sunulan çalışma Kanada Sağlık Araştırma Enstitüleri tarafından finanse edilerek bölgelerinde desteklenmiştir / RD Kanada ortaklık programının Frajil X Araştırma Vakfı, RD, Üniversite Erlangen-Nürnberg Interdisziplinäres Zentrum für klinische Forschung Jerome Lejeune Vakfı RD ve Deutsche Forschungsgemeinschaft gelen RD ve RE. Yazarlar pCMV5-FLAG vektör kullanılabilir hale getirmek için teknik hücre kültürü bakımı ile ilgili yardım yanı sıra Prof. Dr. M. Wegner için Ingrid Zenger&#39;i teşekkür etmek istiyorum. Yazarlar ayrıca, özellikle video çekimi de yararlı destek için Nadja Schroeder teşekkür etmek istiyorum.

1. Hazır Çözümler <ol><li> 2 ug / ml bir son konsantrasyona kadar steril H liyofilize aktif motifi toz 2 O yeniden süspanse edin. Karıştırma için dikkatlice dokunun. </li><li> (Örneğin, 12 ul, her biri 2 ul reaksiyon başına zorunludur) az miktar hazırlayın ve -20 ° C&#39;de saklayın. </li></ol> Hücreler 2. Hazırlık <ol><li> Bu tür antibiyotikler de dahil olmak üzere, bir 24 yuvalı plaka 500 ul büyüme ortamı üzerinde HEK293 hücreleri veya birincil nöronlar gibi tohum memeli hücreleri. NOT: nöronlar kullanırken, düşük yoğunluklu hücreleri tohum ve 24 plaka sadece iki orta satır kullanın. Her iyi ve böylece inkübasyon (adım 4.2) sırasında orta liman boş muadili olacaktır. nöronlar ele aldığınızda her zaman hücreler bir kaç dakikadan fazla inkübatör dışında tutulur değil emin olun. </li><li> Hücreler uygulama kadar kültür, uygun koşullar altında hücrelerin (nemlendirilmiş% 5 CO2 ve 37 ° C). % 50konfluent. NOT: nöronlar kullanırken, kültür istenilen gelişimsel aşamaya kadar hücreler ulaştı ve uygulamayı değiştirecek. ortamda iki kez% 25-50 haftada. Orta (1 x B27, 5 mM L-glutamin ve 1 x penisilin ve streptomisin içeren; ref 16 karşılaştırın), Neuro temelli vasat. </li></ol> 3. Chariot Kompleks Oluşum <ol><li> reaksiyon başına PBS 50 ul sırasıyla özel hedef veya ilgili antikor, bunun yanısıra kontrol proteini veya kontrol antikoru, 0,1-2 ug seyreltin. NOT: Bu teknik aynı zamanda makromoleküler kompleksleri öncesi bağlı primer ve sekonder antikor (. Cp &#39;Temsilcisi Sonuçlar&#39;, Şekil 1) oluşan çalışır. Mix ve sıkma. </li><li> Her numune için, Chariot kendi aralarında birleşmelerini engellemek için ayrı bir tüp kullanarak PBS, 50 ul 2 ul Chariot stok solüsyonu seyreltin. Mix ve sıkma. </li><li> pipet ile Chariot seyreltme seyreltilmiş POI veya antikor (adım 3.1) aktarınting. Mix ve sıkma. </li><li> Oda sıcaklığında 30 dakika süreyle inkübe karışımı (Chariot-PO kompleksleri oluşturacaktır). </li></ol> 4. Hücrelerin transfeksiyonu <ol><li> Chariot kompleksleri, önceden ısıtılmış bir büyüme ortamı 100 ul (37 ° C, katkı maddeleri) (aşama 3.4) ile seyreltilir. Ortamı çıkarın ve bir kez önceden ısıtılmış 1 x PBS kullanarak hücreleri yıkayın. NOT: nöronlar kullanırken, orta atmayın, bu yeniden yapılacaktır. 37 ° C&#39;de tutun. Yıkama için, PBS, 0.5 mM MgCI2 ve CaCl2 ihtiva eden kullanımı. Öneri: plakasını (adım 4.5) kuluçkaya ederken boş kuyularda ortamı tutmak. </li><li> hücrelere Chariot kompleksi çözeltisi (aşama 4.1) uygulayın ve çözeltinin eşit bir dağılımını sağlamak için yavaşça hücreler sallayın. 1 saat boyunca standart koşullar (adım 2.2) altında hücreleri büyür. % 10 oranında bir nihai konsantrasyona kadar serum ekleyin. nöronlar kullanıldığında, Adım 4.1 orta ekleyin. 1 ila 2 saat daha hücreleri büyütün. NOT:Optimal inkübasyon süresi büyüklüğü ve yük özelliklerine bağlıdır ve tecrübi olarak ayarlanması gerekebilir. Aşağıdaki öneriler bir rehber olarak hizmet edebilir: peptitler için, 1 saat 1-2 saat antikorları için, 2 saat tavsiye ve antikorlar ile nöronların transfeksiyon 4 saat için olan proteinler için, genellikle yeterli olur. </li><li> Her zamanki gibi analiz için işlem hücreleri. Lütfen dikkat: teknik sabitleme protokolleri yanı sıra canlı görüntüleme kullanarak deneyler ile uyumludur. </li></ol> 5. Görüntüleme <ol><li> bir lazer tarama mikroskobu kullanılarak, yaklaşık 0.25-0.5 mikron mesafe kullanarak hücreleri ve / veya ilgi hücre bölümlerinde z-yığınları alır. hassas tabaka mesafesi yeniden inşa edilmesi yapı büyüklüğüne bağlıdır ve ayrı ayrı tespit edilmesi gerekmektedir. </li></ol> 6. İmar <ol><li> Aşağıdaki adımlarda, o tıklayarak birden fazla nesneyi seçmek için, seçmek ve gezinmek, Cntr arasında geçiş yapmak için Esc altını kullanmakn onlara ve shift tuşu nesneleri (krş. 6.10 adım) kesmek için. </li><li> Imaris içinde LSM-dosyasını açın. </li><li> parlak yapıları doygunluk ulaşana kadar her kanal için görüntü Ayarı kullanarak, resim kontrast. Yüzey inşaat etkilemeyeceğini bu ayarlama, sadece görüntü eşikleme de deneyci yardımcı olmak için hizmet ettiğini unutmayınız. </li><li> Yeni Yüzeyler Ekle simgesini tıklayın. Bir sihirbaz açılacaktır. </li><li> Seçin: İlgi Segment sadece bir bölge. Bir sonraki adıma geçin. </li><li> ilgilendiğiniz nesne sığdırmak için seçim kutusunun kenar boşluklarını ayarlayın. Görüntü 3 boyutu vardır akılda ve seçimi de z düzlemde ayarlanması gerektiğini Lütfen ayı. Seçim kutusunun dikdörtgen şekli düzgün ilgi nesnesi eşleşmiyor gerekiyorsa, nesneyi açmak ve / veya gerekli tüm yapılar gömülü kadar kutuyu büyütmek. İstenmeyen nesneler daha sonra kaldırılmış olabilir (cp. 6.10 adım). İlerlemek. </li><li> Uygun ch seçinAnnel. Yüzölçümü Detay Seviyesi veya küre çapı yapıları yeniden düzenlenmesi eşleşecek şekilde ayarlanması gerekir. Sinyal özelliklerine bağlı olarak, Mutlak Yoğunluk veya Yerel Kontrast da kullanılabilir. Genellikle, Yerel Kontrast yaygın sinyalleri için daha iyi çalışır. Herhangi bir durumda, ayarlar, sırasıyla, ilgi konusu her bir sinyalin ya da yapı için ayrı ayrı ayarlanabilir olması gerekir. </li><li> Eşik aracını kullanarak, ilgi yapısını yeniden. </li><li> yeşil ok alt tıklayarak yeniden tamamlamak. </li><li> kesme ve gerektiği gibi yüzeylere kaldırmak için kalem aracını kullanarak daha nesne ayarlayın. Istenilen nesneyi kesmek için görüntüyü, bu nedenle, kuzey-güney yönünde kesilir yatırmak için mümkündür. </li><li> seçme İstatistik, Ayrıntılı İstatistiklere ve Ortalama Değerler her yüzey için hacim, alan ölçümleri ve yoğunluklarını alın. </li><li> Renk kodlu istatistikleri gözlemlemek için (İsteğe bağlı adım) (cp. Şekil 2E </strong&gt; ve F), ilgili yüzey rengi sekmesini seçin ve yerine &#39;Base&#39; ve &#39;İstatistik Kodlu&#39; işaretleyin. Çeşitli seçenekler görüntülenir. </li></ol>

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The authors have nothing to disclose.

Burada, bir doz tahrik şekilde hücre işlevlerini kontrol protein sentezleme seviyeleri önemini incelemek için bir protokol mevcut. Açıklanan protokol, dolayısıyla hücresel seviyede protein fonksiyonunun ayrıntılı çalışmalar sağlayarak hipokampal nöronlar dahil olmak üzere çeşitli memeli hücre tipleri, protein ekspresyonunun bir ince ayar manipülasyon sağlar. RNA interferans özel hedefleri aşağı regüle etmek için bilinen bir strateji temsil eder, ancak, dezavanta...

her canlı organizma kendi gelişimini komuta yanı sıra çevresel sinyallere tepki için protein ifade Sıkı denetim esastır. Bu nedenle, mekanizmaların çok sayıda hassas uygulama tarafından kodlanan her protein ifade düzeyini düzenleyen evrim sırasında icat edilmiştir. ömrünün herhangi bir zamanda herhangi bir ökaryotik hücre içinde mevcut olan 20.000 genler. Protein üretimi farklı aşamalarında yer alarak, düzenleyici mekanizmaların posttranslasyonel modifikasyonlar proteini, taşıma ve bozulma yönüne taşıma kromatin yapısının, transkripsiyonu ve RNA yönetim arasında değişir. Moleküler makine ve değiştirilmiş protein ekspresyon düzeyleri altta yatan de arızaları, kanser ya da zihinsel engelli gibi çeşitli hastalıklar ile ilişkili olduğunu, bu nedenle şaşırtıcı değildir. Nitekim, nöronal gelişim ve memeli beyin fonksiyonlarının olağanüstü karmaşıklığı bakarak, sensiProtein ifadesindeki değişiklikler bu sofistike sistemlerin bilirlik Alzheimer ve Frajil X Sendromu (FXS) gibi Parkinson hastalığı (AH ve PH) yanı sıra otizm spektrum bozukluğu (ASD) dahil olmak üzere birçok tanınmış entelektüel açıkları kendini gösterir. Ikinci hastalığı tek için düzenleyici protein, FMRP (Kırılgan X mental retardasyon Protein) 1-4 kaybına bağlı olan bir çok protein, geniş bir misexpression ile karakterize edilir. Nokta mutasyonları zihinsel engelli olan hastalarda tespit değil iken 5 kapatma mRNA değiştirerek mRNA stabilitesini ve çeviri yönetir Ayrıca, Değişken ücret etkileyen kromozomal yeniden düzenlenmeler bağlantılı x protein A (VCXA), bir protein, son zamanlarda, entelektüel açıkları ile ilişkilendirilmiştir şimdi olduğu gibi 6, 7, gözlenen ruhsal bozuklukları değişmiş VCXA ifadesi ve hedef prote düzensiz ifadesi kaynaklanan düşündürenins. Bu bulgulara, de novo ASD ile ilişkili genlerin kopya sayısı varyasyonları, böylece yüksek veya azalmış protein ekspresyon düzeyleri neden olabileceğini fikrini desteklemektedir, yeni gen duplikasyonlar ve silmeler ASD 8 için önemli bir risk faktörü olduğunun tespit edip araştıran bir çalışma doğrultusunda entelektüel açıkları. Dikkate değer olarak, son araştırmalar başka bir proteinin ifade seviyesi, tam hemen hiçbir güvenlik marjları 9 yüksek protein miktarlarının bir sonucu olarak, toplanmayı önlemek için ayarlanır ilişkin kanıtlar sağlamıştır. Nedenle, daha küçük artışlarla, AD ve PD 9 gibi hastalıkları indüklemek için yeterli olduğu önerilmiştir. Protein ekspresyon kontrol katkıda moleküler makine çeşitliliği bu bulgular ışığında karmaşık bir düzenleme planı desteklemesine rağmen, 5000&#39;den fazla memeli genlerinden 10 ekspresyon seviyesini araştıran bir çalışma göstermiştirdoğa daha cimri düzeni tercih: Proteinlerin hücresel bolluğu ağırlıklı böylece RNA kullanılabilirlik yönetimi esas ince ayar protein ifadesini hizmet ettiğini gösteren, çeviri 10 seviyesinde düzenlenir gösterilmiştir. ilgi proteinlerin dozunu incelenmesi (POI) bu nedenle sadece bir proteinin endojen fonksiyonlarının anlaşılmasında önemli değil, aynı zamanda birçok hastalığın araştırılması ve tedavilerin geliştirilmesine değil. Böylece, son on yıl POI dozajı işlemek için RNA girişimi kullanarak çeşitli stratejiler ilerleyişini gördük. RNA enterferans yaygın protein fonksiyonunu incelemek için kullanılır ve hatta kanser ya da göz hastalıkları tedavi etmek yanı sıra hastalarda 11-13 antiviral tedavileri sürdürmeye klinik çalışmalarda uygulanan olmasına rağmen, bazı zorluklar strateji imkansız hale olabilir ortaya çıkabilir. Örneğin, homoloji ile demonte tahrik tohum sırası compara olduğuble kısa, dolayısıyla teşvik dışı hedef etkileri. Yüksek verimli dizileri nadirdir ve zaman alıcı ve masraflı olabilir sağ dizisini tanımlayan, (14 gözden) seçenekler binlerce arasında bulunan gerekir, ancak o zamandan beri sonuçlar yine hayal kırıklığı olabilir. Alternatif bir strateji doğrudan antikorlar tarafından POI hedef etmektir. Burada, hücresel protein durumunu azaltmak için (Aktif Motif tarafından imal edilmiş), protein taşıyıcı Chariot kullanımını göstermektedir ve üç boyutlu rekonstrüksiyon istihdam knock-down aşağıdaki protein fonksiyonunu incelemek için. Chariot, kendisi 2.8 kDa peptidinin, aktif motifi memeli hücreleri 15 membranları boyunca Servisi peptidler, proteinler ve antikorlar için kullanılır. Chariot-POI kompleksleri, bunun üzerine hidrofobik etkileşimler, kullanan olmayan kovalent makromoleküler kompleksler oluşturarak İÇN ile peptid ilişkilendirir bir endosome-ind hücrelere içselleştirmiş olanependent şekilde. Önemli olarak, Chariot mekik proteinlerin hücre içi lokalizasyonu etkiler de sitotoksik etkilerini uygulamak için veya yük 15 biyolojik aktivitesini etkilediği de işaret etti.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chariot</td>
			<td>Active Motif</td>
			<td>30025</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life technologies</td>
			<td>21103-049</td>
			<td>warm up to 37&#176;C before using</td>
		</tr>
		<tr>
			<td>1xB27</td>
			<td>Life technologies</td>
			<td>17504044</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life technologies</td>
			<td>25030-149</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>&#160;Penicilline and Streptomycine</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>n.a.</td>
			<td>expensive, but unmatched</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>n.a.</td>
			<td></td>
		</tr>
	</tbody>
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studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

protein ekspresyonunun bir sıkı yönetim canlı her organizmada gerekli değil, aynı zamanda hücresel modellerinde protein fonksiyonlarını araştırmak için önemli bir strateji değil sadece. Bu nedenle, son araştırmalar RNA ve antikor enterferans dahil memeli hücre hatları, hatta hayvan modellerinde, protein ifadesini hedef için farklı araçlar icat etti. İlk strateji son yirmi yılda çok dikkat toplamıştır ederken, hücresel membranlar boyunca ve hücre içine antikor kargoların bir translokasyon aracılık peptidler, çok daha az ilgi aldı. Bu yayında olarak, antikor müdahale deneyleri gerçekleştirmek için insan embriyonik böbrek hücrelerinde ve primer hipokampal nöronlar Chariot isimli bir peptit taşıyıcı kullanır ve daha fazla protein işlevi analiz üç boyutlu rekonstrüksiyon uygulanmasını göstermek için nasıl ayrıntılı protokol sağlar. Bulgularımız Chariot muhtemelen nükleer lokalizasyon sinyali, Particula için olduğunu göstermektedirrly soma ve çekirdeğinde bulunan proteinleri hedef için çok uygundur. Primer hipokampal kültürlere Chariot uygularken Enteresandır, reaktif şaşırtıcı derecede iyi ayrışmış nöronlar tarafından kabul edilmesi ortaya çıktı.

Aşağıdaki paragraflarda, POI fonksiyonel demonte gösteren örnek sonuçları Chariot reaktif ve antikor girişim sunulmaktadır kullanarak (Simiate, daha fazla ayrıntı için 16, 17 bakınız). Bulgular Simiate azalmış ekspresyon transkripsiyonel aktivite bozar, ve doz bağımlı bir şekilde, daha yüksek antikor miktarları (&gt; 1 ug antikoru) uygulanması durumunda% 99 üzerinde mortalite oranları ile sonuçlanan, apoptosisi teşvik göstermektedir (Bu keşifler, daha önce y...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

Incelenmesi Protein Fonksiyonu ve Antikor Girişim ve üç boyutlu Rekonstrüksiyonunda tarafından değiştirilmiş Protein Ekspresyonu Rolü

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

studying-protein-function-role-altered-protein-expression-antibody

Research

JoVE Journal

Bioengineering

6.6K Görüntüleme.  University of Erlangen-Nuremberg. Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

Video: Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Membran Protein katlanması, Termodinamik Floresans Spektroskopisi ile ölçülür.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Biyomühendislik

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Yumurtalıklar ve fallop borularında Üç Boyutlu Organ Kültür Aljinatta Hidrojeller

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

İletken Mürekkepler düzlemsel ve Üç Boyutlu Baskı

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Bir mikroakışkan Platform kullanarak Yüksek throughput Protein Ekspresyonu Jeneratör

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Protein Katlama Öğrenmenin mikroakışkan Mikserler

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Model Vocal Fold Polip'in Neden Olduğu Üç Boyutlu Akış Ayrımının Araştırılması

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Bir Vaka Çalışması Olarak Tütün Geçici Protein Expression: Deneyler Yaklaşımı Tasarım Kullanma Karmaşık Sistemler Karakterizasyonu

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Üç boyutlu Mürekkep Püskürtmeli Baskı Teknolojisi Kullanımı İnsan Kıkırdak Doku Fabrikasyon

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Üç-boyutlu Tam Kalınlığı Deri Üretimi Eşdeğer ve Otomatik Yaralama

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.