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Published in Chad A. Mirkin, Spherical Nucleic Acids, 2020
Dwight S. Seferos, Andrew E. Prigodich, David A. Giljohann, Pinal C. Patel, Chad A. Mirkin
We prepared DNA-AuNPs and studied their nuclease stability using fluorescence spectroscopy and literature methods [16]. The DNA-AuNPs consist of a 13 ± 1 nm AuNP functionalized with a dense monolayer of oligonucleotides composed of a 20-base DNA sequence, a 10-base DNA linker, and propylthiol anchor. Each probe was allowed to hybridize with fluorescein-labeled DNA complements, and the enzyme deoxyribonuclease I (DNase I), a common endonuclease [20], was added to interrogate DNA-AuNP stability. Since DNase I is known to bind ssDNA (although with much lower affinity than dsDNA), we initially varied the nanoparticle surface coverage using duplex and single-stranded DNA. DNA-AuNPs were allowed to hybridize with different molar ratios of fluorophore-labeled complements (5, 10, 20, and 30 complements per DNA-AuNP). Hybridization was achieved by heating to 70°C and cooling for 12 h. After hybridization, the total dsDNA in each sample was adjusted to 50 nM. Next, the samples were treated with DNase I, and the rate of degradation was measured by a fluorescence-based assay. The results of these experiments reveal similar reaction rates in each sample. We conclude that dsDNA is the substrate of DNase I and the effect of ssDNA or dsDNA on the nanoparticle surface is similar. This is consistent with the ~500 times lower activity of DNase I for ssDNA [21].
Biologic Drug Substance and Drug Product Manufacture
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
Ajit S. Narang, Mary E. Krause, Shelly Pizarro, Joon Chong Yee
Biologic drug products are marketed in several different configurations, including liquid (solution) filled vials or prefilled syringes, lyophilized powder in a vial, or concentrated solutions intended for either dilution in a parenteral fluid before administration or for direct subcutaneous injection using novel delivery devices. Proteins and peptides for parenteral administration are typically formulated as ready-to-use aqueous solutions or as a lyophilized solid mass that is reconstituted with water, isotonic dextrose solution, or isotonic sodium chloride solution immediately before administration; while proteins and peptides for inhalation and nasal routes of administration are typically formulated as dry powders (Mahato and Narang 2018). For stable molecules, nebulization of aqueous solutions is also a viable strategy. For example, Pulmozyme® (dornase alpha) is a solution which is aerosolized by a nebulizer during administration. It contains a CHO-produced recombinant human deoxyribonuclease I, an enzyme which cleaves the DNA present in the mucus of cystic fibrosis patients and reduces viscosity in the lungs, resulting in better clearance of secretions.
Materials for Tissue Engineering
Published in Joseph W. Freeman, Debabrata Banerjee, Building Tissues, 2018
Joseph W. Freeman, Debabrata Banerjee
Detergents work by sulublizing the lipid membranes of cells, unfortunately detergents can also be harmful to signaling proteins (which can help with differentiation of the seeded cells) and structural proteins such as growth factors (which are necessary for maintaining tissue mechanical properties). Sodium dodecyl sulfate (SDS) is a common detergent for dense tissues that has been used on a variety of tissues and organs including kidney, heart, vein, and the temporomandibular joint.6–9 If it is used for too long a period of time or at too high a concentration, SDS can alter tissue mechanical properties and decrease growth factor and glycosaminoglycan content. The ionic nature of SDS makes it difficult to remove from the tissue without extensive washing. Triton X-100 is another detergent that is used in thick tissues such as heart valves.10 Triton X-100 is not ionic, so it is not as harsh to proteins as SDS and therefore does a better job of preserving tissue structure and mechanical properties than SDS. Sodium deoxycholate (SD) is another ionic surfactant, like SDS, but it is milder to proteins than SDS. Unfortunately, its use can lead to the aggregation of DNA at the tissue surface; this can be alieviated by combining SD with a deoxyribonuclease I to break down the DNA.11 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) is zwitterionic (a dipole ion)m is not as harsh on protein as SDS, and is most effective on thinner tissues.12
Functionalized acellular periosteum guides stem cell homing to promote bone defect repair
Published in Journal of Biomaterials Science, Polymer Edition, 2023
Guoqing Zhu, Yidi Zhou, Yichang Xu, Lingjun Wang, Meng Han, Kun Xi, Jincheng Tang, Ziang Li, Yu Kou, Xindie Zhou, Yu Feng, Yong Gu, Liang Chen
The decellularization preparation steps were based on the method described by Chen et al. [7]. The natural periosteum was frozen at −80 °C, and then thawed in a 37 °C water bath; the above procedures were repeated three times. The resulting sample was stirred overnight at room temperature in deionized water at a constant speed of 120 rpm. The sample was then placed in a 2% polyethylene glycol octyl phenyl ether (Triton X-100) solution at room temperature for 12 h, washed with deionized water to remove residual Triton X-100, and placed in a 1% dodecane sodium dodecyl sulfate (SDS) solution at room temperature for 4 h. After being washed with deionized water again, the sample was placed in 100 U/ml deoxyribonuclease I (DNase I) solution for 12 h at 37 °C, followed by further washes in deionized water for two days at room temperature; the above procedures were carried out under uniform stirring at 120 rpm. Finally, the prepared acellular periosteum was placed in PBS containing 100 U/ml penicillin and 100 μg/ml streptomycin for subsequent cryopreservation; these samples could then be thawed for future use.
Study on the characterization of endosulfan-degrading bacterial strains isolated from contaminated rhizospheric soil
Published in Journal of Environmental Science and Health, Part C, 2022
Vandana Singh, Shubhi Srivastava, Namrata Singh, Suchi Srivastava, Alok Lehri, Nandita Singh
The results of the biochemical tests show that all the strains have shown positive reaction for the Voges–Proskauer, nitrate, deoxyribonuclease, and catalase tests and negative for the indole, starch hydrolysis, and urease tests. Strains EAG-EC-12, 13, and 14 have shown negative result for arginine dihydrolase and oxidase tests, while the same strains have shown positive response for citrate, lysine decarboxylase, esculin hydrolysis, and lypase tests. Strain EAG-EC-15 has shown negative response to methyl red, citrate, lysin decarboxylate, ornithine decarboxylase, gelatin hydrolysis, esculin hydrolysis, and lipase and positive response to Voges–Proskauer test, arginine dihydrolase, and oxidase test. Biochemical response in terms of acid production showed that all four strains have shown positive response toward xylose and fructose test, while negative toward maltose test. Strains EAG-EC-12, 13, and 14 have shown negative response toward galactose and dextrose test, whereas EAG-EC-15 has shown negative response to trehalose and positive to dextrose and galactose test. Based on the results of morphological, physiological, and biochemical characterization tests, three strains were identified as Serratia sp. and one as Ochrobactrum daejeonensis.
Bayesian probability of agreement for comparing survival or reliability functions with parametric lifetime regression models
Published in Quality Engineering, 2020
Nathaniel T. Stevens, Lu Lu, Christine M. Anderson-Cook, Steven E. Rigdon
To illustrate how the BPA approach may be used to compare two lifetime distributions in a medical context, we consider the cystic fibrosis study reported in Therneau and Hamilton (1997). This is an example of a recurrent event analysis that examines the effectiveness of a treatment with a highly purified recombinant deoxyribonuclease I (rhDNase) compared to a placebo. Patients with cystic fibrosis experience difficulty clearing their lungs because of thickened mucus caused by extracellular DNA, which is released by leukocytes. These accumulate in the airways in response to chronic bacterial infection. In the data considered, some patients experienced multiple episodes, but the primary endpoint was the number of days until the first pulmonary exacerbation. The definition of an exacerbation is an infection occurring that required the use of intravenous antibiotics. A single covariate, forced expiratory volume (FEV), measured lung capacity with larger scores corresponding to patients with generally better lung health.