Serveur d'exploration sur les relations entre la France et l'Australie

Attention, ce site est en cours de développement !
Attention, site généré par des moyens informatiques à partir de corpus bruts.
Les informations ne sont donc pas validées.

A microfluidic-based neurotoxin concentration gradient for the generation of an in vitro model of Parkinson’s disease

Identifieur interne : 006238 ( Main/Exploration ); précédent : 006237; suivant : 006239

A microfluidic-based neurotoxin concentration gradient for the generation of an in vitro model of Parkinson’s disease

Auteurs : Azadeh Seidi [Japon] ; Hirokazu Kaji [États-Unis, Japon] ; Nasim Annabi [Australie] ; Serge Ostrovidov [Japon] ; Murugan Ramalingam [Japon, France] ; Ali Khademhosseini [Japon, États-Unis]

Source :

RBID : PMC:3145239

Abstract

In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an in vitro model of Parkinson’s disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 μM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an in vitro model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating in vitro models of disease for drug discovery applications.


Url:
DOI: 10.1063/1.3580756
PubMed: 21799720
PubMed Central: 3145239


Affiliations:


Links toward previous steps (curation, corpus...)


Le document en format XML

<record>
<TEI>
<teiHeader>
<fileDesc>
<titleStmt>
<title xml:lang="en">A microfluidic-based neurotoxin concentration gradient for the generation of an
<italic>in vitro</italic>
model of Parkinson’s disease</title>
<author>
<name sortKey="Seidi, Azadeh" sort="Seidi, Azadeh" uniqKey="Seidi A" first="Azadeh" last="Seidi">Azadeh Seidi</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Kaji, Hirokazu" sort="Kaji, Hirokazu" uniqKey="Kaji H" first="Hirokazu" last="Kaji">Hirokazu Kaji</name>
<affiliation wicri:level="1">
<nlm:aff id="a2">Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139</wicri:regionArea>
<wicri:noRegion>Massachusetts 02139</wicri:noRegion>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="a3">Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai 980-8579</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Annabi, Nasim" sort="Annabi, Nasim" uniqKey="Annabi N" first="Nasim" last="Annabi">Nasim Annabi</name>
<affiliation wicri:level="4">
<nlm:aff id="a4">School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, New South Wales 2006, Australia</nlm:aff>
<country xml:lang="fr">Australie</country>
<wicri:regionArea>School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, New South Wales 2006</wicri:regionArea>
<orgName type="university">Université de Sydney</orgName>
<placeName>
<settlement type="city">Sydney</settlement>
<region type="état">Nouvelle-Galles du Sud</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Ostrovidov, Serge" sort="Ostrovidov, Serge" uniqKey="Ostrovidov S" first="Serge" last="Ostrovidov">Serge Ostrovidov</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Ramalingam, Murugan" sort="Ramalingam, Murugan" uniqKey="Ramalingam M" first="Murugan" last="Ramalingam">Murugan Ramalingam</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="a5">Faculte de Medecine, National Institute of Health and Medical Research U977, Université de Strasbourg, Strasbourg, Cedex 67085, France</nlm:aff>
<country xml:lang="fr">France</country>
<wicri:regionArea>Faculte de Medecine, National Institute of Health and Medical Research U977, Université de Strasbourg, Strasbourg, Cedex 67085</wicri:regionArea>
<wicri:noRegion>67085</wicri:noRegion>
<orgName type="university">Université de Strasbourg</orgName>
<placeName>
<settlement type="city">Strasbourg</settlement>
<region type="region" nuts="2">Grand Est</region>
<region type="old region" nuts="2">Alsace (région administrative)</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Khademhosseini, Ali" sort="Khademhosseini, Ali" uniqKey="Khademhosseini A" first="Ali" last="Khademhosseini">Ali Khademhosseini</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
<affiliation wicri:level="1">
<nlm:aff id="a2">Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139</wicri:regionArea>
<wicri:noRegion>Massachusetts 02139</wicri:noRegion>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="a6">Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115</wicri:regionArea>
<orgName type="university">Université Harvard</orgName>
<placeName>
<settlement type="city">Cambridge (Massachusetts)</settlement>
<region type="state">Massachusetts</region>
</placeName>
</affiliation>
</author>
</titleStmt>
<publicationStmt>
<idno type="wicri:source">PMC</idno>
<idno type="pmid">21799720</idno>
<idno type="pmc">3145239</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145239</idno>
<idno type="RBID">PMC:3145239</idno>
<idno type="doi">10.1063/1.3580756</idno>
<date when="2011">2011</date>
<idno type="wicri:Area/Pmc/Corpus">000679</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000679</idno>
<idno type="wicri:Area/Pmc/Curation">000678</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000678</idno>
<idno type="wicri:Area/Pmc/Checkpoint">002423</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">002423</idno>
<idno type="wicri:Area/Ncbi/Merge">000B33</idno>
<idno type="wicri:Area/Ncbi/Curation">000B33</idno>
<idno type="wicri:Area/Ncbi/Checkpoint">000B33</idno>
<idno type="wicri:doubleKey">1932-1058:2011:Seidi A:a:microfluidic:based</idno>
<idno type="wicri:Area/Main/Merge">006614</idno>
<idno type="wicri:Area/Main/Curation">006238</idno>
<idno type="wicri:Area/Main/Exploration">006238</idno>
</publicationStmt>
<sourceDesc>
<biblStruct>
<analytic>
<title xml:lang="en" level="a" type="main">A microfluidic-based neurotoxin concentration gradient for the generation of an
<italic>in vitro</italic>
model of Parkinson’s disease</title>
<author>
<name sortKey="Seidi, Azadeh" sort="Seidi, Azadeh" uniqKey="Seidi A" first="Azadeh" last="Seidi">Azadeh Seidi</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Kaji, Hirokazu" sort="Kaji, Hirokazu" uniqKey="Kaji H" first="Hirokazu" last="Kaji">Hirokazu Kaji</name>
<affiliation wicri:level="1">
<nlm:aff id="a2">Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139</wicri:regionArea>
<wicri:noRegion>Massachusetts 02139</wicri:noRegion>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="a3">Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai 980-8579</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Annabi, Nasim" sort="Annabi, Nasim" uniqKey="Annabi N" first="Nasim" last="Annabi">Nasim Annabi</name>
<affiliation wicri:level="4">
<nlm:aff id="a4">School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, New South Wales 2006, Australia</nlm:aff>
<country xml:lang="fr">Australie</country>
<wicri:regionArea>School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, New South Wales 2006</wicri:regionArea>
<orgName type="university">Université de Sydney</orgName>
<placeName>
<settlement type="city">Sydney</settlement>
<region type="état">Nouvelle-Galles du Sud</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Ostrovidov, Serge" sort="Ostrovidov, Serge" uniqKey="Ostrovidov S" first="Serge" last="Ostrovidov">Serge Ostrovidov</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Ramalingam, Murugan" sort="Ramalingam, Murugan" uniqKey="Ramalingam M" first="Murugan" last="Ramalingam">Murugan Ramalingam</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="a5">Faculte de Medecine, National Institute of Health and Medical Research U977, Université de Strasbourg, Strasbourg, Cedex 67085, France</nlm:aff>
<country xml:lang="fr">France</country>
<wicri:regionArea>Faculte de Medecine, National Institute of Health and Medical Research U977, Université de Strasbourg, Strasbourg, Cedex 67085</wicri:regionArea>
<wicri:noRegion>67085</wicri:noRegion>
<orgName type="university">Université de Strasbourg</orgName>
<placeName>
<settlement type="city">Strasbourg</settlement>
<region type="region" nuts="2">Grand Est</region>
<region type="old region" nuts="2">Alsace (région administrative)</region>
</placeName>
</affiliation>
</author>
<author>
<name sortKey="Khademhosseini, Ali" sort="Khademhosseini, Ali" uniqKey="Khademhosseini A" first="Ali" last="Khademhosseini">Ali Khademhosseini</name>
<affiliation wicri:level="4">
<nlm:aff id="a1">WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan</nlm:aff>
<country xml:lang="fr">Japon</country>
<wicri:regionArea>WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577</wicri:regionArea>
<orgName type="university">Université du Tōhoku</orgName>
<placeName>
<settlement type="city">Sendai</settlement>
<region type="province">Région de Tōhoku</region>
</placeName>
</affiliation>
<affiliation wicri:level="1">
<nlm:aff id="a2">Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139</wicri:regionArea>
<wicri:noRegion>Massachusetts 02139</wicri:noRegion>
</affiliation>
<affiliation wicri:level="4">
<nlm:aff id="a6">Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115</wicri:regionArea>
<orgName type="university">Université Harvard</orgName>
<placeName>
<settlement type="city">Cambridge (Massachusetts)</settlement>
<region type="state">Massachusetts</region>
</placeName>
</affiliation>
</author>
</analytic>
<series>
<title level="j">Biomicrofluidics</title>
<idno type="ISSN">1932-1058</idno>
<idno type="eISSN">1932-1058</idno>
<imprint>
<date when="2011">2011</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc>
<textClass></textClass>
</profileDesc>
</teiHeader>
<front>
<div type="abstract" xml:lang="en">
<p>In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an
<italic>in vitro</italic>
model of Parkinson’s disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 μM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an
<italic>in vitro</italic>
model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating
<italic>in vitro</italic>
models of disease for drug discovery applications.</p>
</div>
</front>
</TEI>
<affiliations>
<list>
<country>
<li>Australie</li>
<li>France</li>
<li>Japon</li>
<li>États-Unis</li>
</country>
<region>
<li>Alsace (région administrative)</li>
<li>Grand Est</li>
<li>Massachusetts</li>
<li>Nouvelle-Galles du Sud</li>
<li>Région de Tōhoku</li>
</region>
<settlement>
<li>Cambridge (Massachusetts)</li>
<li>Sendai</li>
<li>Strasbourg</li>
<li>Sydney</li>
</settlement>
<orgName>
<li>Université Harvard</li>
<li>Université de Strasbourg</li>
<li>Université de Sydney</li>
<li>Université du Tōhoku</li>
</orgName>
</list>
<tree>
<country name="Japon">
<region name="Région de Tōhoku">
<name sortKey="Seidi, Azadeh" sort="Seidi, Azadeh" uniqKey="Seidi A" first="Azadeh" last="Seidi">Azadeh Seidi</name>
</region>
<name sortKey="Kaji, Hirokazu" sort="Kaji, Hirokazu" uniqKey="Kaji H" first="Hirokazu" last="Kaji">Hirokazu Kaji</name>
<name sortKey="Khademhosseini, Ali" sort="Khademhosseini, Ali" uniqKey="Khademhosseini A" first="Ali" last="Khademhosseini">Ali Khademhosseini</name>
<name sortKey="Ostrovidov, Serge" sort="Ostrovidov, Serge" uniqKey="Ostrovidov S" first="Serge" last="Ostrovidov">Serge Ostrovidov</name>
<name sortKey="Ramalingam, Murugan" sort="Ramalingam, Murugan" uniqKey="Ramalingam M" first="Murugan" last="Ramalingam">Murugan Ramalingam</name>
</country>
<country name="États-Unis">
<noRegion>
<name sortKey="Kaji, Hirokazu" sort="Kaji, Hirokazu" uniqKey="Kaji H" first="Hirokazu" last="Kaji">Hirokazu Kaji</name>
</noRegion>
<name sortKey="Khademhosseini, Ali" sort="Khademhosseini, Ali" uniqKey="Khademhosseini A" first="Ali" last="Khademhosseini">Ali Khademhosseini</name>
<name sortKey="Khademhosseini, Ali" sort="Khademhosseini, Ali" uniqKey="Khademhosseini A" first="Ali" last="Khademhosseini">Ali Khademhosseini</name>
</country>
<country name="Australie">
<region name="Nouvelle-Galles du Sud">
<name sortKey="Annabi, Nasim" sort="Annabi, Nasim" uniqKey="Annabi N" first="Nasim" last="Annabi">Nasim Annabi</name>
</region>
</country>
<country name="France">
<region name="Grand Est">
<name sortKey="Ramalingam, Murugan" sort="Ramalingam, Murugan" uniqKey="Ramalingam M" first="Murugan" last="Ramalingam">Murugan Ramalingam</name>
</region>
</country>
</tree>
</affiliations>
</record>

Pour manipuler ce document sous Unix (Dilib)

EXPLOR_STEP=$WICRI_ROOT/Wicri/Asie/explor/AustralieFrV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 006238 | SxmlIndent | more

Ou

HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 006238 | SxmlIndent | more

Pour mettre un lien sur cette page dans le réseau Wicri

{{Explor lien
   |wiki=    Wicri/Asie
   |area=    AustralieFrV1
   |flux=    Main
   |étape=   Exploration
   |type=    RBID
   |clé=     PMC:3145239
   |texte=   A microfluidic-based neurotoxin concentration gradient for the generation of an in vitro model of Parkinson’s disease
}}

Pour générer des pages wiki

HfdIndexSelect -h $EXPLOR_AREA/Data/Main/Exploration/RBID.i   -Sk "pubmed:21799720" \
       | HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd   \
       | NlmPubMed2Wicri -a AustralieFrV1 

Wicri

This area was generated with Dilib version V0.6.33.
Data generation: Tue Dec 5 10:43:12 2017. Site generation: Tue Mar 5 14:07:20 2024