Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.
Identifieur interne : 002550 ( PubMed/Corpus ); précédent : 002549; suivant : 002551Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.
Auteurs : Hongbo Hu ; Li Li ; Richard Y. Kao ; Binbin Kou ; Zhanguo Wang ; Liang Zhang ; Huiyuan Zhang ; Zhiyong Hao ; Wayne H. Tsui ; Anping Ni ; Lianxian Cui ; Baoxing Fan ; Feng Guo ; Shuan Rao ; Chengyu Jiang ; Qian Li ; Manji Sun ; Wei He ; Gang LiuSource :
- Journal of combinatorial chemistry [ 1520-4766 ]
English descriptors
- KwdEn :
- Amino Acid Sequence, Antigens, Viral (chemistry), Antigens, Viral (genetics), Antigens, Viral (immunology), Combinatorial Chemistry Techniques, Epitopes, B-Lymphocyte (chemistry), Epitopes, B-Lymphocyte (genetics), Epitopes, B-Lymphocyte (immunology), Molecular Sequence Data, Peptide Library, SARS Virus (chemistry), SARS Virus (immunology), Severe Acute Respiratory Syndrome (immunology).
- MESH :
- chemical , chemistry : Antigens, Viral, Epitopes, B-Lymphocyte.
- chemical , genetics : Antigens, Viral, Epitopes, B-Lymphocyte.
- chemical , immunology : Antigens, Viral, Epitopes, B-Lymphocyte.
- chemistry : SARS Virus.
- immunology : SARS Virus, Severe Acute Respiratory Syndrome.
- Amino Acid Sequence, Combinatorial Chemistry Techniques, Molecular Sequence Data, Peptide Library.
Abstract
A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.
DOI: 10.1021/cc0500607
PubMed: 16153058
Links to Exploration step
pubmed:16153058Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.</title>
<author><name sortKey="Hu, Hongbo" sort="Hu, Hongbo" uniqKey="Hu H" first="Hongbo" last="Hu">Hongbo Hu</name>
<affiliation><nlm:affiliation>Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, P. R. China.</nlm:affiliation>
</affiliation>
</author>
<author><name sortKey="Li, Li" sort="Li, Li" uniqKey="Li L" first="Li" last="Li">Li Li</name>
</author>
<author><name sortKey="Kao, Richard Y" sort="Kao, Richard Y" uniqKey="Kao R" first="Richard Y" last="Kao">Richard Y. Kao</name>
</author>
<author><name sortKey="Kou, Binbin" sort="Kou, Binbin" uniqKey="Kou B" first="Binbin" last="Kou">Binbin Kou</name>
</author>
<author><name sortKey="Wang, Zhanguo" sort="Wang, Zhanguo" uniqKey="Wang Z" first="Zhanguo" last="Wang">Zhanguo Wang</name>
</author>
<author><name sortKey="Zhang, Liang" sort="Zhang, Liang" uniqKey="Zhang L" first="Liang" last="Zhang">Liang Zhang</name>
</author>
<author><name sortKey="Zhang, Huiyuan" sort="Zhang, Huiyuan" uniqKey="Zhang H" first="Huiyuan" last="Zhang">Huiyuan Zhang</name>
</author>
<author><name sortKey="Hao, Zhiyong" sort="Hao, Zhiyong" uniqKey="Hao Z" first="Zhiyong" last="Hao">Zhiyong Hao</name>
</author>
<author><name sortKey="Tsui, Wayne H" sort="Tsui, Wayne H" uniqKey="Tsui W" first="Wayne H" last="Tsui">Wayne H. Tsui</name>
</author>
<author><name sortKey="Ni, Anping" sort="Ni, Anping" uniqKey="Ni A" first="Anping" last="Ni">Anping Ni</name>
</author>
<author><name sortKey="Cui, Lianxian" sort="Cui, Lianxian" uniqKey="Cui L" first="Lianxian" last="Cui">Lianxian Cui</name>
</author>
<author><name sortKey="Fan, Baoxing" sort="Fan, Baoxing" uniqKey="Fan B" first="Baoxing" last="Fan">Baoxing Fan</name>
</author>
<author><name sortKey="Guo, Feng" sort="Guo, Feng" uniqKey="Guo F" first="Feng" last="Guo">Feng Guo</name>
</author>
<author><name sortKey="Rao, Shuan" sort="Rao, Shuan" uniqKey="Rao S" first="Shuan" last="Rao">Shuan Rao</name>
</author>
<author><name sortKey="Jiang, Chengyu" sort="Jiang, Chengyu" uniqKey="Jiang C" first="Chengyu" last="Jiang">Chengyu Jiang</name>
</author>
<author><name sortKey="Li, Qian" sort="Li, Qian" uniqKey="Li Q" first="Qian" last="Li">Qian Li</name>
</author>
<author><name sortKey="Sun, Manji" sort="Sun, Manji" uniqKey="Sun M" first="Manji" last="Sun">Manji Sun</name>
</author>
<author><name sortKey="He, Wei" sort="He, Wei" uniqKey="He W" first="Wei" last="He">Wei He</name>
</author>
<author><name sortKey="Liu, Gang" sort="Liu, Gang" uniqKey="Liu G" first="Gang" last="Liu">Gang Liu</name>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PubMed</idno>
<date when="????"><PubDate><MedlineDate>2005 Sep-Oct</MedlineDate>
</PubDate>
</date>
<idno type="RBID">pubmed:16153058</idno>
<idno type="pmid">16153058</idno>
<idno type="doi">10.1021/cc0500607</idno>
<idno type="wicri:Area/PubMed/Corpus">002550</idno>
<idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002550</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en">Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.</title>
<author><name sortKey="Hu, Hongbo" sort="Hu, Hongbo" uniqKey="Hu H" first="Hongbo" last="Hu">Hongbo Hu</name>
<affiliation><nlm:affiliation>Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, P. R. China.</nlm:affiliation>
</affiliation>
</author>
<author><name sortKey="Li, Li" sort="Li, Li" uniqKey="Li L" first="Li" last="Li">Li Li</name>
</author>
<author><name sortKey="Kao, Richard Y" sort="Kao, Richard Y" uniqKey="Kao R" first="Richard Y" last="Kao">Richard Y. Kao</name>
</author>
<author><name sortKey="Kou, Binbin" sort="Kou, Binbin" uniqKey="Kou B" first="Binbin" last="Kou">Binbin Kou</name>
</author>
<author><name sortKey="Wang, Zhanguo" sort="Wang, Zhanguo" uniqKey="Wang Z" first="Zhanguo" last="Wang">Zhanguo Wang</name>
</author>
<author><name sortKey="Zhang, Liang" sort="Zhang, Liang" uniqKey="Zhang L" first="Liang" last="Zhang">Liang Zhang</name>
</author>
<author><name sortKey="Zhang, Huiyuan" sort="Zhang, Huiyuan" uniqKey="Zhang H" first="Huiyuan" last="Zhang">Huiyuan Zhang</name>
</author>
<author><name sortKey="Hao, Zhiyong" sort="Hao, Zhiyong" uniqKey="Hao Z" first="Zhiyong" last="Hao">Zhiyong Hao</name>
</author>
<author><name sortKey="Tsui, Wayne H" sort="Tsui, Wayne H" uniqKey="Tsui W" first="Wayne H" last="Tsui">Wayne H. Tsui</name>
</author>
<author><name sortKey="Ni, Anping" sort="Ni, Anping" uniqKey="Ni A" first="Anping" last="Ni">Anping Ni</name>
</author>
<author><name sortKey="Cui, Lianxian" sort="Cui, Lianxian" uniqKey="Cui L" first="Lianxian" last="Cui">Lianxian Cui</name>
</author>
<author><name sortKey="Fan, Baoxing" sort="Fan, Baoxing" uniqKey="Fan B" first="Baoxing" last="Fan">Baoxing Fan</name>
</author>
<author><name sortKey="Guo, Feng" sort="Guo, Feng" uniqKey="Guo F" first="Feng" last="Guo">Feng Guo</name>
</author>
<author><name sortKey="Rao, Shuan" sort="Rao, Shuan" uniqKey="Rao S" first="Shuan" last="Rao">Shuan Rao</name>
</author>
<author><name sortKey="Jiang, Chengyu" sort="Jiang, Chengyu" uniqKey="Jiang C" first="Chengyu" last="Jiang">Chengyu Jiang</name>
</author>
<author><name sortKey="Li, Qian" sort="Li, Qian" uniqKey="Li Q" first="Qian" last="Li">Qian Li</name>
</author>
<author><name sortKey="Sun, Manji" sort="Sun, Manji" uniqKey="Sun M" first="Manji" last="Sun">Manji Sun</name>
</author>
<author><name sortKey="He, Wei" sort="He, Wei" uniqKey="He W" first="Wei" last="He">Wei He</name>
</author>
<author><name sortKey="Liu, Gang" sort="Liu, Gang" uniqKey="Liu G" first="Gang" last="Liu">Gang Liu</name>
</author>
</analytic>
<series><title level="j">Journal of combinatorial chemistry</title>
<idno type="ISSN">1520-4766</idno>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Antigens, Viral (chemistry)</term>
<term>Antigens, Viral (genetics)</term>
<term>Antigens, Viral (immunology)</term>
<term>Combinatorial Chemistry Techniques</term>
<term>Epitopes, B-Lymphocyte (chemistry)</term>
<term>Epitopes, B-Lymphocyte (genetics)</term>
<term>Epitopes, B-Lymphocyte (immunology)</term>
<term>Molecular Sequence Data</term>
<term>Peptide Library</term>
<term>SARS Virus (chemistry)</term>
<term>SARS Virus (immunology)</term>
<term>Severe Acute Respiratory Syndrome (immunology)</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Antigens, Viral</term>
<term>Epitopes, B-Lymphocyte</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Antigens, Viral</term>
<term>Epitopes, B-Lymphocyte</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antigens, Viral</term>
<term>Epitopes, B-Lymphocyte</term>
</keywords>
<keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>SARS Virus</term>
</keywords>
<keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>SARS Virus</term>
<term>Severe Acute Respiratory Syndrome</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term>
<term>Combinatorial Chemistry Techniques</term>
<term>Molecular Sequence Data</term>
<term>Peptide Library</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en">A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.</div>
</front>
</TEI>
<pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">16153058</PMID>
<DateCompleted><Year>2005</Year>
<Month>11</Month>
<Day>07</Day>
</DateCompleted>
<DateRevised><Year>2007</Year>
<Month>11</Month>
<Day>14</Day>
</DateRevised>
<Article PubModel="Print"><Journal><ISSN IssnType="Print">1520-4766</ISSN>
<JournalIssue CitedMedium="Print"><Volume>7</Volume>
<Issue>5</Issue>
<PubDate><MedlineDate>2005 Sep-Oct</MedlineDate>
</PubDate>
</JournalIssue>
<Title>Journal of combinatorial chemistry</Title>
<ISOAbbreviation>J Comb Chem</ISOAbbreviation>
</Journal>
<ArticleTitle>Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.</ArticleTitle>
<Pagination><MedlinePgn>648-56</MedlinePgn>
</Pagination>
<Abstract><AbstractText>A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.</AbstractText>
</Abstract>
<AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Hu</LastName>
<ForeName>Hongbo</ForeName>
<Initials>H</Initials>
<AffiliationInfo><Affiliation>Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, P. R. China.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Li</LastName>
<ForeName>Li</ForeName>
<Initials>L</Initials>
</Author>
<Author ValidYN="Y"><LastName>Kao</LastName>
<ForeName>Richard Y</ForeName>
<Initials>RY</Initials>
</Author>
<Author ValidYN="Y"><LastName>Kou</LastName>
<ForeName>Binbin</ForeName>
<Initials>B</Initials>
</Author>
<Author ValidYN="Y"><LastName>Wang</LastName>
<ForeName>Zhanguo</ForeName>
<Initials>Z</Initials>
</Author>
<Author ValidYN="Y"><LastName>Zhang</LastName>
<ForeName>Liang</ForeName>
<Initials>L</Initials>
</Author>
<Author ValidYN="Y"><LastName>Zhang</LastName>
<ForeName>Huiyuan</ForeName>
<Initials>H</Initials>
</Author>
<Author ValidYN="Y"><LastName>Hao</LastName>
<ForeName>Zhiyong</ForeName>
<Initials>Z</Initials>
</Author>
<Author ValidYN="Y"><LastName>Tsui</LastName>
<ForeName>Wayne H</ForeName>
<Initials>WH</Initials>
</Author>
<Author ValidYN="Y"><LastName>Ni</LastName>
<ForeName>Anping</ForeName>
<Initials>A</Initials>
</Author>
<Author ValidYN="Y"><LastName>Cui</LastName>
<ForeName>Lianxian</ForeName>
<Initials>L</Initials>
</Author>
<Author ValidYN="Y"><LastName>Fan</LastName>
<ForeName>Baoxing</ForeName>
<Initials>B</Initials>
</Author>
<Author ValidYN="Y"><LastName>Guo</LastName>
<ForeName>Feng</ForeName>
<Initials>F</Initials>
</Author>
<Author ValidYN="Y"><LastName>Rao</LastName>
<ForeName>Shuan</ForeName>
<Initials>S</Initials>
</Author>
<Author ValidYN="Y"><LastName>Jiang</LastName>
<ForeName>Chengyu</ForeName>
<Initials>C</Initials>
</Author>
<Author ValidYN="Y"><LastName>Li</LastName>
<ForeName>Qian</ForeName>
<Initials>Q</Initials>
</Author>
<Author ValidYN="Y"><LastName>Sun</LastName>
<ForeName>Manji</ForeName>
<Initials>M</Initials>
</Author>
<Author ValidYN="Y"><LastName>He</LastName>
<ForeName>Wei</ForeName>
<Initials>W</Initials>
</Author>
<Author ValidYN="Y"><LastName>Liu</LastName>
<ForeName>Gang</ForeName>
<Initials>G</Initials>
</Author>
</AuthorList>
<Language>eng</Language>
<GrantList CompleteYN="Y"><Grant><GrantID>U01 AI 061092-01</GrantID>
<Acronym>AI</Acronym>
<Agency>NIAID NIH HHS</Agency>
<Country>United States</Country>
</Grant>
</GrantList>
<PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType>
<PublicationType UI="D052061">Research Support, N.I.H., Extramural</PublicationType>
<PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType>
<PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType>
</PublicationTypeList>
</Article>
<MedlineJournalInfo><Country>United States</Country>
<MedlineTA>J Comb Chem</MedlineTA>
<NlmUniqueID>100886263</NlmUniqueID>
<ISSNLinking>1520-4766</ISSNLinking>
</MedlineJournalInfo>
<ChemicalList><Chemical><RegistryNumber>0</RegistryNumber>
<NameOfSubstance UI="D000956">Antigens, Viral</NameOfSubstance>
</Chemical>
<Chemical><RegistryNumber>0</RegistryNumber>
<NameOfSubstance UI="D018985">Epitopes, B-Lymphocyte</NameOfSubstance>
</Chemical>
<Chemical><RegistryNumber>0</RegistryNumber>
<NameOfSubstance UI="D019151">Peptide Library</NameOfSubstance>
</Chemical>
</ChemicalList>
<CitationSubset>IM</CitationSubset>
<MeshHeadingList><MeshHeading><DescriptorName UI="D000595" MajorTopicYN="N">Amino Acid Sequence</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D000956" MajorTopicYN="N">Antigens, Viral</DescriptorName>
<QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName>
<QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName>
<QualifierName UI="Q000276" MajorTopicYN="N">immunology</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D020650" MajorTopicYN="N">Combinatorial Chemistry Techniques</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D018985" MajorTopicYN="N">Epitopes, B-Lymphocyte</DescriptorName>
<QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName>
<QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName>
<QualifierName UI="Q000276" MajorTopicYN="Y">immunology</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D008969" MajorTopicYN="N">Molecular Sequence Data</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D019151" MajorTopicYN="Y">Peptide Library</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D045473" MajorTopicYN="N">SARS Virus</DescriptorName>
<QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName>
<QualifierName UI="Q000276" MajorTopicYN="Y">immunology</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D045169" MajorTopicYN="N">Severe Acute Respiratory Syndrome</DescriptorName>
<QualifierName UI="Q000276" MajorTopicYN="N">immunology</QualifierName>
</MeshHeading>
</MeshHeadingList>
</MedlineCitation>
<PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2005</Year>
<Month>9</Month>
<Day>13</Day>
<Hour>9</Hour>
<Minute>0</Minute>
</PubMedPubDate>
<PubMedPubDate PubStatus="medline"><Year>2005</Year>
<Month>11</Month>
<Day>8</Day>
<Hour>9</Hour>
<Minute>0</Minute>
</PubMedPubDate>
<PubMedPubDate PubStatus="entrez"><Year>2005</Year>
<Month>9</Month>
<Day>13</Day>
<Hour>9</Hour>
<Minute>0</Minute>
</PubMedPubDate>
</History>
<PublicationStatus>ppublish</PublicationStatus>
<ArticleIdList><ArticleId IdType="pubmed">16153058</ArticleId>
<ArticleId IdType="doi">10.1021/cc0500607</ArticleId>
</ArticleIdList>
</PubmedData>
</pubmed>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/PubMed/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002550 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/PubMed/Corpus/biblio.hfd -nk 002550 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= SrasV1 |flux= PubMed |étape= Corpus |type= RBID |clé= pubmed:16153058 |texte= Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library. }}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/PubMed/Corpus/RBID.i -Sk "pubmed:16153058" \ | HfdSelect -Kh $EXPLOR_AREA/Data/PubMed/Corpus/biblio.hfd \ | NlmPubMed2Wicri -a SrasV1
![]() | This area was generated with Dilib version V0.6.33. | ![]() |