<ol>
	<li>Zhang, Y. Q., Zhang, M., Wang, Z. L., Qiao, X., Ye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+plant-derived+C-glycosides:+phytochemistry,+bioactivities,+and+biotechnological+production.">Advances in plant-derived C-glycosides: phytochemistry, bioactivities, and biotechnological production.</a> Biotechnol Adv. 60, 108030 (2022).</li><li>Franz, G., Grun, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry,+occurrence+and+biosynthesis+of+C-glycosyl+compounds+in+plants.">Chemistry, occurrence and biosynthesis of C-glycosyl compounds in plants.</a> Planta Med. 47 (3), 131-140 (1983).</li><li>Cai, 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=Antiviral+flavonoid-type+C-glycosides+from+the+flowers+of+Trollius+chinensis.">Antiviral flavonoid-type C-glycosides from the flowers of Trollius chinensis.</a> Chem Biodivers. 3 (3), 343-348 (2006).</li><li>Shi, 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=A+flavone+C-glycoside+from+Styela+plicata.">A flavone C-glycoside from Styela plicata.</a> Biochem Syst Ecol. 86, 103924 (2019).</li><li>Xiao, J., Capanoglu, E., Jassbi, A. R., Miron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advance+on+the+flavonoid+C-glycosides+and+health+benefits.">Advance on the flavonoid C-glycosides and health benefits.</a> Crit Rev Food Sci Nutr. 56 (Suppl 1), S29-S45 (2016).</li><li>Ma, R., Cheng, S., Sun, J., Zhu, W., Fu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+gilvocarcin-type+aryl-C-glycosides+from+a+soil-derived+Streptomyces+species.">Antibacterial gilvocarcin-type aryl-C-glycosides from a soil-derived Streptomyces species.</a> J Nat Prod. 85 (10), 2282-2289 (2022).</li><li>Le Dang, Q., 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=Desmodinosides+A-E:+New+flavonoid+C-glycosides+from+Desmodium+heterocarpon+var.+stigosum+with+hepatoprotective+and+antifungal+activity.">Desmodinosides A-E: New flavonoid C-glycosides from Desmodium heterocarpon var. stigosum with hepatoprotective and antifungal activity.</a> Fitoterapia. 169, 105609 (2023).</li><li>Choi, J. 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=Effects+of+C-glycosylation+on+antidiabetic,+anti-Alzheimers+disease+and+anti-inflammatory+potential+of+apigenin.">Effects of C-glycosylation on antidiabetic, anti-Alzheimers disease and anti-inflammatory potential of apigenin.</a> Food Chem Toxicol. 64, 27-33 (2014).</li><li>Courts, F. L., Williamson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence,+fate+and+biological+activities+of+C-glycosyl+flavonoids+in+the+human+diet.">The occurrence, fate and biological activities of C-glycosyl flavonoids in the human diet.</a> Crit Rev Food Sci Nutr. 55 (10), 1352-1367 (2015).</li><li>Thao, N. 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=Anti-inflammatory+flavonoid+C-glycosides+from+Piper+aduncum+leaves.">Anti-inflammatory flavonoid C-glycosides from Piper aduncum leaves.</a> Planta Med. 82 (17), 1475-1481 (2016).</li><li>Kim, M. K., Yun, K. J., Lim, D. H., Kim, J., Jang, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+properties+of+flavone+di-C-glycosides+as+active+principles+of+camellia+mistletoe,+Korthalsella+japonica.">Anti-inflammatory properties of flavone di-C-glycosides as active principles of camellia mistletoe, Korthalsella japonica.</a> Biomol Ther (Seoul). 24 (6), 630-637 (2016).</li><li>Chen, Y. L., Chen, C. Y., Lai, K. H., Chang, Y. C., Hwang, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+and+antiviral+activities+of+flavone+C-glycosides+of+Lophatherum+gracile+for+COVID-19.">Anti-inflammatory and antiviral activities of flavone C-glycosides of Lophatherum gracile for COVID-19.</a> J Funct Foods. 101, 105407 (2023).</li><li>Wang, 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=Flavone+C-glycosides+from+the+leaves+of+Lophatherum+gracile+and+their+in+vitro+antiviral+activity.">Flavone C-glycosides from the leaves of Lophatherum gracile and their in vitro antiviral activity.</a> Planta Med. 78 (1), 46-51 (2012).</li><li>Devkota, H. P., Fukusako, K., Ishiguro, K., Yahara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavone+C-glycosides+from+Lychnis+senno+and+their+antioxidative+activity.">Flavone C-glycosides from Lychnis senno and their antioxidative activity.</a> Nat Prod Commun. 8 (10), 1413-1414 (2013).</li><li>Stark, T. 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=Isolation+and+structure+elucidation+of+highly+antioxidative+3,8-linked+biflavanones+and+flavanone-C-glycosides+from+Garcinia+buchananii+bark.">Isolation and structure elucidation of highly antioxidative 3,8-linked biflavanones and flavanone-C-glycosides from Garcinia buchananii bark.</a> J Agric Food Chem. 60 (8), 2053-2062 (2012).</li><li>Stark, T. D., Germann, D., Balemba, O. B., Wakamatsu, J., Hofmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+highly+in+vitro+antioxidative+3,8-linked+biflav(an)ones+and+flavanone-C-glycosides+from+Garcinia+buchananii+stem+bark.">New highly in vitro antioxidative 3,8-linked biflav(an)ones and flavanone-C-glycosides from Garcinia buchananii stem bark.</a> J Agric Food Chem. 61 (51), 12572-12581 (2013).</li><li>Xiao, J., Capanoglu, E., Jassbi, A. R., Miron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advance+on+the+flavonoid+C-glycosides+and+health+benefits.">Advance on the flavonoid C-glycosides and health benefits.</a> Crit Rev Food Sci Nutr. 56 (Suppl 1), 29-45 (2016).</li><li>Xie, K., Zhang, X., Sui, S., Ye, F., Dai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+and+applying+the+substrate+promiscuity+of+a+C-glycosyltransferase+in+the+chemo-enzymatic+synthesis+of+bioactive+C-glycosides.">Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides.</a> Nat Commun. 11 (1), 5162 (2020).</li><li>Liu, 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=Characterization+of+the+intestinal+absorption+of+seven+flavonoids+from+the+flowers+of+Trollius+chinensis+using+the+Caco-2+cell+monolayer+model.">Characterization of the intestinal absorption of seven flavonoids from the flowers of Trollius chinensis using the Caco-2 cell monolayer model.</a> PLoS One. 10 (3), e0119263 (2015).</li><li>Xu, 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=Mechanism+and+application+of+microbial+transformation+of+glycosides+in+traditional+Chinese+medicine.">Mechanism and application of microbial transformation of glycosides in traditional Chinese medicine.</a> World Sci Technol. 2, 24-27 (2006).</li><li>Xie, 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=Comparison+of+flavonoid+O-glycoside,+C-glycoside+and+their+aglycones+on+antioxidant+capacity+and+metabolism+during+in+vitro+digestion+and+in+vivo.">Comparison of flavonoid O-glycoside, C-glycoside and their aglycones on antioxidant capacity and metabolism during in vitro digestion and in vivo.</a> Foods. 11 (6), 882 (2022).</li><li>Hostetler, G. L., Ralston, R. A., Schwartz, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavones:+Food+sources,+bioavailability,+metabolism,+and+bioactivity.">Flavones: Food sources, bioavailability, metabolism, and bioactivity.</a> Adv Nutr. 8 (3), 423-435 (2017).</li><li>Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+flavonoid+aglycones+and+their+glycosides:+which+show+better+biological+significance.">Dietary flavonoid aglycones and their glycosides: which show better biological significance.</a> Crit Rev Food Sci Nutr. 57 (9), 1874-1905 (2017).</li><li>Hasanah, U., Miki, K., Nitoda, T., Kanzaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerobic+bioconversion+of+C-glycoside+mangiferin+into+its+aglycone+norathyriol+by+an+isolated+mouse+intestinal+bacterium.">Aerobic bioconversion of C-glycoside mangiferin into its aglycone norathyriol by an isolated mouse intestinal bacterium.</a> Biosci Biotechnol Biochem. 85 (4), 989-997 (2021).</li><li>Yang, X., Xu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+model+and+standard+operation+procedure+for+biotransformation+of+chemical+constituents+of+traditional+Chinese+medicine+by+human+intestinal+bacteria.">Establishment of model and standard operation procedure for biotransformation of chemical constituents of traditional Chinese medicine by human intestinal bacteria.</a> China J Chin Mater Med. 36 (1), 19-26 (2011).</li></ol>

<table><tbody><tr><td>Acetonitrile</td><td>Thermo Fisher Scientific</td><td>F2408R205</td><td>HPLC</td></tr><tr><td>Anaerobic Incubator</td><td>Shanghai CIMO Medical Instrument Manufacturing Co., LTD</td><td></td><td>YQX- II</td></tr><tr><td>Beef Extract</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-009</td><td>BR</td></tr><tr><td>Digestive Serum Powder</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-087</td><td>BR</td></tr><tr><td>Dipotassium Hydrogen Phosphate</td><td>Beijing Chemical Works</td><td>M26298</td><td>AR</td></tr><tr><td>Disposable Sterile Stool Collection Tube</td><td>Lang Fu Co., LTD</td><td></td><td>5 mL</td></tr><tr><td>Distilled Water</td><td>Department of Biopharmaceutical, Beijing University of Traditional Chinese Medicine</td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich Corporation</td><td>WXBD2861V</td><td>AR</td></tr><tr><td>EP Tube</td><td>Beijing Biodee Biotechnology Co., LTD</td><td></td><td>10 mL/1.5 mL</td></tr><tr><td>Eppendorf Centrifuge</td><td>Eppendorf AG</td><td></td><td>5418</td></tr><tr><td>Glucose</td><td>Beijing Chemical Works</td><td>GC205003</td><td>AR</td></tr><tr><td>High Performance Liquid Chromatograph</td><td>Shimadzu Corporation</td><td></td><td>LC-20</td></tr><tr><td>High-pressure Steam Sterilizer</td><td>Sanyo Denki Shanghai Co., LTD</td><td></td><td>MLS-3780</td></tr><tr><td>Innoval C&lt;sub&gt;18 &lt;/sub&gt;Chromatographic Column</td><td>Agela Technologies Co., LTD</td><td></td><td>4.6 mm &amp;times; 250 mm, 5 &amp;micro;m</td></tr><tr><td>L-cysteine Hydrochloride</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>BGASY01</td><td>BR</td></tr><tr><td>Liver Extract Powder</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-085</td><td>BR</td></tr><tr><td>Luteolin</td><td>National Institutes for Food and Drug Control</td><td></td><td>&gt;98%</td></tr><tr><td>Magnetic Stirrer</td><td>Ika Werke Co., LTD</td><td></td><td>RCT basic</td></tr><tr><td>Methanol</td><td>Thermo Fisher Scientific</td><td>20240901312</td><td>AR</td></tr><tr><td>Millipore Filter Membrane</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td></td><td>0.22 &amp;micro;L &amp;times; 50 mm</td></tr><tr><td>Orientin</td><td>Yishiming (Beijing) Biotechnology Co., LTD</td><td>19120601</td><td>&gt;98%</td></tr><tr><td>Peptone</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>1685787</td><td>BR</td></tr><tr><td>Petri Dish</td><td>Beijing Biodee Biotechnology Co., LTD</td><td></td><td>150 mm</td></tr><tr><td>Sodium Chloride</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>BN20008</td><td>AR</td></tr><tr><td>Sodium Thioglycolate</td><td>Shanghai Jianglai Biotechnology Co., LTD</td><td>J031S219019</td><td>AR</td></tr><tr><td>Soluble Starch</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>S9765</td><td>BR</td></tr><tr><td>Soya Peptone</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>2147955</td><td>BR</td></tr><tr><td>Tryptone</td><td>Agela Technologies Co., LTD</td><td>1685787</td><td>BR</td></tr><tr><td>Ultrasonic Cleaner</td><td>Kun Shan Ultrasonic Instruments Co., LTD</td><td></td><td>KQ-500DE</td></tr><tr><td>Yeast Extract</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-014</td><td>BR</td></tr></tbody></table>

screening and isolation of c-glycoside-cleaving intestinal bacteria

Screening e isolamento dei batteri intestinali che scindono il glicoside C

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, ...

screening-and-isolation-of-c-glycoside-cleaving-intestinal-bacteria

Research

JoVE Journal

Biochemistry

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

144 Views.  Beijing University of Chinese Medicine. Questo studio stabilisce una serie di procedure operative standard (SOP) per lo screening e l'isolamento efficiente dei batteri intestinali in grado di scindere i glicosidi C.

Video: Screening e isolamento dei batteri intestinali che scindono il glicoside C

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Display interno-membrana batterica per lo screening di una libreria di frammenti di anticorpi

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Ricerca

Biochimica

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Isolamento e misure respiratoria dei mitocondri da Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

In Situ Misurazione e la correlazione di densità delle cellule ed emissione di luce di batteri bioluminescenti

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Rilevamento e isolamento di corpi apoptotici di elevata purezza

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

Screening dell'inibitore della kinasi in microarray proteici umane auto-assemblati

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Screening cinetico dell'attività nucleana utilizzando sonde con acido Nucleic

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Screening per inibitori di Pirofosofasi rilegati a Thermotoga

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

Preconcentrazione su carta e isolamento di microvescicoli ed esosomi

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)&#8212;two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 &#177; 3.6 &#181;M and a Ki of 23.2 &#177; 3.2 &#181;M. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

Screening spettrofotometrico per potenziali inibitori di Glutathione Cytosolic S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.