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 os experimentos cumprir com os regulamentos oficiais, incluindo animais de procedimentos cirúrgicos e não cirúrgicos realizados in vivo e estão em plena conformidade com os protocolos IACUC. 1. Bioprodução de ácido Polymalic <ol><li> Crescer uma cultura de sementes de esférulas em agar e transferir plasmódios a 100 ml de meio de cultura com a 25 ° C thermo afirmou incubadora e meio de cultura basal 8,9. </li><li> Produzir uma quantidade de repleto de gravidade de 500 ml plasmódios micro, com exclusão de luz, a fim de evitar a esporulação. </li><li> Prepare 80 g CaCO3 suspensas em 8 L meio basal em 10 L navio biorreator. Transferir 500 ml de micro plasmódio embalados dentro do recipiente de reacção e permitem a montagem de bioprocessos para 75 horas a 25 ° C, de 10 L / min, fluxo de ar filtrado e 150 rpm de agitação por um agitador segmento. </li><li> Termina quando o caldo de cultura tem pH 4,8, indicando o fim da produção de MPLA e medir o teor de PMLA por tele hidroxamato / Fe (III) de ensaio. </li><li> Arrefece-se o caldo a 17 ° C e permitir que as células assentar por gravidade. </li><li> Arrefece-se até 4 ° C e o pH ajustado a 7,5, utilizar NaOH 2 M. Bombear o sobrenadante através de uma coluna cheia com 1,5 L de DEAE-celulose, em direcção ao fundo para o topo (DEAE grãos grosseiros, equilibrada com 20 mM Tris-HCl pH 7,5 a 5 ° C). </li><li> Lavar com 3 colunas de tampão contendo 0,3 M de NaCl, após a alteração da direcção de cima para baixo e eluir PMLA na presença de 0,7 M de NaCl. </li><li> Ajuste a 0,1 M CaCl 2 e precipitar PMLA-Cálcio com 80% de gelo etanol frio. Fraccionar sobre Sephadex G25 em PMLA-cálcio de 80-300 kDa, 50 - 80 kDa, e 10 - 50 kDa, usar água na ausência de tampão e de sal. </li><li> Passar cada fracção através de uma Amberlite IR 120H + coluna, e congelar imediatamente o ácido polymalic fluxo através de azoto líquido para a liofilização. </li><li> Dissolve-se em acetona seca. Após filtração, remover solventeno fluxo de ar seco, Liofilizar e loja a menos 20 ° C. </li></ol> 2. Síntese de base de ácido Polymalic Nano droga <ol><li> Activar os grupos carboxilo de MPLA (P) através da mistura de 116 mg (1 mmol unidades malil) de MPLA-H em 1 ml de acetona anidra e 1 mmol de N-hidroxissuccinimida e 1 mmol de diciclo-carbodiimida em 2 ml de dimetil formamida (DMF). Agita-se a 20 ° C durante 3 hr. Adicionar 0,05 mmol de mPEG 5000-NH 2 (0,05% em moles de unidades malil) em 1 mL de DMF seguido de 0,05 mmol de trietilamina (TEA). </li><li> Adicionar gota a gota, dissolvido em DMF de 0,4 mmoles de éster etílico leucina (Loet) (40% em mol de unidades malil), 0,1 mmol de 2-tiol-1-etilamina (2-MEA) (10% em mol de unidades malil) e 0,5 mmol de TEA, tudo dissolvido em DMF. Após cada adição, deixe 1 hora e verificar a existência de final da reacção negativa por ninidrina resposta (layer-cromatografia de camada delgada, TLC). </li><li> Não remover restos de NHS-éster por hidrólise espontânea com solução salina tamponada com fosfato (30 min, pH 6,8). Dessalinizar sobre PD-10 Coluna, obter &quot;preconjugate&quot;, como um pó branco por liofilização. Seco loja a menos 20 ° C. </li><li> Dissolve-se 30 mg de anticorpos (mAb, IgG2a-κ) em 4,5 ml de fosfato de sódio 100 mM, NaCl 150 mM, pH 5,5. </li><li> Reduzir ligações dissulfureto com 5 mM de tris (etil-2-carboxi) de cloridrato de fosfina durante 30 min a 20 ° C e purifica-se sobre coluna de PD-10. </li><li> Dissolve-Mal-PEG 3400-Mal em 2 ml do mesmo tampão e adicionar o mAb reduzida a um volume final de 10 ml e agitou-se durante a noite a 4 ° C. Concentre-se para 2,5 ml sobre Vivaspin 20, a exclusão de 30 kDa, e purificar sobre Sephadex G75. Verifique produto sobre seg-HPLC e medir quantidade fotometria no comprimento de onda de 280 nm. </li><li> Anexar Mal-PEG-Mal-mAb &quot;preconjugate&quot; tióis obtenção P / PEG 5000 (5%) / Loet (40%) / mAb (0,2%) / 2-MEA (10%). Para fazer isso, a mistura de 5 ml de 200 nmol de mAb 3400-PEG-MAL de 50 mg (P / mPEG 5000 / LOEt/2-MEA) no tampão de pH acima de 5,5, ajustar a concentração de sulfhydryl a 2 mM e incuba-se a 20 ° C durante 3 horas (rendimento de 98%). </li><li> Solubiliza-3&#39;-H2N-AON em DMF / PBS (pH 7,2) e reagem com N-succinimidil-3-(2-piridilditio) propionato (SPDP), em (1:1, v / v) de DMF / metanol (teste da ninidrina ) e purificar AON-PDP sobre Sephadex-LH20 em metanol, em seguida, em água e Liofilizar. </li><li> Incubar 800 nmol AONs activados no tampão de pH 5,5 com o acima preparado Mal-PEG 3400-Mal-Acm-preconjugate durante a noite, até a reacção de permuta de dissulfureto é terminada. </li><li> Incorporar fluorescente Alexa Fluor 680-maleimide formando tioer com polímero-2-MEA-SH. Bloqueie sulfídrilos excesso com 3-piridiltio-propionato (PDP) e purificar sobre Sephadex G75. Armazenar a 4 ° C ou congelados rapidamente à temperatura de -80 ° C até à sua utilização. </li></ol> 3. Ensaios e Propriedades <ol><li> Realizar testes de pureza e estabilidade em PBS e soro a 4 ° C e 37 ° C por meio de análise de SEC-HPLC. Use sulfonato de poliestireno como MolecOs padrões de peso Ular. Medir a distribuição de tamanho de nano-zeta e potenciais. Utilizar sistemas comerciais. </li><li> Adicionar a 320 mL de amostra 160 mL de 10% (w / v) de cloreto de hidroxilamónio e 160 mL de 10% (w / v) de NaOH. Depois de 10-15 minutos, se misturar com 160 ul de 5% (w / v), Fe (III) Cl 3 em 12% (v / v) de HCl e 540 de ler um hidroxamato / Fe (III). Calcular PMLA assumindo 1 mg / ml PMLA corresponde a 2,5 A 540. </li><li> Hidrolisar a droga nano durante a noite em HCl 2M a 110 ° C e de ácido málico no ensaio de cromatografia em fase inversa, com referência a amostras contendo padrões de ácido málico. Cleave a droga nano com 100 mM ditiotreitol e medir Morfolino-AON por HPLC de fase reversa quantitativa em relação aos padrões Morfolino AON. </li><li> Tome drogas nano sem clivagem e medir anticorpo usando um kit de ensaio de proteína. Quantificar mPEG, permitindo sua reação com ferrothiocyanate de amônio, em seguida, extrair com clorofórmio e ler a absorvância a 510 nm de comprimento de onda de 10 </sup&gt;. </li><li> Realize ELISA com 5 mL de droga nano (1-10 mg mAb) por poço testar atividade de anticorpos e coligação. Use 0,5 ug / poço receptor de transferrina e de HER2, respectivamente, como os antigénios revestidos por placa e anticorpos secundários acoplados com peroxidase. </li><li> Calcule% obtidos para AON, MPEG e mAb no que diz respeito ao ácido málico (100%) e comparar com o% pretendida pelo projeto (ver exemplos Tabelas 1 e 2). </li></ol> 4. Testes in vitro <ol><li> Incubar a droga nano com células cancerígenas cultivadas e medida de células sobreviventes ao longo do tempo, pela sua actividade para reduzir amarelo reagente [3 - (4,5-dimetiltiazol-2-il) -5 - (3-carboximetoxifenil) -2 - (4-sulfofenil )-2H-tetrazólio, sal interno] (MTS) de um corante azul e ler a absorbância em relação com um fotómetro. Siga as instruções do fornecedor kit. </li><li> Preparar extractos de células in vitro ou ex vivo para a transferência de Western. Use gelada extração bpode ser prejudicado que contém SDS e cocktail inibidor de protease. Proteínas separadas por electroforese em gel de redução de SDS-poliacrilamida. </li><li> Do western blotting para as proteínas de interesse nos extratos das amostras. Use anticorpos secundários conjugados com a fosfatase alcalina. </li><li> Use microscopia confocal para demonstrar a captação celular e co-localização de nano plataforma polímero de drogas, AON e endosomes usando rotulagem de fluorescência. Anexar Alexa Fluor 680 para a plataforma conjugado nano e Lissamine para AONs como rótulos de fluorescência. Usar um scanner de 5X espectral microscópio e as células cancerosas vivas para visualizar a distribuição das moléculas marcadas </li><li> Membranas endossoma mancha com tinta fluorescente Estiril Red e demonstrar co-localização endosome ou liberação. </li></ol> 5. Testes in vivo <ol><li> Prepare o modelo do rato de câncer de mama HER2-positivo humano (Tac nu mouse: Cr: (MCR)-Foxnnm). Insira um 0,72 mg 17β-estradiol liberando pellet (liberação de 90 dias) subcutaneously no pescoço de cada rato. </li><li> Em seguida, injetar 1 x 10 7 células tumorais BT-474 por via subcutânea no flanco direito e deixar o tumor crescer. </li><li> Iniciar o tratamento quando os tumores são de 120 mm 3 de tamanho através da injecção na veia da cauda de 150 conjugado nano ul contendo 2,5 mg / kg de AON e / ou 4,5 mg / kg de cada anticorpo. Para o tratamento injetar duas vezes por semana, num total de seis vezes. </li><li> Meça o tamanho do tumor com pinças de duas vezes o fraco e calcular volumes usando a fórmula (comprimento x largura x profundidade) x (π / 6). Eutanásia animais 2 semanas após a última injecção e após sedação com uma solução de cetamina e dexmedetomidina, seguido por deslocação cervical. </li><li> Isolar tumores e seções mancha com H &amp; E para análise morfológica. </li><li> Para avaliar a distribuição de medicamentos em escala animal, usar um sistema de imagens de fluorescência. Injectar a Alexa Fluor 680 nano rotulado de drogas e de imagem em 24 e 48 horas. </li><li> Eutanásia do animal e detectar o fluorescence em tumores e órgãos isolados e perfundidos. </li></ol>

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Os autores Julia Y. Ljubimova, Keith L. Preto, e Eggehard Holler são acionistas da Arrogene Technology Inc.

A via experimental para a preparação de medicamentos de nano a partir de um polímero natural biodegradável é apresentado que possa ser usado na síntese de medicina personalizada. A descrição inicia-se com a produção controlada e purificação de ácido polymalic, que é uma plataforma versátil para a síntese de drogas nano. Usando técnicas reprodutíveis, o polímero é obtido com elevado peso molecular e em extrema pureza adequada para a síntese de produtos farmacêuticos. A síntese é descrita para uma droga nano que é mostrada para o tratamento eficaz do cancro da mama HER2-positivos. A descrição pode ser traduzida em síntese da maioria dos outros medicamentos nano para o tratamento de cancro. Direccionamento envolve anticorpos, tais como Herceptin que se ligam a um antigénio específico de tumor, tais como a proteína HER2 ou qualquer outro marcador de tumor que é eficientemente interiorizado. A droga nano proporciona uma selecção de oligonucleótidos anti-sentido e quimioterápicos que irá inibir eficientemente o crescimento do cancro sob tretamento. No exemplo, a ligação Herceptin para HER2 e recozimento oligonucleotídeo antisense específico com mRNA resultou no bloqueio sustentada de HER2-sinalização e redução grave de câncer de mama HER2-positivo de codificação HER2. Com base no princípio subjacente de segmentação tumor ea inibição da expressão gênica por oligonucleotídeos antisense temos a data sintetizados vários outros medicamentos nano e com sucesso inibido glioblastoma humano pré-clínico e triplo-negativo câncer de mama 11,14-18. O trabalho de síntese começa com a preparação de altamente purificada plataforma droga nano, que é ácido polymalic do sobrenadante da cultura de Physarum polycephalum (uma espécie de família &quot;fungo&quot;). A preparação enfatiza elevados pesos moleculares do polímero, permitindo que, em princípio, a ligação de vários anticorpos, péptidos, oligonucleótidos e outras moléculas que funcionam na entrega da droga activa e inibição do crescimento do tumor. Following a cultura controlada e purificação, qualidade reprodutível de polímero em rendimentos previsíveis foi produzido. O polímero é armazenado em condições convenientes para a qualquer momento. A síntese de conjugados de PMLA nano começando com a activação química dos carboxilatos de polímero-pendentes é realizada em alguns passos de síntese. Entre passos, a síntese pode ser opcionalmente colocados em espera que permite a preparação de quantidades arbitrárias de intermediários e, assim, pode ser utilizado na extrapolação. O progresso da síntese é seguida por TLC e os seg-HPLC, e tanto a composição e actividade da droga nano é controlado por ensaios específicos para grupos químicos quantitativo, ELISA, e uma variedade de medições físicas. Nossa experiência é que essas sínteses foram progrediu bem e reproduzível com excelentes rendimentos e pureza. Ao escolher a especificidade de anticorpos e anti-sentido oligonucleótidos qualquer variante de droga nano é sintetizado favoravelmente como necessário em eprsonalized medicina. As modalidades do sucesso do tratamento do câncer de mama HER2-positivo humano são representantes válidos para elaboração de modelos de câncer mouse, aplicação de medicamentos nano, imagem e análise do crescimento do tumor. Os resultados dos testes de viabilidade in vitro são úteis na escolha da linha de células e o líder da droga a ser utilizada na experiência animal. Xenogen imagiologia in vivo confirma que a droga nano é efectivamente entregues no tumor. Os resultados de western blotting revela se o nível de certas proteínas respondeu da maneira prevista durante o tratamento do câncer. Medição do tamanho do tumor informa sobre o sucesso do tratamento, ou seja, cerca de inibição, recessão ou de regressão. O exemplo descrito reflecte a nossa experiência com outras polymalic à base de ácido nano drogas indicando um elevado grau de previsibilidade e reprodutibilidade notável ausência de toxicidade. Os resultados recentes sobre a toxicidade ea eficácia de vários targeted conjugados de ácido polymalic para o tratamento triplo-negativo câncer de mama estão em forte apoio a esta hipótese 18. Uma característica fundamental é que nossa droga nano é capaz de penetrar bio-barreiras: a barreira endotelial por extravasamento para o interstício tumoral, a membrana da célula tumoral por captação endosome e ruptura da membrana endosome pela ação dos grupos etílico leucina ou trileucina. Extravasamento e absorção endosome são realizadas de forma confiável pelos anticorpos específicos associados à droga nano. Anticorpo para o receptor da transferrina anti-ratinho medeia influxo eficiente para o tumor por transcitose, provavelmente, porque o receptor está sobre-expresso na maioria dos vasos do tumor. Um anticorpo diferente ligado nas mesmas funções da molécula de conjugado em nano especificamente dirigir a droga nano na célula tumoral destinatário. A presença de ambos os anticorpos, o outro para a transcitose e o outro para a absorção do endossoma, é essencial para o funcionamento óptimo. A análise quantitativa de mácido álicos e anticorpo é altamente recomendado, a fim de controlar a composição ótima da droga nano. Finalmente, deve notar-se que a droga covalente nano tudo-em-um liberta o fármaco na forma quimicamente ligada rota através da vasculatura para o hospedeiro de células de tumor receptor. Fixação química torna a maioria das drogas inativo (pró-fármacos), até que sejam reconstituídos como medicamentos gratuitos por clivagem da plataforma conjugado nano no local alvejado. Isto é importante porque a modalidade reactivação fornece um alto grau de segurança durante a distribuição e a oportunidade mínima de evocar efeitos secundários nocivos. Versatilidade, eficácia e segurança são atributos indispensáveis ​​de um bom remédio personalizado.

Na era pós-genômica quando genomas de câncer já foram desvendados (Centro Nacional de Biotecnologia e The Cancer Genome Atlas), o tratamento futuro de câncer serão responsáveis ​​pela diversidade genética dos tumores, muitas vezes dentro de um mesmo tumor 1-4. Bioinformática e rápido, não é caro sequenciamento de DNA permite a aquisição de genes malignos / mutações em um nível pessoal 2,4,5. Uma vez que os genes foram identificados, os pacientes serão tratados com a medicina personalizada para modificar ou silenciar a expressão de genes malignas 6. A necessidade de atingir as células cancerosas e entregar drogas para essas células exige sistemas de entrega polifuncionais. Obviamente, as drogas nano pode atender a essa exigência 7. Em uma onda de afluência de descobertas as nanopartículas têm provado adequado para trazer cargas de fármacos quimioterapêuticos, proteínas e / ou de material geneticamente activa para células cancerosas. No entanto, os efeitos adversos continuam a ser advestida. Um deles relacionado com a ausência de biodegradabilidade pode causar deposição de material no tecido e órgãos saudáveis ​​com o risco de provocar doenças. Para minimizar a deposição, é introduzido ácido polymalic não-tóxico e não imunogénico, o que é de origem microbiana e biodegradável a H 2 O e CO 2 8. Nós usamos o polímero para sintetizar um tipo de droga nano covalente tudo-em-um. Ela contém substâncias quimioterápicas quimicamente ligados, como Temozolomida, doxorrubicina, ou oligonucleótidos anti-sentido e os grupos funcionais que servem extravasamento, segmentação do tecido, entrega endosomolytic. As drogas estão intrinsecamente separado da plataforma nano quando chegaram na célula tumoral alvo, regenerando assim a sua actividade farmacêutica integral. Descreve-se o método de produção microbiana da plataforma nano polímero, a sua purificação, e a síntese química de um fármaco que contém nano Trastuzumab (Herceptin) para o cancroA segmentação e um oligonucleótido anti-sentido para a inibição da sobreprodução de HER2. Ao aplicar o medicamento nano para HER2-positivo câncer de mama humano xenogénicos em camundongos nude, demonstramos alta eficácia do tratamento do câncer. Os princípios de direccionamento de tumor e o silenciamento do gene introduzido aqui para polymalic drogas nano ácido pode ser aplicado no tratamento de outros casos de cancro.

<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?

Um exemplo de uma droga baseada em nano ácido polymalic é apresentado para o desenho racional de medicina personalizada que é aplicável para o cancro. Ela descreve a síntese de um fármaco nano para tratar o cancro da mama humano HER2-positivos em um ratinho nu.

À base de ácido Polymalic Nano Biopolímeros para Segmentação de múltiplos marcadores tumorais: uma oportunidade para a 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 visualizações.  Cedars-Sinai Medical Center.  Um exemplo de uma droga baseada em nano ácido polymalic é apresentado para o desenho racional de medicina personalizada que é aplicável para o cancro. Ela descreve a síntese de um fármaco nano para tratar o cancro da mama humano HER2-positivos em um ratinho nu. 

Vídeo: 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.

Glicanos Análise Node: uma abordagem de baixo para cima para Glycomics

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

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 estável, funcional Hairy Nanopartículas e Biopolímeros de fibras de madeira: Rumo a nanotecnologia Sustentável

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.

Síntese de Bioconjugados de proteínas<em&gt; via</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).

Catodoluminescência baixa energia para (oxi) Phosphors nitreto

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.

Preparação e Avaliação de<sup&gt; 99m</sup&gt; Tc-rotulados tridentadas Quelatos de Pré-alvo 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.

Um sensor baseado em Aptamer para não quelatada gadolínio (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.

Uma enzima de Split-TET2 projetado para Hydroxymethylation de DNA químico-inducible e remodelação epigenéticas

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.

Co de expressão de várias proteínas da fusão fluorescente quimérico de forma eficiente em 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.

Fabricação de nanowire do ferro pelo alumínio anodizado nano-poroso e por sua caracterização

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.

Síntese livre de agentes de direção de estrutura orgânica para *membrana zeolite tipo BEA

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