ETM37 ve Birleşik Krallık Kanser Araştırma Deneysel Kanser Tıp Merkezi tarafından desteklenen: Baş Bilim Ofisi, hibe sayısına göre finanse edilmektedir Yukarıdaki yazarların FCO, DF, JN, DJH ve GDS çalışması bahsettiniz. AL çalışmaları Edinburgh Robertson Güven, bakım ve kanser ve Tıbbi Araştırma Konseyi tedavi için Melville Trust Cerrahlar Kraliyet Koleji tarafından finanse edilmektedir. IO Marie Curie Eylemleri ve İngiltere&#39;de Tıbbi Araştırma Konseyi tarafından cofunded Edinburgh İskoç Hükümeti Bursu Royal Society tarafından desteklenmektedir. Yazarlar bu çalışmada tartışılan konulardan bazıları üzerinde yararlı tartışmalar için SCOTRRCC ortak başvuru ve işbirlikçileri teşekkür etmek istiyorum.

1. Morfolojik ve Moleküler Tümör heterojenliği tanımlanması <ol><li> Tümörler -80 ° C derin dondurucu kaldırıldı ve kuru buz üzerinde tutulmalıdır. </li><li> Yaklaşık 1 cm 3 bölüme tümörleri bölün. Her bir tümör, birbirlerine göre göreceli bölüm ve bir benzersiz ismi ile etiket ilk konumuna Harita. Mağaza örnekleri -80 bireysel kriyotüplerin ° C Kullanıma hazır oluncaya dek. </li><li> Coat örnekleri OCT ve -22 ° C&#39;de kriyostat kesilmiş </li><li> Örnekleri Hematoksilen-Eozin counterstaining yöntemi kullanılarak boyandı. </li><li> Dondurulmuş kesitlerin Mikroskopi analizi onlar ccRCC doğa, canlı tümör ve dereceleme (frozen Fuhrman sınıflarda 1 ila 4 ayırt etmek zor düşük dereceli, yüksek dereceli veya karışık yüksek / düşük dereceli,) için vardır sağlamak. Şekil 1 a ve b (H &amp; E boyama yüksek, düşük ve karma sınıf). </li><li> Protein ekstraksiyonu için her tümör içindeki her morfolojik farklı bölgeden 4 örnekleri kadar seçin. </li></ol> 2. Tümör Örneklerinde Protein Ekstraksiyonu <ol><li> OCT tümör örneği kesin. </li><li> Lizis tamponu, 990 ul ile 2 ml&#39;lik tüp içine doku il 50-75 mg [50 mM Tris-HCl (pH 7.5), 5 mM EGTA (pH 8.5), 150 mM NaCl] aprotinin (Sigma A6279) (10 mg ile desteklenir / ml), fosfataz inhibitörü kokteyl 2 (Sigma P5716), fosfataz inhibitörü kokteyl 3 (Sigma P0044) ve bir proteaz inhibitörü kokteyl (Roche, 11836153001). </li><li> Her bir tüp için tek bir 5 mm çelik top ekleyin ve iki kez her 5 dk süre sonra, bir TissueLyser kullanılarak homojenizasyon seviyesini kontrol 5 dk için 50Hz&#39;de homojenize. </li><li> Arkasında çelik topu bırakarak pipet kullanarak yeni 2 ml tüp homojenize örnek aktarın. </li><li> 4 ° C&#39;de 30 dakika boyunca 13,000 x g&#39;de santrifüj önce her bir numune için Triton X-100, 10 ul ekle </li><li> Taze mikrosantrifüj tüpleri için süpernatan aktarın. </li><li> BCA assay (Şekil 2) kullanarak protein konsantrasyonu tayin edilir. </li><li>1 mg / ml protein konsantrasyonu normalize edilir. </li></ol> 3. Antikor Doğrulama <ol><li> Western blot için protein örneklerinin (uygun hücre hatları veya doku çıkarılır) hazırlayın ve% 10&#39;luk SDS-PAGE jel üzerinde çalıştırın. </li><li> 4 gecede nitroselüloz membran üzerinde örnekleri aktarın ° C. </li><li> Oda sıcaklığında 1 saat boyunca Li-Cor Odyssey engelleme tamponu (PBS içerisinde seyreltilmiş, 50:50) içinde membran engelleme. </li><li> Üreticileri de Li-Cor Odyssey Engelleme Tampon birincil antikorlar (PBS 50:50 seyreltilmiş) seyreltin seyreltme 1,000 tipik 1 önerilir. </li><li> 4 ° C&#39;de gece boyunca birincil antikor olarak zar inkübe </li><li> % 0.1 PBS-Tween20 (; 1 ml Tween20 / 1L PBS PBS-T) Makyaj. </li><li> 5 dakika (x3) ile, oda sıcaklığında PBS-T içinde zar yıkayın. </li><li> Odyssey Engelleme Tamponu (PBS 50:50 seyreltilmiş) 0.01% 1:10,000 dilusyondaki SDS (1.5 μl/15 ml) sekonder antikor floresan etiketli seyreltin. </li><li> Ikincil membran inkübehafif sallama ile 45 dakika için oda sıcaklığında antikorları - bu şekilde sonunda taranmıştır zamana kadar, ışıktan korumak için zar önemlidir. </li><li> Karanlıkta zar tutma 5 dk (x3) ile, oda sıcaklığında PBS-T içinde zar yıkayın. </li><li> Yine karanlıkta zar tutma kalıntı Tween20 kaldırmak için 5 dak (x3), oda sıcaklığında PBS içinde zar yıkayın. </li><li> Karanlıkta filtre kağıt parçası düz membran yatın ve kurumaya bırakın - kuru membran sinyal geliştirmek ve arka plan azaltmak ancak sıyırma ve yeniden tarama için işe yaramaz hale gelebilir sağlar. </li><li> Licor Odyssey tarayıcının membran tarayın. Bu tarandıktan gibi zamana kadar karanlıkta membran tutun. </li><li> Sadece doğru moleküler ağırlık tek bir bant oluşturmak baskın antikorları seçmek. Şekil 3 RPPA ile kullanım için kabul edilebilir ve kabul edilemez antikorların örnekleri gösterilmektedir. </li></ol> 4. RPPA Printing <ol><li> Protein lizatları bir MicroGrid II robotik gözcü kullanarak nitroselüloz kaplı cam slaytlar (Fastslides-whatman) üzerine tespit edildi. Kullanılan slaytlar örnekleri tespit edildiği üzerine 2 pedleri içeriyordu. Her ped Bu durumda 100 örnekleri, özdeş örnekleri ile fark edildi. Diğer kullanılabilir biçimler 1, 8 ve 16 ped slaytları içerir. Pedleri sayısı arttıkça daha küçük her biri üzerine tespit edilebilir numune sayısıdır. </li><li> Beş 2 kat dilüsyonlarının bir dizi her örnekten yapılmış ve her sonra (örnek başına 15 noktalar toplam sonuçlanan) üç nüsha olarak görüldü. </li></ol> 5. RPPA Protein Algılama <ol><li> Aşırı Licor Engelleme Tampon Islak slaytlar (PBS 50:50 seyreltilmiş). Bir sallanan platform üzerinde 1 saat oda sıcaklığında inkübe edin. </li><li> Istenen konsantrasyonlarda Licor Engelleme Tamponu (PBS 50:50 seyreltilmiş) primer antikor 800 ul hazırlayın ve buz üzerinde tutmak. </li><li> Ya (i) tek bir kare Chip Klip slaytlar monteveya (ii) &#39;FastFrame&#39; dört defne slayt tutucu yüzden sıkı bir mühür slayt ve inkübasyon odası arasında oluşan olduğunu. </li><li> Kuyulardan rezidüel tamponu çıkarın ve ilgili kuyulara 600 ul primer antikor ekleyin. </li><li> 4 gecede platformu sallanan bir kapalı ıslak kutu ve inkübe içine slayt ve odasına yerleştirin ° C </li><li> % 0.1 PBS-Tween20 (; 100 ul Tween 20/100 ml PBS PBS-T) Makyaj. </li><li> Soğuk oda slaytları çıkarın ve dikkatle her kuyudan primer antikorlar çıkarın. </li><li> PBS-T 600 ul ekleyin ve 5 dakika (X3) için RT platformu sallanan ilgili slaytlar yıkayın. </li><li> Odyssey Engelleme Tamponu (PBS 50:50 seyreltilmiş) 0.01% ilk etapta 1:2,000 seyreltme (1 ul / 2 ml) SDS içinde seyreltilmesi ile floresan etiketli sekonder antikor hazırlayın. </li><li> Kuyulardan tamponu çıkarın ve ilgili kuyulara 600 ul floresan etiketli sekonder antikor ekleyin. Hafif sallama ile 45 dakika için oda sıcaklığında sekonder antikor inkübe - buNihayet tarandı zamana kadar, ışıktan korumak için zar önemlidir. </li><li> Oda sıcaklığında 600 ul PBS-T iyi ve kısaca yıkama (x3) ikincil antikorlar çıkarın. Taşıyıcı slayt çıkarın ve uygun bir kaba aktarın ve karanlıkta membran tutarak, 15 dakika boyunca aşırı PBS-T yıkayın. </li><li> Yine karanlıkta zar tutarak, kalıntı Tween-20 çıkarmak için PBS-T ile 15 dakika süreyle oda sıcaklığında PBS ile yıkayın daha fazla zar çıkarın. </li><li> 10 dakika için 50 ° C fırın içinde Fastslide kurutun ve sonra Li-Cor Odyssey tarayıcı üzerinde tarama. Tarandiktan kadar karanlıkta slayt tutun. </li><li> Sekonder antikor / kullanılan antikorlara bağlı 680 nm ve / veya 800 nm&#39;de slayt tarayın. İki renk algılama için daima çapraz reaksiyon en aza indirmek için yüksek çapraz adsorbe sekonder antikorlar kullanır. Primer ve sekonder antikor dikkatli seçilmesi iki renk algılama için gereklidir. Birincil öneme seçimidiriki primer antikorları için farklı konak türler (örneğin tavşan ve fare). Bu kolayca ayırt emisyon spektrumları ile boya etiketli anti-tavşan ve anti-fare sekonder antikor tarafından ayrımcılık sağlar. </li><li> Resim dosyaları. Tiff dosyası olarak kaydedilir. Şekil 4 (taranan dosyaların görüntü). </li></ol> 6. Veri Analizi <ol><li> MicroVigene Yazılım (VigeneTech, Carlisle, MA, ABD) başlatın. </li><li> Açın. RPPA slayt tarama içeren tiff görüntü dosyası. </li><li> RPPA slayt görüntü üzerinde bindirmek için bir kılavuz olacak bir önceden tanımlanmış şablon dosyası seçin. </li><li> Click Izgara getirmek için, faiz getirisi (ROI) düğmesinin Bölgeleri tanımlayın. </li><li> RPPA noktalar üzerinde Izgara konumlandırın. Şekil 6a (resmin üzerine ızgara görüntüsü). </li><li> Tüm ROI vurgulamak için Tümünü Seç düğmesini tıklatın. </li><li> Tümünü Bul&#39;u tıklatın. MicroVigene autom olacakatically ROI bulmak noktalar bulmak, plan çıkarma, herhangi bir toz kaldırmak ve noktalar ölçmek. </li><li> RPPA Slayttaki tüm örnekler için sonuçlar getirmek için Görünüm Seyreltme Eğri düğmesini tıklatın. </li><li> Seyreltme Veri Kaydet&#39;i tıklatın. </li><li> Her numune 5 seyreltme noktalar üzerinde yazdırılır olarak üç nüsha halinde, her analiz için 15 puan, hata riskini azaltır ve eğri uydurma kalitesini artırır vardır. MicroVigene antijen-antikor bağlanma kinetik bir Sigmoid eğrisi üretmek için tüm noktalar içeren bir 4-parametreli lojistik-log modeli &quot;Supercurve&quot; algoritması (Şekil 6b), üretir. Varsayım aynı antikor-antijen bağlanma kinetik ortak bir yanıt eğrisi sığdırmak için bir dizi tüm noktalar alarak böylece, hatta farklı örneklerdeki, her numune noktada gerçekleşiyor eğri uydurma 10,11 güvenini artırabilir olduğunu Y = a + ((ba) / (1 + e (c * d-ln (x))) x dilutio olduğuK faktörü ve Y sinyal yoğunluğudur. Numuneler nispeten analizimize supercurve üzerine bu eşleme sonra x değerlerinin orta noktasından y değeri karşılık y0 değeri kullanılarak analiz edilebilir. </li><li> Şekil 7&#39;deki gibi Microsoft Excel ve arsa y0 verileri aktarın. </li><li> İntratümöral protein varyans bir ANOVA çerçevede işlenmemiş ve işlenmiş hastalar için ayrı ayrı hesaplanmıştır. Tüm analiz proteinlerden gelen verilerin birleştirilmesi Varyans dağıtımları Mann-Whitney testi (MWT) ile karşılaştırıldı. Yanlış keşif hızı (FDR) düzeltme 12 uygulandı; bireysel proteinlerin İntratümöral varyansları normallik ve homoscedasticity varsayımları Lillefours ve Fligner testleri kullanılarak sırasıyla, ölçme-değerlendirmeye düzenlenen bir F-testi ile karşılaştırıldı. Işlenmemiş ve işlenmiş hasta numuneleri arasındaki Diferansiyel protein ekspresyonu student t-testi kullanılarak her protein için test nerede normallik ve homoscedasticity varsayımlar aksi MWT yapıldı karşılandı; FDR kombine t-testi ve MWT değerler üzerinde 12 uygulandı. Proteinler için protein ekspresyonu ve varyans Pearson korelasyon Önemi standart bir yaklaşım kullanılarak hesaplanmıştır [R referans] ve FDR 12 uygulanır. </li></ol>

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Çıkar çatışması ilan etti.

Burada sunulan RPPA yöntemi protein analizleri yaygın olarak kullanılan ancak nispeten düşük verim western blot için yüksek kapasiteli bir alternatif temsil eder. Yöntem örnekleri yüzlerce analiz edilmiş ve aynı anda hücre hatları ve doku örnekleri geniş bir yelpazede yer alan önemli proteinlerinin doğrudan karşılaştırma için izin karşılaştırıldığında yarı kantitatif olmasına imkan verir. Farklı antikor türleri ile Multiplexing daha çok antikor aynı anda kullanılmasına izin tekni?...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Aprotinin </td><td> Sigma </td><td> A6279 </td></tr><tr><td> fosfataz inhibitörü kokteyli 2 </td><td> Sigma </td><td> P5726 </td></tr><tr><td> fosfataz inhibitörü kokteyli 3 </td><td> Sigma </td><td> P0044 </td></tr><tr><td> proteaz inhibitörü kokteyl </td><td> Roche </td><td> 11836153001 </td></tr><tr><td> Triton X-100 </td><td> Triton-X </td><td> T8787 </td></tr><tr><td> Li-Cor Odyssey Tampon Engelleme </td><td> Li-Cor </td><td> 927-40000 </td></tr><tr><td> TissueLyser </td><td> Qiagen </td><td> 85600 </td></tr><tr><td> MicroGrid II robotik gözcü </td><td> Biorobotics </td><td></td></tr><tr><td> FastFrame &#39;dört defne slayt tutucu </td><td> Whatman </td><td> 10486001 </td></tr><tr><td> FAST Slayt - 2-Pad </td><td> Whatman </td><td> 10485317 </td></tr><tr><td> IRDye 680LT keçi anti-fare IgG </td><td> Licor </td><td> 926-68020 </td></tr><tr><td> IRDye 800CW keçi anti-tavşan IgG </td><td> Licor </td><td> 926-32211 </td></tr></tbody></table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

Bulunduğu metastatik şeffaf hücreli renal hücreli kanser için hiçbir iyileştirici tedavi, hastalığın yaygın varyantı vardır. Bu tedaviye direnç önemli bir faktör hastalığı 1 moleküler karmaşıklığı olduğu düşünülmektedir. Gibi tirozin kinaz inhibitörü (TKİ)-sunitinib olarak Hedefli tedavi kullanılmıştır, ancak hastaların sadece% 40 1 yıl 2 içinde relaps bu hastaların büyük çoğunluğu ile cevap verecektir. Renal hücreli kanser hastalarında içsel ve kazanılmış direnç gibi soru 3 derece alakalı olduğu gibi. Etkili, kişiselleştirilmiş tedavilerin geliştirilmesi nihai hedefi ile, TKI direnç incelemek için, hedeflenmiş tedavinin belirli bir süre sonra sıralı dokusu, kronik myeloid lösemi 4 başarılı olduğunu kanıtlamıştı bir yaklaşım gereklidir. Ancak renal hücreli karsinom, böyle bir stratejinin uygulama b yüksek düzeyde nedeniyle karmaşıklaşırrenal hücre karsinomu 5,6 yanı sıra diğer katı tümörler içinde bir 7 özelliği oth arası ve intratumoral heterojen. Transkriptomik ve genetik farklılıklar nedeniyle Intertumoral heterojenite iyi bile benzer sunum, evre ve tümör derecesi olan hastalarda kurulmuştur. Buna ek olarak daha büyük moleküler heterojenite temsil etmek olasıdır BİK büyük morfolojik (intratümöral) heterojenite, var olduğu açıktır. Kombine morfolojik analiz ve Fuhrman derecelendirme ile BİK tümörlerin detaylı haritalama ve kategorizasyon proteomik analiz için temsili alanların seçimini sağlar. BİK 8 Protein temelli analiz nedeniyle patoloji laboratuvarlarında yaygın durumuna cazip olmakla birlikte, uygulama spesifik antikorlar 9 sınırlı durumu nedeniyle sorunlu olabilir. Ters Faz Protein Dizilerin dot blot doğa (RPPA), antikor zorunluluktur Borçlar be ön doğrulanmış; kullanılan antikorların böyle sıkı kalite kontrol olarak büyük önem taşımaktadır. Bu kısıtlamaya rağmen dot blot biçimi tek bir nitroselüloz slayda örnekleri yüzlerce baskı sağlayan, tahlil minyatür izin vermiyor. Baskılı slaytlar sonra çoğullama sağlayan, hedefe özgü primer antikor ve fluoresan etiketli ikincil antikoru kullanılarak Batı analizi ile benzer bir şekilde analiz edilebilir. Slayttaki tüm örneklerin genelinde Diferansiyel protein ekspresyonu sonra bir daha maliyet-etkin ve yüksek-throughput şekilde floresans göreli düzeyi karşılaştırılarak aynı anda analiz edilebilir.

Taranan RPPA kayar bir örnek 680 ile gösterilen kanallar 800 nm her ikisi de, Şekil 4 (i) &#39;de görülebilir. Dalga boyu, Şekil 4 (ii) ile görüntü ayırma analiz edilmesi için RPPA kayar ve Şekil 4, (iii) tespit bireysel protein ekspresyonu üzerindeki her bir yastık sağlar. Şekil 4&#39;te görüldüğü gibi (iii) örnek üzerinde tek proteinlerin ekspresyonu gelsolin çok daha düşük bir protein ifade vardır cMYC gö...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA floresan etiketli antikorlar kullanılarak, aynı anda sorgulanmak üzere nitroselüloz slaytlar basılı örnekleri yüzlerce, protein ekspresyonu sağlar. Bu teknik, şeffaf hücreli böbrek karsinomu olan ilaç tedavisi heterojenliğin etkisini araştırmak için uygulanmıştır.

Bireysel Renal Hücreli Kanserlerde içinde Protein Ekspresyonu Varyasyon kaynakları için Ters Faz Protein Diziler (RPPA) ve Kullanımı

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

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Medicine

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

33.5K Görüntüleme.  University of Edinburgh. RPPA floresan etiketli antikorlar kullanılarak, aynı anda sorgulanmak üzere nitroselüloz slaytlar basılı örnekleri yüzlerce, protein ekspresyonu sağlar. Bu teknik, şeffaf hücreli böbrek karsinomu olan ilaç tedavisi heterojenliğin etkisini araştırmak için uygulanmıştır.

Video: The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

İzole Renal Arter Tıkanıklığı Asılı ağırlık Sistemi

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

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Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Kantitatif İmmünofloresan kullanarak Tümörler Protein İfade heterojenite Haritalama

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Kanserler Üretim ve reaktif oksijen türlerinin (ROS Algılama)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

TSSB, Nöral Hematolojik ve Davranış Bağlantılı Revealing için Hayvan Modelinde Biyobelirteçler

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amiloid prekürsör protein intrasellüler Domain saflaştırılması ve toplama

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Prostat Kanserinde Biyomarker Discovery Uygulama: Bireysel herv Lokusları İfade Mikroarray tabanlı Kimlik

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Kanser hücre istilası ve Uydu Tümör oluşumu keşfedin Kültür Testi Üç Boyutlu

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Yetişkin sıçan böbrek üzerinden olduğu gibi Glomeruli yalıtmak için verimli bir eleme yöntemi

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

İnsan İnflamatuar Yanıtında Bireysel Değişimi Değerlendirmek Için Minimal Eritem Dozu (MED) Testinin Hassas Uygulanması

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

Böbrek Atardamarı Ile Böbreğe Doğrudan İlaç Verilmesi

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...