Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan

Cowman, Mary K.; Chen, Cherry C.; Pandya, Monika; Han, Yuan; Ramkishun, Dianne; LoBello, Jaclyn; Bhilocha, Shardul; Russell-Puleri, Sparkle; Skendaj, Eraldi; Mijović, Jovan; Wei, Jing

doi:10.1016/j.ab.2011.05.023

Cited by 61 publications

(43 citation statements)

References 68 publications

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“…pH adjustments were performed with sterile cell culture graded 1 N NaOH and HCl. Digested samples were evaluated by electrophoresis on a 0.9% agarose gel (25 V, 1 h followed by 35 V, 5 h), followed by overnight staining in 0.005% Stains-All (Sigma) in 50% ethanol/water and further destaining as described previously (54 4 cells per well in 12-well plates for 24 h before transfection. siRNA constructs were transfected into RAW 264.7 cells at a final concentration of 50 nM using 2.5 l of Dharmafect 4 transfection reagent per well (1 ml), according to the manufacturer's protocol (Dharmacon).…”

Section: Methodsmentioning

confidence: 99%

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Sokołowska

Chen

Eberlein

et al. 2014

Journal of Biological Chemistry

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Background: Fragmented hyaluronan (a major extracellular matrix component) and eicosanoids (potent lipid mediators) are associated with chronic inflammatory diseases and cancer. Results: Fragmented hyaluronan stimulates lipid mediator production in human monocytes and macrophages and influences macrophage differentiation toward a distinct activation pattern. Conclusion: These findings reveal a novel link between hyaluronan-mediated inflammation and lipid metabolism. Significance: This link may provide new targets for disease therapeutics.

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Section: Methodsmentioning

confidence: 99%

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Sokołowska

Chen

Eberlein

et al. 2014

Journal of Biological Chemistry

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“…The M distribution of this combined sample was determined by polyacrylamide gel and agarose gel electrophoresis, with detection using Stains-All™ dye. These methods have been previously shown to give accurate molecular mass determinations for HA within appropriate M ranges for each gel type [13,14]. Figure 2 shows the polyacrylamide gel image, and corresponding densitometric profiles.…”

Section: Methodsmentioning

confidence: 99%

Determination of hyaluronan molecular mass distribution in human breast milk

Han

Amin

et al. 2015

Analytical Biochemistry

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Hyaluronan (HA) in human milk mediates host responses to microbial infection, via TLR4- and CD44-dependent signaling. Signaling by HA is generally size-specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low M component. Here we report the size distribution of HA in human milk samples from twenty unique donors. A new method for HA analysis, employingion exchange (IEX) chromatography to fractionate HA by size, and specific quantification of each size fraction by competitive Enzyme Linked Sorbent Assay (ELSA), was developed. When separated into four fractions, milk HA with M ≤ 20 kDa, M ≈20-60 kDa, and M ≈ 60-110 kDa comprised an average of 1.5%, 1.4% and 2% of the total HA, respectively. The remaining 95% was HA with M≥110 kDa. Electrophoretic analysis of the higher M HA from thirteen samples showed nearly identical M distributions, with an average M of ∼440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low M HA components.

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“…Gels were then photobleached for 20 min on an LED light box and scanned with an Epson Perfection V600 photo scanner (Epson, Nagano, Japan). Scanned images were analyzed using ImageJ (60), and data analysis was performed as previously described (59). All samples were analyzed in duplicate.…”

Section: Figmentioning

confidence: 99%

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

Westbrook

Moo‐Young

Chou

2016

Appl Environ Microbiol

160

156

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The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for Escherichia coli and Saccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery. IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in Bacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of B. subtilis strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.

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Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan

Cited by 61 publications

References 68 publications

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Determination of hyaluronan molecular mass distribution in human breast milk

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

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