Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation.

BACKGROUND: O-GlcNAcylation is the enzymatic addition of a sugar, O-linked ß-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak. METHODS: O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2. RESULTS: We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak. CONCLUSIONS: These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

Correlative imaging using super-resolution fluorescence microscopy and soft X-ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development.

Active virosomes (AVs) are derivatives of viruses, broadly similar to 'parent' pathogens, with an outer envelope that contains a bespoke genome coding for four to five viral proteins capable of eliciting an antigenic response. AVs are essentially novel vaccine formulations that present on their surface selected viral proteins as antigens. Once administered, they elicit an initial 'anti-viral' immune response. AVs are also internalised by host cells where their cargo viral genes are used to express viral antigen(s) intracellularly. These can then be transported to the host cell surface resulting in a second wave of antigen exposure and a more potent immuno-stimulation. A new 3D correlative microscopy approach is used here to provide a robust analytical method for characterisation of Zika- and Chikungunya-derivatised AV populations including vesicle size distribution and variations in antigen loading. Manufactured batches were compared to assess the extent and nature of batch-to-batch variations. We also show preliminary results that verify antigen expression on the surface of host cells. We present here a reliable and efficient high-resolution 3D imaging regime that allows the evaluation of the microstructure and biochemistry of novel vaccine formulations such as AVs.

A novel combination of microscopies involving X-ray and laser light has been developed at the correlative cryo-imaging beamline B24 of the UK synchrotron which can be used to analyse across- and within-batch variability of active virosome vaccine formulations. We use 3D fluorescence imaging to localise viral components within vaccine vesicles and soft X-ray tomography to characterise sample variability and impact upon delivery to cells. Moreover, we offer the next step in automation of data processing and evaluation to further enable rapid assessment of exosome-based vaccines. Active virosome vaccines are suspensions of membrane-bounded vesicles that carry antigens and genetic material from select viral pathogens. These elicit both an initial immune response through their introduction and a subsequent sustained antigenic potential via gene expression in host cells. In this case, as in all novel vaccine formulations, rapid assessment and batch standardisation are of paramount importance for the medical community and the methods described here provide a robust way of quick and efficient assessment and validation of formulations during research and development and at the production stages.

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells.

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

Protein O-GlcNAcylation in the heart.

O-GlcNAcylation is a ubiquitous post-translational modification that is extremely labile and plays a significant role in physiology, including the heart. Sustained activation of cardiac O-GlcNAcylation is frequently associated with alterations in cellular metabolism, leading to detrimental effects on cardiovascular function. This is particularly true during conditions such as diabetes, hypertension, cardiac remodelling, heart failure and arrhythmogenesis. Paradoxically, transient elevation of cardiac protein O-GlcNAcylation can also exert beneficial effects in the heart. There is compelling evidence to suggest that a complex interaction between O-GlcNAcylation and phosphorylation also exists in the heart. Beyond direct functional consequences on cardiomyocytes, O-GlcNAcylation also acts indirectly by altering the function of transcription factors that affect downstream signalling. This review focuses on the potential cardioprotective role of protein O-GlcNAcylation during ischaemia-reperfusion injury, the deleterious consequences of chronically elevated O-GlcNAc levels, the interplay between O-GlcNAcylation and phosphorylation in the cardiomyocytes and the effects of O-GlcNAcylation on other major non-myocyte cell types in the heart.

Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures.

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

FKBPs facilitate the termination of spontaneous Ca2+ release in wild-type RyR2 but not CPVT mutant RyR2.

FK506-binding proteins 12.6 (FKBP12.6) and 12 (FKBP12) tightly associate with the cardiac ryanodine receptor (RyR2). Studies suggest that dissociation of FKBP12.6 from mutant forms of RyR2 contributes to store overload-induced Ca(2+) release (SOICR) and Ca(2+)-triggered arrhythmias. However, these findings are controversial. Previous studies focused on the effect of FKBP12.6 on the initiation of SOICR and did not explore changes in the termination of Ca(2+) release. Less is known about FKBP12. We aimed to determine the effect of FKBP12.6 and FKBP12 on the termination of SOICR. Using single-cell imaging, in cells expressing wild-type RyR2, we found that FKBP12.6 and FKBP12 significantly increase the termination threshold of SOICR without changing the activation threshold of SOICR. This effect, dependent on the association of each FKBP with RyR2, reduced the magnitude of Ca(2+) release but had no effect on the propensity for SOICR. In contrast, neither FKBP12.6 nor FKBP12 was able to regulate an arrhythmogenic variant of RyR2, despite a conserved protein interaction. Our results suggest that both FKBP12.6 and FKBP12 play critical roles in regulating RyR2 function by facilitating the termination of SOICR. The inability of FKBPs to mediate a similar effect on the mutant RyR2 represents a novel mechanism by which mutations within RyR2 lead to arrhythmia.

HEXANE EXTRACT OF Dacryodes edulis FRUITS POSSESSES ANTI-DIABETIC AND HYPOLIPIDAEMIC POTENTIALS IN ALLOXAN DIABETES OF RATS.

BACKGROUND: The fruit extract of Dacryodes edulis (D. edulis), the African pear or plum, a tree indigenous to the humid tropics has been used for managing wounds, skin diseases, sickle cell anaemia, dysentery and fever in some African nations. In South Eastern Nigeria, 'herbal doctors' include its marshed fruit in decoctions administered to diabetic patients. However no scientific substantiation of their claim and use exist in literature. Hence, the need to evaluate the antidiabetic and hypolipidaemic activity of hexane extracts of D. edulis fruit in alloxanised animal model. MATERIALS AND METHODS: Sub-toxic doses between 400 and 1600mg/kg were orally administered sub-chronically to alloxan-induced diabetic rats for 15 days and compared to glibenclamide (2.5mg/kg). The glycaemia levels, body weights, lipid profile, blood urea, creatinine and liver enzyme levels were measured. Basic histology of the pancreatic tissue was also performed to examine the effects on the pancreas as possible mechanistic lead. RESULTS: Oral acute dosing of D. edulis hexane extract decreased blood glucose levels, while sub-chronic treatment of the extract down-regulated significantly hyperglycaemia, total cholesterol, triglycerides, LDL-C, ALT and ALP levels. However, the HDL-C levels increased significantly. Histopathological examination of the pancreatic tissues after sub-chronic treatment showed that glibenclamide and the highest dose of the extract 1600mg/kg restored the afore-damaged pancreatic ß-cell architecture. CONCLUSION: Our findings portend that D. edulis hexane fruit extract possesses hypoglycaemic and hypolipidaemic activities as well as restoration of the pancreatic architecture without any obvious untoward hepatic damages, suggesting that its use in the management of the diabetes may be valid. List of Non-standard abbreviations:D. edulis = dacryode edulis, DEnH = Dacryodes edulis n-hexane fruit extract, B.wt. = Body weight, Per os = Oral administration, NC = normal control, DC =Diabetic control, SC = Standard control, LDL-C = low density lipoprotein cholesterol, HDL-C = High density lipoprotein cholesterol, TG = Triglyceride, TC = Total cholesterol.