Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi.
Identifieur interne : 000041 ( Main/Corpus ); précédent : 000040; suivant : 000042Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi.
Auteurs : Justyna Sulej ; Monika Osi Ska-Jaroszuk ; Magdalena Jaszek ; Marcin Gr Z ; Jolanta Kutkowska ; Anna Pawlik ; Agata Chudzik ; Renata BancerzSource :
- Fungal biology [ 1878-6146 ] ; 2019.
English descriptors
- KwdEn :
- Anti-Infective Agents (isolation & purification), Anti-Infective Agents (pharmacology), Antioxidants (isolation & purification), Antioxidants (pharmacology), Basidiomycota (enzymology), Basidiomycota (isolation & purification), Biphenyl Compounds (metabolism), Carbohydrate Dehydrogenases (isolation & purification), Carbohydrate Dehydrogenases (metabolism), Carbohydrate Dehydrogenases (pharmacology), Cellobiose (metabolism), Enzymes, Immobilized (pharmacology), Escherichia coli (drug effects), Escherichia coli (growth & development), Lactose (metabolism), Microbial Sensitivity Tests (MeSH), Picrates (metabolism), Pseudomonas aeruginosa (drug effects), Pseudomonas aeruginosa (growth & development), Staphylococcus aureus (drug effects), Staphylococcus aureus (growth & development), Wood (microbiology).
- MESH :
- chemical , isolation & purification : Anti-Infective Agents, Antioxidants, Carbohydrate Dehydrogenases.
- chemical , metabolism : Biphenyl Compounds, Carbohydrate Dehydrogenases, Cellobiose, Lactose, Picrates.
- chemical , pharmacology : Anti-Infective Agents, Antioxidants, Carbohydrate Dehydrogenases, Enzymes, Immobilized.
- drug effects : Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus.
- enzymology : Basidiomycota.
- growth & development : Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus.
- isolation & purification : Basidiomycota.
- microbiology : Wood.
- Microbial Sensitivity Tests.
Abstract
Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC50 (10.04 ± 0.75 μg/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 μg/ml). The EC50value reached 12.6 ± 1.51 μg/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.
DOI: 10.1016/j.funbio.2019.09.007
PubMed: 31733730
Links to Exploration step
pubmed:31733730Le document en format XML
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Electronic address: justyna.sulej@poczta.umcs.lublin.pl.</nlm:affiliation></affiliation></author><author><name sortKey="Osi Ska Jaroszuk, Monika" sort="Osi Ska Jaroszuk, Monika" uniqKey="Osi Ska Jaroszuk M" first="Monika" last="Osi Ska-Jaroszuk">Monika Osi Ska-Jaroszuk</name><affiliation><nlm:affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</nlm:affiliation></affiliation></author><author><name sortKey="Jaszek, Magdalena" sort="Jaszek, Magdalena" uniqKey="Jaszek M" first="Magdalena" last="Jaszek">Magdalena Jaszek</name><affiliation><nlm:affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</nlm:affiliation></affiliation></author><author><name sortKey="Gr Z, Marcin" sort="Gr Z, Marcin" uniqKey="Gr Z M" first="Marcin" last="Gr Z">Marcin Gr Z</name><affiliation><nlm:affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</nlm:affiliation></affiliation></author><author><name sortKey="Kutkowska, Jolanta" sort="Kutkowska, Jolanta" uniqKey="Kutkowska J" first="Jolanta" last="Kutkowska">Jolanta Kutkowska</name><affiliation><nlm:affiliation>Department of Genetics and Microbiology, Maria Curie-Sklodowska University, Akademicka 19, Lublin 20-033, Poland.</nlm:affiliation></affiliation></author><author><name sortKey="Pawlik, Anna" sort="Pawlik, Anna" uniqKey="Pawlik A" first="Anna" last="Pawlik">Anna Pawlik</name><affiliation><nlm:affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</nlm:affiliation></affiliation></author><author><name sortKey="Chudzik, Agata" sort="Chudzik, Agata" uniqKey="Chudzik A" first="Agata" last="Chudzik">Agata Chudzik</name><affiliation><nlm:affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</nlm:affiliation></affiliation></author><author><name sortKey="Bancerz, Renata" sort="Bancerz, Renata" uniqKey="Bancerz R" first="Renata" last="Bancerz">Renata Bancerz</name><affiliation><nlm:affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Fungal biology</title><idno type="ISSN">1878-6146</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Anti-Infective Agents (isolation & purification)</term><term>Anti-Infective Agents (pharmacology)</term><term>Antioxidants (isolation & purification)</term><term>Antioxidants (pharmacology)</term><term>Basidiomycota (enzymology)</term><term>Basidiomycota (isolation & purification)</term><term>Biphenyl Compounds (metabolism)</term><term>Carbohydrate Dehydrogenases (isolation & purification)</term><term>Carbohydrate Dehydrogenases (metabolism)</term><term>Carbohydrate Dehydrogenases (pharmacology)</term><term>Cellobiose (metabolism)</term><term>Enzymes, Immobilized (pharmacology)</term><term>Escherichia coli (drug effects)</term><term>Escherichia coli (growth & development)</term><term>Lactose (metabolism)</term><term>Microbial Sensitivity Tests (MeSH)</term><term>Picrates (metabolism)</term><term>Pseudomonas aeruginosa (drug effects)</term><term>Pseudomonas aeruginosa (growth & development)</term><term>Staphylococcus aureus (drug effects)</term><term>Staphylococcus aureus (growth & development)</term><term>Wood (microbiology)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Anti-Infective Agents</term><term>Antioxidants</term><term>Carbohydrate Dehydrogenases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Biphenyl Compounds</term><term>Carbohydrate Dehydrogenases</term><term>Cellobiose</term><term>Lactose</term><term>Picrates</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Anti-Infective Agents</term><term>Antioxidants</term><term>Carbohydrate Dehydrogenases</term><term>Enzymes, Immobilized</term></keywords><keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>Escherichia coli</term><term>Pseudomonas aeruginosa</term><term>Staphylococcus aureus</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Basidiomycota</term></keywords><keywords scheme="MESH" qualifier="growth & development" xml:lang="en"><term>Escherichia coli</term><term>Pseudomonas aeruginosa</term><term>Staphylococcus aureus</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Basidiomycota</term></keywords><keywords scheme="MESH" qualifier="microbiology" xml:lang="en"><term>Wood</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Microbial Sensitivity Tests</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC<sub>50</sub> (10.04 ± 0.75 μg/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 μg/ml). The EC<sub>50</sub>value reached 12.6 ± 1.51 μg/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">31733730</PMID><DateCompleted><Year>2020</Year><Month>05</Month><Day>21</Day></DateCompleted><DateRevised><Year>2020</Year><Month>05</Month><Day>21</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">1878-6146</ISSN><JournalIssue CitedMedium="Internet"><Volume>123</Volume><Issue>12</Issue><PubDate><Year>2019</Year><Month>12</Month></PubDate></JournalIssue><Title>Fungal biology</Title><ISOAbbreviation>Fungal Biol</ISOAbbreviation></Journal><ArticleTitle>Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi.</ArticleTitle><Pagination><MedlinePgn>875-886</MedlinePgn></Pagination><ELocationID EIdType="pii" ValidYN="Y">S1878-6146(19)30128-X</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.funbio.2019.09.007</ELocationID><Abstract><AbstractText>Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC<sub>50</sub> (10.04 ± 0.75 μg/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 μg/ml). The EC<sub>50</sub>value reached 12.6 ± 1.51 μg/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.</AbstractText><CopyrightInformation>Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Sulej</LastName><ForeName>Justyna</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland. Electronic address: justyna.sulej@poczta.umcs.lublin.pl.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Osińska-Jaroszuk</LastName><ForeName>Monika</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Jaszek</LastName><ForeName>Magdalena</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Grąz</LastName><ForeName>Marcin</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kutkowska</LastName><ForeName>Jolanta</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Department of Genetics and Microbiology, Maria Curie-Sklodowska University, Akademicka 19, Lublin 20-033, Poland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Pawlik</LastName><ForeName>Anna</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chudzik</LastName><ForeName>Agata</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Bancerz</LastName><ForeName>Renata</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>09</Month><Day>24</Day></ArticleDate></Article><MedlineJournalInfo><Country>Netherlands</Country><MedlineTA>Fungal Biol</MedlineTA><NlmUniqueID>101524465</NlmUniqueID></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000890">Anti-Infective Agents</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000975">Antioxidants</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D001713">Biphenyl Compounds</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004800">Enzymes, Immobilized</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D010851">Picrates</NameOfSubstance></Chemical><Chemical><RegistryNumber>16462-44-5</RegistryNumber><NameOfSubstance UI="D002475">Cellobiose</NameOfSubstance></Chemical><Chemical><RegistryNumber>DFD3H4VGDH</RegistryNumber><NameOfSubstance UI="C004931">1,1-diphenyl-2-picrylhydrazyl</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.1.-</RegistryNumber><NameOfSubstance UI="D002237">Carbohydrate Dehydrogenases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.1.99.18</RegistryNumber><NameOfSubstance UI="C019859">cellobiose-quinone oxidoreductase</NameOfSubstance></Chemical><Chemical><RegistryNumber>J2B2A4N98G</RegistryNumber><NameOfSubstance UI="D007785">Lactose</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000890" MajorTopicYN="N">Anti-Infective Agents</DescriptorName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName><QualifierName UI="Q000494" MajorTopicYN="Y">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D000975" MajorTopicYN="N">Antioxidants</DescriptorName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName><QualifierName UI="Q000494" MajorTopicYN="Y">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D001487" MajorTopicYN="N">Basidiomycota</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D001713" MajorTopicYN="N">Biphenyl Compounds</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D002237" MajorTopicYN="N">Carbohydrate Dehydrogenases</DescriptorName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName><QualifierName UI="Q000494" MajorTopicYN="Y">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D002475" MajorTopicYN="N">Cellobiose</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004800" MajorTopicYN="N">Enzymes, Immobilized</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="N">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000187" MajorTopicYN="N">drug effects</QualifierName><QualifierName UI="Q000254" MajorTopicYN="N">growth & development</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D007785" MajorTopicYN="N">Lactose</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008826" MajorTopicYN="N">Microbial Sensitivity Tests</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010851" MajorTopicYN="N">Picrates</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011550" MajorTopicYN="N">Pseudomonas aeruginosa</DescriptorName><QualifierName UI="Q000187" MajorTopicYN="N">drug effects</QualifierName><QualifierName UI="Q000254" MajorTopicYN="N">growth & development</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013211" MajorTopicYN="N">Staphylococcus aureus</DescriptorName><QualifierName UI="Q000187" MajorTopicYN="N">drug effects</QualifierName><QualifierName UI="Q000254" MajorTopicYN="N">growth & development</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014934" MajorTopicYN="N">Wood</DescriptorName><QualifierName UI="Q000382" MajorTopicYN="N">microbiology</QualifierName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">Alginate</Keyword><Keyword MajorTopicYN="Y">Antibacterial properties</Keyword><Keyword MajorTopicYN="Y">Immobilisation</Keyword><Keyword MajorTopicYN="Y">Lactobionic acid</Keyword><Keyword MajorTopicYN="Y">Scavenging effect</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2019</Year><Month>06</Month><Day>07</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2019</Year><Month>09</Month><Day>06</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2019</Year><Month>09</Month><Day>17</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>11</Month><Day>18</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2019</Year><Month>11</Month><Day>18</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate 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