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Background and Purpose: Given the accepted multistep process of disease causation in amyotrophic lateral sclerosis (ALS), the present study was undertaken to determine the number of steps required for disease onset across each of the ALS phenotypes., Methods: Clinical and demographic data were prospectively accumulated using the Australian Motor Neurone Disease Registry (2005-2016), and age-specific incidence rates were calculated. Poisson regression was utilized to assess the relationship between log age-specific incidence and log age of onset, with McFadden's R 2 used to assess the goodness of fit of the model., Results: In total, 2647 ALS patients were included, with mean disease-onset age being 62.2 ± 12.1 years. A linear relationship between log incidence and log age was established across ALS phenotypes, with variable slope estimates: bulbar 5.1 (95% confidence interval [CI] 4.6-5.6); cervical 2.7 (95% CI 2.3-3.0); lumbar 3.5 (95% CI 3.2-3.9); flail arm 4.7 (95% CI 3.9-5.5); flail leg 3.6 (95% CI 2.6-4.5); primary lateral sclerosis 2.7 (95% CI 1.8-3.7). Slope estimates were significantly higher in the bulbar compared to the cervical, lumbar and primary lateral sclerosis phenotypes. McFadden's R 2 values were >0.4 for all phenotypes indicating excellent model fit., Discussion: A multistep process has been established across all ALS phenotypes with variable slope estimates, suggesting that the number of steps to develop disease is different across clinical presentations. Identification of mechanisms underlying slope estimate variability could exert pathophysiological significance., (© 2024 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

An estimated 65 million people globally suffer from post-acute sequelae of COVID-19 (PASC), with many experiencing cardiovascular symptoms (PASC-CVS) like chest pain and heart palpitations. This study examines the role of chronic inflammation in PASC-CVS, particularly in individuals with symptoms persisting over a year after infection. Blood samples from three groups-recovered individuals, those with prolonged PASC-CVS and SARS-CoV-2-negative individuals-revealed that those with PASC-CVS had a blood signature linked to inflammation. Trace-level pro-inflammatory cytokines were detected in the plasma from donors with PASC-CVS 18 months post infection using nanotechnology. Importantly, these trace-level cytokines affected the function of primary human cardiomyocytes. Plasma proteomics also demonstrated higher levels of complement and coagulation proteins in the plasma from patients with PASC-CVS. This study highlights chronic inflammation's role in the symptoms of PASC-CVS., Competing Interests: Competing interests: K.R.S. has historically been a consultant for Sanofi, Pfizer, Roche and NovoNordisk. The opinions and data presented in this manuscript are of the authors and are independent of these relationships. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Background: Amyotrophic lateral sclerosis (ALS) is a complex, late-onset, neurodegenerative disease with a genetic contribution to disease liability. Genome-wide association studies (GWAS) have identified ten risk loci to date, including the TNIP1/GPX3 locus on chromosome five. Given association analysis data alone cannot determine the most plausible risk gene for this locus, we undertook a comprehensive suite of in silico, in vivo and in vitro studies to address this., Methods: The Functional Mapping and Annotation (FUMA) pipeline and five tools (conditional and joint analysis (GCTA-COJO), Stratified Linkage Disequilibrium Score Regression (S-LDSC), Polygenic Priority Scoring (PoPS), Summary-based Mendelian Randomisation (SMR-HEIDI) and transcriptome-wide association study (TWAS) analyses) were used to perform bioinformatic integration of GWAS data (N cases = 20,806, N controls = 59,804) with 'omics reference datasets including the blood (eQTLgen consortium N = 31,684) and brain (N = 2581). This was followed up by specific expression studies in ALS case-control cohorts (microarray N total = 942, protein N total = 300) and gene knockdown (KD) studies of human neuronal iPSC cells and zebrafish-morpholinos (MO)., Results: SMR analyses implicated both TNIP1 and GPX3 (p < 1.15 × 10 -6 ), but there was no simple SNP/expression relationship. Integrating multiple datasets using PoPS supported GPX3 but not TNIP1. In vivo expression analyses from blood in ALS cases identified that lower GPX3 expression correlated with a more progressed disease (ALS functional rating score, p = 5.5 × 10 -3 , adjusted R 2 = 0.042, B effect = 27.4 ± 13.3 ng/ml/ALSFRS unit) with microarray and protein data suggesting lower expression with risk allele (recessive model p = 0.06, p = 0.02 respectively). Validation in vivo indicated gpx3 KD caused significant motor deficits in zebrafish-MO (mean difference vs. control ± 95% CI, vs. control, swim distance = 112 ± 28 mm, time = 1.29 ± 0.59 s, speed = 32.0 ± 2.53 mm/s, respectively, p for all < 0.0001), which were rescued with gpx3 expression, with no phenotype identified with tnip1 KD or gpx3 overexpression., Conclusions: These results support GPX3 as a lead ALS risk gene in this locus, with more data needed to confirm/reject a role for TNIP1. This has implications for understanding disease mechanisms (GPX3 acts in the same pathway as SOD1, a well-established ALS-associated gene) and identifying new therapeutic approaches. Few previous examples of in-depth investigations of risk loci in ALS exist and a similar approach could be applied to investigate future expected GWAS findings., (© 2021. The Author(s).)

The availability of high-field-strength magnetic resonance imaging (MRI) systems has brought about the development of techniques that aim to map myelination via the exploitation of various contrast mechanisms. Myelin mapping techniques have the potential to provide tools for the diagnosis and treatment of diseases, such as multiple sclerosis. In this study, we evaluated the sensitivity of T 2 *, frequency shift and susceptibility measures to myelin levels in a cuprizone mouse model of demyelination. The model was supplemented with two different dosages of fingolimod, a drug known to positively affect demyelination. A decrease in grey-white matter contrast with the cuprizone diet was observed for T 2 *, frequency shift and susceptibility measures, together with myelin basic protein antibody findings. These results indicate that T 2 *, frequency shift and susceptibility measures have the potential to act as biomarkers for myelination. Susceptibility was found to be the most sensitive measure to changes in grey-white matter contrast. In addition, fingolimod treatment was found to reduce the level of demyelination, with a larger dosage exhibiting a greater reduction in demyelination for the in vivo MRI results. Overall, susceptibility mapping appears to be a more promising tool than T 2 * or frequency shift mapping for the early diagnosis and treatment of diseases in which myelination is implicated., (Copyright © 2017 John Wiley & Sons, Ltd.)

The 1.8 A resolution structure of the glycosyl-enzyme intermediate formed on the retaining beta-1,4-xylanase from Bacillus circulans has been determined using X-ray crystallographic techniques. The 2-fluoro-xylose residue bound in the -1 subsite adopts a 2,5B (boat) conformation, allowing atoms C5, O5, C1, and C2 of the sugar to achieve coplanarity as required at the oxocarbenium ion-like transition states of the double-displacement catalytic mechanism. Comparison of this structure to that of a mutant of this same enzyme noncovalently complexed with xylotetraose [Wakarchuk et al. (1994) Protein Sci. 3, 467-475] reveals a number of differences beyond the distortion of the sugar moiety. Most notably, a bifurcated hydrogen bond interaction is formed in the glycosyl-enzyme intermediate involving Heta of Tyr69, the endocyclic oxygen (O5) of the xylose residue in the -1 subsite, and Oepsilon2 of the catalytic nucleophile, Glu78. To gain additional understanding of the role of Tyr69 at the active site of this enzyme, we also determined the 1.5 A resolution structure of the catalytically inactive Tyr69Phe mutant. Interestingly, no significant structural perturbation due to the loss of the phenolic group is observed. These results suggest that the interactions involving the phenolic group of Tyr69, O5 of the proximal saccharide, and Glu78 Oepsilon2 are important for the catalytic mechanism of this enzyme, and it is proposed that, through charge redistribution, these interactions serve to stabilize the oxocarbenium-like ion of the transition state. Studies of the covalent glycosyl-enzyme intermediate of this xylanase also provide insight into specificity, as contacts with C5 of the xylose moiety exclude sugars with hydroxymethyl substituents, and the mechanism of catalysis, including aspects of stereoelectronic theory as applied to glycoside hydrolysis.

The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pKa values. 13C-NMR titrations of xylanase, labeled with [delta-13C]glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032]. A similar pKa is measured for Glu172 when a glutamine is present at position 78. This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases.

A new mechanism-based inactivator of beta-1,4-xylanases, 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside, has been synthesized and used to trap the covalent intermediate formed during catalysis by Bacillus subtilis xylanase. Electrospray mass spectrometry confirmed the 1:1 stoichiometry of the incorporation of inactivator into the enzyme. Inactivation of xylanase followed the expected pseudo-first-order kinetic behavior, and kinetic parameters were determined. The intermediate trapped was relatively stable toward hydrolytic turnover (t1/2 = 350 min). However, turnover could be facilitated by transglycosylation following the addition of the acceptor benzyl thio-beta-xylobioside, thus demonstrating the catalytic competence of the trapped intermediate. Reactivation kinetic parameters for this process of kre = 0.03 min-1 and Kre = 46 mM were determined. The nucleophilic amino acid was identified as Glu78 by a tandem mass spectrometric technique which does not require the use of radiolabels. The peptic digest of the labeled enzyme was separated by high-performance liquid chromatography and the eluent fed into a tandem mass spectrometer via an electrospray ionization device. The labeled peptide was identified as one of m/z = 826 (doubly charged) which fragmented in the collision chamber between the mass analyzers with loss of the mass of a 2-fluoroxylobiosyl unit. Confirmation of the peptide identity was obtained both by tandem mass spectrometric sequencing and by Edman degradation of the purified peptide. Glu78 is completely conserved in all members of this xylanase family and indeed is shown to be located in the active site in the recently determined X-ray crystal structure.

The syntheses are described of methyl 4'-O-[4-S-(3-azi-4-alpha-D-glucopyranosyloxybutyl)-6-deoxy-4 -thio-alpha-D-xylo-hex-5-enopyranosyl]-alpha-maltoside (28), methyl 4-O-[4-S-(3-azi-4-alpha-maltosyloxybutyl)-6-deoxy-4-thio- alpha-D-xylo-hex-5-enopyranosyl]-alpha-D-glucopyranoside (29), and methyl 4-S-(3-azi-4-alpha-maltotriosyloxybutyl)-6-deoxy-4-thio- alpha-D-xylo-hex-5-enopyranoside (30), which are analogues of maltopentaose in which a central glucosyl unit is replaced by an acyclic, four-membered hydrocarbon spacer carrying a photolabile azi group. Only 30 was slowly cleaved by high concentrations of pancreatic alpha-amylase. The Ki values (mM) were 0.15 (28), 2.1 (29), and 2.5 (30). Deactivation of the enzyme by irradiation in the presence severely of 28-30 was 96.4%, 98.1%, and 40%, respectively. There is an indication for regiospecific photoaffinity labelling of the binding subsites of the enzyme.

2,3,6,2',3',4',6'-Hepta-O-methylmaltose diethyl dithioacetal (2), obtained under mild conditions from N-methyl-hepta-O-methyl-N-p-nitrophenyl-beta-maltosylamine (1), was converted into the disulfone 4. Storage of a solution of 4 in aqueous tetrahydrofuran gave 1,2-dideoxy-1,1-bis(ethylsulfonyl)hepta-O-methylmalt-1-en itol (5). Fragmentation of 5 with aqueous ammonia released 2,3,4,6-tetra-O-methyl-D-glucose. The degradation procedure has potential for the sequencing of branched oligosaccharides.

(1/2,5,6)-2-(3-Azibutylthio)-5,6-epoxy-3-cyclohexen-1-ol (1) was synthesized and was found to irreversibly inactivate beta-D-galactosidase (Escherichia coli). The inactivation was prevented by the presence of isopropyl 1-thio-beta-D-galactopyranoside (IPTG). The vinyloxirane group of 1 reacted with water and other nucleophiles, especially at higher pH values. Reaction of 1 with beta-D-galactosidase was slow enough so that a competitive-inhibition constant (Ki) of 29mM could be determined. The inhibition constant for (1,2/3,6)-6-(3-azibutylthio)-2-bromo-4-cyclohexene-1,3-diol (2), the precursor of the bireactant inhibitor 1, was 13 mM, while that of (1,3/2,4)-3-(3-azibutylthio)-5-cyclohexene-1,2,4-triol (3), the product formed when the reactant is allowed to react with water, was 23mM. After irradiation by light, beta-D-galactosidase that had initially been treated with the bireactant compound and then digested with trypsin, showed a new pattern of elution from h.p.l.c., indicating that there was reaction at two regions of the beta-D-galactosidase molecule.

O-Deacylation and S-deacylation of the diastereomers of 2-azido-4-S-benzoyl-4-mercaptobutyl 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranoside (9) with methanolic sodium methoxide and coupling of the resulting thiol to methyl 3,4-anhydro-6-deoxy-beta-L-arabino-hex-5-enopyranoside (2) gave the corresponding diastereomers of the spacer-modified disaccharide methyl 4-S-(3-azido-4-alpha-D-glucopyranosyloxybutyl)-6-deoxy-4-thio-alph a-D-xylo-hex-5-enopyranoside (10). Glucosylation of the diastereomers of 10 with alpha-cyclo-dextrin-CGTase and treatment of the products with beta-amylase gave the diastereomers of the spacer-modified oligosaccharides methyl 4-S-(3-azido-4-alpha-maltosyloxybutyl)-6-deoxy-4-thio-alpha-D-xylo -hex-5-enopyranosides (11) and 4-S-(3-azido-4-alpha-maltotriosyloxybutyl)-6-deoxy-4-thio-alpha-D- hex-5-enopyranosides (12). The diastereomers of 10 each had a good affinity for pancreatic alpha-amylase and the maltose-binding protein from E. coli. The affinities of the diastereomers of 11 and 12 were higher by at least one order of magnitude.

(R)- and (S)-2-Azido-1-methoxyethyl beta-D-glucopyranosides (16) and (17), (R)- and (S)-3-azido-1-methoxypropyl beta-D-glucopyranosides (18) and (19), (R,S)-4-azido-1-methoxybutyl beta-D-glucopyranoside (20), and (R,S)-5-azido-1-methoxypentyl beta-D-glucopyranoside (22) were synthesized from omega-substituted dimethyl acetals of acetaldehyde, propanal, butanal, and pentanal by trimethylsilyl triflate-catalysed transacetalation using 1-O-trimethysilyl 2,3,4,6-tetra-O-acetyl-beta-D-glucose (1) as acceptor. Most of the acetylated (R,S)-epimers could be resolved into pure compounds by column chromatography. Preliminary tests showed that the deacetylated acetal glucosides carrying omega-bromo, azido, or acetamido substituents in the aglycon are good substrates for beta-D-glucosidase from sweet almonds. The corresponding omega-amino derivatives of compounds 16, 19, 20, and 22, (R)-2-amino-1-methoxyethyl beta-D-glucopyranoside (23), (S)-3-amino-1-methoxypropyl beta-D-glucopyranoside (24), (R,S)-4-amino-1-methoxybutyl beta-D-glucopyranoside (25), and (R,S)-5-amino-1-methoxypentyl beta-D-glucopyranoside (26) proved almost completely resistant to beta-D-glucosidase. The stability of the glucosides against enzyme hydrolysis is dependent on the distance between the amino group and the anomeric center.