A rapid extraction method for glycogen from formalin-fixed liver

Sullivan, Mitchell A.; Li, Shihan; Aroney, Samuel T N; Deng, Bin; Li, Cheng; Roura, Eugeni; Schulz, Benjamin L.; Harcourt, Brooke E.; Forbes, Josephine M.; Gilbert, Robert G.

doi:10.1016/j.carbpol.2014.11.005

Cited by 27 publications

(24 citation statements)

References 34 publications

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“…Peptides were analyzed as described previously70 by LC-ESI–MS/MS using a Prominence nanoLC system (Shimadzu, Japan) and TripleTOF 5600 mass spectrometry with a Nanospray III interface (Ab Sciex, USA). Identical LC conditions were used for SWATH-MS, with an MS-TOF scan from an m / z of 350–1,800 for 0.05 sec followed by high sensitivity information-independent acquisition with 26 m / z isolation windows with 1 m / z window overlap each for 0.1 sec across an m / z range of 400–1,250.…”

Section: Methodsmentioning

confidence: 99%

Sirtuins in the phylum Basidiomycota: A role in virulence in Cryptococcus neoformans

Arras

Chitty

Wizrah

et al. 2017

Sci Rep

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Virulence of Cryptococcus neoformans is regulated by a range of transcription factors, and is also influenced by the acquisition of adaptive mutations during infection. Beyond the temporal regulation of virulence factor production by transcription factors and these permanent microevolutionary changes, heritable epigenetic modifications such as histone deacetylation may also play a role during infection. Here we describe the first comprehensive analysis of the sirtuin class of NAD+ dependent histone deacetylases in the phylum Basidiomycota, identifying five sirtuins encoded in the C. neoformans genome. Each sirtuin gene was deleted and a wide range of phenotypic tests performed to gain insight into the potential roles they play. Given the pleiotropic nature of sirtuins in other species, it was surprising that only two of the five deletion strains revealed mutant phenotypes in vitro. However, cryptic consequences of the loss of each sirtuin were identified through whole cell proteomics, and mouse infections revealed a role in virulence for SIR2, HST3 and HST4. The most intriguing phenotype was the repeated inability to complement mutant phenotypes through the reintroduction of the wild-type gene. These data support the model that regulation of sirtuin activity may be employed to enable a drastic alteration of the epigenetic landscape and virulence of C. neoformans.

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Section: Methodsmentioning

confidence: 99%

Sirtuins in the phylum Basidiomycota: A role in virulence in Cryptococcus neoformans

Arras

Chitty

Wizrah

et al. 2017

Sci Rep

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“…The method was further improved using cold Tris buffer, as this can inhibit the glucosidase activity during the extraction procedure. 28 However, the formalin extraction technique was significantly faster compared to the sucrosegradient centrifugation method. 8,26,27 For liver samples preserved in formalin solution, a homogenization step followed by precipitation of the supernatant using absolute ethanol resulted in higher yields but a lower purity of glycogen with size distributions similar to the extraction for frozen samples using the sucrose-gradient centrifugation approach.…”

Section: Glycogen Extraction Methodologiesmentioning

confidence: 99%

“…45 In this review, we showed that glycogen α particles are molecularly fragile in diabetes patients when compared to healthy individuals: an effect that correlated with the loss of blood-sugar homeostasis in diabetes. 92 Studies based on this technique are currently in progress. In addition, an imbalance theory of GS/GBE activity ratio was proposed (Figure 7), which, if existed, is hypersensitive, because a slight decrease in the postprandial blood concentration by consuming TCM can resemble diabetic glycogen particles as healthy glycogen particles.…”

Section: Perspectivesmentioning

confidence: 99%

Is liver glycogen fragility a possible drug target for diabetes?

Cheng

2019

FASEB j.

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Liver glycogen α particles are molecularly fragile in diabetic mice, and readily form smaller β particles, which degrade more rapidly to glucose. This effect is well associated with the loss of blood-glucose homeostasis in diabetes. The biological mechanism of such fragility is still unknown; therefore, there are perceived opportunities that could eventually lead to new means to manage type 2 diabetes. The hierarchical structures of glycogen particles are controlled by the underlying biosynthesis/degradation process that involves various enzymes, including, for example, glycogen synthase (GS) and glycogen-branching enzyme (GBE). Recent studies have shown that fragile glycogen α particles in diabetic mice have longer chains and a higher molecular density compared to wild-type mice, indicating an enhanced enzymatic activity ratio of GS to GBE in diabetes. Furthermore, it has been shown that with an improved blood glucose homeostasis, the glycogen fragility in diabetic mice can be restored by treatment with active ingredients from traditional Chinese medicine, yet the underlying mechanism is unknown. In this review, we summarize recent advances in understandings glycogen fragility from the perspectives of glycogen biosynthesis/degradation, glycogen hierarchical structures, and its relation to diabetes. Importantly, we for the first time set GS/GBE activity ratio as the therapeutic target for diabetes. K E Y W O R D Schain-length distribution, glycogen biosynthesis, glycogen degradation, particle size distribution 4 | LI and HU ACKNOWLEDGMENTSWe would like to thank Prof. Robert G. Gilbert (The University of Queensland) for his mentor guidance and intelligent work in the research field of studying molecular structure of glycogen. We would also like to thank the financial support from

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“…Glycogen was extracted following a previous procedure [15]. Approximately 1.5 g of rat liver was homogenized in 25 mL of glycogen isolation buffer (50 mM Tris, pH 8, 150 mM NaCl, 2 mM EDTA, 50 mM NaF and 5 mM sodium pyrophosphate).…”

Section: Purification and Extraction Of Glycogenmentioning

confidence: 99%