Biological Procedures Online最新文献

{"title":"Combination of Size-Exclusion Chromatography and Ultracentrifugation Improves the Proteomic Profiling of Plasma-Derived Small Extracellular Vesicles.","authors":"Rui Wei,&nbsp;Libo Zhao,&nbsp;Guanyi Kong,&nbsp;Xiang Liu,&nbsp;Shengtao Zhu,&nbsp;Shutian Zhang,&nbsp;Li Min","doi":"10.1186/s12575-020-00125-5","DOIUrl":"https://doi.org/10.1186/s12575-020-00125-5","url":null,"abstract":"<p><strong>Background: </strong>Circulating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins and plasma proteins. Previous studies suggested combinations of different sEVs isolation methods could improve the yield and purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC), and their combination in a proteomic perspective.</p><p><strong>Results: </strong>Plasma samples were collected from healthy individuals, and sEVs were separated by one-step SEC, one-step UC, and combining SEC with UC, respectively. Here we exhibited that the purity of sEVs was improved by SEC in contrast to traditional UC. Furthermore, by conducting a SEC procedure followed by UC, we separated sEVs with the highest purity. In the proteomic analysis, 992 protein species were identified in the plasma sEVs isolated by our novel separation method, of which several proteins are sEVs-associated proteins but hitherto never been identified in the previous studies and database, much more than plasma sEVs isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, plasma sEVs isolated by the new procedure kept 584 previously identified sEVs-associated proteins and 360 other proteins that have not been detected before. Detailed analysis suggested that more kinds of sEVs biomarkers, such as CD9, ALIX, and FLOT1, could be identified in plasma sEVs isolated by the novel isolation method as compared to one-step UC/SEC. Furthermore, the lower abundance ranks of common contaminants, such as lipoproteins and IgG chains, in the sEVs fractions obtained by our new method as compared to one-step UC/SEC also demonstrated the purity of sEVs had been improved.</p><p><strong>Conclusions: </strong>Combining SEC with UC could significantly improve the performance of mass spectrometry-based proteomic profiling in analyzing plasma-derived sEVs.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"12"},"PeriodicalIF":6.4,"publicationDate":"2020-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00125-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38092057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of System Biology to Explore the Association of Neprilysin, Angiotensin-Converting Enzyme 2 (ACE2), and Carbonic Anhydrase (CA) in Pathogenesis of SARS-CoV-2.","authors":"Reza Zolfaghari Emameh, Reza Falak, Elham Bahreini","doi":"10.1186/s12575-020-00124-6","DOIUrl":"10.1186/s12575-020-00124-6","url":null,"abstract":"<p><strong>Background: </strong>Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears with common symptoms including fever, dry cough, and fatigue, as well as some less common sysmptoms such as loss of taste and smell, diarrhea, skin rashes and discoloration of fingers. COVID-19 patients may also suffer from serious symptoms including shortness of breathing, chest pressure and pain, as well as loss of daily routine habits, pointing out to a sever reduction in the quality of life. COVID-19 has afftected almost all countries, however, the United States contains the highest number of infection (> 1,595,000 cases) and deaths cases (> 95,000 deaths) in the world until May 21, 2020. Finding an influential treatment strategy against COVID-19 can be facilitated through better understanding of the virus pathogenesis and consequently interrupting the biochemical pathways that the virus may play role in human body as the current reservoir of the virus.</p><p><strong>Results: </strong>In this study, we combined system biology and bioinformatic approaches to define the role of coexpression of angiotensin-converting enzyme 2 (ACE2), neprilysin or membrane metallo-endopeptidase (MME), and carbonic anhydrases (CAs) and their association in the pathogenesis of SARS-CoV-2. The results revealed that ACE2 as the cellular attachment site of SARS-CoV-2, neprilysin, and CAs have a great contribution together in the renin angiotensin system (RAS) and consequently in pathogenesis of SARS-CoV-2 in the vital organs such as respiratory, renal, and blood circulation systems. Any disorder in neprilysin, ACE2, and CAs can lead to increase of CO₂ concentration in blood and respiratory acidosis, induction of pulmonary edema and heart and renal failures.</p><p><strong>Conclusions: </strong>Due to the presence of ACE2-Neprilysin-CA complex in most of vital organs and as a receptor of COVID-19, it is expected that most organs are affected by SARS-CoV-2 such as inflammation and fibrosis of lungs, which may conversely affect their vital functions, temporary or permanently, sometimes leading to death. Therefore, ACE2-Neprilysin-CA complex could be the key factor of pathogenesis of SARS-CoV-2 and may provide us useful information to find better provocative and therapeutic strategies against COVID-19.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"11"},"PeriodicalIF":3.7,"publicationDate":"2020-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302923/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38073699","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deep Insights in Circular RNAs: from biogenesis to therapeutics.","authors":"Peerzada Tajamul Mumtaz,&nbsp;Qamar Taban,&nbsp;Mashooq Ahmad Dar,&nbsp;Shabir Mir,&nbsp;Zulfkar Ul Haq,&nbsp;Sajad Majeed Zargar,&nbsp;Riaz Ahmad Shah,&nbsp;Syed Mudasir Ahmad","doi":"10.1186/s12575-020-00122-8","DOIUrl":"https://doi.org/10.1186/s12575-020-00122-8","url":null,"abstract":"<p><strong>Abstract: </strong>Circular RNAs (circRNAs) have emerged as a universal novel class of eukaryotic non-coding RNA (ncRNA) molecules and are becoming a new research hotspot in RNA biology. They form a covalent loop without 5' cap and 3' tail, unlike their linear counterparts. Endogenous circRNAs in mammalian cells are abundantly conserved and discovered so far. In the biogenesis of circRNAs exonic, intronic, reverse complementary sequences or RNA-binding proteins (RBPs) play a very important role. Interestingly, the majority of them are highly conserved, stable, resistant to RNase R and show developmental-stage/tissue-specific expression. CircRNAs play multifunctional roles as microRNA (miRNA) sponges, regulators of transcription and post-transcription, parental gene expression and translation of proteins in various diseased conditions. Growing evidence shows that circRNAs play an important role in neurological disorders, atherosclerotic vascular disease, and cancer and potentially serve as diagnostic or predictive biomarkers due to its abundance in various biological samples. Here, we review the biogenesis, properties, functions, and impact of circRNAs on various diseases.</p><p><strong>Graphical abstract: </strong></p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"10"},"PeriodicalIF":6.4,"publicationDate":"2020-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00122-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37986547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reverse Genetics Assembly of Newcastle Disease Virus Genome Template Using Asis-Sal-Pac BioBrick Strategy.","authors":"Amin Tavassoli,&nbsp;Safoura Soleymani,&nbsp;Alireza Haghparast,&nbsp;Gholamreza Hashemi Tabar,&nbsp;Mohammad Reza Bassami,&nbsp;Hesam Dehghani","doi":"10.1186/s12575-020-00119-3","DOIUrl":"https://doi.org/10.1186/s12575-020-00119-3","url":null,"abstract":"<p><strong>Background: </strong>The BioBrick construction as an approach in synthetic biology provides the ability to assemble various gene fragments. To date, different BioBrick strategies have been exploited for assembly and cloning of a variety of gene fragments. We present a new BioBrick strategy, here referred as Asis-Sal-Pac BioBrick, which we used for the assembly of NDV as a candidate for single-stranded non-segmented, negative-sense RNA genome viruses.</p><p><strong>Results: </strong>In the present study, we isolated three NDVs from clinical samples which were classified into the VIId genotype based on their pathogenicity and phylogenetic analyses. Then, SalI, AsisI, and PacI enzymes were used to design and develop a novel BioBrick strategy, which enabled us to assemble the NDV genome, adopting the \"rule of six\". In this method, in each assembly step, the restriction sites in the newly formed destination plasmid are reproduced, which will be used for the next insertion. In this study using two overlapping PCRs, the cleavage site of the F gene was also modified from ¹¹²RRQKRF¹¹⁷to ¹¹²GRQGRL¹¹⁷ in order to generate the attenuated recombinant NDV. Finally, in order to construct the recombinant NDV viruses, the plasmids harboring the assembled full-length genome of the NDV and the helper plasmids were co-transfected into T7-BHK cells. The rescue of the recombinant NDVwas confirmed by RT-PCR and HA tests.</p><p><strong>Conclusions: </strong>These findings suggest that the combination of reverse genetic technology and BioBrick assembly have the potential to be applied for the development of novel vaccine candidates. This promising strategy provides an effective and reliable approach to make genotype-matched vaccines against specific NDV strains or any other virus.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"9"},"PeriodicalIF":6.4,"publicationDate":"2020-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00119-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37908119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combination of Biodata Mining and Computational Modelling in Identification and Characterization of ORF1ab Polyprotein of SARS-CoV-2 Isolated from Oronasopharynx of an Iranian Patient.","authors":"Reza Zolfaghari Emameh,&nbsp;Hassan Nosrati,&nbsp;Ramezan Ali Taheri","doi":"10.1186/s12575-020-00121-9","DOIUrl":"https://doi.org/10.1186/s12575-020-00121-9","url":null,"abstract":"<p><strong>Background: </strong>Coronavirus disease 2019 (COVID-19) is an emerging zoonotic viral infection, which was started in Wuhan, China, in December 2019 and transmitted to other countries worldwide as a pandemic outbreak. Iran is one of the top ranked countries in the tables of COVID-19-infected and -mortality cases that make the Iranian patients as the potential targets for diversity of studies including epidemiology, biomedical, biodata, and viral proteins computational modelling studies.</p><p><strong>Results: </strong>In this study, we applied bioinformatic biodata mining methods to detect CDS and protein sequences of ORF1ab polyprotein of SARS-CoV-2 isolated from oronasopharynx of an Iranian patient. Then through the computational modelling and antigenicity prediction approaches, the identified polyprotein sequence was analyzed. The results revealed that the identified ORF1ab polyprotein belongs to a part of nonstructural protein 1 (nsp1) with the high antigenicity residues in a glycine-proline or hydrophobic amino acid rich domain.</p><p><strong>Conclusions: </strong>The results revealed that nsp1 as a virulence factor and crucial agent in spreading of the COVID-19 among the society can be a potential target for the future epidemiology, drug, and vaccine studies.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"8"},"PeriodicalIF":6.4,"publicationDate":"2020-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00121-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37874385","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations.","authors":"Verena Hiebl,&nbsp;Daniel Schachner,&nbsp;Angela Ladurner,&nbsp;Elke H Heiss,&nbsp;Herbert Stangl,&nbsp;Verena M Dirsch","doi":"10.1186/s12575-020-00120-w","DOIUrl":"https://doi.org/10.1186/s12575-020-00120-w","url":null,"abstract":"<p><strong>Background: </strong>The human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. As the intestine is a major interface in cholesterol turnover and represents a non-biliary pathway for cholesterol excretion, Caco-2 cells are also a valuable model for studying cholesterol homeostasis, including cholesterol uptake and efflux. Currently available protocols are, however, either sketchy or not consistent among different laboratories. Our aim was therefore to generate a collection of optimized protocols, considering the different approaches of the different laboratories and to highlight possibilities and limitations of measuring cholesterol transport with this cell line.</p><p><strong>Results: </strong>We developed comprehensive and quality-controlled protocols for the cultivation of Caco-2 cells on filter inserts in a single tight monolayer. A cholesterol uptake as well as a cholesterol efflux assay is described in detail, including suitable positive controls. We further show that Caco-2 cells can be efficiently transfected for luciferase reporter gene assays in order to determine nuclear receptor activation, main transcriptional regulators of cholesterol transporters (ABCA1, ABCB1, ABCG5/8, NPC1L1). Detection of protein and mRNA levels of cholesterol transporters in cells grown on filter inserts can pose challenges for which we highlight essential steps and alternative approaches for consideration. A protocol for viability assays with cells differentiated on filter inserts is provided for the first time.</p><p><strong>Conclusions: </strong>The Caco-2 cell line is widely used in the scientific community as model for the intestinal epithelium, although with highly divergent protocols. The herein provided information and protocols can be a common basis for researchers intending to use Caco-2 cells in the context of cellular cholesterol homeostasis.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"7"},"PeriodicalIF":6.4,"publicationDate":"2020-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00120-w","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37849570","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.","authors":"Niina Aaltonen,&nbsp;Prosanta K Singha,&nbsp;Hermina Jakupović,&nbsp;Thomas Wirth,&nbsp;Haritha Samaranayake,&nbsp;Sanna Pasonen-Seppänen,&nbsp;Kirsi Rilla,&nbsp;Markku Varjosalo,&nbsp;Laura E Edgington-Mitchell,&nbsp;Paulina Kasperkiewicz,&nbsp;Marcin Drag,&nbsp;Sara Kälvälä,&nbsp;Eemeli Moisio,&nbsp;Juha R Savinainen,&nbsp;Jarmo T Laitinen","doi":"10.1186/s12575-020-00118-4","DOIUrl":"https://doi.org/10.1186/s12575-020-00118-4","url":null,"abstract":"<p><strong>Background: </strong>Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes.</p><p><strong>Results: </strong>Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification.</p><p><strong>Conclusions: </strong>Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"6"},"PeriodicalIF":6.4,"publicationDate":"2020-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00118-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37753219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Adjuvant Chemotherapy with Chinese Herbal Medicine Formulas Versus Placebo in Patients with Lung Adenocarcinoma after Radical Surgery: a Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.","authors":"Qin Wang,&nbsp;Lijing Jiao,&nbsp;Shengfei Wang,&nbsp;Peiqi Chen,&nbsp;Ling Bi,&nbsp;Di Zhou,&nbsp;Jialin Yao,&nbsp;Jiaqi Li,&nbsp;Liyu Wang,&nbsp;Zhiwei Chen,&nbsp;Yingjie Jia,&nbsp;Ziwen Zhang,&nbsp;Weisheng Shen,&nbsp;Weirong Zhu,&nbsp;Jianfang Xu,&nbsp;Yong Gao,&nbsp;Ling Xu,&nbsp;Yabin Gong","doi":"10.1186/s12575-020-00117-5","DOIUrl":"https://doi.org/10.1186/s12575-020-00117-5","url":null,"abstract":"<p><strong>Background: </strong>The toxicity and side effects caused by adjuvant chemotherapy (ACT) after radical surgery for lung adenocarcinoma (LAC) lead to early termination frequently. This study was conducted to provide an objective basis for the effect of Chinese herbal medicine formulas (CHMFs) combined with chemotherapy in reducing toxicity and enhancing efficacy of ACT.</p><p><strong>Method: </strong>From February 17th, 2012 to March 20th, 2015, 233 patients from 7 hospitals diagnosed with LAC in IB~IIIA stage were randomly assigned into ACT + CHMF group (116 patients) and ACT + placebo group (117 patients). CHMF was taken orally until the end of chemotherapy. Chemotherapy-related toxic, side effects were investigated as the primary outcome. Disease-free survival (DFS) and overall survival (OS) were used as the secondary outcome.</p><p><strong>Results: </strong>At one week following chemotherapy, the incidence of dry mouth, diarrhea and thrombocytopenia significantly decreased in CHMF group (<i>P</i> = 0.017, <i>P</i> = 0.033, <i>P</i> = 0.019, respectively). At two weeks following chemotherapy, fatigue and diarrhea were more obvious in the placebo group (<i>P</i> = 0.028, <i>P</i> = 0.025, respectively). In addition, patients in the CHMF group showed an increase in median DFS from 37.1 to 51.5 months compared with placebo group although there was no statistical significance (<i>P</i> = 0.16). In the stage IB subgroup, the CHMF group had a significantly better DFS (HR (95% CI) = 0.53 (0.28-0.99), <i>P</i> = 0.046). There was no significant difference in OS between the groups (<i>P</i> = 0.72).</p><p><strong>Conclusion: </strong>For patients with LAC, ACT combined with CHMF after radical surgery can prolong the DFS time especially in the early stage, and reduces the chemotherapy-related toxic and side effects.</p><p><strong>Trial registration: </strong>NCT01441752. Registered 14 July, 2011.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"5"},"PeriodicalIF":6.4,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00117-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37710507","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploring G Protein-Coupled Receptor Signaling in Primary Pancreatic Islets.","authors":"Juliane Röthe,&nbsp;Robert Kraft,&nbsp;Torsten Schöneberg,&nbsp;Doreen Thor","doi":"10.1186/s12575-019-0116-y","DOIUrl":"https://doi.org/10.1186/s12575-019-0116-y","url":null,"abstract":"<p><strong>Background: </strong>Targeting G protein-coupled receptors (GPCRs) in pancreatic cells is feasible to modulate glucose-induced insulin secretion. Because pancreatic islets consist of several cell types and GPCRs can couple to more than one G-protein family, results obtained in pancreatic cell lines do not always match the response in primary cells or intact islets. Therefore, we set out to establish a protocol to analyze second messenger activation in mouse pancreatic islets.</p><p><strong>Results: </strong>Activation of Gq/11-coupled receptor expressed in primary β cells increased the second messenger IP1 in an accumulation assay. Applying a Gq/11 protein inhibitor completely abolished this signal. Activation of the V1 vasopressin and ghrelin receptors, predominantly expressed in the less abundant alpha and delta cells, was not sufficient to induce a significant IP1 increase in this assay. However, fura-2-based fluorescence imaging showed calcium signals upon application of arginine vasopressin or ghrelin within intact pancreatic islets. Using the here established protocol we were also able to determine changes in intracellular cAMP levels induced by receptors coupling to Gs and Gi/o proteins.</p><p><strong>Conclusions: </strong>Detection of the second messengers IP1, cAMP, and calcium, can be used to reliably analyze GPCR activation in intact islets.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"4"},"PeriodicalIF":6.4,"publicationDate":"2020-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-019-0116-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37664382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A microcarrier-based spheroid 3D invasion assay to monitor dynamic cell movement in extracellular matrix.","authors":"Hui Liu,&nbsp;Tao Lu,&nbsp;Gert-Jan Kremers,&nbsp;Ann L B Seynhaeve,&nbsp;Timo L M Ten Hagen","doi":"10.1186/s12575-019-0114-0","DOIUrl":"https://doi.org/10.1186/s12575-019-0114-0","url":null,"abstract":"<p><strong>Background: </strong>Cell invasion through extracellular matrix (ECM) is a critical step in tumor metastasis. To study cell invasion in vitro, the internal microenvironment can be simulated via the application of 3D models.</p><p><strong>Results: </strong>This study presents a method for 3D invasion examination using microcarrier-based spheroids. Cell invasiveness can be evaluated by quantifying cell dispersion in matrices or tracking cell movement through time-lapse imaging. It allows measuring of cell invasion and monitoring of dynamic cell behavior in three dimensions. Here we show different invasive capacities of several cell types using this method. The content and concentration of matrices can influence cell invasion, which should be optimized before large scale experiments. We also introduce further analysis methods of this 3D invasion assay, including manual measurements and homemade semi-automatic quantification. Finally, our results indicate that the position of spheroids in a matrix has a strong impact on cell moving paths, which may be easily overlooked by researchers and may generate false invasion results.</p><p><strong>Conclusions: </strong>In all, the microcarrier-based spheroids 3D model allows exploration of adherent cell invasion in a fast and highly reproducible way, and provides informative results on dynamic cell behavior in vitro.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"3"},"PeriodicalIF":6.4,"publicationDate":"2020-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-019-0114-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37612030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}