropyl methylcellulose (HPMC) (3 cP)-0.5% Tween 80. Notably, it was found that the molecular weight of HPMC influenced not only particle size but also the stability of nanosuspensions and they were stable for 4 weeks at 5°C. The nanosuspensions increased in vitro dissolution rates and provided 3.9 and 3.0 times higher Cmax and 4.4 and 1.6 times higher area under the concentration-time curve from 0-24 h (AUC0-24 h) in rats (oral dose of 300 mg/kg) for cilostazol and danazol, respectively. In conclusion, applying a wet media milling method with the combination of HPMC of a small molecular weight and Tween 80 as a dispersing agent, nanosuspensions can be practically prepared and conveniently utilized for enhancing the oral absorption of poorly water-soluble compounds in toxicology studies in the discovery stage.

The in vitro micronucleus (MN) test is widely used for screening genotoxic compounds, but it often produces false-positive results. To consider the significance of positive results, it is important to know whether DNA adducts are formed in the cells treated with the test compound. Recently, Matsuda et al. developed the DNA adductome approach to detect DNA adducts comprehensively ([4] Kanaly, et al., Antioxid. Redox Signal., 2006, 8, 993-1001). We applied this method to assess the DNA-damaging capability of in vitro MN test-positive compounds. CHL/IU cells were treated with compounds from three categories: (1) carcinogens causing DNA alkylation, ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; (2) carcinogens producing DNA bulky adducts, 2-amino-6-phenyl-1-methylimidazo[4,5-b]pyrene, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and 4-nitroquinoline-1-oxide, and (3) non-carcinogens, caffeine, maltol, and sodium chloride, with or without metabolic activation. With the conditions in which all test compounds gave positive results in the MN tests, DNA was extracted from the cells and hydrolyzed to deoxyribonucleosides, which were subsequently subjected to LC/ESI-MS/MS analysis. All carcinogens (categories 1 and 2) produced various DNA adduct peaks, and some of the m/z peak values corresponded to known adducts. No non-carcinogens produced DNA adducts, indicating that these compounds produced MN through different mechanisms from the adduct formation. These results indicate that the adductome approach is useful to demonstrate DNA damage formation of MN test-positive compounds and to understand their mechanisms of action.

Compared to a Si (Silicon) power device, an SiC (Silicon Carbide) power device is regarded as an indispensable power device for realizing the next generation power converter, because of its low switching loss, higher speed, and capability to operate at high temperatures. Toshiba developed an SiC hybrid IGBT (Insulated gate type bipolar transistor) with a withstand voltage of 3.3 kV for 1,500 V DC catenary systems [1] [2]. This is applied to the traction inverter of Nishi-Nippon Railroad Co., Ltd.'s 3000 Series train cars. By utilizing the reduced switching loss characteristic of SiC hybrid IGBT, improvements in the inverter output current and carrier frequency of PWM control are now possible while retaining the performance of a conventional inverter cooler. With the purpose of conserving energy, this paper introduces the development and verification test results of the traction control focusing on: (1) Enhancing regenerative brake force in high speed region (2) Reducing motor harmonics loss by multi-pulsing of PWM control.

The olfactory system is indispensable to the survival of animals in finding foods and for the reproductive process. Odorant signals are conveyed through olfactory sensory neurons to the olfactory bulb, which modifies the signals and relays them to the neocortex. In the present study, a "full-length" cDNA library was constructed from the main and accessory olfactory bulbs of 5-week-old male pigs, in order to elucidate the expressed genes. The average insert size of the library was estimated to be 1.7 kb based on 54 randomly-selected clones. One thousand randomly selected clones were subjected to sequencing, and the resulting 883 sequences were then clustered into 753 sequences based on similarity. Since 723 of the 753 sequences had sufficient sequence information for homology analysis, the 723 sequences were subjected to BLAST analysis against GenBank/EMBL/DDBJ; 655 out of the 723 sequences showed similarities with known genes, and the remaining 68 were indicated to be novel sequences. The full-length rate of the library was estimated to be ca. 80%, using 70 sequences corresponding to human full-length cDNAs. The full-length cDNA sequences of a single gene appearing more than 6 times in the analysis were aligned to determine major transcription initiation sites for SLC25A, CKB, TUBB4, TUBB, YWHAH, TUBB2, and CNP genes.

The present study aimed to develop a practical method for preparing nanosuspension formulations of poorly water-soluble compounds for enhancing oral absorption in toxicology studies in the discovery stage. To obtain a suitable nanosuspension formulation for the intended purpose, formulations were optimized with a focus on the following characteristics: i) containing a high drug concentration, ii) consisting of commonly used excipient types in proper quantities for toxicology studies, iii) having long-term stability, and iv) having versatility for use with diverse compounds. Test compounds were milled with various excipients by wet media milling methods using a mixer mill (10 mg/batch) and a rotation/revolution mixer (0.5 g/batch). As a result, 100 mg/mL nanosuspensions of all 11 test compounds could be prepared with an optimized dispersing agent, 0.5% hydroxypropyl methylcellulose (HPMC) (3 cP)-0.5% Tween 80. Notably, it was found that the molecular weight of HPMC influenced not only particle size but also the stability of nanosuspensions and they were stable for 4 weeks at 5°C. The nanosuspensions increased in vitro dissolution rates and provided 3.9 and 3.0 times higher Cmax and 4.4 and 1.6 times higher area under the concentration-time curve from 0-24 h (AUC0-24 h) in rats (oral dose of 300 mg/kg) for cilostazol and danazol, respectively. In conclusion, applying a wet media milling method with the combination of HPMC of a small molecular weight and Tween 80 as a dispersing agent, nanosuspensions can be practically prepared and conveniently utilized for enhancing the oral absorption of poorly water-soluble compounds in toxicology studies in the discovery stage.

A 17-year-old woman presented to our hospital with a complaint of pain around the anus and was admitted on suspicion of perianal cystoma. The cystoma was found to be a parenchymal tumor, and examination of a biopsy specimen revealed cystic rhabdomyosarcoma that appeared to have arisen in the anal sphincter. Abdominoperineal resection of the rectum was performed and followed by adjuvant chemotherapy (VAC therapy) and radiation therapy. The patient remains free of relapse as of 6 years 9 months after treatment. The other patient was a 21-year-old woman who presented with swelling of the left inguinal lymph nodes and perianal pain. Colostomy was performed to relieve anal obstruction caused by a perianal tumor. Biopsy revealed cystic rhabdomyosarcoma. The patient had bone metastasis and lymph node metastasis and died 3 months after the operation. Twenty-five cases of perianal rhabdomyosarcoma have been reported in Japan. A review of the literature suggests that although recent advances in chemotherapy have contributed to improving the survival rate, it appeares necessary to completely resect the tumor, and follow it by appropriate adjuvant chemotherapy.

Two-dimensional (2-D) DNA fingerprinting was used to investigate genomic changes in human low-grade gliomas of different subtypes. DNA variations were identified in the 2-D hybridization patterns as spot losses or gains. Computer-aided matching of spot patterns from different patients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cloning protocol which includes the preparation of a duplicate and a master gel. The DNA fragments of the 2-D gels were transferred to DEAE and nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot was determined with reference to the lambda DNA fragments used as external markers in both gels. The gel spot DNA was recovered from the DEAE membrane by high salt elution and was polymerase chain reaction (PCR)-amplified after ligation of adaptor oligo cassettes. The PCR products were cloned and used as locus-specific probe for the rehybridization of the 2-D blots. One of these probes detected a spot loss in 7 of 28 low-grade gliomas of different subtypes analyzed. Another probe revealed a characteristic intensity shift in 8 of 9 pilocytic astrocytomas between two neighboring spots. The target sequence of this highly specific effect was assigned to chromosome 11q14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successfully established a spot cloning procedure for the generation of locus-specific probes that may be instrumental in the discovery of the ciritical early events of glioma pathogenesis.

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUCI8, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15%, of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis . Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Waking EEGs of 150 normal children aged from 20 days to 15 years were subjected to analysis. Discrete time series of an artifact free segment of the EEG record at 50 samples/sec for twenty seconds was generated and autoregressive (AR) and component analyses were carried out with a minicomputer PDP 11/40 (DEC). The results may be summarized as follows: 1) In the group of 1 year old and less, the power increased with the monthly age, whereas the bio-informing activity amount decreased. In those older than 1 year, both parameters showed maximal values at 1 year and then decreased with age, and the decreases were marked from 1 to 3 years. 2) The first- and second-order component activities of 129 and 677 waves, respectively, were obtained by applying component analysis to 152 EEG records. The frequency polygons of natural frequency of second-order component waves verified several modes, each of which was enhanced in the frequency range of the well-known delta 0, delta 1, theta 1, theta 2, alpha 1, alpha 2, beta 1, beta 2 and beta 3 waves, respectively. 3) The average percent-power of the delta wave (delta 0 + delta 1) decreased with age, especially from 1 to 3 years old, whereas those of beta- and alpha-waves increased with advancing age. That of the theta wave tended to increase from 2 to 4 years of age, and thereafter decreased gradually with increasing age. 4) With increasing age, the durations of damped oscillations were significantly lengthened in delta 1, alpha 1 and beta 3 waves, whereas that in the theta 1 wave was significantly shortened. 5) The bio-informing activity amounts of alpha waves increased from 1 to 3 years with increasing age, whereas those of delta and theta waves decreased. No significant developmental change in the parameters, however, was observed in the beta wave. The results indicate that AR-power spectral and component analyses of EEG are sensitive methods for obtaining valuable information regarding the electrical brain maturation in childhood.

Autoregressive (AR) analysis and component analysis were done on EEGs during sleep stage 2 in normal and autistic children, since AR-coefficients of EEG characterize the autocovariance and power spectral density of EEG. In addition, as the generalized distance of the average AR-coefficient vector of an EEG group at one side from that at the opposite side exhibits an F-distribution, the statistical inferences were determined, as to whether there is hemispheric specialization in EEG in 21 right-handed autistic children and whether there is a difference between the AR-spectral pattern of EEG in the autistic children and that of 28 normal children of the same age range. The results obtained are as follows: 1. Significant hemispheric lateralization in EEG at the significant level of 0.01 was observed in normal children, whereas no lateralization was confirmed in autistic children. 2. Random click stimulation had cumulative effects on the hemispheric lateralization in the normal children, but not in the autistic children. On the contrary, the effects of random flash light stimulation on hemispheric asymmetry were verified in the autistic children as well as in the normal children. 3. The EEG components of autistic children were significantly different from the normal ones in the frequency range of alpha-2 component waves at bilateral frontal (Fp1 and Fp2) regions and the activity of these component waves was higher in the autistic children than in the normal ones over 5 years old. 4. These findings suggest that autistic children are not only less responsive to external stimuli, but also neurophysiologically less active in the internal regulatory mechanism.

Synthese des alcaloides du titre a partir de pyrrolidinone-2 via des pyrrolidinecarboxylates-1 α-substituees