قبل الجراحة تجريب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويمكن في الاشعة المقطعية في البطن يمكن ان ينظر اليها بسهولة القناة الصفراوية (الظهور داخل لحمة الكبد مع قتامة لون) الموسع على نطاق واسع على الجانب الأيسر من الكبد D التعمير 0.3 أثبتت وجود الحق الشريان الكبدي الأصلي مباشرة من الشريان الأورطي و لا مزيد من التشوهات الوعائية. </li><li style=";text-align:right;direction:rtl"> ويمكن تقسيم الكبد وظيفيا إلى 8 قطاعات كما هو مبين في الفيديو وبالنظر إلى أن اقتصر على توسع الجزء 2 و 3 (قطاعات اليسار) ، وكان يخطط لsectoriectomy بالمنظار اليسار. </li></ol> جراحة داخلية الداخلي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يوضع المريض في موقف ضعيف والوقوف الجراح بين ساقي المريض. وتستخدم 5 trocars ووضعها كما هو مبين في الفيديو </li><li style=";text-align:right;direction:rtl"> يتم وضع المشبك الأوعية الدموية على ثالوث المدخل من أجل الحد من النزيف (manouvre برينغل ل) </li><li style=";text-align:right;direction:rtl"> يتم إجراء تقسيم لحمة الكبد كاو كهربي باستخدام مزيج متناسق أو القطبين ومشرط على طول الرباط المنجلي بين الجزء الثالث والرابع </li><li style=";text-align:right;direction:rtl"> مرة واحدة يتم تشريح كامل الجزء الثالث عنيق يمكن تقسيمها باستخدام إندو 45mm GIA بخرطوشة الأوعية الدموية. </li><li style=";text-align:right;direction:rtl"> انه يحمل على إجراء مزيد من التقسيم مع لحمة حتى وصلت الرئيسي هو عنيق الجزء الثاني ، معزولة ومقسمة أخيرا باستخدام endoGIA 45mm مع خرطوشة الأوعية الدموية </li><li style=";text-align:right;direction:rtl"> وينقسم الآن في الرباط غادر الثلاثي باستخدام كاو كهربي وعزل الوريد الكبدي الأيسر وتنقسم الأوعية الدموية باستخدام دباسة </li><li style=";text-align:right;direction:rtl"> يتم فحص السطح لوقف نزيف في الكبد ؛ يطبق الأرجون الشعاع على سطح الكبد ويتم إزالة المشبك الأوعية الدموية. في هذه المرحلة هو تطبيق الغراء الليفين على النظام للحد من خطر تسرب الصفراء بعد العمليات الجراحية. يتم وضع العينة في endobag وإزالتها </li></ol>

<ol>
	<li>Nguyen, K. T., Gamblin, T. C., Geller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection+for+cancer.">Laparoscopic liver resection for cancer.</a> Future Oncol. 4 (5), 661-670 (2008).</li><li>Gumbs, A. A., Gayet, B., Gagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection:+when+to+use+the+laparoscopic+stapler+device.">Laparoscopic liver resection: when to use the laparoscopic stapler device.</a> HPB (Oxford). 10 (4), 296-303 (2008).</li><li>Pulitan&#242;, C., Catena, M., Arru, M., Guzzetti, E., Comotti, L., Ferla, G., Aldrighetti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection+without+portal+clamping:+a+prospective+evaluation.">Laparoscopic liver resection without portal clamping: a prospective evaluation.</a> Surg Endosc. 22 (10), 2196-2200 (2008).</li><li>Yonem, O., Bayraktar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+characteristics+of+Caroli's+disease.">Clinical characteristics of Caroli's disease.</a> World J Gastroenterol. 13 (13), 1934-1937 (2007).</li></ol>

يمكن أن تكون عملية جراحية بالمنظار لاستئصال بأمان أيضا لأمراض الكبد على حد سواء الحميدة والخبيثة. الحد من الصدمة الجراحية باستخدام نهج مينيملي يسمح للتقليل من حدوث مضاعفات ما بعد الجراحة وبعد العملية البقاء في المستشفى.

laparoscopic left liver sectoriectomy of caroli's disease limited to segment ii and iii

يتم تعريف المرض Caroli باعتبارها توسع غير طبيعي في القناة الصفراوية داخل الكبدية : حدوثه منخفضة للغاية (1 في 1000000 نسمة) ، وفي معظم الحالات الكبد كله مهتم وزرع الكبد هو العلاج الأمثل. في حالة توسع تقتصر على الفص الأيمن أو الأيسر ، يمكن إجراء استئصال الكبد. لسنوات عديدة وقد نهج موحد لاستئصال الكبد البطن رسمية عن طريق شق البطن الكبيرة التي يتميز المرض بعد operatie كبيرة. في الآونة الأخيرة ، وقد اقترح مينيملي ، والنهج والاسلوب الجراحي بالمنظار لاستئصال الكبد ممكنة سواء بالنسبة للأمراض الحميدة والخبيثة. تتمثل الفوائد الرئيسية لنهج مينيملي بانخفاض كبير من الصدمة الجراحية التي يسمح أسرع الانتعاش أقل مضاعفات ما بعد الجراحة. هذا الفيديو يظهر حالة المرض Caroli ق وقع في 58 سنة من الذكور واعترف في قسم أمراض الجهاز الهضمي لظهور مفاجئ لألم في البطن مصحوب بحمى (&gt; 38C °) ، والغثيان ويرتجف. الموجات فوق الصوتية أظهرت البطن a توسع كبير في داخل القناة الصفراوية الكبدية اليسرى يقع مع عدم وجود أدلة من المرارة أو القناة الصفراوية المشتركة الحجارة. وتأكدت هذه النتائج في البطن وارتفاع القرار التصوير المقطعي الكمبيوتر. وكان من المقرر بالمنظار sectoriectomy اليسار. وأظهر التنقيب في تجويف البطن trocars خمسة و 30 درجة واستخدمت البصرية ، أي التصاقات أو أدلة من الأمراض الأخرى. وكان من أجل السيطرة على تدفق الدم الى الكبد والأوعية الدموية وضع المشبك على كبدي العنيقة (برينغل manouvre ق) ، ويتم تقسيم متني خارجا مع الجمع بين استخدام ملقط 5 ملم و 5 القطبين مشرح بالموجات فوق الصوتية ملم. تم عزل والمتوسعة بشدة القناة الكبدية اليسرى وتقسم باستخدام 45mm دباسة الوعائية بالمنظار. تشريح الكبد واستمر حتى بمعزل عن فرع المدخل الرئيسي اليسار أنه تم تقسيم ثم مع خرطوشة مزيد من الأوعية الدموية دباسة 45 ملم. في وجهة نظره في الكبد اليسار لا يزال يعلق فقط من الوريد الكبدي اليسار : تم تنفيذ التقسيم الثلاثي في ​​الرباط باستخدام ربط أحادي القطب ، والوريد الكبدي معزولة ومقسمة باستخدام دباسة الأوعية الدموية. وقد تم صقل Haemostatis بتطبيق الأرجون شعاع التخثر وكشفت عدم وجود نزيف حتى بعد إزالة المشبك الأوعية الدموية (الوقت الإجمالي برينغل ق 27 دقيقة). كان هادئا ، ارتفاع الحد الأدنى للاختبارات وظائف الكبد طبعا تم تسجيلها في ما بعد الجراحة بعد الجراحة يوم 1 لكنه عاد الى طبيعته في تصريفها على بعد 3 ايام من المنطوق.

Watch this Scientific Journal Video about Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III at JoVE.com

Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III

هذا الفيديو يمثل حالة من الكبد بالمنظار sectoriectomy اليسار perfomed في رجل يبلغ من العمر 53 عاما يعاني من مرض Caroli المحدودة على الجزء الثاني والثالث من الكبد.

Sectoriectomy الكبد بالمنظار بقي من الأمراض Caroli المحدودة على الجزء الثاني والثالث

Caroli's disease is defined as a abnormal dilatation of the intra-hepatica bile ducts: Its  incidence is extremely low  (1 in 1,000,000 population) and in ...

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14.1K Views.  University of Insubria. هذا الفيديو يمثل حالة من الكبد بالمنظار sectoriectomy اليسار perfomed في رجل يبلغ من العمر 53 عاما يعاني من مرض Caroli المحدودة على الجزء الثاني والثالث من الكبد.

Video: Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

بوغ زقي zeae Gibberella الإنتاج والتحصيل للتجارب ميكروأري.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

تشكيل الأنبوبة Matrigel القائم على الفحص لتقييم نشاط الخلايا السرطانية مكون للأوعية

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

في الإلتقام الكبد المجراة تلاه تنقية خلايا الكبد الكبد الإرواء

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

افعل ذلك بنفسك جهاز لاستخراج العينات Cryopreserved سقطت سهوا في صهاريج التخزين المبردة

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

عزلة البروتينية ريبوسوم الوليدة منضم<em&gt; في المختبر</em&gt; لتحديد المواقع وقفة بالحركة على طول مرنا

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

بروتوكول لLentiviral تنبيغ وتحليل المصب من المعوية Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

استخدام<em&gt; فيفو السابقين</em&gt; التصوير الجامع بالأعضاء والأنسجة الكمي الأنسجة لتحديد بيو توزيع الجزيئات Fluorescently إعتبر

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

بروتوكولات لاختبار السمية لرواية كيمياء مبيدات الحشرات للبعوض

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

والعزلة وتصوير الأسفار لتعمير واحد-الجسيمات Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

عزل خلايا الصمام الأولي البشري لنمذجة الأمراض في المختبر

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...