Bu çalışma, Ulusal Sağlık Enstitüsü, hibe No NIH / NCRR P41-RR 016.292 (ACERT) tarafından desteklenmiştir.

1. Malzemeler Bu çalışmada kullanılan tüm çözücüler saflıkta. Spin N-tert-butil-α-phenylnitrone (PBN) ve standart spin etiketi dahil olmak üzere, tuzak, 2,2,6,6-tetramethyl-1 piperinyoxyl (TEMPO) Sigma firmasından temin ve tedarik edildiği şekliyle kullanılmıştır. Bitki antioksidanlar Swanson, Inc USA ticari olarak elde edilmiştir. 2. Serbest radikaller, sigara dumanı ve analiz hazırlanması <ol><li> Antioksidanlar filtre içine tanıtmak için, likopen aseton içinde çözünmüş iken, antioksidanlar Pycnogenol ve üzüm çekirdeği ekstresi,% 95 etanol içinde çözülür. Çözücü hacimleri antioksidan çözünürlük bağlı olarak farklı idi. </li><li> Antioksidan miktarı 0.4 mg / filtre kullanılmıştır. Daha sonra 10 mg aktif karbon ile kaplanmıştır. Bu amaçla aktif karbon filtre edilir ve vakum altında kurutulur anaerobik koşullarda antioksidan çözümü ile ~ 12 saat, karıştırıldı. </li&gt; <li> Antioksidanlar sonra geleneksel asetat filtre (CA filtre) haline getirilmiştir. Bu amaçla, filtre, iki parça halinde kesilmiş oldu. Antioksidan filtre sandviç (Şekil 1A) - kaplı bitki antioksidanlar filtresi ve bir parça bant ile sarılmış iki adet arasında bir filtre oluşturmak için yerleştirildi. Bu kombine filtre daha sonra tütün içeren sigara çubuklar bağlanmıştır. Filtre, herhangi bir antioksidandır dışında antioksidan filtre olarak aynı şekilde yapıldı kontrolü eklendi. </li><li> Önce sigara simülasyon, araştırma sigara ambalajsız ve 2 gün en az doymuş bir NaBr çözümü kullanarak sabit nem ortamında (20 ° C,% 60 bağıl nem) tutuldu. </li><li> Rutin analiz için tütsüleme simülasyonu Şekil 1&#39;de gösterildiği gibi, bir tek portlu sigara cihazı kullanılarak ortam sıcaklığında yapıldı, su aspiratör veya GAST DOA-P104-BN Vakum Pompa / Kompresör (Benton Harbor, Mich) bağlı oluşur spin-yakalamabir ucunu açık bir T-kavşak ile montaj. Puflar ponponları arasında açık kaldı açık ucuna takarak yapıldı. Gaz akış debimetre tarafından kontrol edilir ve ~ 2.2 SCFH = 17.5 mL / s pompa ve havalandırma arasına yerleştirilen bir vana ayarlayarak. </li><li> 2 saniyelik bir süre için 1 &#39;e benzer her 60 saniyede tekrarlanan araştırma sigara puf hacmi 35 mL koşul altında tüketilmiştir. Şekil gösterildiği gibi spin yakalama sistemi ile birlikte gaz faz serbest radikallerin kantitatif tahmini için sigara simülasyonu yapıldı. 1. </li><li> On yoğun puf (35 ml / puf) her sigara için alınmıştır. Gaz-faz, serbest radikaller Cambridge filtresi pad aracılığıyla MCS geçen tarafından toplanan ve daha sonra spin yakalama çözüm (benzen, 2.0 ml 0.05 M PBN) haline getirilmiştir. </li><li> Son puf sonra, kabarmış yakalama çözüm aynı benzen ile ilk hacmi (2.0 mL) için yeniden düzenlenebilir. Bir kısım ~ 25 aktarılır.0 mm uzunluğunda, bir ucu kapatılmış 3 mm ID cam tüp. </li><li> Yakalama çözüm donma-pompa-çözülme yordamı kullanarak deoxygenated oldu. Sıvı azot altında dondurulmuş ve bir vakum uygulandı. Sonra, argon atmosferi altında hapsolmuş gaz kabarcıkları kaçmak için izin çözülmüş oldu ve tekrar dondurulmuş. Bu döngü üç kez tekrar edildikten sonra, tüpün alev sızdırmaz, vakum altında ve daha fazla ESR ölçümleri kullanılır. Benzen iyi çözünür oksijen, organik radikallerin ESR hatları genişletiyor çünkü bu adım gereklidir. Deoxygenation sinyal-gürültü oranı önemli ölçüde geliştirir. </li><li> X-Band ESR spektrumları Bruker EMX spektrometre, 9.34 GHz standart koşullar altında bir frekansında kaydedildi. Çoğu deneylerde kullanılan spektrometre ayarlarını merkezi alanında 3312.5G, tarama genişliği 80G, modülasyon genliği 0.5G, zaman sabiti 82 mikrosaniye, tarama süresi 40 sn. </li><li> Spin trap adducts bazı durumlarda req deney koşulları, nispeten istikrarlı bir25 birikimleri, genelde ~ 20 dakika aldı uired. Ancak, 12 saat sonra benzen çözüm ESR sinyalleri ~ 5 faktör yoğunluğu azalmıştır. </li><li> Yakalama adduct konsantrasyonunu ölçmek için, ilk birinci türev ESR spektrumu entegre edildi. Absorbsiyon spektrumunun duman bulunan kurum / katranlı ürünler nedeniyle büyük olasılıkla geniş bir tekli arka plan gösterir. Bu arka plana çıkarma sonra yakalama adduct ayrılmış üçlü bir kez daha entegre edilmiştir (Şekil 2). </li></ol> 3. Temsilcisi Sonuçlar Yanan sigara duman (gaz fazı) serbest radikallerin çoğu anlık ve istikrarsız. Bu radikallerin gözlemlemek için bir spin tuzak tekniği kullanılmaktadır. Bu daha istikrarlı ve ESR (Şekil 1) tarafından tespit edilebilir bir spin adduct dönüştürerek gaz faz serbest radikalleri yakalar. 0.05 Bu çalışmada, spin-trap çözümM PBN duman gazı, oksijen ve karbon merkezli radikallerin 15 ayrı zor bir karışımı olan serbest radikaller, faz toplamak için kullanıldı . Bizim durumumuzda ise, gözlenen aşırı ince bölme sabitleri N = 13.7G ve H = 1.95G alkoxyl serbest radikaller (RO.) 2 ana ürün olduğunu öne sürerek, yakalama adducts karşılık gelen değerler birbirine çok benzer. Biz duman akış nem nedeniyle bu zayıf ESR sinyalleri (Şekil 2) bizim ilk ölçümlerde gözlenen düşük tekrarlanabilirlik gösterdi. Bu sorunu gidermek için, Cambridge filtresi ve spin trap çözümü arasında bir sıvı nitrojen (LN2) tuzak ekledi. LN 2 tuzak, hızlı dondurma, MCS akış su kaldırılır ve cam tüp iç duvara yakalamak. Bu ESR sinyalleri büyük ölçüde geliştirilmiş ve yüksek tekrarlanabilir sonuçları (Şekil 3) için izin. Sıkışıp serbest radikallerin miktarını tespit edildireferans örnek. Kontrol örnekleri için antioksidanlar olmadan, çift ayrılmaz spektrum TEMPO bilinen konsantrasyon için bir çift integral spektrumu ile karşılaştırılması tahmin tipik adduct benzen konsantrasyonu 1.24 mcM (Şekil 2). ~ 350 ml hava miktarı sigara boyunca her sigara geçti bu yana, bu ~ 7.1X10 -9 M MCS gaz fazında serbest radikal konsantrasyonu için bir tahmin sağlar ve gaz fazı sıkışıp radikallerin toplam sayısı bir bütün 1.5X10 15 ~ sigara. Gaz ve partikül faz de dahil olmak üzere bütün bir sigara, duman serbest radikallerin toplam tutarı için bir tahmin, ~ 10 16 serbest radikallerin 9 oldu. Gaz-faz ana akım sigara dumanı serbest radikaller üzerinde bitki antioksidan süpürücü etkisi farklı düzeylerde gözlendi. Onların süpürücü oranları Şekil sunuldu. 4. Likopen ve üzüm çekirdeği ekstresi, en yüksek oranları düşük ikener oranı Pycnogenol gözlenmiştir (Şekil 4). <img alt="Şekil 1" src="/files/ftp_upload/3406/3406fig1.jpg" /> Şekil 1. Dönüş tuzağı kullanarak ana sigara dumanı (MCS) gaz faz serbest radikalleri toplayarak sigara simülasyon tasarımı için geliştirilmiş bir diyagram. MCS sonra CA filtresi, Cambridge filtresi (sarı filtre) ile su aspiratör tarafından çekilen ve H 2 O kaldırmak için sıvı azot tuzağı ile geçti Gaz faz ürünleri nihayet tuzak spin gitti ve spin trap çözüm yoluyla kabarmış. Bitki antioksidan MCS serbest radikalleri temizleyebileceği sigaraya bağlı iki adet konvansiyonel asetat filtreler (daire bir Genişlemiş) arasına yerleştirilir. <img alt="Şekil 2" src="/files/ftp_upload/3406/3406fig2.jpg" /> Şekil 2 benzen spin-yakalama adduct konsantrasyon için bir ilk integra kantitatif tahmin çıkarılarak geniş bir arka plan sinyali gerektirirl spektrum. Spin-yakalama adduct aşırı ince bölme parametreleri H = 1.95 G, N = 13.7G. <img alt="Şekil 3" src="/files/ftp_upload/3406/3406fig3.jpg" /> Şekil 3 LN2 tuzak geçerek MCS spin-yakalama ile elde edilen ESR sinyalleri kalitesini önemli ölçüde geliştirir . <img alt="Şekil 4" src="/files/ftp_upload/3406/3406fig4.jpg" /> Şekil 4. Doğal antioksidanlar serbest radikallerin MCS konsantrasyonuna etkisi. Göreceli sinyal şiddetleri vardır: Kontrol -% 100, Pycnogenol -% 55, üzüm çekirdeği ekstresi -% 12, likopen -% 10.

<ol>
	<li>Baum, S. L., Anderson, I. G. M., Baker, R. R., Murphy, D. M., Rowlands, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+spin+resonance+and+spin+trap+investigation+of+free+radicals+in+cigarette+smoke:+development+of+a+quantification+procedure.">Electron spin resonance and spin trap investigation of free radicals in cigarette smoke: development of a quantification procedure.</a> Analytica Chimica. Acta. 481, 1-13 (2003).</li><li>Bluhm, A. L., Weinstein, J., Sousa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+tobacco+smoke.">Free radicals in tobacco smoke.</a> Nature. 229, 500-500 (1971).</li><li>Church, D. F., Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-radical+chemistry+of+cigarette+smoke+and+its+toxicological+implications.">Free-radical chemistry of cigarette smoke and its toxicological implications.</a> Environ. Health. Perspect. 64, 111-126 (1985).</li><li>Deliconstantinos, G., Villiotou, V., Stavrides, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenging+effects+of+hemoglobin+and+related+heme+containing+compounds+on+nitric+oxide,+reactive+oxidants+and+carcinogenic+volatile+nitrosocompounds+of+cigarette+smoke.+a+new+method+for+protection+against+the+dangerous+cigarette+constituents.">Scavenging effects of hemoglobin and related heme containing compounds on nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke. a new method for protection against the dangerous cigarette constituents.</a> Anticancer. Res. 14, 2717-2726 (1994).</li><li>Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+carcinogens+and+lung+cancer.">Tobacco smoke carcinogens and lung cancer.</a> Journal of the National Cancer Institute. 91, 1194-1210 (1999).</li><li>Kodama, M., Kaneko, M., Aida, M., Inoue, F., Nakayama, T., Akimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radical+chemistry+of+cigarette+smoke+and+its+implication+in+human+cancer.">Free radical chemistry of cigarette smoke and its implication in human cancer.</a> Anticancer. Res. 17, 433-437 (1997).</li><li>Nair, A. K., Brandt, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+smoking+on+health+care+costs.">Effects of smoking on health care costs.</a> Journal of Oklahoma State Medical Association. 93, 245-250 (2000).</li><li>Nishizawa, M., Kohno, M., Nishimura, M., Kitagawa, A., Niwano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+Peroxyradicals+in+Cigarette+Smoke+and+the+Scavenging+Effect+of+Shikonin,+a+Naphthoquinone+Pigment.">Presence of Peroxyradicals in Cigarette Smoke and the Scavenging Effect of Shikonin, a Naphthoquinone Pigment.</a> Chem. Pharm. Bull. 53, 796-799 (2005).</li><li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+and+the+involvement+of+free+radical+reactions+in+chemical+carcinogenesis.">Cigarette smoke and the involvement of free radical reactions in chemical carcinogenesis.</a> Br. J. Cancer. 8, 19-23 (1987).</li><li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+radicals+and+the+role+of+free+radicals+in+chemical+carcinogenicity.">Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity.</a> Environ. Health. Perspect. 105, 875-882 (1997).</li><li>Rozzi, N. I., Singh, R. K., Vierling, R. A., Watkins, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+extraction+of+lycopene+from+tomato+processing+byproducts.">Supercritical fluid extraction of lycopene from tomato processing byproducts.</a> J. Agric. Food. Chem. 50, 2638-2643 (2002).</li><li>Shopland, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+use+and+its+contribution+to+early+cancer+mortality+with+a+special+emphesis+on+cigarette+smoking.">Tobacco use and its contribution to early cancer mortality with a special emphesis on cigarette smoking.</a> Environmental. Health. Perspectives. 103, 131-142 (1995).</li><li>Shrikhandle, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wine+products+with+health+benefits.">Wine products with health benefits.</a> Food Res. Int. 33, 469-474 (2000).</li><li>Valavanidis, A., Haralambous, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+by+electron+paramagnetic+resonance+of+free+radical+species+in+the+mainstream+and+sidestream+smoke+of+cigarettes+with+conventional+acetate+filters+and+'bio-filters'.">A comparative study by electron paramagnetic resonance of free radical species in the mainstream and sidestream smoke of cigarettes with conventional acetate filters and 'bio-filters'.</a> Redox. Rep. 6, 161-171 (2001).</li><li>Valavanidis, A., Vlachogianni, T., Fiotakis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke:+involvement+of+reactive+oxygen+species+and+stable+free+radicals+in+mechanisms+of+oxidative+damage,+carcinogenesis+and+synergistic+effects+with+other+respirable+particles.">Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles.</a> International Journal of Environmental Research and Public Health. 6, 445-462 (2009).</li></ol>

Çıkar çatışması ilan etti.

Güvenilir bir tahminin, tütün dumanı, farklı serbest radikalleri yok eden enzimleri etkisi serbest radikallere kantitatif tespiti için tekrarlanabilir bir teknik gerektirir. Daha önce 1, non-polar çözücüler içinde yüksek konsantrasyonda spin trap çözümleri küçük miktarlarda tütün dumanı serbest radikalleri yakalama en etkili olduğu kanıtlanmıştır. Sigara dumanı her zaman organik bileşiklerin yanma ve bindirme çözücü sonuna kadar tütün kalan nem ve su buharı içerir. PBN spin trap çözüm su bu karışıma spin yakalama adducts ömrü ve kendi ESR sinyalleri yoğunluğu önemli ölçüde azalır. Gaz-faz radikallerin bazı fraksiyon da donmuş yüzeyinde kapana kısılmış olsa bile, sıvı nitrojen ile soğutulan bir U-şekilli bir tüp aracılığıyla basit geçen MCS bu nem çıkarma dramatik deneylerde ESR spektrumları kalitesi artar. T Kullanımıonun tekniği, sigara filtreleri haline getirilen doğal bileşikler farklı radikal süpürücü göreceli etkinliği karşılaştırıldı. Biz likopen ve üzüm çekirdeği, sigara filtreleri içine içeren sonra serbest radikallerin% 90 kadar gaz fazı MSC scavenge hemen ayıklamak bulundu. Bu tür yüksek süpürücü kapasitesi bu ucuz hazır doğal bileşikler, hemoglobin ve shikonine 8,14 gibi en etkili serbest radikal koruyucuları arasında sırada yer alıyor . Ancak, çalışılan doğal antioksidanlar ile yüklenen deneyler, sigara filtreleri, depolama oda sıcaklığında bir hafta sonra atma kapasitesi gözle görülür bir bölümünü kaybetti. Bu problem çözme, likopen ve ticari sigara filtrelerinde üzüm çekirdeği ekstresi gelecekteki uygulama uyarabilir.

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

Sigara içmek insan kanseri ile ilişkilidir. Akciğer kanseri ölümlerinin çoğu sigara 5,6,7,12 neden olduğu bildirilmiştir. , Sigara dumanı, partikül aşamasında tütün katran ve ilgili ürünlerin karsinojenik ve mutajenik ilgili hastalıkların başlıca nedenleri olmasına rağmen, sigara dumanı, serbest radikallerin de önemli bir grup 9,10 kanserojen olarak kabul edilen önemli miktarda içerir. Serbest radikallerin zararlı protein yapısı, lipidler ve DNA dizilerinin hücre bileşenleri saldırı ve çeşitli kanser türlerine yakalanma riski artar. İnhale radikaller, tütün dumanının akciğer 3 olumsuz sağlık etkileri pek çok katkıda adducts üretir . Çalışmalar, sigara kaynaklı hasar riskleri azaltmak için sigara dumanı serbest radikaller azaltmak için yapılmıştır. Hemoglobin ve heme içeren bileşikler kısmen nitrik oksit, reaktif scavenge olabilir bildirilmiştiroksidanlar ve sigara dumanı 4 kanserojen uçucu nitrosocompounds. Hemoglobin ve aktif karbon, serbest radikallerin scavenge ve serbest radikaller, sigara dumanı 14% 90 yukarı kaldırmak için kullanılan bir &#39;biyo-filtre&#39; oluşuyordu. Ancak, maliyet etkisizliği nedeniyle, başarılı bir şekilde ticari hale değildir. Başka bir çalışmada, Çin bitkisel ilaç 8 bir bileşeni shikonin iyi bir süpürücü verimliliği gösterdi . Bu çalışmada, sigara filtresi spin-yakalama kullanarak ana sigara dumanı, gaz fazında serbest radikallerin scavenge etkisi (MCS) sigara dumanı ve ölçüm gaz fazı, serbest radikallerin temizlenmesi için ortak doğal antioksidan özleri tanıtmak için bir protokol rapor Elektron Spin Rezonans (ESR) Spektroskopisi 1,2,14. Biz likopen ve sigara filtrelerinde gelecekteki uygulama noktası olabilecek üzüm çekirdeği ekstresi yüksek süpürücü kapasitesi gösterdi. Bu profesyonellerin önemli bir avantajpective temizleyiciler sırasıyla domates ya da şarap endüstrisi yan 11,13 büyük miktarlarda elde edilebilir.

Watch this Scientific Journal Video about A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke at JoVE.com

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Spin-yakalama ESR spektroskopi, bitki antioksidan likopen, sigara dumanı, gaz-faz, serbest radikallerin temizlenmesi Pycnogenol ve üzüm çekirdeği ekstresi etkisini araştırmak için kullanılmıştır.

Serbest Radikaller Mainstream Sigara Duman algılama ve gaz faz Atma Protokolü

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

a-protocol-for-detecting-scavenging-gas-phase-free-radicals

Research

JoVE Journal

Bioengineering

19.2K Görüntüleme.  CDCF-AOX Lab. Spin-yakalama ESR spektroskopi, bitki antioksidan likopen, sigara dumanı, gaz-faz, serbest radikallerin temizlenmesi Pycnogenol ve üzüm çekirdeği ekstresi etkisini araştırmak için kullanılmıştır.

Video: A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Protein ve hücre Etrafının İki Boyutlu Desenli Yüzeyler Oluşturma

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Biyomühendislik

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Mikro-ayağı ile bir mikroakışkan Aygıt Biyofilm Çıtası Formasyonu Protokolü

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Düşük Bolluk Proteinler saptamak için plazma veya idrar hidrojel Nanoparçacık Hasat

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Tüm Sigara Dumanına Organotipik 3D Bronşiyal ve Burun Doku Kültürü Modelleri Tekrarlanan Pozlama Etki Değerlendirmesi

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

Sitokrom Kapsüllenen<em&gt; C</em&gt; Metal Nanopartiküller olmadan Silika Aerojel Nanoarchitectures Gaz faz biyoaktivitesinin İstinat ederken

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Sıvı özleri geleneksel sigara duman ve elektronik sigara Aerosol gelen nesil için aygıt mikroişlemci işletilen

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Katı, sabit ve lekeli, genel olarak, milimetre ölçekli örnekler için bölüm ücretsiz 3D Histoloji mikro bilgisayarlı tomografi tarafından katıştırma

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Anyonik Seryum oksit nano tanecikleri tarafından bitki reaktif oksijen türleri içinde Vivo katalitik kayıt atma

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Bireysel Lipozomlar Arasındaki Bileşimsel Homojenliği Saptamak için Kantitatif Floresan Mikroskopesi Bazlı Tek Lipozom Testi

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Avustralya Senkrotron'da Seri Kristalografi Ölçümleri için Lipidico Enjeksiyon Protokolü

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...