We isolated an extreme thermophilic bacterium strain SS, which grew between 55 and 82°C (optimum 70°C), from sewage sludge in Kagoshima City, Japan. Morphological and physiological properties, as well as 16S rRNA gene sequence indicated that strain SS belongs to the genus Thermaerobacter. To confirm the distribution of Thermaerobacter spp. in sewage sludge, genus-specific detection by nested PCR of 16S rRNA gene was performed. As a result, the existence of Thermaerobacter spp. in six sewage sludge samples including composted sludge was demonstrated by sequencing of the nested PCR products. However, enrichment cultures with sewage sludge were needed to detect the target products, indicating that population of Thermaerobacter spp. may be significantly low. As far as we know, this is the first report on occurrence of extreme thermophilic bacteria in a municipal wastewater treatment process under mesophilic condition.
{"title":"Occurrence of Extreme Thermophiles, Thermaerobacter spp., in Sewage Sludge","authors":"Kota Shiotsuka, A. Tanaka, K. Sakai","doi":"10.3118/JJSE.9.67","DOIUrl":"https://doi.org/10.3118/JJSE.9.67","url":null,"abstract":"We isolated an extreme thermophilic bacterium strain SS, which grew between 55 and 82°C (optimum 70°C), from sewage sludge in Kagoshima City, Japan. Morphological and physiological properties, as well as 16S rRNA gene sequence indicated that strain SS belongs to the genus Thermaerobacter. To confirm the distribution of Thermaerobacter spp. in sewage sludge, genus-specific detection by nested PCR of 16S rRNA gene was performed. As a result, the existence of Thermaerobacter spp. in six sewage sludge samples including composted sludge was demonstrated by sequencing of the nested PCR products. However, enrichment cultures with sewage sludge were needed to detect the target products, indicating that population of Thermaerobacter spp. may be significantly low. As far as we know, this is the first report on occurrence of extreme thermophilic bacteria in a municipal wastewater treatment process under mesophilic condition.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"166 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2010-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123295159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"「好鹽菌」、「嗜?菌」から「好嗜塩菌」へ","authors":"正博 亀倉","doi":"10.3118/JJSE.9.57","DOIUrl":"https://doi.org/10.3118/JJSE.9.57","url":null,"abstract":"","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"25 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2010-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115587277","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Y. Zhang, R. An, R. Yatsunami, M. Sato, K. Orishimo, Y. Hatori, T. Fukui, Satoshi Nakamura
A chitinase (ChiN1) from Halobacterium salinarum NRC-1 belongs to the glycoside hydrolase family 18. According to the three-dimensional structure model of ChiN1, Asn239 and Gln242 on a small helix between Phe237 and Arg247 were targeted for mutagenesis. Mutants N239D (Asn239 was replaced by Asp) and Q242E (Gln242 was replaced by Glu) were expressed in Haloarcula japonica and then characterized. Characterization of the mutants revealed that halotolerancy could be improved by introducing an acidic amino acid on the surface of ChiN1.
{"title":"Characterization of a Haloarchaeal GH Family 18 Chitinase with Additional Acidic Amino Acids on Its Protein Surface","authors":"Y. Zhang, R. An, R. Yatsunami, M. Sato, K. Orishimo, Y. Hatori, T. Fukui, Satoshi Nakamura","doi":"10.3118/JJSE.9.72","DOIUrl":"https://doi.org/10.3118/JJSE.9.72","url":null,"abstract":"A chitinase (ChiN1) from Halobacterium salinarum NRC-1 belongs to the glycoside hydrolase family 18. According to the three-dimensional structure model of ChiN1, Asn239 and Gln242 on a small helix between Phe237 and Arg247 were targeted for mutagenesis. Mutants N239D (Asn239 was replaced by Asp) and Q242E (Gln242 was replaced by Glu) were expressed in Haloarcula japonica and then characterized. Characterization of the mutants revealed that halotolerancy could be improved by introducing an acidic amino acid on the surface of ChiN1.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"9 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2010-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130114561","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Acid-extractable cellular polyamines of thermophilic Caldisericum exile belonging to a new bacterial phylum, Caldiserica were analyzed by HPLC and GC. The coexistence of an unusual tertiary brancehed tetra-amine, N4-aminopropylspermidine with spermine, a linear tetra-amine, as the major polyamines in addition to putrescine and spermidine, is first reported in the moderate thermophile isolated from a terrestrial hot spring in Japan. Linear and branched penta-amines were not detected. The novel cellular polyamine profile found in the moderate thermophile has never been observed within various bacteria and archaea previously analyzed.
{"title":"A tertiary-branched tetra-amine, N4-aminopropylspermidine is a major cellular polyamine in an anaerobic thermophile, Caldisericum exile belonging to a new bacterial phylum, Caldiserica","authors":"K. Hamana, H. Hayashi, M. Niitsu","doi":"10.3118/JJSE.9.75","DOIUrl":"https://doi.org/10.3118/JJSE.9.75","url":null,"abstract":"Acid-extractable cellular polyamines of thermophilic Caldisericum exile belonging to a new bacterial phylum, Caldiserica were analyzed by HPLC and GC. The coexistence of an unusual tertiary brancehed tetra-amine, N4-aminopropylspermidine with spermine, a linear tetra-amine, as the major polyamines in addition to putrescine and spermidine, is first reported in the moderate thermophile isolated from a terrestrial hot spring in Japan. Linear and branched penta-amines were not detected. The novel cellular polyamine profile found in the moderate thermophile has never been observed within various bacteria and archaea previously analyzed.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2010-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124039958","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
N. Yoshida, N. Ohmura, N. Matsumoto, K. Sasaki, H. Saiki, H. Arai, M. Ishii, Y. Igarashi
A thermophilic, obligately chemolithoauto- trophic H2-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can utilize O2 or NO3- as an electron acceptor. Here we show that this bacterium can grow autotrophically by the reduction of Fe(III)-EDTA or Fe(III)-DTPA (diethylene-triamine-pentaacetic acid) as an electron acceptor, through oxidation of H2 as an electron donor. A 7.6 kDa c-type cytochrome was purified from soluble fraction as the iron reducing protein and the protein was identified as cytochrome c-552. A rate constant for the reduction of Fe(III)-EDTA by the cytochrome was kinetically determined to be 1.73 x 107 s-1 by stopped-flow spectrophotometry, which is more than 20-fold higher than the constants of other iron reductases. The redox potential was measured as E = +0.27 V (vs NHE [Normal Hydrogen Electrode]), which is high enough to reduce Fe(III)-EDTA. These results suggest that cytochrome c-552 works as a terminal electron donor for Fe(III) in the periplasmic space.
{"title":"Dissimilatory Fe (III) reduction by cytochrome c-552 in a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6","authors":"N. Yoshida, N. Ohmura, N. Matsumoto, K. Sasaki, H. Saiki, H. Arai, M. Ishii, Y. Igarashi","doi":"10.3118/JJSE.9.61","DOIUrl":"https://doi.org/10.3118/JJSE.9.61","url":null,"abstract":"A thermophilic, obligately chemolithoauto- trophic H2-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can utilize O2 or NO3- as an electron acceptor. Here we show that this bacterium can grow autotrophically by the reduction of Fe(III)-EDTA or Fe(III)-DTPA (diethylene-triamine-pentaacetic acid) as an electron acceptor, through oxidation of H2 as an electron donor. A 7.6 kDa c-type cytochrome was purified from soluble fraction as the iron reducing protein and the protein was identified as cytochrome c-552. A rate constant for the reduction of Fe(III)-EDTA by the cytochrome was kinetically determined to be 1.73 x 107 s-1 by stopped-flow spectrophotometry, which is more than 20-fold higher than the constants of other iron reductases. The redox potential was measured as E = +0.27 V (vs NHE [Normal Hydrogen Electrode]), which is high enough to reduce Fe(III)-EDTA. These results suggest that cytochrome c-552 works as a terminal electron donor for Fe(III) in the periplasmic space.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"35 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2010-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124017843","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
I report the successful culture of the tardigrade Ramazzottius varieornatus by supplying the green alga Chlorella vulgaris as a food. A standard strain named YOKOZUNA-1 has been isolated from single egg of R. varieornatus. The reared animals have anhydrobiotic ability throughout their life cycle in eggs, juveniles, and adults. Body water content of adult R. varieornatus drops from 78.6% wt./wt. in hydrated state to 2.5% wt./wt. in anhydrobiotic state. Reared adults, while in an anhydrobiotic state, are tolerant of exposure to 100°C, 1 GPa, 99.8% acetonitrile, and 5000 Gy of 4He ions. Furthermore, hydrated active adults survive freezing at -2 to -20°C at a cooling rate of 1°C/min. Based on useful culture systems and extraordinary tolerance to extreme environmental conditions of R. varieornatus, this species is expected to be a suitable model for extremophile research focusing on multicellular organisms.
{"title":"The tardigrade Ramazzottius varieornatus as a model of extremotolerant animals","authors":"D. Horikawa","doi":"10.3118/JJSE.7.2.25","DOIUrl":"https://doi.org/10.3118/JJSE.7.2.25","url":null,"abstract":"I report the successful culture of the tardigrade Ramazzottius varieornatus by supplying the green alga Chlorella vulgaris as a food. A standard strain named YOKOZUNA-1 has been isolated from single egg of R. varieornatus. The reared animals have anhydrobiotic ability throughout their life cycle in eggs, juveniles, and adults. Body water content of adult R. varieornatus drops from 78.6% wt./wt. in hydrated state to 2.5% wt./wt. in anhydrobiotic state. Reared adults, while in an anhydrobiotic state, are tolerant of exposure to 100°C, 1 GPa, 99.8% acetonitrile, and 5000 Gy of 4He ions. Furthermore, hydrated active adults survive freezing at -2 to -20°C at a cooling rate of 1°C/min. Based on useful culture systems and extraordinary tolerance to extreme environmental conditions of R. varieornatus, this species is expected to be a suitable model for extremophile research focusing on multicellular organisms.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"40 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131287847","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Minegishi, Takeshi Miura, Yasuhiko Yoshida, R. Usami, F. Abe
Deep-sea yeast strains were isolated from mud samples collected in Sagami Bay (1,100-1,400 m) and the Japan Trench (4,500-6,500 m). All of the 46 yeast isolates were capable of growth at 24°C and atmospheric pressure, suggesting that these strains might originate neritic regions or land but survived in deep sea. Based on sequencing of 26S rRNA gene, 15 strains were classified into basidiomycetous yeasts including Cryptococcus liquefaciens, Kondoa aeria, Rhodosporidium diobovatum, Rhodosporidium sphaerocarpum, Rhodotorula mucilaginosa, Rhodotorula dairenensis and Rhodotorula slooffiae, and 8 strains were classified into ascomycetous yeasts including Aureobasidium pullulans, Candida zeylanoides, Kluyveromyces nonfermentans, Metschnikowia bicuspidate and Williopsis saturnus. Screening of the 46 isolates appeared to yield a high frequency of polygalacturonase (PGase) producers capable of degrading pectin. We suggest that deep-sea yeasts are new sources of PGase producers.
{"title":"Phylogenetic analysis of pectin degrading yeasts from deep-sea environments","authors":"H. Minegishi, Takeshi Miura, Yasuhiko Yoshida, R. Usami, F. Abe","doi":"10.3118/JJSE.5.21","DOIUrl":"https://doi.org/10.3118/JJSE.5.21","url":null,"abstract":"Deep-sea yeast strains were isolated from mud samples collected in Sagami Bay (1,100-1,400 m) and the Japan Trench (4,500-6,500 m). All of the 46 yeast isolates were capable of growth at 24°C and atmospheric pressure, suggesting that these strains might originate neritic regions or land but survived in deep sea. Based on sequencing of 26S rRNA gene, 15 strains were classified into basidiomycetous yeasts including Cryptococcus liquefaciens, Kondoa aeria, Rhodosporidium diobovatum, Rhodosporidium sphaerocarpum, Rhodotorula mucilaginosa, Rhodotorula dairenensis and Rhodotorula slooffiae, and 8 strains were classified into ascomycetous yeasts including Aureobasidium pullulans, Candida zeylanoides, Kluyveromyces nonfermentans, Metschnikowia bicuspidate and Williopsis saturnus. Screening of the 46 isolates appeared to yield a high frequency of polygalacturonase (PGase) producers capable of degrading pectin. We suggest that deep-sea yeasts are new sources of PGase producers.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"240 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114337002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cellular polyamines of 61 archaeal newly published strains were analyzed by HPLC to evaluate correlation of polyamine profiles to their extremophily and chemotaxonomy. A quaternary branched penta-amine, N4-bis( aminopropyl )spermidine, was found in hyperthermophilic Methanocaldococcus and Methanotorris belonging to the family Methanocaldococcaceae but not in the family Methanococcaceae nor any other methanogenic archaeal orders. Hyperthermophilic / extremely thermophilic Palaeococcus, Thermococcus and Pyrococcus of the order Thermococcales ubiquitously contained the branched penta-amine. Mesophilic, extreme halophiles including halo-alkaliphiles, belonging to the order Halobacteriales, contained a trace of spermidine, spermine and agmatine. In the phylum Crenarchaeota, extremely thermophilic Caldisphaera of the provisional order 'Caldisphaerales' contained norspermidine, spermidine and agmatine, and hyperthermophilic Pyrobaculum belonging to the order Thermoproteales contained norspermidine, spermidine, norspermine and spermine, but not any penta-amines. Within the order Desulfurococcales, Desulfurococcus and Ignisphaera contained spermidine, norspermidine, homospermidine, norspermine and spermine, whereas Aeropyrum contained caldopentamine and thermopentamine in addition to these triamines and tetra-amines.
{"title":"Polyamine analysis of methanogens, thermophiles and extreme halophiles belonging to the domain Archaea","authors":"K. Hamana, Ryuichi Hosoya, T. Itoh","doi":"10.3118/JJSE.6.25","DOIUrl":"https://doi.org/10.3118/JJSE.6.25","url":null,"abstract":"Cellular polyamines of 61 archaeal newly published strains were analyzed by HPLC to evaluate correlation of polyamine profiles to their extremophily and chemotaxonomy. A quaternary branched penta-amine, N4-bis( aminopropyl )spermidine, was found in hyperthermophilic Methanocaldococcus and Methanotorris belonging to the family Methanocaldococcaceae but not in the family Methanococcaceae nor any other methanogenic archaeal orders. Hyperthermophilic / extremely thermophilic Palaeococcus, Thermococcus and Pyrococcus of the order Thermococcales ubiquitously contained the branched penta-amine. Mesophilic, extreme halophiles including halo-alkaliphiles, belonging to the order Halobacteriales, contained a trace of spermidine, spermine and agmatine. In the phylum Crenarchaeota, extremely thermophilic Caldisphaera of the provisional order 'Caldisphaerales' contained norspermidine, spermidine and agmatine, and hyperthermophilic Pyrobaculum belonging to the order Thermoproteales contained norspermidine, spermidine, norspermine and spermine, but not any penta-amines. Within the order Desulfurococcales, Desulfurococcus and Ignisphaera contained spermidine, norspermidine, homospermidine, norspermine and spermine, whereas Aeropyrum contained caldopentamine and thermopentamine in addition to these triamines and tetra-amines.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"21 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125679001","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K. Hamana, Ryuichi Hosoya, A. Yokota, M. Niitsu, H. Hayashi, T. Itoh
Cellular polyamines of 44 newly validated eubacterial thermophiles growing at 45-80°C, belonging to eight orders (six phyla) of the domain Bacteria, were analyzed by HPLC and GC. A quaternary branched penta-amine, N4-bis(aminopropyl)norspermidine, was found in Hydrogenivirga and Sulfurihydrogenibium belonging to the order of Aquificales. Another quaternary branched penta-amine, N4-bis(aminopropyl) spermidine, was detected in Thermovibrio, Desulfurobacterium (Aquificales), Moorella, Desul-fovirgula (Thermoanaerobacteriales), Thermaerobacter (Clostridiales), Caldalkalibacillus, Thermobacillus (Bacillales), and Desulfothermus (Desulfovibrionales of the phylum Proteobacteria). The two branched penta- amines as well as linear penta- and hexa-amines were produced by Thermus species of the order Thermales. Marinitoga and Fervidobacterium (Thermotogales) contained linear penta-amines. The production of these long polyamines was dependent on culture temperature and remarkable at 70-80°C in the extreme/ hyper-thermophiles and to alkaline medium pH 8.5-9.0 in moderately thermophilic bacilli. The long polyamines were not detected in moderately thermophilic Anaerolinea (Anaerolineales of the phylum Chloroflexi). Distribution of either or both long linear polyamines and/or quaternary branched polyamines in the extreme/hyper-thermophiles is also reflected in their phylogenetical evolved locations.
{"title":"Distribution of long linear and branched polyamines in the thermophiles belonging to the domain Bacteria","authors":"K. Hamana, Ryuichi Hosoya, A. Yokota, M. Niitsu, H. Hayashi, T. Itoh","doi":"10.3118/JJSE.7.1.10","DOIUrl":"https://doi.org/10.3118/JJSE.7.1.10","url":null,"abstract":"Cellular polyamines of 44 newly validated eubacterial thermophiles growing at 45-80°C, belonging to eight orders (six phyla) of the domain Bacteria, were analyzed by HPLC and GC. A quaternary branched penta-amine, N4-bis(aminopropyl)norspermidine, was found in Hydrogenivirga and Sulfurihydrogenibium belonging to the order of Aquificales. Another quaternary branched penta-amine, N4-bis(aminopropyl) spermidine, was detected in Thermovibrio, Desulfurobacterium (Aquificales), Moorella, Desul-fovirgula (Thermoanaerobacteriales), Thermaerobacter (Clostridiales), Caldalkalibacillus, Thermobacillus (Bacillales), and Desulfothermus (Desulfovibrionales of the phylum Proteobacteria). The two branched penta- amines as well as linear penta- and hexa-amines were produced by Thermus species of the order Thermales. Marinitoga and Fervidobacterium (Thermotogales) contained linear penta-amines. The production of these long polyamines was dependent on culture temperature and remarkable at 70-80°C in the extreme/ hyper-thermophiles and to alkaline medium pH 8.5-9.0 in moderately thermophilic bacilli. The long polyamines were not detected in moderately thermophilic Anaerolinea (Anaerolineales of the phylum Chloroflexi). Distribution of either or both long linear polyamines and/or quaternary branched polyamines in the extreme/hyper-thermophiles is also reflected in their phylogenetical evolved locations.","PeriodicalId":204480,"journal":{"name":"Journal of Japanese Society for Extremophiles","volume":"20 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127822585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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