<ol>
	<li>Sharndama, H. C., Mba, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori:+an+up-to-date+overview+on+the+virulence+and+pathogenesis+mechanisms.">Helicobacter pylori: an up-to-date overview on the virulence and pathogenesis mechanisms.</a> Braz J Microbiol. 53 (1), 33-50 (2022).</li><li>Ravikumara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+in+children:+think+before+you+kill+the+bug.">Helicobacter pylori in children: think before you kill the bug.</a> Therap Adv Gastroenterol. 16 (6), 1-10 (2023).</li><li>Reshetnyak, V. I., Burmistrov, A. I., Maev, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori:+Commensal,+symbiont+or+pathogen.">Helicobacter pylori: Commensal, symbiont or pathogen.</a> World J Gastroenterol. 27 (7), 545-560 (2021).</li><li>Al-Ouqaili, M. T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+vacuolating+cytotoxin+A+(vacA)+genotypes+of+ulcerogenic+and+non-ulcerogenic+strains+of+Helicobacter+pylori+and+its+association+with+gastric+disease.">Study of vacuolating cytotoxin A (vacA) genotypes of ulcerogenic and non-ulcerogenic strains of Helicobacter pylori and its association with gastric disease.</a> Saudi J Biol Sci. 30 (12), 103867 (2023).</li><li>Hussein, R. A., Al-Ouqaili, M. T. S., Majeed, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+alcohol+consumption,+cigarette+smoking,+and+Helicobacter+pylori+infection+in+Iraqi+patients+submitted+to+gastrointestinal+endoscopy.">Association between alcohol consumption, cigarette smoking, and Helicobacter pylori infection in Iraqi patients submitted to gastrointestinal endoscopy.</a> J Emerg Med Trauma Acute Care. 6, 12 (2022).</li><li>Thrift, A. P., Wenker, T. N., El-Serag, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+gastric+cancer:+Epidemiological+trends,+risk+factors,+screening+and+prevention.">Global burden of gastric cancer: Epidemiological trends, risk factors, screening and prevention.</a> Nat Rev Clin Oncol. 20 (5), 338-349 (2023).</li><li>Liou, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+eradication+of+Helicobacter+pylori+for+gastric+cancer+prevention:+The+Taipei+global+consensus.">Screening and eradication of Helicobacter pylori for gastric cancer prevention: The Taipei global consensus.</a> Gut. 69 (12), 2093-2112 (2020).</li><li>Gao, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+burden+and+risk+in+the+Chinese+population+aged+55+years+and+above:+A+systematic+analysis+and+comparison+with+the+USA+and+Western+Europe.">Cancer burden and risk in the Chinese population aged 55 years and above: A systematic analysis and comparison with the USA and Western Europe.</a> J Glob Health. 14, 04014 (2024).</li><li>Hussein, R. A., Al-Ouqaili, M. T. S., Majeed, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Helicobacter+pylori+infection+by+invasive+and+non-invasive+techniques+in+patients+with+gastrointestinal+diseases+from+Iraq:+A+validation+study.">Detection of Helicobacter pylori infection by invasive and non-invasive techniques in patients with gastrointestinal diseases from Iraq: A validation study.</a> PLoS One. 16 (8), e0256393 (2021).</li><li>Thung, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+article:+The+global+emergence+of+Helicobacter+pylori+antibiotic+resistance.">Review article: The global emergence of Helicobacter pylori antibiotic resistance.</a> Aliment Pharmacol Ther. 43 (4), 514-533 (2016).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+antibiotic+target+genes+related+to+clarithromycin,+metronidazole+and+levofloxacin+resistance+in+Helicobacter+pylori+strains+from+children+in+China.">Mutations in the antibiotic target genes related to clarithromycin, metronidazole and levofloxacin resistance in Helicobacter pylori strains from children in China.</a> Infect Drug Resist. 13 (1), 311-322 (2020).</li><li>Saranathan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+Infections+in+the+Bronx,+New+York:+Surveying+antibiotic+susceptibility+and+strain+lineage+by+whole-genome+sequencing.">Helicobacter pylori Infections in the Bronx, New York: Surveying antibiotic susceptibility and strain lineage by whole-genome sequencing.</a> J Clin Microbiol. 58 (3), e01591-e01619 (2020).</li><li>Malfertheiner, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+Helicobacter+pylori+infection+-+The+Maastricht+IV/+Florence+consensus+report.">Management of Helicobacter pylori infection - The Maastricht IV/ Florence consensus report.</a> Gut. 61 (5), 646-664 (2012).</li><li>Palma, G. Z. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+mutations+in+the+antimicrobial+target+genes+related+to+levofloxacin,+clarithromycin,+and+amoxicillin+resistance+in+Helicobacter+pylori+isolates+from+Buenos+Aires+city.">Occurrence of mutations in the antimicrobial target genes related to levofloxacin, clarithromycin, and amoxicillin resistance in Helicobacter pylori isolates from Buenos Aires city.</a> Microb Drug Resist. 23 (3), 351-358 (2017).</li><li>Zhang, L., Zhao, M., Fu, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+microbiota+dysbiosis+and+Helicobacter+pylori+infection.">Gastric microbiota dysbiosis and Helicobacter pylori infection.</a> Front Microbiol. 14, 1153269 (2023).</li><li>Peng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+juice-based+real-time+PCR+for+tailored+Helicobacter+Pylori+treatment:+A+practical+approach.">Gastric juice-based real-time PCR for tailored Helicobacter Pylori treatment: A practical approach.</a> Int J Med Sci. 14 (6), 595-601 (2017).</li><li>Weingart, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+a+novel+stool+antigen+test+for+detection+of+Helicobacter+pylori+in+adult+outpatients+before+and+after+eradication+therapy.">Sensitivity of a novel stool antigen test for detection of Helicobacter pylori in adult outpatients before and after eradication therapy.</a> J Clin Microbiol. 42 (3), 1319-1321 (2004).</li><li>Chi, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+high-throughput+multiplex+genetic+detection+assay+for+detection,+semi-quantification+and+virulence+genotyping+of+Helicobacter+pylori+in+non-invasive+oral+samples.">A rapid and high-throughput multiplex genetic detection assay for detection, semi-quantification and virulence genotyping of Helicobacter pylori in non-invasive oral samples.</a> Front Cell Infect Microbiol. 13, 1267288 (2023).</li><li>Zhang, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+high-throughput+multiplex+genetic+detection+system+for+Helicobacter+pylori+identification,+quantification,+virulence,+and+resistance+analysis.">Validation of a high-throughput multiplex genetic detection system for Helicobacter pylori identification, quantification, virulence, and resistance analysis.</a> Front Microbiol. 7, 1401 (2016).</li><li>Gisbert, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirical+or+susceptibility-guided+treatment+for+Helicobacter+pylori+infection?+A+comprehensive+review.">Empirical or susceptibility-guided treatment for Helicobacter pylori infection? A comprehensive review.</a> Therap Adv Gastroenterol. 13, 1756284820968736 (2020).</li><li>B&#233;n&#233;jat, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+for+Helicobacter+pylori+diagnosis.+The+best+tools+available.">Real-time PCR for Helicobacter pylori diagnosis. The best tools available.</a> Helicobacter. 23 (5), e12512 (2018).</li><li>Vasapolli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+assessment+of+H.+pylori+infection+to+guide+molecular+antibiotic+resistance+testing:+A+combined+endoscopy-gastric+juice+analysis+approach.">Real-time assessment of H. pylori infection to guide molecular antibiotic resistance testing: A combined endoscopy-gastric juice analysis approach.</a> Aliment Pharmacol Ther. 61 (3), 465-471 (2025).</li><li>Hussein, R. A., Al-Ouqaili, M. T. S., Majeed, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+clarithromycin+resistance+and+23SrRNA+point+mutations+in+clinical+isolates+of+Helicobacter+pylori+isolates:+Phenotypic+and+molecular+methods.">Detection of clarithromycin resistance and 23SrRNA point mutations in clinical isolates of Helicobacter pylori isolates: Phenotypic and molecular methods.</a> Saudi J Biol Sci. 29 (1), 513-520 (2022).</li><li>Wang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+and+meta-analysis+of+genotypic+methods+for+detecting+antibiotic+resistance+in+Helicobacter+pylori.">A systematic review and meta-analysis of genotypic methods for detecting antibiotic resistance in Helicobacter pylori.</a> Helicobacter. 23 (2), e12467 (2018).</li><li>Egli, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+diagnostic+performance+of+qPCR,+Sanger+sequencing,+and+whole-genome+sequencing+in+determining+clarithromycin+and+levofloxacin+resistance+in+Helicobacter+pylori.">Comparison of the diagnostic performance of qPCR, Sanger sequencing, and whole-genome sequencing in determining clarithromycin and levofloxacin resistance in Helicobacter pylori.</a> Front Cell Infect Microbiol. 10, 596371 (2020).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+Helicobacter+pylori+infection+and+colorectal+polyp/colorectal+cancer.">Relationship between Helicobacter pylori infection and colorectal polyp/colorectal cancer.</a> World J Gastrointest Surg. 16 (4), 1008-1016 (2024).</li><li>Oliver, A. S., Wu, F., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+gastric+microbiota+in+gastric+cancer.">The role of gastric microbiota in gastric cancer.</a> Gut Microbes. 11 (5), 1220-1230 (2020).</li><li>Hulten, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+culture+with+antibiogram+to+next-generation+sequencing+using+bacterial+isolates+and+formalin-fixed,+paraffin+embedded+gastric+biopsies.">Comparison of culture with antibiogram to next-generation sequencing using bacterial isolates and formalin-fixed, paraffin embedded gastric biopsies.</a> Gastroenterology. 161 (5), 1433-1442 (2021).</li></ol>

<table><tbody><tr><td>Bath Incubator</td><td>ALLSHENG</td><td>MK2000-2</td><td>Provide a constant temperature environment</td></tr><tr><td>Biosafety cabinet</td><td>Haier</td><td>HR1500-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/67704/67704eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;B2</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Fisher Scientific</td><td>THERMO ST16R</td><td>Centrifuge the residual liquid off the wall of the tube.</td></tr><tr><td>Chelex-100&amp;nbsp;</td><td>sigma</td><td>C7901</td><td>Resin</td></tr><tr><td>Gastroscope biopsy forceps</td><td>Boston Scientific Corporation</td><td></td><td>Sampling of the gastric mucosa</td></tr><tr><td>Helicobacter pylori 23S rRNA gene and gyrA gene mutation detection kit</td><td>Jiangsu Mole Bioscience</td><td>CFDA 20223400137</td><td>qPCR detection kit for &lt;em&gt;H. pylori &lt;/em&gt;and drug-resistance genes (clarithromycin and quinolones)</td></tr><tr><td>Nucleic acid extraction reagent</td><td>Jiangsu Mole Bioscience</td><td>SEDA 20150076</td><td>For DNA extraction</td></tr><tr><td>SDS Software</td><td>Applied Biosystems</td><td>7300/7500</td><td>Data analysis</td></tr><tr><td>Thermocylcer</td><td>Thermo Fisher Scientific</td><td>ABI 7500</td><td>For qPCR detection of H. pylori and drug-resistance genes (clarithromycin and quinolones)</td></tr><tr><td>Vortex mixer</td><td>JOANLAB</td><td>VM-5005</td><td>For mixing reagent</td></tr></tbody></table>

gastric mucosa quantitative polymerase chain reaction analysis for detecting helicobacter pylori and antibiotic resistance

헬리코박터 파일로리균 및 항생제 내성 검출을 위한 위 점막 정량적 중합효소 연쇄 반응 분석

Helicobacter pylori is a main pathogen that infects nearly half of the global population and is threatening public health due to its increasing antibiotic ...

gastric-mucosa-quantitative-polymerase-chain-reaction-analysis-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Gastric Mucosa Quantitative Polymerase Chain Reaction Analysis for Detecting Helicobacter pylori and Antibiotic Resistance

217 조회수.  Southern Medical University. 헬리코박터 파일로리균 검출 및 약물 내성 검사를 위한 빠르고 정확한 방법은 임상 실습에서 헬리코박터 파일로리균을 효율적으로 박멸하는 데 매우 중요합니다. 이 프로토콜은 H. pylori 및 항생제 내성의 신속한 검출을 위해 위 점막 정량적 중합효소 연쇄 반응(qPCR)과 관련된 특정 방법론을 제시하는 것을 목표로 ?...

비디오: 헬리코박터 파일로리균 및 항생제 내성 검출을 위한 위 점막 정량적 중합효소 연쇄 반응 분석

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

조류 엠브료 모델을 사용하여 암 전이의 정량 분석

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

연구

의학

Roux-en-Y Gastric Bypass Operation in Rats

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12.
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

쥐에서 Roux-EN-Y 위장 바이패스 수술

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

질병의 염색질 Proteomes의 정량 분석

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

세포의 기원, 종류, 그리고 상호 작용의 시각화를위한 형광 조직 이식에 따라 정량 멀티 스펙트럼 분석

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

쥐의 대동맥 부비동의 동맥 경화 병변의 정량 분석​​ 및 특성

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

구현<em&gt; 체외</em&gt; 약물 저항 분석법 : 임상 적 저항 메커니즘을 폭로 가능성을 극대화

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can improve strength in individuals with MS, however; it remains unclear the duration of RST that is needed to make strength gains and how to adapt hip strengthening exercises for individuals of varying strength using only resistance bands. This paper describes the methodology to set up and implement an adapted resistance strength training program, using resistance bands, for individuals with MS. Directions for pre- and post-strength tests to evaluate efficacy of the strength-training program are included. Safety features and detailed instructions outline the weekly program content and progression. Current evidence is presented showing that significant strength gains can be made within 8 weeks of starting a RST program. Evidence is also presented showing that resistance strength training can be successfully adapted for individuals with MS of varying strength with little equipment.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

적응 저항 훈련은 다발성 경화증을 가진 개인에 여덟 주에서 강도를 향상

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis에서 Vivo에서 CaspaseTracker 바이오 센서에 의해 감지

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

파괴 강도 시험 및 정량 CT 기반의 유한 요소 분석을위한 근위 대퇴골 사체 준비

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life.
The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI.
We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.

Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

헬리코박터 파일로리균 및 항생제 내성 검출을 위한 위 점막 정량적 중합효소 연쇄 반응 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

조류 엠브료 모델을 사용하여 암 전이의 정량 분석

쥐에서 Roux-EN-Y 위장 바이패스 수술

질병의 염색질 Proteomes의 정량 분석

세포의 기원, 종류, 그리고 상호 작용의 시각화를위한 형광 조직 이식에 따라 정량 멀티 스펙트럼 분석

쥐의 대동맥 부비동의 동맥 경화 병변의 정량 분석 및 특성

구현

적응 저항 훈련은 다발성 경화증을 가진 개인에 여덟 주에서 강도를 향상

Anastasis에서 Vivo에서 CaspaseTracker 바이오 센서에 의해 감지

파괴 강도 시험 및 정량 CT 기반의 유한 요소 분석을위한 근위 대퇴골 사체 준비

개정 공동 교체에 새로운 진단: 쥡니다 및 다중 연쇄 반응 임 플 란 트

헬리코박터 파일로리균 및 항생제 내성 검출을 위한 위 점막 정량적 중합효소 연쇄 반응 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

조류 엠브료 모델을 사용하여 암 전이의 정량 분석

쥐에서 Roux-EN-Y 위장 바이패스 수술

질병의 염색질 Proteomes의 정량 분석

세포의 기원, 종류, 그리고 상호 작용의 시각화를위한 형광 조직 이식에 따라 정량 멀티 스펙트럼 분석

쥐의 대동맥 부비동의 동맥 경화 병변의 정량 분석​​ 및 특성

구현

적응 저항 훈련은 다발성 경화증을 가진 개인에 여덟 주에서 강도를 향상

Anastasis에서 Vivo에서 CaspaseTracker 바이오 센서에 의해 감지

파괴 강도 시험 및 정량 CT 기반의 유한 요소 분석을위한 근위 대퇴골 사체 준비

개정 공동 교체에 새로운 진단: 쥡니다 및 다중 연쇄 반응 임 플 란 트

쥐의 대동맥 부비동의 동맥 경화 병변의 정량 분석 및 특성