Novel High-Throughput Deoxyribonuclease 1 Assay

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z’ ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

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Abstract 782: Development of a high throughput in vitro screening platform to identify novel inducers of immunological cell death

10.1158/1538-7445.am2018-782 ◽

2018 ◽

Author(s):

Didier Grillot ◽

Akanksha Gangar ◽

Raphaelle Guillard-Huet ◽

Eric Boursier ◽

Florent Potvain ◽

...

Keyword(s):

Cell Death ◽

High Throughput ◽

In Vitro Screening ◽

Screening Platform

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A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

Journal of Cell Death ◽

10.4137/jcd.s3660 ◽

2009 ◽

Vol 2 ◽

pp. JCD.S3660

Author(s):

Hang Fai Kwok ◽

Julie A. Gormley ◽

Christopher J. Scott ◽

James A. Johnston ◽

Shane A. Olwill

Keyword(s):

Cell Death ◽

High Throughput ◽

High Throughput Screening ◽

False Negative ◽

Death Receptor ◽

Annexin V ◽

Fas Receptor ◽

Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

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High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0805130105 ◽

2008 ◽

Vol 105 (46) ◽

pp. 17670-17675 ◽

Cited By ~ 138

Author(s):

Luca Mannocci ◽

Yixin Zhang ◽

Jörg Scheuermann ◽

Markus Leimbacher ◽

Gianluca De Bellis ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

High Throughput ◽

High Throughput Sequencing ◽

Organic Molecules ◽

Relative Quantification ◽

Dna Encoding ◽

Chemical Library ◽

Chemical Libraries ◽

Matrix Metalloproteinase 3 ◽

Individual Reaction

DNA encoding facilitates the construction and screening of large chemical libraries. Here, we describe general strategies for the stepwise coupling of coding DNA fragments to nascent organic molecules throughout individual reaction steps as well as the first implementation of high-throughput sequencing for the identification and relative quantification of the library members. The methodology was exemplified in the construction of a DNA-encoded chemical library containing 4,000 compounds and in the discovery of binders to streptavidin, matrix metalloproteinase 3, and polyclonal human IgG.

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Detection of deoxyribonuclease I and II activities in Japanese quail oocytes

Zygote ◽

10.1017/s0967199401001010 ◽

2001 ◽

Vol 9 (1) ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Urszula Stepińska ◽

Bozenna Olszańska

Keyword(s):

Japanese Quail ◽

Dna Degradation ◽

Dnase I ◽

Early Cleavage ◽

Experimental Conditions ◽

Dnase Ii ◽

Exogenous Dna ◽

Deoxyribonuclease I ◽

Female Pronucleus

Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation. However, only one of them unites with the female pronucleus to form a zygote nucleus; the supernumerary spermatozoal nuclei degenerate at the early cleavage stages. To establish a factor responsible for spermatozoal degeneration, the presence of DNase activity was studied in vitro in extracts of Japanese quail oocytes using λ DNA/HindIII as a substrate. The experimental conditions were designed to reveal the presence of either DNase I or DNase II activities, separately. Degradation of the substrate DNA was evaluated by electrophoresis on agarose gels stained with ethidium bromide. High activities of DNase I and DNase II were found in the germinal discs of the largest vitellogenic oocytes. DNase I activity was estimated to be about 3 × 10−3 Kunitz units and DNase II about 4 × 10−2 Kunitz units per germinal disc. DNase I activity in an oocyte seems to increase during oogenesis since DNA degradation by the extracts from the germinal discs of the largest vitellogenic oocytes was much higher than by those from previtellogenic and small vitellogenic oocytes. The presence of high DNase I and II activities in the largest vitellogenic oocytes would point to their role in degradation of DNA from supernumerary spermatozoa entering the ovum during polyspermic fertilisation in birds. The enzymes could be a factor, or one of the factors, in the late block to polyspermy in the cytoplasm of avian eggs. It is suggested here that the DNase activities might also be responsible for poor efficiency in obtaining transgenic birds by microinjection of exogenous DNA into the fertilised chick ovum.

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Differences in the sensitivity of classically and alternatively activated macrophages to TAK1 inhibitor-induced necroptosis

Cancer Immunology Immunotherapy ◽

10.1007/s00262-020-02623-7 ◽

2020 ◽

Vol 69 (11) ◽

pp. 2193-2207 ◽

Cited By ~ 2

Author(s):

Zsófia Varga ◽

Tamás Molnár ◽

Anett Mázló ◽

Ramóna Kovács ◽

Viktória Jenei ◽

...

Keyword(s):

Cell Death ◽

Glycogen Synthase ◽

Therapeutic Option ◽

Cell Types ◽

Therapeutic Benefit ◽

Alternatively Activated Macrophages ◽

Highly Sensitive ◽

Anti Inflammatory ◽

Macrophage Populations

Abstract Controlling the balance of pro-inflammatory M1 versus anti-inflammatory M2 macrophages may have paramount therapeutic benefit in cardiovascular diseases, infections, cancer and chronic inflammation. The targeted depletion of different macrophage populations provides a therapeutic option to regulate macrophage-mediated functions. Macrophages are highly sensitive to necroptosis, a newly described regulated cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like pseudokinase. Antagonists of inhibitors of apoptosis proteins (SMAC mimetics) block RIPK1 ubiquitination, while TGF-activated kinase 1 (TAK1) inhibitors prevent the phosphorylation of RIPK1, resulting in increased necroptosis. We compared the sensitivity of monocyte-derived human M1 and M2 cells to various apoptotic and necroptotic signals. The two cell types were equally sensitive to all investigated stimuli, but TAK1 inhibitor induced more intense necroptosis in M2 cells. Consequently, the treatment of co-cultured M1 and M2 cells with TAK1 inhibitor shifted the balance of the two populations toward M1 dominance. Blockage of either Aurora Kinase A or glycogen synthase kinase 3β, two newly described necroptosis inhibitors, increased the sensitivity of M1 cells to TAK1-inhibitor-induced cell death. Finally, we demonstrated that in vitro differentiated tumor-associated macrophages (TAM-like cells) were as highly sensitive to TAK1 inhibitor-induced necroptosis as M2 cells. Our results indicate that at least two different necroptotic pathways operate in macrophages and the targeted elimination of different macrophage populations by TAK1 inhibitor or SMAC mimetic may provide a therapeutic option to regulate the balance of inflammatory/anti-inflammatory macrophage functions.

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Use of Differential Scanning Fluorimetry as a High-Throughput Assay to Identify Nuclear Receptor Ligands

Nuclear Receptor Signaling ◽

10.1621/nrs.10002 ◽

2012 ◽

Vol 10 (1) ◽

pp. nrs.10002 ◽

Cited By ~ 23

Author(s):

Kara DeSantis ◽

Aaron Reed ◽

Raneen Rahhal ◽

Jeff Reinking

Keyword(s):

Nuclear Receptors ◽

Nuclear Receptor ◽

High Throughput ◽

Initial Step ◽

Estrogen Receptor Α ◽

Chemical Library ◽

Differential Scanning Fluorimetry ◽

Nuclear Receptor Ligands

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10μL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.

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DNase-I-dependent dissociation of erythrocyte cytoskeletons.

The Journal of Cell Biology ◽

10.1083/jcb.81.1.266 ◽

1979 ◽

Vol 81 (1) ◽

pp. 266-270 ◽

Cited By ~ 19

Author(s):

M P Sheetz

Keyword(s):

Human Erythrocyte ◽

Actin Filaments ◽

Cytoplasmic Membrane ◽

Protein Complexes ◽

Membrane Surface ◽

Dnase I ◽

Large Protein ◽

Deoxyribonuclease I ◽

Peripheral Membrane Proteins

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.

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Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057116658646 ◽

2016 ◽

Vol 21 (9) ◽

pp. 989-997 ◽

Cited By ~ 4

Author(s):

Bryan Severyn ◽

Thi Nguyen ◽

Michael D. Altman ◽

Lixia Li ◽

Kumiko Nagashima ◽

...

Keyword(s):

Gene Expression ◽

High Throughput ◽

Mapk Pathway ◽

Gene Signature ◽

Mapk Signaling ◽

Performance Measurements ◽

Chemical Library ◽

True Negative ◽

Recurrent Mutations

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression–based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose–response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway.

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Novel Angiogenic Compounds for Targeted Drug Delivery

MRS Proceedings ◽

10.1557/proc-845-aa5.47 ◽

2004 ◽

Vol 845 ◽

Author(s):

Kristen A. Wieghaus ◽

Scott M. Capitosti ◽

Milton L. Brown ◽

Edward A. Botchwey

Keyword(s):

Cell Death ◽

Wound Repair ◽

Culture Conditions ◽

Hypoxic Cell ◽

Growth Factor Delivery ◽

Microvascular Networks ◽

Highly Sensitive ◽

Endothelial Cell Death ◽

Graft Reconstruction

ABSTRACTInduction of angiogenesis is necessary for the success of engineered implantable tissues in order to meet oxygen and nutrient requirements of cells during tissue repair. Insufficient vascularization in bone graft reconstruction may impede healing and initiate hypoxic cell death at the interior of the implant. As a result, endogenous growth factors have been studied to enhance angiogenesis during wound repair. However, these peptide-based molecules are highly sensitive to processing that occurs during scaffold biomaterial fabrication and treatment for tissue engineering purposes. We report here the development of new small molecule regulators of angiogenesis that may circumvent the impediments associated with protein-based growth factor delivery. In this study, we report the design and evaluation of SC–3–143 as a regulator of endothelial function. We show that the compound significantly increases the formation of microvascular networks in vitro, and selectively enhances endothelial survivability by reducing endothelial cell death under serum deprived culture conditions.

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