The Experts below are selected from a list of 94683 Experts worldwide ranked by ideXlab platform
Scott Bailey - One of the best experts on this subject based on the ideXlab platform.
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in Vitro reconstitution of an escherichia coli rna guided immune system reveals unidirectional atp dependent Degradation of dna target
Journal of Biological Chemistry, 2013Co-Authors: Sabin Mulepati, Scott BaileyAbstract:Abstract Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. in Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid Degradation in Vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.
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in Vitro reconstitution of an escherichia coli rna guided immune system reveals unidirectional atp dependent Degradation of dna target
Journal of Biological Chemistry, 2013Co-Authors: Sabin Mulepati, Scott BaileyAbstract:Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. in Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid Degradation in Vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner. Background: The CRISPR-Cas immune system protects E. coli against invasive DNA. Results: We have reconstituted this immune system in Vitro using recombinant proteins. Conclusion: Degradation of invasive DNA is tightly regulated and unidirectional. Significance: Reconstitution provides an invaluable tool for understanding the CRISPR-Cas immune system.
Sabin Mulepati - One of the best experts on this subject based on the ideXlab platform.
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in Vitro reconstitution of an escherichia coli rna guided immune system reveals unidirectional atp dependent Degradation of dna target
Journal of Biological Chemistry, 2013Co-Authors: Sabin Mulepati, Scott BaileyAbstract:Abstract Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. in Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid Degradation in Vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.
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in Vitro reconstitution of an escherichia coli rna guided immune system reveals unidirectional atp dependent Degradation of dna target
Journal of Biological Chemistry, 2013Co-Authors: Sabin Mulepati, Scott BaileyAbstract:Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. in Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid Degradation in Vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner. Background: The CRISPR-Cas immune system protects E. coli against invasive DNA. Results: We have reconstituted this immune system in Vitro using recombinant proteins. Conclusion: Degradation of invasive DNA is tightly regulated and unidirectional. Significance: Reconstitution provides an invaluable tool for understanding the CRISPR-Cas immune system.
Donald R Griffin - One of the best experts on this subject based on the ideXlab platform.
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activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing
Nature Materials, 2020Co-Authors: Donald R Griffin, Maani M Archang, Chenhsiang Kuan, Westbrook M Weaver, Jason S Weinstein, An Chieh Feng, Amber Ruccia, Elias Sideris, Vasileios RagkousisAbstract:Microporous annealed particle (MAP) scaffolds are flowable, in situ crosslinked, microporous scaffolds composed of microgel building blocks and were previously shown to accelerate wound healing. To promote more extensive tissue ingrowth before scaffold Degradation, we aimed to slow MAP Degradation by switching the chirality of the crosslinking peptides from L- to D-amino acids. Unexpectedly, despite showing the predicted slower enzymatic Degradation in Vitro, D-peptide crosslinked MAP hydrogel (D-MAP) hastened material Degradation in vivo and imparted significant tissue regeneration to healed cutaneous wounds, including increased tensile strength and hair neogenesis. MAP scaffolds recruit IL-33 type 2 myeloid cells, which is amplified in the presence of D-peptides. Remarkably, D-MAP elicited significant antigen-specific immunity against the D-chiral peptides, and an intact adaptive immune system was required for the hydrogel-induced skin regeneration. These findings demonstrate that the generation of an adaptive immune response from a biomaterial is sufficient to induce cutaneous regenerative healing despite faster scaffold Degradation.
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activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing
bioRxiv, 2020Co-Authors: Donald R Griffin, Maani M Archang, Westbrook M Weaver, Jason S Weinstein, An Chieh Feng, Amber Ruccia, Chen H Kuan, Elias SiderisAbstract:Biomaterial scaffolds represent a promising approach for material-based tissue regeneration. We previously developed microporous annealed particle (MAP) hydrogels - a flowable, microparticle-based hydrogel in which neighboring hydrogel particles are linked in situ to form a porous scaffold that accelerates wound healing. To promote more extensive tissue ingrowth before scaffold Degradation, we aimed to slow scaffold Degradation by switching the chirality of the crosslinking peptides from L-peptides to D-peptides. Unexpectedly, despite showing the predicted slower enzymatic Degradation in Vitro, D-peptide crosslinked MAP hydrogel (D-MAP) hastened material Degradation in vivo and imparted significant tissue regeneration to healed cutaneous wounds, including increased tensile strength and hair neogenesis. By themselves, D-chiral peptides were poor activators of macrophage innate immune signaling in vivo, but MAP particles elicit IL-33 type 2 myeloid cell recruitment which is amplified in vivo in the presence of D-peptides. Remarkably, D-MAP elicited significant antigen-specific immunity against the D-chiral peptides, and an intact adaptive immune system was required for the hydrogel-induced skin regeneration. These findings demonstrate that the generation of an adaptive immune response from a biomaterial is sufficient to induce cutaneous regenerative healing despite faster scaffold Degradation.
Andreas Bernkopschnurch - One of the best experts on this subject based on the ideXlab platform.
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oral peptide drug delivery polymer inhibitor conjugates protecting insulin from enzymatic Degradation in Vitro
Biomaterials, 2000Co-Authors: Michaela K. Marschütz, Andreas BernkopschnurchAbstract:Abstract A drug-carrier matrix has been developed which protects embedded insulin from Degradation by the luminally secreted serine-proteases trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) in Vitro. increasing amounts of the Bowman–Birk inhibitor (BBI) and elastatinal, respectively, were thereby covalently bound to the mucoadhesive polymer sodium carboxymethylcellulose (Na-CMC). The inhibitory efficacy of resulting polymers was evaluated. On the one hand, all polymer–BBI conjugates showed a strong inhibitory activity towards trypsin and chymotrypsin whereas it was markedly lower towards elastase. The polymer–elastatinal conjugates, on the other hand, displayed a comparatively higher inhibitory activity towards elastase. in an artificial intestinal fluid containing trypsin, chymotrypsin and elastase in physiological concentrations insulin, being incorporated in unmodified Na-CMC, was rapidly degraded at 37°C. Within 1 h 98.7±0.4% (mean±SD, n=3) of the peptide drug were thereby metabolized. On the contrary, the incorporation of insulin in a mixture of the two polymer–inhibitor conjugates CMC–BBI (40%; w/w) and CMC–elastatinal conjugate (60%; w/w) led to a peptide Degradation of 22.3±2.5% (mean±SD, n=3) within the same time period. Even after 4 h of incubation, 33.6±3.2% (mean±SD, n=3) of the therapeutic agent remained stable towards enzymatic attack. Hence, the polymer–inhibitor conjugates described in this study seem to be a useful tool in overcoming the luminal enzymatic barrier in peroral insulin delivery.
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oral peptide drug delivery polymer inhibitor conjugates protecting insulin from enzymatic Degradation in Vitro
Biomaterials, 2000Co-Authors: Michaela K. Marschütz, Andreas BernkopschnurchAbstract:A drug-carrier matrix has been developed which protects embedded insulin from Degradation by the luminally secreted serine-proteases trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) in Vitro. increasing amounts of the Bowman-Birk inhibitor (BBI) and elastatinal, respectively, were thereby covalently bound to the mucoadhesive polymer sodium carboxymethylcellulose (Na-CMC). The inhibitory efficacy of resulting polymers was evaluated. On the one hand, all polymer-BBI conjugates showed a strong inhibitory activity towards trypsin and chymotrypsin whereas it was markedly lower towards elastase. The polymer-elastatinal conjugates, on the other hand, displayed a comparatively higher inhibitory activity towards elastase. in an artificial intestinal fluid containing trypsin, chymotrypsin and elastase in physiological concentrations insulin, being incorporated in unmodified Na-CMC, was rapidly degraded at 37 degrees C. Within 1 h 98.7 +/- 0.4% (mean +/- SD, n = 3) of the peptide drug were thereby metabolized. On the contrary, the incorporation of insulin in a mixture of the two polymer-inhibitor conjugates CMC-BBI (40%; w/w) and CMC-elastatinal conjugate (60%; w/w) led to a peptide Degradation of 22.3 +/- 2.5% (mean +/- SD, n = 3) within the same time period. Even after 4 h of incubation, 33.6 +/- 3.2% (mean +/- SD, n = 3) of the therapeutic agent remained stable towards enzymatic attack. Hence, the polymer-inhibitor conjugates described in this study seem to be a useful tool in overcoming the luminal enzymatic barrier in peroral insulin delivery.
Vasileios Ragkousis - One of the best experts on this subject based on the ideXlab platform.
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activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing
Nature Materials, 2020Co-Authors: Donald R Griffin, Maani M Archang, Chenhsiang Kuan, Westbrook M Weaver, Jason S Weinstein, An Chieh Feng, Amber Ruccia, Elias Sideris, Vasileios RagkousisAbstract:Microporous annealed particle (MAP) scaffolds are flowable, in situ crosslinked, microporous scaffolds composed of microgel building blocks and were previously shown to accelerate wound healing. To promote more extensive tissue ingrowth before scaffold Degradation, we aimed to slow MAP Degradation by switching the chirality of the crosslinking peptides from L- to D-amino acids. Unexpectedly, despite showing the predicted slower enzymatic Degradation in Vitro, D-peptide crosslinked MAP hydrogel (D-MAP) hastened material Degradation in vivo and imparted significant tissue regeneration to healed cutaneous wounds, including increased tensile strength and hair neogenesis. MAP scaffolds recruit IL-33 type 2 myeloid cells, which is amplified in the presence of D-peptides. Remarkably, D-MAP elicited significant antigen-specific immunity against the D-chiral peptides, and an intact adaptive immune system was required for the hydrogel-induced skin regeneration. These findings demonstrate that the generation of an adaptive immune response from a biomaterial is sufficient to induce cutaneous regenerative healing despite faster scaffold Degradation.