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Biological Procedures Online

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https://www.readbyqxmd.com/read/29434528/antitumor-efficacy-of-human-monocyte-derived-dendritic-cells-comparing-effects-of-two-monocyte-isolation-methods
#1
Graça S Marques, Zélia Silva, Paula A Videira
Background: Dendritic cells (DCs), which can be used as anti-cancer vaccines, are generally obtained in vitro from isolated CD14+ monocytes (MoDCs). This generates high cell numbers and allows instructing DCs to guarantee effective antitumor responses. However, the impact of the monocyte isolation step in the antitumor effectiveness of the generated MoDCs is still unknown. Here, we compared the most used immunomagnetic technologies for monocyte isolation: magnetic activated cell sorting (MACS) from Miltenyi Biotec and EasySep from STEM CELL...
2018: Biological Procedures Online
https://www.readbyqxmd.com/read/29434527/visualisation-of-multiple-tight-junctional-complexes-in-human-airway-epithelial-cells
#2
Alysia G Buckley, Kevin Looi, Thomas Iosifidis, Kak-Ming Ling, Erika N Sutanto, Kelly M Martinovich, Elizabeth Kicic-Starcevich, Luke W Garratt, Nicole C Shaw, Francis J Lannigan, Alexander N Larcombe, Graeme Zosky, Darryl A Knight, Paul J Rigby, Anthony Kicic, Stephen M Stick
Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this...
2018: Biological Procedures Online
https://www.readbyqxmd.com/read/29375275/gibson-deletion-a-novel-application-of-isothermal-in-vitro-recombination
#3
Swara Kalva, Jef D Boeke, Paolo Mita
Background: Recombinant DNA technology is today a fundamental tool for virtually all biological research fields. Among the many techniques available for the construction of a "custom DNA" molecule, the isothermal in vitro assembly, or Gibson assembly, allows for an efficient, one-step, scarless recombination-based assembly. Results: Here, we apply and characterize the use of Gibson assembly for the deletion of DNA sequences around a DNA cut. This method, that we named "Gibson Deletion", can be used to easily substitute or delete one or more restriction sites within a DNA molecule...
2018: Biological Procedures Online
https://www.readbyqxmd.com/read/29321719/cancer-chemoprevention-and-therapy-using-chinese-herbal-medicine
#4
REVIEW
Lijing Jiao, Ling Bi, Yan Lu, Qin Wang, Yabin Gong, Jun Shi, Ling Xu
Traditional Chinese medicine (TCM) plays an indispensable role in cancer prevention and treatment. Chinese herbal medicine (CHM) is a key component of TCM and has been practiced for thousands of years. A number of naturally occurring products from Chinese herbs extracts exhibit strong inhibitory properties against carcinogenesis, including CHM single-herb extracts, CHM-derived active components, and CHM formulas (the polyherbal combinations), which regulate JAK/STAT, MAPK, and NF-ҡB pathways. The present review aims to report the cancer-preventive effect of CHM with evidence from cell-line, animal, epidemiological, and clinical experiments...
2018: Biological Procedures Online
https://www.readbyqxmd.com/read/29255379/maintaining-rna-integrity-for-transcriptomic-profiling-of-ex-vivo-cultured-limbal-epithelial-stem-cells-after-fluorescence-activated-cell-sorting-facs
#5
Lei Liu, Frederik Mølgaard Nielsen, Simone Elkjær Riis, Jeppe Emmersen, Trine Fink, Jesper Østergaard Hjortdal, Chris Bath, Vladimir Zachar
Background: Transcriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS)...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/29151819/production-of-monoclonal-antibodies-against-human-trefoil-factor-3-and-development-of-a-modified-sandwich-elisa-for-detection-of-trefoil-factor-3-homodimer-in-saliva
#6
Saichit Khummuang, Waraporn Phanphrom, Witida Laopajon, Watchara Kasinrerk, Ponlatham Chaiyarit, Supansa Pata
Background: Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/29075153/sv40-large-t-antigen-disrupts-embryogenesis-of-canine-and-porcine-somatic-cell-nuclear-transfer-embryo
#7
Kiyoung Eun, Seon-Ung Hwang, Yeon Woo Jeong, Sunyoung Seo, Seon Yong Lee, Woo Suk Hwang, Sang-Hwan Hyun, Hyunggee Kim
BACKGROUND: Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28959142/enhanced-mutant-screening-in-one-step-pcr-based-multiple-site-directed-plasmid-mutagenesis-by-introduction-of-silent-restriction-sites-for-structural-and-functional-study-of-proteins
#8
Ting-Yu Kuo, Chung-Che Tsai, Hua-Wen Fu
Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3' end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28932173/efficient-and-inexpensive-transient-expression-of-multispecific-multivalent-antibodies-in-expi293-cells
#9
Xiaotian T Fang, Dag Sehlin, Lars Lannfelt, Stina Syvänen, Greta Hultqvist
BACKGROUND: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.. METHODS: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28855851/an-assessment-of-fixed-and-native-chromatin-preparation-methods-to-study-histone-post-translational-modifications-at-a-whole-genome-scale-in-skeletal-muscle-tissue
#10
Sarah-Anne David, Benoît Piégu, Christelle Hennequet-Antier, Maëlle Pannetier, Tiphaine Aguirre-Lavin, Sabine Crochet, Thierry Bordeau, Nathalie Couroussé, Aurélien Brionne, Yves Bigot, Anne Collin, Vincent Coustham
BACKGROUND: Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28814944/cell-counting-and-viability-assessment-of-2d-and-3d-cell-cultures-expected-reliability-of-the-trypan-blue-assay
#11
Filippo Piccinini, Anna Tesei, Chiara Arienti, Alessandro Bevilacqua
BACKGROUND: Whatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28785173/an-initial-investigation-of-an-alternative-model-to-study-rat-primordial-germ-cell-epigenetic-reprogramming
#12
Isabelle Hernandez Cantão, Renato Borges Tesser, Taiza Stumpp
BACKGROUND: Primordial germ cells (PGC) are the precursors of the gametes. During pre-natal development, PGC undergo an epigenetic reprogramming when bulk DNA demethylation occurs and is followed by sex-specific de novo methylation. The de novo methylation and the maintenance of the methylation patterns depend on DNA methyltransferases (DNMTs). PGC reprogramming has been widely studied in mice but not in rats. We have previously shown that the rat might be an interesting model to study germ cell development...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28690429/3d-printer-generated-tissue-imolds-for-cleared-tissue-using-single-and-multi-photon-microscopy-for-deep-tissue-evaluation
#13
Sean J Miller, Jeffrey D Rothstein
BACKGROUND: Pathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28674481/pre-microporation-improves-outcome-of-pancreatic-islet-labelling-for-optical-and-19-f-mr-imaging
#14
Vít Herynek, Andrea Gálisová, Mangala Srinivas, Eric A W van Dinther, Lucie Kosinová, Jiri Ruzicka, Markéta Jirátová, Jan Kriz, Daniel Jirák
BACKGROUND: In vitro labelling of cells and small cell structures is a necessary step before in vivo monitoring of grafts. We modified and optimised a procedure for pancreatic islet labelling using bimodal positively charged poly(lactic-co-glycolic acid) nanoparticles with encapsulated perfluoro crown ethers and indocyanine green dye via microporation and compared the method with passive endocytosis. RESULTS: Pancreatic islets were microporated using two pulses at various voltages...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28596713/performances-of-different-fragment-sizes-for-reduced-representation-bisulfite-sequencing-in-pigs
#15
Xiao-Long Yuan, Zhe Zhang, Rong-Yang Pan, Ning Gao, Xi Deng, Bin Li, Hao Zhang, Per Torp Sangild, Jia-Qi Li
BACKGROUND: Reduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome. RESULTS: We found that the MspI-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28490975/comparison-of-transepithelial-resistance-measurement-techniques-chopsticks-vs-endohm
#16
Rebecca A Sheller, Maria E Cuevas, Maria C Todd
BACKGROUND: TER measurements across confluent cellular monolayers provide a useful indication of TJ strength between epithelial and endothelial cells in culture. Having a reliable and accurate method of measuring cell-to-cell adhesion is critical to studies in pathophysiology and cancer metastasis. However, the use of different technical approaches to measure TER has reportedly yielded inconsistent measurements within the same cell lines. METHODS: In the current study, we compared the peak TER values for the MDCK (canine kidney) and MCF-7 (human breast cancer) epithelial cell lines using two common approaches (Chopstick and Endohm) and two types of polymer inserts (PC and PET)...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28465674/lncrnas-and-immunity-watchdogs-for-host-pathogen-interactions
#17
REVIEW
Peerzada Tajamul Mumtaz, Shakil Ahmad Bhat, Syed Mudasir Ahmad, Mashooq Ahmad Dar, Raashid Ahmed, Uneeb Urwat, Aadil Ayaz, Divya Shrivastava, Riaz Ahmad Shah, Nazir Ahmad Ganai
Immune responses combat various infectious agents by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. The polygenic responses to these external stimuli are temporally and coordinately regulated. Specific lncRNAs are induced to modulate innate and adaptive immune responses which can function through various target interactions like RNA-DNA, RNA-RNA, and RNA-protein interaction and hence affect the immunogenic regulation at various stages of gene expression. LncRNA are found to be present in various immune cells like monocytes, macrophages, dendritic cells, neutrophils, T cells and B cells...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28331435/methylated-dna-rna-in-body-fluids-as-biomarkers-for-lung-cancer
#18
REVIEW
Yan Lu, Shulin/Sl Li, Shiguo/Sg Zhu, Yabin/Yb Gong, Jun/J Shi, Ling/L Xu
DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/28100961/auxin-extraction-and-purification-based-on-recombinant-aux-iaa-proteins
#19
Yi Su, Weigui Luo, Xiaofei Chen, Huizhen Liu, Yueqing Hu, Wanhuang Lin, Langtao Xiao
BACKGROUND: Indole-3-acetic acid (IAA) extraction and purification are of great importance in auxin research, which is a hot topic in the plant growth and development field. Solid-phase extraction (SPE) is frequently used for IAA extraction and purification. However, no IAA-specific SPE columns are commercially available at the moment. Therefore, the development of IAA-specific recognition materials and IAA extraction and purification methods will help researchers meet the need for more precise analytical methods for research on phytohormones...
2017: Biological Procedures Online
https://www.readbyqxmd.com/read/27895534/an-automated-perifusion-system-for-modifying-cell-culture-conditions-over-time
#20
Nicholas B Whitticar, Elisha W Strahler, Parthiban Rajan, Savas Kaya, Craig S Nunemaker
BACKGROUND: Cells are continuously exposed to changes in their environment. Endocrine systems, in particular, communicate by rhythms and feedback loops. In this study, we developed an automated system to produce such conditions for cultured cells in a precisely timed manner. We utilized a programmable pair of syringe pumps for inflow and a peristaltic pump for outflow to create rhythmic pulses at 5-min intervals in solutions that mimic the endogenous patterns of insulin produced by pancreatic islets as a test case...
2016: Biological Procedures Online
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