Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

National Taiwan University

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.

Nuclear export of the large (60S) ribosomal subunit depends on the adapter protein Nmd3 to provide a nuclear export signal (NES). The leucine-rich NES is recognized by the export receptor Crm1 to mediate export via interaction with the nuclear pore complex (NPC). Here, we show that certain mutant Nmd3 proteins that are impaired for binding to the 60S subunit accumulate at the nuclear envelope. These mutant proteins also show enhanced binding to Crm1, both in vivo and in vitro. Although their interaction with the NPC is dependent on recognition of the NES by Crm1, their interaction with Crm1 is not strictly dependent on RanGTP. Using a collection of GFP-tagged nucleoporin mutants, we identified several nucleoporins, including components of the Nup82 complex that copurified with the mutant Nmd3. The Nup82 complex is on the cytoplasmic face of the NPC and has previously been shown to be important as a terminal binding site for Crm1-mediated export. Mutations in the Nup82 complex led to accumulation of wild-type Nmd3 in the nucleoplasm, suggesting that the interaction of mutant Nmd3 with the Nup82 complex reflects a defect in the bona fide export pathway for the 60S subunit. These results suggest that in the absence of the ribosome, Nmd3 is not efficiently released from Crm1 at the NPC.

Novel interaction of the 60S ribosomal subunit export adapter Nmd3 at the nuclear pore complex.

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

Arx1 is a nuclear export receptor for the 60S ribosomal subunit in yeast.

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages.

Reengineering ribosome export.

The ribosome stalk is essential for recruitment of translation factors. In yeast, P0 and Rpl12 correspond to bacterial L10 and L11 and form the stalk base of mature ribosomes, whereas Mrt4 is a nuclear paralogue of P0. In this study, we show that the dual-specificity phosphatase Yvh1 is required for the release of Mrt4 from the pre-60S subunits. Deletion of YVH1 leads to the persistence of Mrt4 on pre-60S subunits in the cytoplasm. A mutation in Mrt4 at the protein-RNA interface bypasses the requirement for Yvh1. Pre-60S subunits associated with Yvh1 contain Rpl12 but lack both Mrt4 and P0. These results suggest a linear series of events in which Yvh1 binds to the pre-60S subunit to displace Mrt4. Subsequently, P0 loads onto the subunit to assemble the mature stalk, and Yvh1 is released. The initial assembly of the ribosome with Mrt4 may provide functional compartmentalization of ribosome assembly in addition to the spatial separation afforded by the nuclear envelope.

Ribosome stalk assembly requires the dual-specificity phosphatase Yvh1 for the exchange of Mrt4 with P0.

In eukaryotic cells the final maturation of ribosomes occurs in the cytoplasm, where trans-acting factors are removed and critical ribosomal proteins are added for functionality. Here, we have carried out a comprehensive analysis of cytoplasmic maturation, ordering the known steps into a coherent pathway. Maturation is initiated by the ATPase Drg1. Downstream, assembly of the ribosome stalk is essential for the release of Tif6. The stalk recruits GTPases during translation. Because the GTPase Efl1, which is required for the release of Tif6, resembles the translation elongation factor eEF2, we suggest that assembly of the stalk recruits Efl1, triggering a step in 60S biogenesis that mimics aspects of translocation. Efl1 could thereby provide a mechanism to functionally check the nascent subunit. Finally, the release of Tif6 is a prerequisite for the release of the nuclear export adaptor Nmd3. Establishing this pathway provides an important conceptual framework for understanding ribosome maturation.

Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit.

&#x4F20;&#x7EDF;&#x7684;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x7ECF;&#x5E38;&#x88AB;&#x7528;&#x6765;&#x63A2;&#x6D4B;&#x901A;&#x8FC7;&#x5171;&#x8F6D;&#x8367;&#x5149;&#x6297;&#x4F53;&#x7684;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6807;&#x5FD7;&#x7269;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x8367;&#x5149;&#x5171;&#x8F6D;&#x7684;&#x6297;&#x4F53;&#x4F1A;&#x7ED1;&#x5B9A;&#x5C5E;&#x6027;&#xFF0C;&#x5982;&#x5F3A;&#x5EA6;&#x548C;&#x7279;&#x5F02;&#x6027;&#x6297;&#x4F53;&#x7684;&#x6539;&#x53D8;&#x5F80;&#x5F80;&#x65E0;&#x65B9;&#x5F0F;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x63D0;&#x51FA;&#x4E00;&#x79CD;&#x5229;&#x7528;&#x659C;&#x5165;&#x5C04;&#x53CD;&#x5C04;&#x7387;&#x5DEE;&#x5F02; (&#x53CB;&#x7231;&#x8DEF;) &#x663E;&#x5FAE;&#x955C;&#x7684;&#x65E0;&#x6807;&#x7B7E;&#x7684;&#x65B9;&#x6CD5;&#x5B9E;&#x65F6;&#x68C0;&#x6D4B;&#x7684;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6807;&#x8BB0;&#xFF0C;&#x5E76;&#x5C06;&#x5176;&#x5E94;&#x7528;&#x5230;&#x7279;&#x5B9A;&#x9636;&#x6BB5;&#x7684;&#x80DA;&#x80CE;&#x6297;&#x539F; 1 (SSEA1) &#x5BF9;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x5206;&#x6790;&#x3002;&#x5C0F;&#x9F20;&#x5E72;&#x7EC6;&#x80DE;&#x5176;&#x8868;&#x9762;&#x548C; SSEA1 &#x8DCC;&#x5E45;&#x7684;&#x6C34;&#x5E73;&#x8868;&#x793A; SSEA1&#xFF0C;&#x5F53;&#x7EC6;&#x80DE;&#x5F00;&#x59CB;&#x5206;&#x5316;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#xFF0C;&#x6211;&#x4EEC;&#x56FA;&#x5B9A;&#x5316;&#x5C0F;&#x9F20;&#x5E72;&#x7EC6;&#x80DE;&#x548C;&#x975E;-&#x5E72;&#x7EC6;&#x80DE; &#xFF08;&#x63A7;&#x5236;&#xFF09; &#x4F5C;&#x4E3A;&#x82AF;&#x7247;&#x7684;&#x73BB;&#x7483;&#x8868;&#x9762;&#x548C;&#x7EC6;&#x80DE;&#x82AF;&#x7247;&#x4E0E;&#x672A;&#x6807;&#x8BB0; SSEA1 &#x6297;&#x4F53;&#x7684;&#x53CD;&#x5E94;&#x3002;&#x901A;&#x8FC7;&#x4E0E;&#x4EC1;&#x7231;&#x8DEF;&#x663E;&#x5FAE;&#x955C;&#x5728;&#x5B9E;&#x65F6;&#x76D1;&#x89C6;&#x53CD;&#x5E94;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x5B9E; SSEA1 &#x6297;&#x4F53;&#x7ED1;&#x5B9A;&#x53EA;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x548C;&#x975E;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x8868;&#x9762;&#x3002;&#x4ECE;&#x7ED1;&#x5B9A;&#x7684;&#x66F2;&#x7EBF;&#xFF0C;&#x6211;&#x4EEC;&#x51B3;&#x5FC3;&#x4E0E;&#x5E72;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x4E0A;&#x7684; SSEA1 &#x6807;&#x8BB0;&#x6297;&#x4F53;&#x7684;&#x5E73;&#x8861;&#x89E3;&#x79BB;&#x5E38;&#x6570; &#xFF08;Kd&#xFF09;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x5728;&#x4EC1;&#x7231;&#x8DEF;&#x663E;&#x5FAE;&#x955C;&#x53EF;&#x4EE5;&#x7528;&#x4E8E;&#x68C0;&#x6D4B;&#x7EA6;&#x675F;&#x529B;&#x7684;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x6807;&#x5FD7;&#x7269;&#x548C;&#x672A;&#x6807;&#x8BB0;&#x7684;&#x6297;&#x4F53;&#x4E4B;&#x95F4;&#x7684;&#x4EB2;&#x7F18;&#x5173;&#x7CFB;&#x7ED1;&#x5B9A;&#x5230;&#x5355;&#x5143;&#x683C; &#xFF1B;&#x6B64;&#x4FE1;&#x606F;&#x53EF;&#x7528;&#x4E8E;&#x6D4B;&#x5B9A;&#x7684;&#x5E72;&#x7EC6;&#x80DE;&#x9636;&#x6BB5;&#x3002;

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Conventional fluorescence microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in often uncharacterized ways. Here we present a method using an oblique-incidence reflectivity difference (OI-RD) microscope for label-free, real-time detection of cell surface markers, and apply it to analysis of stage-specific embryonic antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces, and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies bind only to the surface of the stem cells and not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. Thus, the OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells; this information could be useful for determination of stem cell stages.

Label-free detection of surface markers on stem cells by oblique-incidence reflectivity difference microscopy.

Conventionnel microscopie &#xE0; fluorescence est couramment utilis&#xE9;e pour d&#xE9;tecter les marqueurs de surface cellulaire par le biais fluorophore anticorps conjugu&#xE9;s. Toutefois, un fluorophore conjugaison d&#x26;#39;anticorps alt&#xE8;re les propri&#xE9;t&#xE9;s de liaison tels que la force et la sp&#xE9;cificit&#xE9; de l&#x26;#39;anticorps de mani&#xE8;re souvent non caract&#xE9;ris&#xE9;s. Nous pr&#xE9;sentons ici une m&#xE9;thode utilisant une barre oblique d&#x26;#39;incidence diff&#xE9;rence de r&#xE9;flectivit&#xE9; (OI-RD) microscope pour sans &#xE9;tiquette, en temps r&#xE9;el de d&#xE9;tection de marqueurs de surface cellulaire, et l&#x26;#39;appliquer &#xE0; l&#x26;#39;analyse de l&#x26;#39;&#xE9;tape de l&#x26;#39;antig&#xE8;ne sp&#xE9;cifique embryonnaire 1 (SSEA1) sur les cellules souches . Les cellules souches de souris express SSEA1 sur leurs surfaces, et le niveau de SSEA1 diminue lorsque les cellules commencent &#xE0; se diff&#xE9;rencier. Dans cette &#xE9;tude, nous avons immobilis&#xE9; cellules souches de souris et non de ces cellules souches (de contr&#xF4;le) sur une surface de verre comme une puce et a r&#xE9;agi le micror&#xE9;seau de cellules non marqu&#xE9;es avec des anticorps SSEA1. En surveillant la r&#xE9;action avec un microscope PO-RD en temps r&#xE9;el, on confirme que les anticorps se lient SSEA1 seulement &#xE0; la surface des cellules souches et non &#xE0; la surface de cellules souches non. &#xC0; partir des courbes de liaison, on d&#xE9;termine la constante de dissociation &#xE0; l&#x26;#39;&#xE9;quilibre (Kd) de l&#x26;#39;anticorps avec les SSEA1 marqueurs sur la surface de cellules souches. Ainsi, le microscope OI-RD peut &#xEA;tre utilis&#xE9; pour d&#xE9;tecter des affinit&#xE9;s de liaison entre les marqueurs de surface cellulaire et des anticorps non marqu&#xE9;s li&#xE9;s aux cellules; cette information pourrait &#xEA;tre utile pour la d&#xE9;termination des stades de cellules souches.

Sans &#xE9;tiquette de d&#xE9;tection de marqueurs de surface sur les cellules souches par obliques incidence Microscopie diff&#xE9;rence de r&#xE9;flectivit&#xE9;

Konventionelle Fluoreszenzmikroskopie wird routinem&auml;&szlig;ig zur Zelle Oberfl&auml;che Marker durch Fluorophor-konjugierten Antik&ouml;rpern erkennen. Jedoch &auml;ndert Fluorophor-Konjugation von Antik&ouml;rpern Bindungseigenschaften wie Festigkeit und Spezifit&auml;t des Antik&ouml;rpers oft uncharakterisiertes M&ouml;glichkeiten. Hier pr&auml;sentieren wir Ihnen eine Methode mit einem schr&auml;g-Inzidenz Reflexionsverm&ouml;gen-Unterschied (OI-RD)-Mikroskop f&uuml;r Etikett-frei, Echtzeit-Erkennung von Zelle Oberfl&auml;che Marker, und wenden sie auf Analyse der embryonalen Phase-spezifisches-Antigen 1 (SSEA1) auf Stammzellen. Maus-Stammzellen schnelles SSEA1 auf ihrer Oberfl&auml;che, und das Niveau der SSEA1 sinkt, wenn die Zellen beginnen zu unterscheiden. In dieser Studie haben wir immobilisiert Maus-Stammzellen und nicht-Stammzellen (Kontrolle) auf einer Glasoberfl&auml;che als ein Microarray und reagiert die Zelle Microarray unbeschriftete SSEA1-Antik&ouml;rper. &Uuml;berwachen Sie die Reaktion mit einem OI-RD-Mikroskop in Echtzeit, best&auml;tigt wir, dass die SSEA1 Antik&ouml;rper nur an der Oberfl&auml;che der Stammzellen und nicht an die Oberfl&auml;che des nicht-Stammzellen binden. Der Bindung-Kurven haben wir die Dissoziation Gleichgewichtskonstante (Kd) des Antik&ouml;rpers mit dem SSEA1-Marker auf der Stammzell-Oberfl&auml;che festgestellt. So k&ouml;nnen Sie das OI-RD-Mikroskop zum erkennen Affinit&auml;ten zwischen Zelle Oberfl&auml;che Marker und unbeschriftete Antik&ouml;rper binden an die Zellen gebunden; Diese Informationen k&ouml;nnten f&uuml;r Bestimmung der Stammzelle Stufen n&uuml;tzlich sein.

Etikett-freie Abfragung der Oberfl&auml;che Marker auf Stammzellen durch schr&auml;g-Inzidenz Reflexionsverm&ouml;gen Unterschied Mikroskopie.

Microscopia di fluorescenza convenzionale viene utilizzata regolarmente per rilevare i marcatori di superficie cella attraverso anticorpi coniugati fluorophore. Tuttavia, fluorophore-coniugazione degli anticorpi altera le propriet&agrave; di associazione come forza e specificit&agrave; dell&#39;anticorpo in modi spesso atipici. Qui vi presentiamo un metodo utilizzando un microscopio di differenza (OI-RD) Incidenza obliqua riflettivit&agrave; per privo di etichetta, rilevazione in tempo reale della cella i marcatori di superficie e si applicano all&#39;analisi della specifica fase embrionale antigene 1 (SSEA1) sulle cellule staminali. Cellule staminali di topo express SSEA1 sulle loro superfici e il livello di SSEA1 diminuisce quando le cellule iniziano a differenziarsi. In questo studio, abbiamo immobilizzato cellule staminali di topo e non-cellule staminali (controllo) su una superficie di vetro come un microarray e ha reagito il microarray delle cellule con anticorpi SSEA1 adenoidi. Monitorando la reazione con un microscopio di OI-RD in tempo reale, abbiamo confermato che i SSEA1 gli anticorpi si legano solo alla superficie delle cellule staminali e non alla superficie delle cellule staminali. Dalle curve associazione, abbiamo determinato la costante di dissociazione di equilibrio (Kd) dell&#39;anticorpo con i marcatori SSEA1 sulla superficie delle cellule staminali. Cos&igrave;, il microscopio OI-RD pu&ograve; essere utilizzato per rilevare l&#39;associazione affinit&agrave; tra i marcatori di superficie delle cellule e degli anticorpi senza etichetta associata alle cellule; Questa informazione potrebbe essere utile per la determinazione delle fasi di cellule staminali.

Rilevamento di marcatori di superficie sulle cellule staminali da microscopia differenza di incidenza obliqua riflettivit&agrave; senza etichetta.

&#x5F93;&#x6765;&#x306E;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1;&#x306F;&#x3001;&#x65E5;&#x5E38;&#x7684;&#x306B;&#x30D5;&#x30EB;&#x30AA;&#x30ED;&#x30D5;&#x30A9;&#x30A2;&#x6A19;&#x8B58;&#x6297;&#x4F53;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x6297;&#x4F53;&#x306E;&#x86CD;&#x5149;&#x4F53;&#x5171;&#x5F79;&#x306F;&#x3001;&#x3053;&#x306E;&#x3088;&#x3046;&#x306A;&#x5F37;&#x3055;&#x3068;&#x3001;&#x3057;&#x3070;&#x3057;&#x3070;&#x672A;&#x77E5;&#x306E;&#x65B9;&#x6CD5;&#x3067;&#x6297;&#x4F53;&#x306E;&#x7279;&#x7570;&#x6027;&#x306A;&#x3069;&#x306E;&#x7D50;&#x5408;&#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x3092;&#x5909;&#x66F4;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306E;&#x30E9;&#x30D9;&#x30EB;&#x30D5;&#x30EA;&#x30FC;&#x3001;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x306E;&#x691C;&#x51FA;&#x306E;&#x305F;&#x3081;&#x306E;&#x659C;&#x5165;&#x5C04;&#x53CD;&#x5C04;&#x7387;&#x5DEE;&#xFF08;OI-RD&#xFF09;&#x9855;&#x5FAE;&#x93E1;&#x3092;&#x7528;&#x3044;&#x305F;&#x65B9;&#x6CD5;&#x3092;&#x63D0;&#x793A;&#x3057;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306B;&#x30B9;&#x30C6;&#x30FC;&#x30B8;&#x56FA;&#x6709;&#x306E;&#x80CE;&#x5150;&#x6027;&#x6297;&#x539F;1&#xFF08;SSEA1&#xFF09;&#x306E;&#x5206;&#x6790;&#x306B;&#x9069;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x304C;&#x5206;&#x5316;&#x3092;&#x958B;&#x59CB;&#x3057;&#x305F;&#x3068;&#x304D;&#x306B;&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x5E79;&#x7D30;&#x80DE;&#x306F;&#x305D;&#x306E;&#x8868;&#x9762;&#x306B;SSEA1&#x3092;&#x8868;&#x73FE;&#x3057;&#x3001;SSEA1&#x6E1B;&#x5C11;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30A2;&#x30EC;&#x30A4;&#x306E;&#x3088;&#x3046;&#x306B;&#x30AC;&#x30E9;&#x30B9;&#x8868;&#x9762;&#x4E0A;&#x3067;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x5E79;&#x7D30;&#x80DE;&#x3068;&#x975E;&#x5E79;&#x7D30;&#x80DE;&#xFF08;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#xFF09;&#x56FA;&#x5B9A;&#x5316;&#x3068;&#x975E;&#x6A19;&#x8B58;SSEA1&#x6297;&#x4F53;&#x3068;&#x7D30;&#x80DE;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30A2;&#x30EC;&#x30A4;&#x3092;&#x53CD;&#x5FDC;&#x3055;&#x305B;&#x305F;&#x3002;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x3067;OI-RD&#x9855;&#x5FAE;&#x93E1;&#x3068;&#x306E;&#x53CD;&#x5FDC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;SSEA1&#x6297;&#x4F53;&#x304C;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x306B;&#x3067;&#x306F;&#x306A;&#x304F;&#x3001;&#x975E;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x306B;&#x306E;&#x307F;&#x7D50;&#x5408;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x305F;&#x3002;&#x7D50;&#x5408;&#x66F2;&#x7DDA;&#x304B;&#x3089;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x4E0A;SSEA1&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068;&#x6297;&#x4F53;&#x306E;&#x5E73;&#x8861;&#x89E3;&#x96E2;&#x5B9A;&#x6570;&#xFF08;Kd&#xFF09;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;OI-RD&#x9855;&#x5FAE;&#x93E1;&#x306F;&#x7D30;&#x80DE;&#x306B;&#x7D50;&#x5408;&#x3057;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068;&#x672A;&#x6A19;&#x8B58;&#x6297;&#x4F53;&#x3068;&#x306E;&#x7D50;&#x5408;&#x89AA;&#x548C;&#x6027;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x60C5;&#x5831;&#x306F;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x6BB5;&#x968E;&#x306E;&#x6C7A;&#x5B9A;&#x306B;&#x6709;&#x7528;&#x3067;&#x3042;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x659C;&#x5165;&#x5C04;&#x53CD;&#x5C04;&#x7387;&#x5DEE;&#x9855;&#x5FAE;&#x93E1;&#x306B;&#x3088;&#x308B;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x9762;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306E;&#x30E9;&#x30D9;&#x30EB;&#x30D5;&#x30EA;&#x30FC;&#x691C;&#x51FA;

&#xAE30;&#xC874;&#xC758; &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; fluorophore conjugated &#xD56D; &#xCCB4;&#xB97C; &#xD1B5;&#xD574; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4;&#xB97C; &#xAC10;&#xC9C0; &#xD558; &#xC77C;&#xC0C1;&#xC801;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xD56D; &#xCCB4;&#xC758; fluorophore-&#xD65C;&#xC6A9; &#xC885;&#xC885; &#xC0C8;&#xB86D;&#xAC70;&#xB098; &#xBC29;&#xBC95;&#xC73C;&#xB85C; &#xAC15;&#xB3C4; &#xBC0F; &#xD56D; &#xCCB4;&#xC758; &#xD2B9;&#xC774;&#xC131; &#xB4F1;&#xC758; &#xBC14;&#xC778;&#xB529; &#xC18D;&#xC131;&#xC744; &#xBCC0;&#xACBD;&#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;&#xC5D0; &#xC6B0;&#xB9AC;&#xAC00; &#xD604;&#xC7AC; &#xB808;&#xC774;&#xBE14; &#xC5C6;&#xB294; &#xB300; &#xD55C; &#xAC04;&#xC811; &#xBC1C;&#xC0DD;&#xB960; &#xBC18;&#xC0AC;&#xB3C4; &#xCC28;&#xC774; (&#xD558;&#xC774;-RD) &#xD604;&#xBBF8;&#xACBD;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xBA54;&#xC11C;&#xB4DC; &#xC140;&#xC758; &#xC2E4;&#xC2DC;&#xAC04; &#xD0D0;&#xC9C0; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4;, &#xADF8;&#xB9AC;&#xACE0; &#xBB34;&#xB300; &#xAD00;&#xB828; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBC30;&#xC544; &#xD56D; &#xC6D0; 1 (SSEA1)&#xC758; &#xBD84;&#xC11D;&#xC5D0; &#xC801;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC6B0;&#xC2A4; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC140;&#xC744; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; &#xB370; &#xC2DC;&#xC791;&#xD560; &#xB54C; &#xADF8;&#xB4E4;&#xC758; &#xD45C;&#xBA74;&#xC5D0; SSEA1 &#xAC10;&#xC18C; &#xC218;&#xC900;&#xC744; SSEA1 &#xC775;&#xC2A4;&#xD504;&#xB808;&#xC2A4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xB9C8;&#xC6B0;&#xC2A4; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBC0F; &#xBE44;-&#xC904;&#xAE30; &#xC138;&#xD3EC; (&#xC81C;&#xC5B4;)&#xB97C; microarray&#xB85C; &#xC720;&#xB9AC; &#xD45C;&#xBA74;&#xC5D0; &#xC6C0;&#xC9C1;&#xC77C; &#xD558; &#xACE0; &#xC140; microarray &#xB808;&#xC774;&#xBE14;&#xC774; &#xC5C6;&#xB294; SSEA1 &#xD56D; &#xCCB4;&#xC640; &#xBC18;&#xC751;. &#xC2E4;&#xC2DC;&#xAC04;&#xC73C;&#xB85C; &#xC624;&#xC774; RD &#xD604;&#xBBF8;&#xACBD;&#xC73C;&#xB85C; &#xBC18;&#xC751;&#xC744; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD568;&#xC73C;&#xB85C;&#xC368; &#xC6B0;&#xB9AC;&#xB294; SSEA1 &#xD56D; &#xCCB4; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xD45C;&#xBA74; &#xC544;&#xB2C8;&#xB77C; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xD45C;&#xBA74;&#xC5D0;&#xB9CC; &#xBC14;&#xC778;&#xB529;&#xD560; &#xD655;&#xC778; &#xD588;&#xB2E4;. &#xBC14;&#xC778;&#xB529; &#xCEE4;&#xBE0C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; SSEA1 &#xD45C;&#xC2DD;&#xC774; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; &#xC788;&#xB294; &#xD56D; &#xCCB4;&#xC758; &#xD3C9;&#xD615; &#xBD84;&#xB9AC; &#xC0C1;&#xC218; (Kd) &#xACB0;&#xC815;. &#xC624;&#xC774; RD &#xD604;&#xBBF8;&#xACBD;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC140;;&#xC5D0; &#xBC14;&#xC778;&#xB529;&#xB41C; &#xBC14;&#xC778;&#xB529; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4; &#xBC0F; &#xB808;&#xC774;&#xBE14; &#xC5C6;&#xB294; &#xD56D; &#xCCB4; &#xC0AC;&#xC774;&#xC758; &#xC120;&#xD638;&#xB3C4; &#xAC10;&#xC9C0; &#xD558;&#xB294; &#xB530;&#xB77C;&#xC11C;, &#xC774; &#xC815;&#xBCF4;&#xB294; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xB2E8;&#xACC4; &#xACB0;&#xC815;&#xC5D0; &#xC720;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xB808;&#xC774;&#xBE14; &#xC5C6;&#xB294; &#xAC04;&#xC811; &#xBC1C;&#xC0DD;&#xB960; &#xBC18;&#xC0AC;&#xB3C4; &#xCC28;&#xC774; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95;&#xC5D0; &#xC758;&#xD574; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xB9C8;&#xCEE4; &#xAC80;&#xCD9C;.

Microscop&#xED;a de fluorescencia convencional se utiliza rutinariamente para detectar marcadores de superficie celular a trav&#xE9;s fluor&#xF3;foro anticuerpos conjugados. Sin embargo, fluor&#xF3;foro-conjugaci&#xF3;n de anticuerpos altera las propiedades de uni&#xF3;n tales como la resistencia y la especificidad del anticuerpo en formas a menudo no caracterizados. Aqu&#xED; presentamos un m&#xE9;todo que utiliza una oblicua de incidencia diferencia de reflectividad (OI-RD) microscopio libre de marca, en tiempo real la detecci&#xF3;n de marcadores de superficie celular, y aplicarlo al an&#xE1;lisis de ant&#xED;geno espec&#xED;fico de la fase embrionaria 1 (SSEA1) sobre las c&#xE9;lulas madre . C&#xE9;lulas madre de ratones expresan SSEA1 en su superficie, y el nivel de SSEA1 disminuye cuando las c&#xE9;lulas comienzan a diferenciarse. En este estudio, que inmoviliz&#xF3; c&#xE9;lulas madre de ratones y las c&#xE9;lulas madre no (control) sobre una superficie de vidrio como los microarrays y reaccionaron los microarrays de c&#xE9;lulas con anticuerpos no marcados SSEA1. Mediante la supervisi&#xF3;n de la reacci&#xF3;n con un microscopio de OI-DR en tiempo real, se confirm&#xF3; que los anticuerpos se unen SSEA1 s&#xF3;lo a la superficie de las c&#xE9;lulas madre y no a la superficie de las c&#xE9;lulas madre no. De las curvas de uni&#xF3;n, se determin&#xF3; la constante de equilibrio de disociaci&#xF3;n (Kd) de los anticuerpos con los marcadores SSEA1 en la superficie de c&#xE9;lulas madre. As&#xED;, el microscopio de OI-RD puede ser utilizado para detectar afinidades de uni&#xF3;n entre los marcadores de la superficie celular y los anticuerpos no marcados unidos a las c&#xE9;lulas; esta informaci&#xF3;n podr&#xED;a ser &#xFA;til para la determinaci&#xF3;n de las etapas de c&#xE9;lulas madre.

Libre de marca de detecci&#xF3;n de marcadores de superficie de las c&#xE9;lulas madre por Microscop&#xED;a de incidencia oblicua reflectividad Diferencia

Reactive oxygen species (ROS) are known to be a key factor in the development of cancer, and many exogenous sources are supposed to be related to the formation of ROS. In this paper, a microfluidic chip was developed for studying the production of ROS in lung cancer cells under different chemical and physical stimuli. This chip has two unique features: (1) five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 are achieved in the culture regions; (2) a shear stress gradient is produced inside each of the five culture areas. Lung cancer cells were seeded inside this biocompatible chip for investigating their response to different concentrations of H2O2, a chemical stimulus known to increase the production of ROS. Then the effect of shear stress, a physical stimulus, on lung cancer cells was examined, showing that the production of ROS was increased in response to a larger shear stress. Finally, two antioxidants, α-tocopherol and ferulic acid, were used to study their effects on reducing ROS. It was found that high-dose α-tocopherol was not able to effectively eliminate the ROS produced inside cells. This counter effect was not observed in cells cultured in a traditional chamber slide, where no shear stress was present. This result suggests that the current microfluidic chip provides an in vitro platform best mimicking the physiological condition where cells are under circulating conditions.

Effects of shear stresses and antioxidant concentrations on the production of reactive oxygen species in lung cancer cells.

Cell migration is an essential process involved in the development and maintenance of multicellular organisms. Electric fields (EFs) are one of the many physical and chemical factors known to affect cell migration, a phenomenon termed electrotaxis or galvanotaxis. In this paper, a microfluidics chip was developed to study the migration of cells under different electrical and chemical stimuli. This chip is capable of providing four different strengths of EFs in combination with two different chemicals via one simple set of agar salt bridges and Ag/AgCl electrodes. NIH 3T3 fibroblasts were seeded inside this chip to study their migration and reactive oxygen species (ROS) production in response to different EF strengths and the presence of β-lapachone. We found that both the EF and β-lapachone level increased the cell migration rate and the production of ROS in an EF-strength-dependent manner. A strong linear correlation between the cell migration rate and the amount of intracellular ROS suggests that ROS are an intermediate product by which EF and β-lapachone enhance cell migration. Moreover, an anti-oxidant, α-tocopherol, was found to quench the production of ROS, resulting in a decrease in the migration rate.

Correlation between cell migration and reactive oxygen species under electric field stimulation.

In the present study, evaluation and optimization of taro waste (TW), which was mainly composed of taro peels that contain many starch residues, as the main carbon source in medium were studied. The flask studies showed the optimal medium was using 170g/L of TW which is about 100g/L of glucose and 9g/L of CGM as alternative nitrogen source. Simultaneous saccharification and fermentation (SSF) exhibited higher bioethanol productivity toward separation hydrolysis and fermentation (SHF). The optimal condition of SSF was 5% of Kluyveromyces marxianus K21 inoculum at 40°C resulting in the maximum ethanol concentration (48.98g/L) and productivity (2.23g/L/h) after 22h of cultivation. The scaling up experiment in a 5L bioreactor demonstrated that K21 can still maintain its capability. After 20h of cultivation, 43.78g/L of ethanol (2.19g/L/h of productivity) was achieved corresponding to a 94.2% theoretical ethanol yield.

Bioethanol production from taro waste using thermo-tolerant yeast Kluyveromyces marxianus K21.

A plastic composite support (PCS) bioreactor was implemented to evaluate the effects on isoflavone deglycosylation in black soymilk fermented by Rhizopus oligosporus NTU 5.

Enrichment of two isoflavone aglycones in black soymilk by Rhizopus oligosporus NTU 5 in a plastic composite support bioreactor.

Collective cell migration plays important roles in many physiological processes such as embryonic development, tissue repair, and angiogenesis. A "wound" occurs when epithelial cells are lost and/or damaged due to some external factors, and collective cell migration takes place in the following wound-healing process. To study this cellular behavior, various kinds of wound-healing assays are developed. In these assays, a "wound," or a "cell-free region," is created in a cell monolayer mechanically, chemically, optically, or electrically. These assays are useful tools in studying the effects of certain physical or chemical stimuli on the wound-healing process. Most of these methods have disadvantages such as creating wounds of different sizes or shapes, yielding batch-to-batch variation, and damaging the coating of the cell culture surface. In this study, we used ultraviolet (UV) lights to selectively kill cells and create a wound out of a cell monolayer. A comparison between the current assay and the traditional scratch assay was made, indicating that these two methods resulted in similar wound-healing rates. The advantages of this UV-created wound-healing assay include fast and easy procedure, high throughput, and no direct contact to cells.

A Wound-Healing Assay Based on Ultraviolet Light Ablation.

Puf6 and Loc1 have two important functional roles in the cells, asymmetric mRNA distribution and ribosome biogenesis. Puf6 and Loc1 are localized predominantly in the nucleolus. They bind ASH1 mRNA, repress its translation, and facilitate the transport to the daughter cells. Asymmetric mRNA distribution is important for cell differentiation. Besides their role in mRNA localization, Puf6 and Loc1 have been shown to be involved in 60S biogenesis. In puf6∆ or loc1∆ cells, pre-rRNA processing and 60S export are impaired and 60S subunits are under-accumulated. The functional studies of Puf6 and Loc1 have focused on ASH1 mRNA pathway, but their roles in 60S biogenesis are still not clear. In this study, we found that Puf6 and Loc1 interact directly with each other and both proteins interact with the ribosomal protein Rpl43 (L43e). Notably, the roles of Puf6 and Loc1 in 60S biogenesis are interdependent and both are required for efficient accommodation of Rpl43. Loc1 is further required to maintain the protein level of Rpl43. Additionally, the recruitment of Rpl43 is required for release of Puf6 and Loc1. We propose that Puf6 and Loc1 facilitate Rpl43 loading and they are sequentially released from 60S after incorporation of Rpl43 into ribosomes in yeast.

Kai-Yin Lo

Department of Agricultural Chemistry

National Taiwan University

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Kai-Yin Lo

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Agricultural Chemistry

National Taiwan University

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Department of Agricultural Chemistry