We greatly acknowledge financial support by NIH R01 CA123495, U01 CA151815, R01 CA136841, grants from the Department of Neurosurgery at Cedars-Cedars Medical Center and Arrogene Technology Inc.

Todos los experimentos se ajustan a las disposiciones oficiales de los animales, incluidos los procedimientos quirúrgicos y no quirúrgicos realizados en vivo y están en plena conformidad con los protocolos del IACUC. 1. Bioproducción de ácido polimálico <ol><li> Haga crecer un cultivo de siembra de esférulas en agar y la transferencia de los plasmodios a 100 ml de medio de cultivo utilizando un 25 ° C Thermo declarado incubadora y medio de cultivo basal 8,9. </li><li> Produce una cantidad de gravedad llena 500 ml plasmodios micro bajo la exclusión de la luz con el fin de evitar la esporulación. </li><li> Preparar 80 g de CaCO 3 en suspensión en 8 L de medio basal en 10 L de recipiente biorreactor. Transferir los 500 ml envasados ​​plasmodios micro en el recipiente de reacción montado y permitir que el bioproceso de 75 horas a 25 ° C, 10 L / min de flujo de aire filtrado y 150 rpm de agitación mediante un agitador segmento. </li><li> Terminar cuando el caldo de cultivo tiene un pH de 4,8 que indica el final de la producción de MPLA y medir el contenido de MPLA por tque hidroxamato / Fe (III) de ensayo. </li><li> Enfriar el caldo a 17 ° C y permiten que las células se asienten por gravedad. </li><li> Se enfría a 4 ° C y ajustar el pH a 7,5, utilice 2 M NaOH. Bombear el sobrenadante a través de una columna llena con 1,5 l de DEAE-celulosa en la parte inferior dirección hacia arriba (DEAE grano grueso, equilibrada con 20 mM de Tris-HCl, pH 7.5 a 5 ° C). </li><li> Lavar con 3 columnas de tampón que contiene NaCl 0,3 M después de cambiar la dirección de arriba a abajo y eluir PMLA en presencia de 0,7 M de NaCl. </li><li> Ajuste a 0,1 M CaCl2 y precipitar PMLA-Calcio con 80% etanol helado. Fraccionar sobre Sephadex G25 en PMLA-calcio de 80 a 300 kDa, 50 - 80 kDa, y 10 - 50 kDa, utilizar agua en ausencia de tampón y sal. </li><li> Pasar cada fracción sobre un 120H + columna de Amberlite IR, y congelar inmediatamente el ácido polimálico de flujo a través en nitrógeno líquido durante la liofilización. </li><li> Disolver en acetona seca. Después de la filtración, el disolvente se retiraen la corriente de aire seco, se liofiliza y se almacena a -20 ° C. </li></ol> 2. Síntesis de a base de ácido polimálico Nano Drogas <ol><li> Activar grupos carboxilo de MPLA (P) mediante la mezcla de 116 mg (1 mmol unidades malyl) de PMLA-H en 1 ml de acetona anhidra y 1 mmol de N-hidroxisuccinimida y 1 mmol diciclohexil carbodiimida en 2 ml de dimetil formamida (DMF). Se agita a 20 ° C durante 3 h. Añadir 0,05 mmol de mPEG 5000-NH 2 (0,05% en moles de unidades malyl) en 1 ml de DMF seguido de 0,05 mmol de trietilamina (TEA). </li><li> Añadir gota a gota disuelto en DMF 0,4 mmol de éster etílico de leucina (Loet) (40% en moles de unidades malyl), 0,1 mmol de ácido 2-tiol-1-etilamina (2-MEA) (10% en moles de unidades malyl) y 0,5 mmoles de TEA, todos disueltos en DMF. Después de cada adición, deje 1 hora y compruebe que no se completó la reacción de ninhidrina negativo de respuesta (capa-cromatografía fina, TLC). </li><li> No retire los restos de éster NHS por hidrólisis espontánea con solución salina tamponada con fosfato (30 min, pH 6,8). Desalar más de PD-10 Columna, obtener &quot;preconjugate&quot; como un polvo blanco por liofilización. Seco tienda a menos 20 ° C. </li><li> Disolver 30 mg de anticuerpo (mAb, IgG2a-κ) en 4,5 ml de fosfato de sodio 100 mM, NaCl 150 mM, pH 5,5. </li><li> Reducir los enlaces disulfuro con Tris 5 mM (acetato de 2-carboxi) de clorhidrato de fosfina durante 30 min a 20 ° C y purificar sobre columna PD-10. </li><li> Disolver Mal-PEG 3400-MAL en 2 ml del mismo tampón y añadir el mAb reducido a un volumen final de 10 ml y se agita durante la noche a 4 ° C. Se concentra hasta 2,5 ml durante Vivaspin 20, la exclusión de 30 kDa, y purificar a través de Sephadex G75. Verifique producto sobre sec-HPLC y medir la cantidad fotométrica a 280 nm de longitud de onda. </li><li> Adjuntar Mal-PEG-MAL-mAb a &quot;preconjugate&quot; tioles obtención de P / PEG 5000 (5%) / Loet (40%) / mAb (0,2%) / 2-MEA (10%). Para ello, mezclar 5 ml de 200 nmol mAb-PEG 3400-Mal a 50 mg (p / mPEG 5000 / LOEt/2-MEA) en el pH por encima de 5,5, ajustar la concentración de sulfhyDryl a 2 mM y se incuba a 20 ° C durante 3 horas (rendimiento 98%). </li><li> Solubilizar 3&#39;-H 2 N-AON en DMF / PBS (pH 7,2) y reaccionar con N-succinimidil-3-(2-piridilditio) propionato (SPDP) en (01:01 v / v) de DMF / metanol (prueba de ninhidrina ) y purificar AON-PDP sobre Sephadex LH20-en metanol, y luego en agua y se liofiliza. </li><li> Incubar 800 nmol AONs activados en el buffer de pH 5,5 con el Mal-PEG 3400-Mal-MAB-preconjugate anteriormente preparado durante toda la noche hasta que se termina la reacción de intercambio de disulfuro. </li><li> Incorporar fluorescente Alexa Fluor 680-maleimida formando tioéter con polímero-2-MEA-SH. Bloquear el exceso de sulfhidrilos con 3-piridilditio-propionato (PDP) y purificar sobre Sephadex G75. Almacenar a 4 ° C o se congeló a -80 ° C hasta su uso posterior. </li></ol> 3. Ensayos y Propiedades <ol><li> Realizar pruebas para la pureza y la estabilidad en PBS y suero a 4 ° C y 37 ° C por análisis SEC-HPLC. Utilice sulfonato poliestireno como molecLos estándares de peso Ular. Mida distribución de tamaño nano y zeta potenciales. Utilice los sistemas comerciales. </li><li> Añadir a 320 l muestra de 160 l de 10% (w / v) de cloruro de hidroxilamonio y 160 l de 10% (w / v) de NaOH. Después de 10-15 minutos, mezclar con 160 l de 5% (w / v) de Fe (III) Cl 3 en el 12% (v / v) de HCl y leer A 540 de hidroxamato / Fe (III). Calcular PMLA asumiendo 1 mg / ml PMLA corresponde a 2,5 A 540. </li><li> Hidrolizar el fármaco de nano durante la noche en HCl 2 M a 110 ° C y ensayo de ácido málico en la cromatografía de fase inversa con referencia a las muestras cargadas con las normas de ácido málico. Cleave la droga nano con ditiotreitol 100 mM y medir Morpholino-AON por HPLC en fase reversa cuantitativa frente a estándares Morpholino AON. </li><li> Tomar drogas nano sin escote y medir anticuerpos usando un kit de ensayo de proteínas. Cuantificar mPEG al permitir que su reacción con ferrothiocyanate amonio, a continuación, se extrae con cloroformo y leer la absorbencia a 510 nm de longitud de onda 10 </sup&gt;. </li><li> Realice ELISA con 5 l de drogas nano (1-10 mg MAB) por pocillo ensayar la actividad de anticuerpos y coligación. Utilice 0,5 mg / pocillo receptor de transferrina y HER2, respectivamente, como antígenos de placa recubierta y anticuerpos secundarios acoplados a peroxidasa. </li><li> Calcular logrado% para AON, MPEG y mAb con respecto a ácido málico (100%) y comparar con el% previsto por diseño (ver ejemplos Tabla 1 y Tabla 2). </li></ol> 4. Pruebas in vitro <ol><li> Incubar el fármaco nano con células cancerosas cultivadas y medida de células supervivientes con el tiempo por su actividad para reducir reactivo amarillo [3 - (4,5-dimetiltiazol-2-il) -5 - (3-carboximetoxifenil) -2 - (4-sulfofenil )-2H-tetrazolio, sal interna] (MTS) a un colorante azul y leer la absorbancia relativa con un fotómetro. Siga las instrucciones del proveedor kit. </li><li> Preparar extractos de células in vitro o ex vivo para la transferencia Western. Utilice helada extracción buffer que contiene SDS y cóctel inhibidor de la proteasa. Proteínas separadas por electroforesis en gel de reducción de SDS-poliacrilamida. </li><li> Realice el Western Blot para las proteínas de interés en los extractos de la muestra. Utilice anticuerpos secundarios conjugados con fosfatasa alcalina. </li><li> Uso de la microscopia confocal para demostrar la captación celular y co-localización de nano plataforma de polímero de drogas, AON y endosomas usando etiquetado de fluorescencia. Adjunte Alexa Fluor 680 a la plataforma conjugado nano y Lissamina a AONs como etiquetas fluorescentes. Utilice un microscopio 5X escáner espectral y células cancerosas vivas para visualizar la distribución de las moléculas marcadas </li><li> Membranas endosoma mancha con tinte fluorescente Estiril Red y demuestran colocalización endosoma o liberación. </li></ol> 5. Ensayo in vivo <ol><li> Preparar el modelo de ratón de cáncer de mama positivo para HER2 humana (Tac de ratón desnudo: Cr: (MCR)-Foxnnm). Inserte un 0,72 mg liberando pellet 17β-estradiol (versión 90-días) subcutaneously en el cuello de cada ratón. </li><li> Luego inyecte 1 x 10 7 células tumorales BT-474 por vía subcutánea en el flanco derecho y se dejó crecer el tumor. </li><li> Iniciar el tratamiento cuando los tumores son 120 mm 3 en tamaño mediante la inyección en la vena de la cola 150 conjugado nano l que contiene 2,5 mg / kg de AON y / o 4,5 mg / kg de cada anticuerpo. Para el tratamiento inyectar dos veces por semana, total de seis veces. </li><li> Mida el tamaño del tumor con calibres dos veces el débil y el cálculo de volúmenes utilizando la fórmula (longitud x profundidad x anchura) x (π / 6). La eutanasia a los animales 2 semanas después de la inyección final y después de la sedación con una solución de ketamina y dexmedetomidina seguido por dislocación cervical. </li><li> Aislar los tumores y las secciones de tinción con H &amp; E para la evaluación morfológica. </li><li> Para la evaluación de la distribución de drogas a escala animal, utilizar un sistema de imágenes de fluorescencia. Inyectar la Alexa Fluor 680 drogas nano etiqueta e imagen a las 24 y 48 h. </li><li> La eutanasia a los animales y detectar el fluorEscence en tumores y órganos aislados y perfundidos. </li></ol>

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Los autores Julia Y. Ljubimova, Keith L. Black, y Eggehard Holler son accionistas de Arrogene Technology Inc.

La ruta experimental para la preparación de medicamentos nano de un polímero natural biodegradable se presentó que se puede utilizar en la síntesis de la medicina personalizada. La descripción comienza con la producción controlada y la purificación de ácido polimálico, que es una plataforma versátil para la síntesis de drogas nano. Utilizando técnicas reproducibles, el polímero se obtiene con alto peso molecular y en la pureza extrema adecuado para síntesis farmacéuticas. La síntesis se describe por un medicamento nano que se muestra para el tratamiento eficaz del cáncer de mama HER2-positivo. La descripción se puede traducir en la síntesis de la mayoría de otros fármacos nano para el tratamiento del cáncer. Orientación implica anticuerpos tales como Herceptin que se unen a un antígeno específico de tumor, tales como proteína HER2 o cualquier otro marcador tumoral que se internaliza de manera eficiente. El fármaco de nano ofrece una selección de oligonucleótidos antisentido y quimioterápicos que inhibe eficazmente el crecimiento del cáncer bajo Tretamiento. En el ejemplo, Herceptin vinculante para HER2 y la hibridación específica de oligonucleótidos antisentido con HER2-mRNA de codificación resultó en el bloqueo sostenido de HER2-señalización y reducción grave de cáncer de mama HER2-positivo. Basado en el principio subyacente de la focalización del tumor y la inhibición de la expresión génica mediante oligonucleótidos antisentido hemos actualizado sintetizado varias otras drogas nano y éxito inhibido glioblastoma humano preclínica y el cáncer de mama triple negativo 11,14-18. El trabajo de síntesis comienza con la preparación de la plataforma de drogas nano altamente purificada, que es el ácido polimálico del sobrenadante del cultivo de Physarum polycephalum (una especie de la familia &quot;moho de fango&quot;). La preparación enfatiza altos pesos moleculares del polímero, en principio, lo que permite la unión de numerosos anticuerpos, péptidos, oligonucleótidos y otras moléculas que funcionan en la entrega de fármaco activo y la inhibición del crecimiento del tumor. Following el cultivo y la purificación controlada, calidad reproducible de polímero en rendimientos predecibles se ha producido. El polímero se almacena bajo condiciones convenientes para cualquier momento. La síntesis de conjugados de nanopartículas PMLA que comienzan con la activación química de los carboxilatos polímero colgante se lleva a cabo en unos pocos pasos sintéticos. En entre los pasos, la síntesis se puede colocar opcionalmente en espera que permite la preparación de cantidades arbitrarias de intermedios y por lo tanto se puede utilizar en un escalado. El progreso de la síntesis se sigue por TLC y SEC-HPLC, y tanto la composición y la actividad del fármaco de nano se controla mediante ensayos específicos para el grupo químico cuantitativo, ELISA, y una variedad de mediciones físicas. Nuestra experiencia es que estas síntesis se han desarrollado sin contratiempos y de manera reproducible con excelentes rendimientos y pureza. Al optar por la especificidad de anticuerpos y oligonucleótidos antisentido cualquier variante de drogas nano se sintetiza favorablemente, según sea necesario en personalized medicamento. Las modalidades del tratamiento exitoso del cáncer de mama HER2-positivo humano son interlocutores válidos para la preparación de modelos de cáncer de ratón, la aplicación de drogas nano, imágenes, y el análisis del crecimiento del tumor. Los resultados de las pruebas de viabilidad in vitro en son útiles en la selección de la línea celular y el fármaco que conduce a ser utilizada en el experimento con animales. En vivo de imágenes de Xenogen valida que el fármaco nano está entregado de hecho en el tumor. Resultados de Western Blot revela si el nivel de ciertas proteínas respondió de la manera prevista durante el tratamiento del cáncer. Medición del tamaño del tumor informa sobre el éxito del tratamiento, es decir, sobre la inhibición, la recesión o la regresión. El ejemplo descrito refleja nuestra experiencia con otros fármacos nano a base de ácido polimálico que indican un alto grado de previsibilidad, la reproducibilidad y la ausencia de toxicidad notables. Resultados recientes sobre la toxicidad y la eficacia de múltiples tconjugados de ácido polimálico argeted para el tratamiento triple negativo cáncer de mama se encuentran en un fuerte apoyo de esta noción 18. Una característica clave es que nuestro fármaco nano es capaz de penetrar barreras biológicas: la barrera endotelial por la extravasación en el intersticial del tumor, la membrana de las células tumorales por absorción endosoma, y ​​la membrana del endosoma interrupción por la acción de los grupos de éster etílico de leucina o trileucina. La extravasación y la captación endosoma se llevan a cabo de forma fiable por los anticuerpos específicos vinculados a la droga nano. Anticuerpo receptor de transferrina anti-ratón de media afluencia eficiente en el tumor por transcitosis, probablemente debido a que el receptor se sobreexpresa en la mayoría de la vasculatura del tumor. Un anticuerpo diferente unido en las mismas funciones nano molécula conjugada en dirigir específicamente el fármaco nano en la célula tumoral receptor. La presencia de ambos anticuerpos, el uno para transcitosis y la otra para la absorción endosoma, es esencial para el funcionamiento óptimo. El análisis cuantitativo de mácido Alic y el anticuerpo es altamente recomendado para el control de la composición óptima del fármaco nano. Por último hay que señalar que el fármaco de nano covalente todo-en-uno administra el fármaco en forma unida químicamente en ruta a través de la vasculatura huésped en la célula tumoral receptor. La unión química hace que la mayoría de los fármacos inactivos (profármacos) hasta que se reconstituyen como medicamentos gratuitos por la escisión de la plataforma conjugado nano en el lugar de destino. Esto es importante porque la modalidad de reactivación proporciona un alto grado de seguridad durante el parto y una mínima posibilidad de evocar efectos secundarios dañinos. La versatilidad, eficacia y seguridad son atributos indispensables de una buena medicina personalizada.

En la era post-genómica en genomas de cáncer se han desentrañado (Centro Nacional de Biotecnología y el Atlas del Genoma del Cáncer), el tratamiento futuro del cáncer representará la diversidad genética de los tumores a menudo dentro del mismo tumor 1-4. Bioinformática y rápido, no es caro secuenciación de ADN permite la adquisición de genes / mutaciones malignas a nivel personal 2,4,5. Una vez que los genes han sido identificados, los pacientes serán tratados con la medicina personalizada para modificar o silenciar la expresión de genes malignos 6. La necesidad de dirigirse a las células cancerosas y liberar fármacos en estas células requiere de sistemas de entrega polifuncionales. Obviamente, las drogas nano pueden cumplir con este requisito 7. En una ola creciente de descubrimientos nanopartículas han demostrado ser adecuado para llevar las cargas útiles de los fármacos quimioterapéuticos, proteínas y / o material genéticamente activa a las células cancerosas. Sin embargo, los efectos adversos siguen siendo advestida. Uno de ellos relacionado con la falta de biodegradabilidad puede causar la deposición de material en el tejido y órganos sanos con la probabilidad de provocar enfermedades. Para minimizar la deposición, se introdujo ácido polimálico no tóxico y no inmunogénico, que es de origen microbiano y biodegradable a H 2 O y CO 2 8. Utilizamos el polímero para sintetizar un tipo covalente todo-en-uno de la droga nano. Contiene agentes quimioterapéuticos unidos químicamente tales como temozolomida, doxorrubicina, o los oligonucleótidos antisentido y los grupos funcionales que sirven extravasación, la orientación del tejido, la entrega endosomolítico. Las drogas están intrínsecamente escinden de la plataforma nano cuando llegaron a la célula tumoral específica, regenerando así su actividad farmacéutica completa. Se describe el método de producción microbiana de la plataforma nano polímero, su purificación, y la síntesis química de un fármaco que contiene nano Trastuzumab (Herceptin) para el cáncerfocalización y un oligonucleótido antisentido para la inhibición de HER2 sobreproducción. En la aplicación de la droga nano a xenogeneico cáncer de mama HER2-positivo humano en ratones desnudos, se demuestra una alta eficacia del tratamiento contra el cáncer. Los principios de la orientación del tumor y el silenciamiento de gen introducido aquí para medicamentos nano ácido polimálico se pueden aplicar en el tratamiento de otros casos de cáncer.

<table border="0" cellpadding="0" cellspacing="0" width="935">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Name of Reagent/Material</td>
			<td style="width:263px;">Company</td>
			<td style="width:187px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercapto-1-ethylamine (Cysteamine (2MEA)</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">30078-25G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-Diamidino-2-phenylindole (DAPI)</td>
			<td colspan="2">Vector Laboratories, Burlingame, CA</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">90-Day release 17&#946;-estradiol pellet&#160;</td>
			<td>Innovative Research of America</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 680 C2 maleimide</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:187px;">A20344</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amberlite IR 120H</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">6428</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Antifoam Y-30 Emulsion</td>
			<td style="width:263px;">Sigam-Aldrich</td>
			<td style="width:187px;">A5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-laminin-411 chain mAbs</td>
			<td style="width:263px;">Santa Cruz</td>
			<td style="width:187px;">SC-59980</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-pAkt mAb</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">9271S</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-PARB mAb</td>
			<td style="width:263px;">BD Biosciences</td>
			<td style="width:187px;">556494</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-von Willebrand Factor antibody&#160;</td>
			<td>Abcam</td>
			<td style="width:187px;">AB6994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Bacto Yeast Extract</td>
			<td style="width:263px;">Bacto, Dickinson/Sparks, MD</td>
			<td style="width:187px;">212720</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-actin</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">3700</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BT-474&#160;</td>
			<td style="width:263px;">ATCC</td>
			<td style="width:187px;">HTB-20</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Calcium Carbonate</td>
			<td style="width:263px;">Alfa Aesar</td>
			<td style="width:187px;">36337</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Proliferation Assay kit</td>
			<td>Promega, Madison, WI, USA</td>
			<td>PR-G3580</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centriplus 100&#160;</td>
			<td>Millipore</td>
			<td style="width:187px;">4414</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dicyclohexylcarbodimide</td>
			<td style="width:263px;">Fluka</td>
			<td style="width:187px;">36650</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">D5796</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin</td>
			<td style="width:263px;">Cardinal Health</td>
			<td style="width:187px;">S7439-4</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galardin (MMP-inhibitors)&#160;</td>
			<td style="width:263px;">Santa Cruz</td>
			<td style="width:187px;">SC-994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH</td>
			<td style="width:263px;">Cell Signalling&#160;</td>
			<td style="width:187px;">2118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxilin</td>
			<td style="width:263px;">Cardinal Health</td>
			<td style="width:187px;">S7439-3</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Hemin from porcine</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">51280</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herceptin</td>
			<td style="width:263px;">Genentech</td>
			<td style="width:187px;">15534</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herceptin (Western blotting)</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">2165S</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">IgG2a-kappa murine malignoma</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:187px;">M77695X5m</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immun-Star AP Substrate Pack</td>
			<td style="width:263px;">Biorad</td>
			<td style="width:187px;">170-5012</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immun-StarTM AP Substrate Pack&#160;</td>
			<td>Biorad</td>
			<td>170-5012</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">LAL Reagent water&#160;</td>
			<td style="width:263px;">Lonza, MD</td>
			<td style="width:187px;">W50-1000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin-411 mAbs</td>
			<td style="width:263px;">Abcam</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leucine ethyl ester (LOEt)</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">61850-10G-F</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Limulus Amebocyte Lysate (LAL) PYROGENT-5000 tests</td>
			<td style="width:263px;">Cambrex BioScience</td>
			<td style="width:187px;">N384</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Lissamin-Morpholino AON</td>
			<td style="width:263px;">Gene Tools</td>
			<td style="width:187px;">Custom made</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-malic acid&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">M6413</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Malate dehydrogenase&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">M2634</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mal-PEG-Mal</td>
			<td style="width:263px;">Laysan</td>
			<td style="width:187px;">mal-PEG-mal-3400</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td style="width:187px;">354248</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milk, nonfat powdered&#160;</td>
			<td style="width:263px;">Proteomics</td>
			<td>M203-10G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Morpholino oligonucleotides&#160;</td>
			<td style="width:263px;">GeneTools</td>
			<td style="width:187px;">custom made</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">mPEG5000</td>
			<td style="width:263px;">Lysan</td>
			<td style="width:187px;">mPEG-NH2-5000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-hydroxysuccinimde (NHS)&#160;</td>
			<td style="width:263px;">ACROS Organics</td>
			<td style="width:187px;">15727100</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Ninhydrin</td>
			<td style="width:263px;">Merck</td>
			<td style="width:187px;">1.06762.0100</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose Membrane Filter Paper Sandwich</td>
			<td>Invitrogen</td>
			<td>LC2001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose Membrane Filter Paper Sandwich, 0.45 &#181;m&#160;</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:187px;">LC2001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Novex &#174; Tris-Glycine Transfer Buffer (25X)&#160;</td>
			<td>Invitrogen</td>
			<td>LC3675</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nude Mice [Tac:Cr:(MCr)-Foxnnm]&#160;</td>
			<td>Taconic</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">PBS pH 7.2 10x</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:187px;">10010-49</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">PBS pH 7.2 1x</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:187px;">70013</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD-10 desalting columns</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0851</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Physarum polycephalum M3CVII</td>
			<td>ATCC</td>
			<td>204388</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce BCA protein assay&#160;</td>
			<td style="width:263px;">Thermo Scientific</td>
			<td style="width:187px;">23225</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor cocktail</td>
			<td style="width:263px;">Roche</td>
			<td style="width:187px;">1.18362E+11</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Detector ELISA Kit</td>
			<td style="width:263px;">KPL</td>
			<td style="width:187px;">54-62-18</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G-25 superfine</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0031</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G-75&#160;</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0050</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex-LH20&#160;</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0090</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKBR-3&#160;</td>
			<td style="width:263px;">ATCC</td>
			<td style="width:187px;">HTB-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPDP</td>
			<td style="width:263px;">Proteochem&#160;</td>
			<td style="width:187px;">C1116</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streamline-DEAE</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Styryl Red FM 1-43&#160;</td>
			<td>Life Technologies&#160;</td>
			<td style="width:187px;">T-3163</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TBS 10x</td>
			<td>Bio-Rad</td>
			<td>170-6435</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCEP</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">C4706-2G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TLC, silica coated aluminia sheets</td>
			<td style="width:263px;">Merck, Darmstadt, DE</td>
			<td style="width:187px;">60F254</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trileucine (LLL)</td>
			<td style="width:263px;">Bachem</td>
			<td style="width:187px;">H-3915</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-114&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">X114</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween&#174;20&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin 20&#160;</td>
			<td style="width:263px;">VWR</td>
			<td style="width:187px;">14005-302</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:407px;">DAPI</td>
			<td style="width:263px;">Vector Laboratories, Burlingame, CA</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dexmedetomidine</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atipamezole</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td style="width:263px;">Foster and Smith, Wisconsin</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

polymalic acid-based nano biopolymers for targeting of multiple tumor markers: an opportunity for personalized medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

Watch this Scientific Journal Video about Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? at JoVE.com

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Un ejemplo de un fármaco de nano a base de ácido polimálico se presenta hacia el diseño racional de la medicina personalizada que es aplicable al cáncer. En él se describe la síntesis de un fármaco nano para tratar el cáncer de mama HER2-positivo humano en un ratón desnudo.

Biopolímeros Nano basados ​​en ácido polimálico de Focalización de múltiples Marcadores tumorales: una oportunidad para la medicina personalizada?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

polymalic-acid-based-nano-biopolymers-for-targeting-multiple-tumor

Research

JoVE Journal

Chemistry

16.5K vistas.  Cedars-Sinai Medical Center.  Un ejemplo de un fármaco de nano a base de ácido polimálico se presenta hacia el diseño racional de la medicina personalizada que es aplicable al cáncer. En él se describe la síntesis de un fármaco nano para tratar el cáncer de mama HER2-positivo humano en un ratón desnudo. 

Video: Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

El análisis de glicanos Nodo: un enfoque de abajo hacia arriba para Glicómica

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Investigación

Química

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Altamente estable, funcional Hairy nanopartículas y biopolímeros con fibras de madera: Hacia Nanotecnología Sostenible

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Síntesis de Proteínas bioconjugados<em&gt; vía</em&gt; Química cisteína-maleimida

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

Catodoluminiscencia de baja energía para (Oxy) Los fósforos de nitruro

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Preparación y Evaluación de<sup&gt; 99m</sup&gt; Tc-etiquetados tridentado Los quelatos de orientación previa Usando Bioorthogonal Química

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

An Aptamer-based Sensor for Unchelated Gadolinium(III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

Un sensor basado en aptámero para no quelado de gadolinio (III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

División ingeniería-TET2 enzima ADN químico-inducible Hydroxymethylation y remodelación epigenéticos

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

La expresión de múltiples proteínas de fusión quimérica de fluorescente de manera eficiente en las plantas

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Iron Nanowire Fabrication by Nano-Porous Anodized Aluminum and its Characterization

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and data storage. We demonstrate a fabrication method for iron (Fe) nanowires&#160;via electrochemical deposition into anodic&#160;alumina oxide (AAO) templates. The templates are fabricated by anodization of aluminum (Al) discs, and the pore length and diameter are controlled by changing the anodizing conditions. Pores with an average diameter of around 120 nm are created using oxalic acid as the electrolyte. Using this method, cylindrical nanowires are synthesized, which are released by dissolving the alumina using a selective chemical etchant.

In this work, we describe a&#160;protocol to fabricate iron nanowires, including the formation of the porous alumina membrane that is used as the template, electrodeposition into templates using electrolyte solution, and the release of the nanowires into the solution.

Fabricación de Nanowire de Hierro de Aluminio Anodizado Nano-Porous y su Caracterización

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and ...

Organic Structure-directing Agent-free Synthesis for *BEA-type Zeolite Membrane

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in petroleum and petrochemical fields because of their high thermal, chemical, and mechanical strength. A *BEA-type zeolite is an interesting membrane material because of its large pore size and wide Si/Al range. This manuscript presents a protocol for *BEA membrane preparation by a secondary growth method that does not use an organic structure-directing agent (OSDA). The preparation protocol consists of four steps: pretreatment of support, seed preparation, dip-coating, and membrane crystallization. First, the *BEA seed crystal is prepared by conventional hydrothermal synthesis using OSDA. The synthesized seed crystal is loaded on the outer surface of a 3 cm long tubular &#945;-Al2O3 support by a dip-coating method. The loaded seed layer is prepared with the secondary growth method using a hydrothermal treatment at 393 K for 7 days without using OSDA. A *BEA membrane having very few defects is successfully obtained. The seed preparation and dip-coating steps strongly affect the membrane quality.

A *BEA seed crystal was loaded on a porous &#945;-Al2O3 support by the dip-coating method, and hydrothermally grown without using an organic structure-directing agent. A *BEA-type zeolite membrane having very few defects was successfully prepared by the secondary growth method.

Síntesis libre de agentes que dirige la estructura orgánica para la membrana de zeolita tipo BEA

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in ...

Biopolímeros Nano basados ​​en ácido polimálico de Focalización de múltiples Marcadores tumorales: una oportunidad para la medicina personalizada?

Biopolímeros Nano basados en ácido polimálico de Focalización de múltiples Marcadores tumorales: una oportunidad para la medicina personalizada?