Tibet Tıbbı Üniversitesi'nin 14. Beş Yıllık Planının (2022ZYYGH12) Çağrışım İnşaatı Projesi'nden, Tibet Tıbbı Üniversitesi Eğitim Bakanlığı'nın Temel Laboratuvarı'nın 2022 Açık Konularından (ZYYJC-22-04), Ningxia'nın Temel Araştırma ve Geliştirme Programından (2023BEG02012) alınan mali destek için içten takdirlerimizi ifade ederiz, ve Chengdu Geleneksel Çin Tıbbı Üniversitesi'nin (XKTD2022013) Xinglin Scholar Araştırma Teşvik Projesi.

Bileşik çözelti, protein çözeltisi ve boya, bir PBS çözeltisine dahil edildi. Daha sonra, numuneler, dijital ısıtma çalkalama kuru banyosu kullanılarak kademeli olarak ısıtıldı ve proteinlerin termal stabilitesi, karmaşık sistem içindeki floresan yoğunluğundaki değişimin ölçülmesiyle değerlendirildi. Ayrıntılı adımlar aşağıda özetlenmiştir ve Şekil 1'de protokole genel bir bakış gösterilmektedir.
1. Çözelti hazırlama
<ol><li>616.75 μL DMSO çözeltisine 0.1 mg umbelliferon ekleyerek 1 mM'lik bir umbelliferon çözeltisi hazırlayın (bkz.</li>
	<li>500 μL ddH 2O çözeltisine 0.1 mg CD40 ekleyerek 200 μg / mLCD40protein çözeltisi hazırlayın (Malzeme Tablosuna bakınız).</li>
	<li>9 μL fosfat tamponlu tuzlu suya (PBS) 1 μL 5000x turuncu boya ekleyerek 500x turuncu boya çözeltisi hazırlayın (bkz. NOT: Yukarıdaki işlemleri buz üzerinde gerçekleştirin ve pipet minimum 0,1 μL aralığına sahip olduğundan, sonraki kullanımı kolaylaştırmak için başlangıçta turuncu protein boyasını 10 kat seyreltin.</li>
</ol>2. Boya performans testi
<ol><li>500x turuncu boyayı bir PBS çözeltisine ekleyin ve boya elde etmek için seyreltin: 1:500, 1:1000, 1:2000 ve 1:4000 PBS oranları. NOT: DMSO'nun nihai konsantrasyonunun %2'yi (h/v) aşmadığından emin olun.</li>
	<li>Floresan mikroplaka okuyucuyu açın ve 10 dakika önceden ısıtın.</li>
	<li>Bilgisayarı ve veri toplama yazılımını sırayla açın (bkz. Malzeme Tablosu).</li>
	<li>Cihaz'a tıklayın, ilgili floresan mikroplaka modelini seçin ve veri toplama yazılımında Tamam'ı seçin. NOT: Cihaz ile yazılım arasında başarılı bir bağlantı sağlamak için veri toplama yazılımını açmadan önce floresan mikroplakanın ön ısıtmayı bitirmesini ve paneldeki sıcaklığı görüntülemesini bekleyin.</li>
	<li> Edinme Ayarları'na tıklayın ve veri toplama yazılımında Floresans'ı seçin.</li>
	<li>Programı Dalga Boyları arayüzünde düzenleyin: Lm1 = 470 nm, 570 nm ve ardından Tamam'ı seçin. NOT: Turuncu boya, 300 nm'de UV ışığı veya 470 nm'de görünür ışıkla uyarılabilir ve emisyon 570 nm'de ölçülebilir.</li>
	<li>100 μL / oyukta 96 oyuklu plakalara farklı konsantrasyonlarda turuncu boya (1:500, 1:1000, 1:2000 ve 1:4000) ve PBS ekleyin. NOT: Çözeltiyi PBS'ye eklemeden önce DMSO'da çözün ve boyanın çökelme eğilimi nedeniyle tam çözünmeyi sağlamak için hazırlama işlemi sırasında 2000 x g'da iyice girdaplayın.</li>
	<li>96 oyuklu plakayı cihazın algılama aşamasına yerleştirin, veri toplama yazılımındaki Oku düğmesine tıklayın ve her boya konsantrasyonunun floresansını her seferinde 2 dakika aralıklarla oda sıcaklığında 13 kez ölçün.</li>
	<li>Her belirlemeden sonra Dışa Aktar düğmesine tıklayın, XML XLS TXT'ye Aktar'ı seçin, Tüm Plakalar'ı seçin, ardından Çıktı Biçiminde Plaka'yı seçin ve son olarak daha fazla analiz için verileri "XLS" formatında bir klasöre kaydetmek için Tamam'a tıklayın. NOT: Bu adımlar, sürekli ölçümün oda sıcaklığında turuncu boyanın otofloresansı üzerindeki etkisini belirlemeye ve optimum boyama konsantrasyonunu belirlemeye yardımcı olur.</li>
	<li>Dijital ısıtmalı çalkalama kuru banyosunu açın, ısıtma sıcaklığını 35 °C'ye ve ısıtma süresini 2 dakikaya ayarlayın.</li>
	<li>Boyanın optimum seyreltme oranını 1,5 mL'lik bir mikrosantrifüj tüpünde hazırlayın ve 2 dakika boyunca dijital ısıtma çalkalama kuru banyosuna yerleştirin.</li>
	<li>PBS çözeltisini ve 35 ° C'de ısıtılan boyama çözeltisini, oyuk başına 100 μL'lik bir hacimde 96 oyuklu plakaya ekleyin.</li>
	<li>96 oyuklu plakayı cihazın algılama aşamasına yerleştirin ve veri toplama yazılımındaki Oku düğmesine tıklayın.</li>
	<li>Isıtma sıcaklığını 40 °C'ye ve ısıtma süresini 2 dakikaya ayarlayın.</li>
	<li>96 oyuklu plakadaki boya solüsyonunu 1.5 mL mikrosantrifüj tüpüne geri pipetleyin ve cihazda 2 dakika ısıtın.</li>
	<li>Boya çözeltisinin absorbansını sırasıyla 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C ve 95 °C'de test etmek için adım 2.12-2.15 ve adım 2.9'daki işlemleri tekrarlayın. NOT: Bu adımlar, 35 °C ila 95 °C arasında bir gradyanda sürekli ısıtma altında turuncu boyanın otofloresan stabilitesinin belirlenmesine yardımcı olur. Hızlı sıcaklık düşüşlerinden veya deneysel hatalara yol açabilecek yanlış sıvının eklenmesinden kaçınmak için hızlı çalışmak çok önemlidir.</li>
</ol>3. Proteinin erime sıcaklığının (Tm) tespiti
<ol><li>Sırasıyla 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL ve 1:500 nihai konsantrasyonlar elde etmek için CD40 proteini ve turuncu boyayı PBS çözeltisi ile birleştirin.</li>
	<li>CD40 protein çözeltisinin sırasıyla 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C ve 95 °C sıcaklıklarda absorbansını değerlendirmek için adım 2.2-2.6 ve adım 2.10-2.16'da belirtilen işlemleri tekrarlayın.</li>
	<li>Verileri kaydetmek ve analiz etmek için adım 2.9'u tekrarlayın ve ardından veri analiz yazılımı kullanarak Tm değerini hesaplayın (bkz. Malzeme Tablosu).</li>
	<li>Sırasıyla 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 10 μM ve 1:500 nihai konsantrasyonlar elde etmek için PBS çözeltisine CD40 proteini, umbelliferon ve portakal boyası ekleyin. NOT: PBS'de boya, CD40 proteini ve umbelliferonun eşit dağılımını sağlamak için çözelti hazırlama sırasında kuvvetli girdaplama gereklidir.</li>
	<li>Sırasıyla 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C ve 95 °C sıcaklıklarda CD40-umbelliferone kompleksleri çözeltisinin absorpsiyonunu ölçmek için adım 3.2-3.3'teki işlemleri tekrarlayın. NOT: Bu adımlar, umbelliferona optimal CD40 protein bağlanma konsantrasyonunu belirlemeyi amaçlayan CD40 proteini ve CD40-umbelliferone komplekslerinin Tm değerlerini belirlemek için gerçekleştirilmiştir.</li>
</ol>

Floresan kullanarak bileşik-protein bağlanmasının yüksek hassasiyetli tespiti

<ol>
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Yazarların ifşa edecek hiçbir şeyi yok.

Termal kayma tahlili veya termal floresan tahlili olarak da bilinen DSF, yavaş, programlanmış bir sıcaklık artışı sırasında test numunesinin veya boyanın floresan sinyalindeki değişiklikleri izleyerek numunelerdeki proteinlerin termal denatürasyon sürecini tespit etmek için kullanılan bir tekniktir. Başlangıçta Pantoliano14 tarafından kurulan DSF, yüksek verimli bir yöntem olarak hizmet vermektedir. Ana prosedür, bilgisayar kontrollü ısıtılmış bir plaka üzerindeki s?...

Umbelliferous familyasından bir bitki olan Vicatia thibetica H. Boissieu, Tibet tıbbında yaygın olarak kullanılır ve beş temel bileşenin (Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew. ve Gymnadenia conopsea (L.) R. Br.) 1. Esas olarak kuzeybatı Yunnan, batı Sichuan, Tibet ve diğer bölgeler gibi güneybatı Çin'de dağıtılan kurutulmuş kökü, Angelica1'in yerel bir ikamesi olarak hizmet eder. Kokusuyla bilinen kök, genellikle güveç baharatı olarak kullanılır ve Tibet kerevizi olarak adlandırılan yapraklar nefis yemeklere katkıda bulunur. Bu nedenle, Vicatia thibetica sadece eşsiz bir şifalı bitki olarak değil, aynı zamanda Tibet'te bir besin kaynağı olarak da önem taşımaktadır.
Hem yerel hem de uluslararası araştırmalar, Vicatia thibetica'nın adet kanamasını düzenlemek için faydalı olan kan yenileyici ve qi (güç veya enerji) canlandırıcı özelliklere sahip olduğunu göstermektedir. Çarpıntı ve kan eksikliğinden kaynaklanan düzensiz adet dismenoresi semptomlarını ele almak için kullanılır ve vücut bağışıklığının antioksidan düzenlenmesi gibi farmakolojik etkiler gösterir2. Vicatia thibetica'nın alkol özütü, siklofosfamid tarafından indüklenen bağışıklığı baskılanmış farelerde vücut kütlesini ve organ indeksini geri kazanma yeteneğini göstermiştir. Ek olarak, serumdaki süperoksit dismutaz aktivitesini arttırır ve malondialdehit içeriğini azaltır, bu da antioksidan kapasitede bir iyileşme ve lipid peroksidasyonu üzerinde dengeleyici bir etki olduğunu düşündürür 2,3. Aynı zamanda, sadece vücudun hematopoietik işlevini objektif olarak yansıtmakla kalmayıp aynı zamanda bağışıklık sisteminde çok önemli bir rol oynayan kan hücrelerinin ve hemoglobinin sayısını artırır 2,3.
Asiküler kristaller ve acı bir tat ile karakterize edilen umbelliferon, küçük bir moleküler ağırlığa sahiptir, uçucudur ve buharla damıtılabilir. Kolayca süblimleşir, suda düşük çözünürlüğe ve organik maddede yüksek çözünürlüğe sahiptir. Vicatia thibetica'nın ana kimyasal bileşenlerinden biri olan umbelliferon, hidrokortizonun neden olduğu immünosupresyon fare modellerinde hücresel bağışıklık, humoral bağışıklık ve spesifik olmayan bağışıklık üzerinde immünomodülatör etkiler sergiler 4,5.
Tümör nekroz faktörü reseptör süper ailesinin bir üyesi olan hücre yüzey molekülü CD40, bağışıklık hücrelerinde yaygın olarak eksprese edilir6. CD40L olarak da bilinen homolog ligandı CD154, aktive edilmiş T lenfositleri tarafından eksprese edilen bir tip II transmembran proteinidir. CD40 aktivasyonu, dendritik hücrelerin (DC) ve monositlerin yüzeyindeki ko-stimülatör moleküllerin ekspresyonunu yukarı regüle etme kapasitesine sahiptir. Bu işlem, majör histouyumluluk kompleksi (MHC) moleküllerinin antijen sunum fonksiyonunu teşvik eder ve ayrıca CD8 + T hücreleriniaktive eder 7.
Makrofajlar, tümör mikroçevresinin oluşumunda ve düzenlenmesinde çok önemli bir rol oynar ve CD40 aktivasyonu, tümör mikroçevresinin makrofajlar tarafından yeniden şekillenmesini artırabilir8. CD40 sinyal aktivasyonu, B hücrelerinin proliferasyonunu ve aktivasyonunu önemli ölçüde etkiler. B hücreleri, CD40 tarafından aktive edildiğinde, etkili antijen sunan hücreler olarak işlev görebilir. Antijenleri sunarlar, efektör T hücresi aktivitesi oluştururlar ve böylece anti-tümör etkilerine katkıda bulunurlar9. Ayrıca, tümör hücrelerinde CD40 aktivasyonu apoptozu indükleyebilir ve tümör büyümesini inhibe edebilir10. CD40 esas olarak Lyn, Fyn, Syk ve diğerleri dahil olmak üzere reseptör olmayan tirozin protein kinazların aktivitesini kontrol ederek sinyalleri iletir. Ek olarak, Bcl-xL, Cdk4 ve Cdk6 proteinlerini uyarma, Rel/NF-kB transkripsiyon faktörlerini aktive etme ve CG-2 ve PI3K10'u fosforile etme yeteneğine sahiptir.
Diferansiyel Taramalı Floresan (DSF) yöntemi, tampon bileşimi, sıcaklık ve küçük moleküllü ligandlar gibi çeşitli çevresel koşulların protein yapılarının termal stabilitesi üzerindeki etkisini değerlendirmek için yaygın olarak kullanılmaktadır. DSF için yaygın olarak kullanılan boya, turuncu, çevreye duyarlı, hidrofobik bir boyadır. Normal koşullar altında, protein yapısı katlanır ve hidrofobik segmentini dahili olarak gizler. Sıcaklık arttıkça, proteinin hidrofobik bölgesi daha fazla açığa çıkar ve bu da doğal protein yapısının kademeli olarak parçalanmasına neden olur. Boya, açıkta kalan bu protein kısmına seçici olarak bağlanır ve floresansını arttırır. Tm değerleri daha sonra floresan sinyali algılamadaki11 değişikliklerin izlenmesiyle hesaplanır. Bir dereceye kadar, Tm değerlerindeki değişiklikler, mutasyonlar, tamponlardaki değişiklikler veya ligand bağlanması nedeniyle protein stabilitesindeki değişimleri ölçebilir. Ayrıca, protein katlama işlemi12 sırasında yapısal değişiklikleri gösterebilir. Bu yaklaşım, kesin veriler, geniş bir sıcaklık aralığı, yüksek hassasiyet ve minimum protein numune kaybısunar 13.
Bu çalışmada, floresan tayini, floresan kantitatif polimeraz zincir reaksiyonu (PCR) aparatı yerine floresan mikroplaka okuyucu kullanılarak gerçekleştirilmiştir. Bu modifikasyon, floresan kantitatif PCR cihazı olmayan laboratuvarlarda DSF tespitini mümkün kılar, bu da yöntemi daha az karmaşık hale getirir ve cihaz kurulumu için gereken adımları azaltır, böylece deneysel süreci basitleştirir. Ancak, bu yaklaşımın bazı dezavantajları vardır. Karmaşıklık azalırken, prosedür daha hantal hale gelir. Farklı sıcaklıklarda manuel floresan algılaması gereklidir ve sistemden otomatik ve sürekli floresan toplanması sağlanamaz. Bu nedenle, bu çalışma, CD40 proteini ve umbelliferon arasındaki etkileşimi araştırmak için DSF tekniğini kullandı ve Tibet tıbbının moleküler mekanizmalarına yeni bakış açıları sağladı.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD40 protein</td>
			<td>MedChemExpress</td>
			<td>HY-P75408</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexStation 3 multifunctional microplate reader</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>FlexStation 3</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro 7.1</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>SoftMax Pro 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSYPRO orange dye</td>
			<td>Sigma</td>
			<td>S5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Umbelliferone</td>
			<td>Shanghai Yuanye Biotechnology Co.</td>
			<td>B21854</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

Farklı moleküller arasındaki etkileşimlerin araştırılması, hastalık patogenezini anlamanın ve ilaç hedeflerinin taranmasının çok önemli bir yönüdür. Tibet tıbbı Vicatia thibetica'da aktif bir bileşen olan umbelliferon, bilinmeyen bir mekanizma ile immünomodülatör bir etki sergiler. CD40 proteini, bağışıklık tepkisinde önemli bir hedeftir. Bu nedenle, bu çalışma, floresan enzim belirteçleri kullanarak CD40 proteini ve umbelliferon arasındaki etkileşimleri analiz etmek için diferansiyel taramalı floresan teknolojisi ilkesini kullanmaktadır. Başlangıçta, protein floresan turuncu boyanın stabilitesi deneysel olarak doğrulandı ve 1:500'lük optimal seyreltme oranı belirlendi. Daha sonra, CD40 proteininin sıcaklıkta erime (Tm) değerinin, konsantrasyondaki bir artışla azalma eğiliminde olduğu gözlendi. İlginç bir şekilde, CD40 proteini ve umbelliferon arasındaki etkileşimin, CD40 proteininin termal stabilitesini arttırdığı bulundu. Bu çalışma, floresan mikroplakalar ve floresan boyalar kullanarak küçük moleküllü bileşiklerin ve proteinlerin bağlanma potansiyelini tespit etmeye yönelik ilk girişimi temsil etmektedir. Teknik, yüksek hassasiyet ve doğruluk, protein stabilitesi, protein yapısı ve protein-ligand etkileşimleri alanlarında umut verici ilerlemeler ile karakterize edilir, böylece daha fazla araştırma ve araştırmayı kolaylaştırır.

Turuncu boya, hem oda sıcaklığında hem de yüksek sıcaklıklarda Ex = 470 nm ve Em = 570 nm'de sürekli olarak kararlı floresan uyarımı sergiledi. 1:500'lük bir optimal seyreltme oranı belirlenmiştir (Şekil 2A,B). CD40 proteininin konsantrasyonu 15 μg/mL'nin altında olduğunda Tm değerinin saptanması zor olmuştur (Şekil 3A,B). Bununla birlikte, 15 μg/mL'lik bir konsantrasyonda, CD40 protein konsantrasyonundak...

Watch this Scientific Journal Video about Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency at JoVE.com

Yazar Spotlight: Gelişmiş Erişilebilirlik ve Verimlilik için PCR Yöntemlerini Kolaylaştırma

Bu protokol, bileşikler ve protein molekülleri arasındaki bağlanmayı tespit etmek için benzersiz bir yöntemi tanımlar ve minimum protein numune kaybı ve yüksek veri doğruluğu avantajları sunar.

Diferansiyel Taramalı Floresan ile CD40 Protein-Umbelliferon Etkileşiminin Tespiti

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

Protokolümüz, özel ekipman olmadan kullanıma izin vererek önemli bir avantaj sağlar. Geleneksel floresan kantitatif PCR yöntemlerinden farklı olarak, deneysel süreci basitleştirir, daha az adım gerektirir ve genel karmaşıklığı azaltır, bu da onu daha güvenli ve yürütülmesi daha kolay hale getirir. Bu deney, operatörün deney prosedürüne ve floresan mikroplaka okuyucusuna çok aşina olmasını ve çözelti ısıtması ve floresan tespiti sırasında çok fazla zaman kaybetmemesini gerektirir.Umbelliferonun bağışıklık etkileri üretmek için CD14 proteinine bağlandığı moleküler mekanizmaya odaklanacağız.

detection-cd40-protein-umbelliferone-interaction-via-differential

Research

JoVE Journal

Medicine

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

378 Görüntüleme.  University of Tibetan Medicine. Protokolümüz, özel ekipman olmadan kullanıma izin vererek önemli bir avantaj sağlar. Geleneksel floresan kantitatif PCR yöntemlerinden farklı olarak, deneysel süreci basitleştirir, daha az adım gerektirir ve genel karmaşıklığı azaltır, bu da onu daha güvenli ve yürütülmesi daha kolay hale getirir. Bu deney, operatörün deney prosedürüne ve floresan mikroplaka okuyucusuna çok aşina olmasını ve çözelti ısıtması ve floresan tespiti sırasında çok fazla zaman ka...

Video: Yazar Spotlight: Gelişmiş Erişilebilirlik ve Verimlilik için PCR Yöntemlerini Kolaylaştırma

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Jinekolojik Onkoloji Sentinel lenf nodu intraoperatif Algılama için Multispektral Gerçek zamanlı Floresans Görüntüleme

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Tıp

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Floresan In-Situ Hibridizasyon ve Konfokal Lazer Tarama Mikroskopi Palatal Genişleticilerden Oral Biyofilm Analizi

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

İnsan Meme Fibroblastlar Karsinom İlişkili Fibroblastlar Deneysel Nesil (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Kanser Tedavisi İzleme Cerenkov Lüminesans Görüntüleme (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms. Oriented structures are analyzed using Gabor filters and phase portraits to detect sites of radiating tissue patterns. Each site is characterized and classified using measures to represent spiculating patterns. The methods should assist in the detection of breast cancer.

Önceki mamogram Mimari Bozulma Algılama<em&gt; Via</emOdaklı Patterns&gt; Analiz

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Floresan-aracılı tomografi algılama ve fare bağırsak iltihabı makrofaj ile ilgili miktar

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Kolesterol parçacıkları morfolojisi oransal olarak Lipid düşürücü ilaçlar farklı etkileri

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. BMD is an important component of bone&#39;s nutritional status and in skeletal remains of both juveniles and adults, and it can provide information about bone quality. For adults remains, it can provide information on pathological conditions or when bone insufficiency may have occurred. In juveniles, it provides a useful metric to elucidate cases of fatal starvation or neglect, which are generally difficult to identify. This paper provides a protocol for the anatomical orientation and analysis of skeletal remains for scanning via dual-energy X-ray absorptiometry (DXA). Three case studies are presented to illustrate when DXA scans can be informative to the forensic practitioner. The first case study presents an individual with observed longitudinal fractures in the weight bearing bones and DXA is used to assess bone insufficiency. BMD is found to be normal suggesting another etiology for the fracture pattern present. The second case study employed DXA to investigate suspected chronic malnutrition. The BMD results are consistent with results from long bone lengths and suggest the juvenile had suffered from chronic malnutrition. The final case study provides an example where fatal starvation in a fourteen-month infant is suspected, which supports autopsy findings of fatal starvation. DXA scans showed low bone mineral density for chronological age and is substantiated by traditional assessments of infant health. However, when dealing with skeletal remains taphonomic alterations should be considered before applying this method.

Bone mineral density (BMD) is an important factor in understanding nutritional intake. For human skeletal remains, it is a useful metric to assess quality of life in both juveniles and adults, particularly in fatal starvation and neglect cases. This paper provides guidelines for scanning human skeletal remains for forensic purposes.

Kemik Mineral Yoğunluğu adli bağlamlarda için tarama iskelet kalıntıları

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Taramalı Elektron Mikroskobu ile Membran Fırfırı Oluşumunu Görselleştirme

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) is a noninvasive tool to facilitate the distinction of composite resin material from sound tooth substance. Compared to conventional illumination, FIT is a very accurate, reliable, and fast diagnostic method. When composite resin is illuminated with a wavelength of approximately 398 &plusmn; 5 nm, certain fluorescent components make the composite resin appear brighter than the tooth structure. Any fluorescence-inducing light source with the appropriate wavelength can be used for this method. Optimally, this technique is used without additional natural or artificial lighting. The application of FIT can be used for diagnostic purposes, for example, dental charts, and additionally for the complete and minimally invasive removal of composite resin restorations, bracket debonding, and trauma splint removal. The assessment of volumetric changes after composite removal can be provided by overlapping pre- and postoperative scans and subsequent calculation using suitable software.

The Fluorescence-aided Identification Technique is a practicable, fast, and reliable approach for the differentiation of composite resin restorations from tooth substance, and facilitates the minimally invasive and complete removal of composite resin restorations and composite bonded trauma splints.

Floresan Destekli Tanımlama Tekniği Kullanılarak Diş Rengindeki Kompozit Reçinenin Tespiti ve Uzaklaştırılması

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) ...