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Glomerular Barrier Behaves as an Atomically Precise Bandpass Filter in a Sub-nanometre Regime *
Published in Valerio Voliani, Nanomaterials and Neoplasms, 2021
Du Bujie, Xingya Jiang, Anindita Das, Qinhan Zhou, Yu Mengxiao, Rongchao Jin, Jie Zheng
To test whether these AuNCs chemically bind to the glycocalyx, we incubated Au18SG14 and Au25SG18 with a 10% (vol/vol) major component of glycocalyx-heparan sulfate (HS) proteoglycan from Sigma-Aldrich in PBS at 37°C for 30 min and 24 h, respectively. To identify the colourless HS proteoglycan band, HS proteoglycan incubated Au18SG14 and Au25SG18 were stained by 10% (vol/vol) Coomassie Brilliant Blue 250 (CBB 250). All the samples were analysed by 2% agarose gel electrophoresis using the Minisub cell GT gel electrophoresis system (Bio-Rad Laboratories). The amount of gold in each band was quantified using ICP-MS.
Cellular and Molecular Mechanisms of Ischemic Acute Renal Failure and Repair
Published in Robin S. Goldstein, Mechanisms of Injury in Renal Disease and Toxicity, 2020
Joseph V. Bonventre, Ralph Witzgall
The pathological features of necrosis are very different from those of programmed cell death (Searle, et al., 1982). The latter has been called “apoptosis”, a term coined by Kerr et al., who attributes the derivation of the term to Professor James Cormack of the University of Aberdeen (Kerr, et al., 1972). “Apoptosis” is a Greek term to describe “dropping off’ or “falling off’ of petals from a flower or leaves from a tree. Pathologically, the early stages of apoptosis are characterized by disappearance of microvilli, chromatin condensation at the periphery of the nucleus, condensation of cytosolic components, breakdown of epithelial desmosomal attachments, followed by cell surface protuberances, and fragmentation of the nucleus. Ultimately there is blebbing off of cell surface protuberances containing condensed cytosol and occasional nuclear fragments, to generate spherical or ovoid “apoptotic bodies” (Wyllie, et al., 1980) which can be phagocytosed by macrophages or epithelial cells. Apoptosis histologically is often associated biochemically with cleavage of double-stranded DNA at the linker regions between nucleosomes, resulting in fragments of approximately 180 to 200 bp (Kerr and Harmon, 1991). On agarose gel electrophoresis there is a characteristic ladder pattern. The endonuclease responsible for this biochemical correlate of apoptosis has not been well characterized. A candidate enzyme is an endonuclease that is activated by calcium in thymocytes (McConkey, et al., 1988). Increases in cellular calcium concentration can induce apoptosis (Kizaki, et al., 1989).
HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
RFLP analysis was first applied to the investigation of HLA class II polymorphism by Wake et al.30 The technique (Figure 1) involves hybridization of labeled, single-stranded DNA sequences (probes) to homologous single-stranded DNA sequences (targets) within genomic DNA. Prior to hybridization, genomic DNA is treated by digestion with a restriction endonuclease; endonucleolytic fragments are then separated according to size by agarose gel electrophoresis and are transferred to a solid support by capillary or vacuum blotting. Although originally advocated as a technique with only a supplementary role in tissue typing, subsequent studies have shown that RFLP analysis permits the accurate genotyping of most DR, DQ, and Dw specificities.1-4-11-12-23-28 These investigations have shown that the correlation between RFLP and phenotypically determined DR, DQ, and Dw specificities varies according to the choice of restriction endonuclease employed.
Use of Decellularized SMILE (Small-Incision Lenticule Extraction) Lenticules for Engineering the Corneal Endothelial Layer: A Proof-of-Concept
Published in Current Eye Research, 2023
Swatilekha Hazra, Jacquelyn Akepogu, Supriya Krishna, SriRavali Pulipaka, Bhupesh Bagga, Charanya Ramachandran
Lenticules, at the end of the incubation period in the different media, were dried at 60 °C for 2 hrs and the dry weight of the lenticules was measured. Dried lenticules were chopped and suspended in 200 µl of lysis buffer containing 1 M Tris pH 8, 10% SDS, and 0.5M EDTA pH 8. After 10 mins of incubation at room temperature, 20 mg/ml Proteinase K (Genei, India) was added and incubated at 56 °C for 2 hrs. 10 µl of 5M NaCl and equal proportions of isopropanol were added and incubated at 4 °C for 10 mins and centrifuged at 12,000 rpm for 15 mins. The supernatant was removed followed by resuspension of the pellet in 70% ethanol and centrifugation at 12,000 rpm for 5 mins. Ethanol was discarded and the air-dried pellet was resuspended in TE buffer (pH 8). DNA was quantified using NanoDrop (Thermo Scientific, USA) and the values were used to calculate DNA concentration per milligram weight of lenticules (N = 3). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was performed on the data using GraphPad Prism V9.0. Significance was set at p < 0.05. Agarose gel electrophoresis was used to visualize the genomic DNA.
Buyang Huanwu decoction improves neural recovery after spinal cord injury in rats through the mTOR signaling pathway and autophagy
Published in The Journal of Spinal Cord Medicine, 2023
Ying Nie, Yujie Fan, Xi Zhang, Xiaosong Li, Jian Yin, Meili Li, Zhaoyong Hu, Liang Li, Xiaoye Wang
Cells were digested and lysed, and total RNA was extracted. A total of 5 μl of RNA was obtained from each group, and ultra-pure water of RNase was added to dilute the substance at a ratio of 1:20. NanoPhotometer ultramicro-ultraviolet spectrophotometer (Biorad, Hercules, CA, USA) was used to measure OD260/280 values for calculating the RNA concentration and purity. The OD 260/280 ratio was kept between 1.8 and 2.0 to ensure RNA purity. Agarose gel electrophoresis (1%) was performed to maintain integrity. The reverse transcription was performed according to the Fermentas kit instructions. Amplification reactions were performed using the Universal SYBR Green Master system (Roche, Basel, Switzerland). Amplification conditions were as follows: denaturation at 95°C for 2 min, denaturation at 95°C for 10 sec, annealing at 57°C for 10 sec, extension at 72°C for 15 sec, a total of 40 cycles of denaturation, annealing and extension. Primer 5.0 software was used for primer design and the primer sequences are shown in Table S1. β-actin (Sangon Biotech, Shanghai, China) was used as an internal control Ct and 2–ΔΔCt were obtained and used for quantitative analysis of Beclin1, LC3, and mTOR mRNA expression.
Fabrication of hesperidin nanoparticles loaded by poly lactic co-Glycolic acid for improved therapeutic efficiency and cytotoxicity
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Saja H. Ali, Ghassan M. Sulaiman, Mohammad M. F. Al-Halbosiy, Majid S. Jabir, Anaheed H. Hameed
Analysis of DNA fragments using agarose gel electrophoresis was performed according to Magnesia tissue culture cell DNA extraction Kit as manufacturer indicated. MCF-7 cells were seeded in suitable conditions and followed the same protocol introduced by Sulaiman et al. [23]. This protocol provides a qualitative method for assessing cell death by detecting DNA fragmentation. In brief, the growing cells were treated with three concentrations (10, 20, 30 and 40 µg mL−1) of modified nanohesperidin for 24 h. Cells at density 1.5 × 106 cell mL−1 were then harvested and suspended in ice-cold PBS. The cell suspension was centrifuged (1200 rpm) at 4 °C for 10 min. The supernatant was removed and the cells was frozen (−20 °C) overnight. The DNA was dissolved with DNA loading buffer, and then applied to 0.8% agarose gel electrophoresis. After staining with ethidium bromide, the DNA was visualized by UV irradiation and photographed by gel documentation system (Sci-Plus, UK). Loading marker was loaded in line parallelled to that of the samples.