A solution state NMR study of the structure and ligand binding properties of the human C-type lectin DC-SIGNR

The protein DC-SIGNR (Dendritic-cell specific ICAM3 grabbing non-integrin related) is a C-type (calcium-dependent) lectin, which binds highly-branched mannose oligosaccharides. DC-SIGNR interacts with a range of deadly diseases via surface glycans on pathogenic glycoproteins, and the ability of DC-SIGNR to increase the rate of infection of viruses including human immunodeficiency virus (HIV) and hepatitis C virus (HCV) makes the study of DC-SIGNR/oligosaccharide interactions very attractive. The research described in this thesis sought to gain insight into the calcium and ligand binding properties of the DC-SIGNR carbohydrate recognition domain (CRD) in solution by utilising solution state nuclear magnetic resonance spectroscopy (NMR). A protocol for the production of uniformly 15N /13C labelled DC-SIGNR CRD was developed, allowing the acquisition of heteronuclear NMR experiments and the first assignment of the calcium-bound (holo) DC-SIGNR CRD to be reported. The assignment has allowed investigation of calcium and glycan binding, as well as the pH dependence of the DC-SIGNR CRD. The data presented in this thesis reveal that the DC-SIGNR CRD is highly dynamic in the calcium-free state, with the addition of calcium resulting in global conformational and dynamic changes throughout the CRD. While calcium binding hinders the protein dynamics (particularly in the calcium binding regions), a large degree of mobility remains. The evidence that ligands are released at low pH suggests that DC-SIGNR may act as an endocytic receptor. In addition to calcium binding, interactions of the DC-SIGNR CRD with a range of ligands were investigated. In particular, interactions with the oligosaccharide Man9GlcNAc (present on the HIV viral envelope) are described, representing the first direct study of the CRD interacting with a diseaseassociated ligand. The glycans employed in this study all bind to the primary calcium binding site, supporting previous crystal data. However, each glycan displays distinct patterns of chemical shift perturbations implying that they each have different, extended binding modes. Particularly striking is the difference between the disease-associated Man9GlcNAc ligand and the ligand present in a previously published crystal structure, (GlcNAc)2Man3. An investigation of the dynamics of the CRD in the holo form and bound to the ligand Man5 shows that the CRD is highly dynamic and that glycan binding further hinders, but does not abolish, the molecular motions. The dynamics data also suggests that a ligand-induced conformational change may occur and indicates potential new binding sites which are not present in any published crystal structures. The dynamic nature of the DC-SIGNR CRD may explain the wide range of ligand specificities and affinities of the C-type lectin scaffold and suggests that the study of the ligand binding properties and dynamics of proteins such as DC-SIGNR in solution is essential to further understanding of this class of proteins

1H NMR-Linked Metabolomics Analysis of Liver from a Mouse Model of NP-C1 Disease

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link

A blood-based metabolomics test to distinguish relapsing–remitting and secondary progressive multiple sclerosis: addressing practical considerations for clinical application

The transition from relapsing–remitting multiple sclerosis (RRMS) to secondary progressive MS (SPMS) represents a huge clinical challenge. We previously demonstrated that serum metabolomics could distinguish RRMS from SPMS with high diagnostic accuracy. As differing sample-handling protocols can affect the blood metabolite profile, it is vital to understand which factors may influence the accuracy of this metabolomics-based test in a clinical setting. Herein, we aim to further validate the high accuracy of this metabolomics test and to determine if this is maintained in a ‘real-life’ clinical environment. Blood from 31 RRMS and 28 SPMS patients was subjected to different sample-handling protocols representing variations encountered in clinics. The effect of freeze–thaw cycles (0 or 1) and time to erythrocyte removal (30, 120, or 240 min) on the accuracy of the test was investigated. For test development, samples from the optimised protocol (30 min standing time, 0 freeze–thaw) were used, resulting in high diagnostic accuracy (mean ± SD, 91.0 ± 3.0%). This test remained able to discriminate RRMS and SPMS samples that had experienced additional freeze–thaw, and increased standing times of 120 and 240 min with accuracies ranging from 85.5 to 88.0%, because the top discriminatory metabolite biomarkers from the optimised protocol remained discriminatory between RRMS and SPMS despite these sample-handling variations. In conclusion, while strict sample-handling is essential for the development of metabolomics-based blood tests, the results confirmed that the RRMS vs. SPMS test is resistant to sample-handling variations and can distinguish these two MS stages in the clinics

Foveal changes in aquaporin‐4 antibody seropositive neuromyelitis optica spectrum disorder are independent of optic neuritis and not overtly progressive

Background and purpose: Foveal changes were reported in aquaporin-4 antibody (AQP4-Ab) seropositive neuromyelitis optica spectrum disorder (NMOSD) patients; however, it is unclear whether they are independent of optic neuritis (ON), stem from subclinical ON or crossover from ON in fellow eyes. Fovea morphometry and a statistical classification approach were used to investigate if foveal changes in NMOSD are independent of ON and progressive. Methods: This was a retrospective longitudinal study of 27 AQP4-IgG + NMOSD patients (49 eyes; 15 ON eyes and 34 eyes without a history of ON [NON eyes]), follow-up median (first and third quartile) 2.32 (1.33-3.28), and 38 healthy controls (HCs) (76 eyes), follow-up median (first and third quartile) 1.95 (1.83-2.54). The peripapillary retinal nerve fibre layer thickness and the volume of combined ganglion cell and inner plexiform layer as measures of neuroaxonal damage from ON were determined by optical coherence tomography. Nineteen foveal morphometry parameters were extracted from macular optical coherence tomography volume scans. Data were analysed using orthogonal partial least squares discriminant analysis and linear mixed effects models. Results: At baseline, foveal shape was significantly altered in ON eyes and NON eyes compared to HCs. Discriminatory analysis showed 81% accuracy distinguishing ON vs. HCs and 68% accuracy in NON vs. HCs. NON eyes were distinguished from HCs by foveal shape parameters indicating widening. Orthogonal partial least squares discriminant analysis discriminated ON vs. NON with 76% accuracy. In a follow-up of 2.4 (20.85) years, no significant time-dependent foveal changes were found. Conclusion: The parafoveal area is altered in AQP4-Ab seropositive NMOSD patients suggesting independent neuroaxonal damage from subclinical ON. Longer follow-ups are needed to confirm the stability of the parafoveal structure over time

In FUS[1−359]‐tg mice O,S-dibenzoyl thiamine reduces muscle atrophy, decreases glycogen synthase kinase 3 beta, and normalizes the metabolome

peer reviewedMutations in the gene encoding the RNA/DNA-binding protein Fused in Sarcoma (FUS) have been detected in familial amyotrophic lateral sclerosis (ALS) patients. FUS has been found to be a critical component of the oxidative damage repair complex that might explain its role in neurodegeneration. Here, we examined what impact antioxidant treatment with thiamine (vitamine B1), or its more bioavailable derivative O,S- dibenzoylthiamine (DBT), would have on the hallmarks of pathology in the FUS[1− 359]-transgenic mouse model of ALS. From 8-weeks old, in the pre-symptomatic phase of disease, animals received either thiamine, DBT (200 mg/kg/day), or vehicle for 6 weeks. We examined physiological, behavioral, molecular and histological outcomes, as well as the serum metabolome using nuclear magnetic resonance (NMR). The DBT-treated mice displayed improvements in physiological outcomes, motor function and muscle atrophy compared to vehicle, and the treatment normalized levels of brain glycogen synthase kinase-3β (GSK-3β), GSK-3β mRNA and IL-1β mRNA in the spinal cord. Analysis of the metabolome revealed an increase in the levels of choline and lactate in the vehicle-treated FUS mutants alone, which is also elevated in the cerebrospinal fluid of ALS patients, and reduced glucose and lipoprotein concentrations in the FUS[1− 359]-tg mice, which were not the case in the DBT- treated mutants. The administration of thiamine had little impact on the outcome measures, but it did normalize circulating HDL levels. Thus, our study shows that DBT therapy in FUS mutants is more effective than thiamine and highlights how metabolomics may be used to evaluate therapy in this model.PhytoAPP E

Determination of CSF GFAP, CCN5, and vWF Levels Enhances the Diagnostic Accuracy of Clinically Defined MS From Non-MS Patients With CSF Oligoclonal Bands

BackgroundInclusion of cerebrospinal fluid (CSF) oligoclonal IgG bands (OCGB) in the revised McDonald criteria increases the sensitivity of diagnosis when dissemination in time (DIT) cannot be proven. While OCGB negative patients are unlikely to develop clinically definite (CD) MS, OCGB positivity may lead to an erroneous diagnosis in conditions that present similarly, such as neuromyelitis optica spectrum disorders (NMOSD) or neurosarcoidosis.ObjectiveTo identify specific, OCGB-complementary, biomarkers to improve diagnostic accuracy in OCGB positive patients.MethodsWe analysed the CSF metabolome and proteome of CDMS (n=41) and confirmed non-MS patients (n=64) comprising a range of CNS conditions routinely encountered in neurology clinics.ResultsOCGB discriminated between CDMS and non-MS with high sensitivity (85%), but low specificity (67%), as previously described. Machine learning methods revealed CCN5 levels provide greater accuracy, sensitivity, and specificity than OCGB (79%, +5%; 90%, +5%; and 72%, +5% respectively) while glial fibrillary acidic protein (GFAP) identified CDMS with 100% specificity (+33%). A multiomics approach improved accuracy further to 90% (+16%).ConclusionThe measurement of a few additional CSF biomarkers could be used to complement OCGB and improve the specificity of MS diagnosis when clinical and radiological evidence of DIT is absent

Chronically elevated branched chain amino acid levels are pro-arrhythmic

Aims. Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. Methods and results. We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6–9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4–5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs—leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. Conclusions. Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states