Kinetic study of the thermal denaturation of a hyperthermostable extracellular α-amylase from <i>Pyrococcus furiosus</i>

AbstractHyperthermophilic enzymes are of industrial importance and interest, especially due to their denaturation kinetics at commercial sterilisation temperatures inside safety indicating time–temperature integrators (TTIs). The thermal stability and irreversible thermal inactivation of native extracellular Pyrococcus furiosus α-amylase were investigated using differential scanning calorimetry, circular dichroism and Fourier transform infrared spectroscopy. Denaturation of the amylase was irreversible above a Tm of approximately 106°C and could be described by a one-step irreversible model. The activation energy at 121°C was found to be 316kJ/mol. Using CD and FT-IR spectroscopy it was shown that folding and stability greatly increase with temperature. Under an isothermal holding temperature of 121°C, the structure of the PFA changes during denaturation from an α-helical structure, through a β-sheet structure to an aggregated protein. Such data reinforces the use of P. furiosus α-amylase as a labile species in TTIs

Variations in Duschinsky rotations in m-fluorotoluene and m-chlorotoluene during excitation and ionization

We investigate Duschinsky rotation/mixing between three vibrations for both m-fluorotoluene (mFT) and m-chlorotoluene (mClT), during electronic excitation and ionization. In the case of mFT, we investigate both the S1 → S0 electronic transition and the D0+ ← S1 ionization, by two-dimensional laser-induced fluorescence (2D-LIF) and zero-electron-kinetic energy (ZEKE) spectroscopy, respectively; for mClT, only the D0+ ← S1 ionization was investigated, by ZEKE spectroscopy. The Duschinsky mixings are different in the two molecules, owing to shifts in vibrational wavenumber and variations in the form of the fundamental vibrations between the different electronic states. There is a very unusual behavior for two of the mFT vibrations, where apparently different conclusions for the identity of two S1 vibrations arise from the 2D-LIF and ZEKE spectra. We compare the experimental observations to the calculated Duschinsky matrices, finding that these successfully pick up the key geometric changes associated with each electronic transition and so are successful in qualitatively explaining the vibrational activity in the spectra. Experimental values for a number of vibrations across the S0, S1, and D0+ states are reported and found to compare well to those calculated. Assignments are made for the observed vibration-torsion (“vibtor”) bands, and the effect of vibrational motion on the torsional potential is briefly discussed

Molecular pathology and synaptic loss in primary tauopathies: A [18F]AV-1451 and [11C]UCB-J PET study

The relationship between in vivo synaptic density and tau burden in primary tauopathies is key to understanding the impact of tauopathy on functional decline and in informing new early therapeutic strategies. In this cross-sectional observational study, we determine the in vivo relationship between synaptic density and molecular pathology, in the primary tauopathies of Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD), as a function of disease severity. Twenty three patients with PSP, and twelve patients with Corticobasal Syndrome (CBS) were recruited from a tertiary referral centre. Nineteen education, sex and gender-matched control participants were recruited from the National Institute for Health Research ‘Join Dementia Research’ platform. Cerebral synaptic density and molecular pathology, in all participants, were estimated using PET imaging with the radioligands [11C]UCB-J and [18F]AV-1451, respectively. Patients with CBS also underwent amyloid PET imaging with [11C]PiB to exclude those with likely Alzheimer’s pathology – we refer to the amyloid negative cohort as having CBD although acknowledge other pathologies exist. Disease severity was assessed with the PSP rating scale; regional non-displaceable binding potentials (BPND) of [11C]UCB-J and [18F]AV-1451 were estimated in regions of interest from the Hammersmith Atlas, excluding those with known off-target binding for [18F]AV-1451. As an exploratory analysis, we also investigated the relationship between molecular pathology in cortical brain regions, and synaptic density in subcortical areas. Across brain regions, there was a positive correlation between [11C]UCB-J and [18F]AV-1451 BPND (ß = 0.4, t = 3.6, p = 0.001), independent of age or time between PET scans. However, this correlation became less positive as a function of disease severity in patients (ß = - 0.02, t = -2.9, p = 0.007, R = -0.41). Between regions, cortical [18F]AV-1451 binding was negatively correlated with synaptic density in subcortical areas (caudate nucleus, putamen). Brain regions with higher synaptic density are associated with a higher [18F]AV-1451 binding in PSP/CBD, but this association diminishes with disease severity. Moreover, higher cortical [18F]AV-1451 binding correlates with lower subcortical synaptic density. Longitudinal imaging is required to confirm the mediation of synaptic loss by molecular pathology. However, the effect of disease severity suggests a biphasic relationship between synaptic density and molecular pathology with synapse rich regions vulnerable to accrual of pathological aggregates, followed by a loss of synapses in response to pathology. Given the importance of synaptic function for cognition, our study elucidates the pathophysiology of primary tauopathies and may inform the design of future clinical trials.Cambridge Centre for Parkinson-Plus (RG95450); the National Institute for Health Research Cambridge Biomedical Research Centre (BRC-1215-20014); the PSP Association (“MAPT-PSP” study), and the Association of British Neurologists, Patrick Berthoud Charitable Trust (RG99368)

The Aortic-Femoral Arterial Stiffness Gradient: An Atherosclerosis Risk in Communities (ARIC) Study

Background: The aortic to femoral arterial stiffness gradient (af-SG) may be a novel measure of arterial health and cardiovascular disease (CVD) risk, but its association with CVD risk factors and CVD status, and whether or not they differ from the referent measure, carotid-femoral pulse-wave velocity (cfPWV), is not known. Method: Accordingly, we compared the associations of the af-SG and cfPWV with (i) age and traditional CVD risk factors and (ii) CVD status. We evaluated 4183 older-aged (75.2 ± 5.0 years) men and women in the community-based Atherosclerosis Risk in Communities (ARIC) Study. cfPWV and femoral-ankle PWV (faPWV) were measured using an automated cardiovascular screening device. The af-SG was calculated as faPWV divided by cfPWV. Associations of af-SG and cfPWV with age, CVD risk factors (age, BMI, blood pressure, heart rate, glucose and blood lipid levels) and CVD status (hypertension, diabetes, coronary heart disease, heart failure, stroke) were determined using linear and logistic regression analyses. Results: (i) the af-SG and cfPWV demonstrated comparable associations with age and CVD risk factors, except BMI. (ii) a low af-SG was associated with diabetes, coronary heart disease, heart failure and stroke, whilst a high cfPWV was only associated with diabetes. Conclusion: Although future studies are necessary to confirm clinical utility, the af-SG is a promising tool that may provide a unique picture of hemodynamic integration and identification of CVD risk when compared with cfPWV

Associations of lower-limb atherosclerosis and arteriosclerosis with cardiovascular risk factors and disease in older adults:The Atherosclerosis Risk in Communities (ARIC) study

BACKGROUND & AIMS Atherosclerosis and arteriosclerosis contribute to vascular aging and cardiovascular disease (CVD) risk. Both processes can be assessed simply in the lower-limbs and reflect systemic pathology. However, only atherosclerosis is routinely assessed, typically via ankle-brachial index (ABI). Arteriosclerosis can be assessed using femoral-ankle pulse wave velocity (faPWV), but no studies have identified whether ABI and faPWV similarly associate with overt CVD and risk factors, nor whether faPWV confers additional information. The aims of this study were to, (i) Compare associations of ABI and faPWV with traditional CVD risk factors, including age, sex, systolic blood pressure (SBP), high-density lipoprotein (HDL), total cholesterol (TC), smoking, and diabetes; and, ii) Determine the independent and additive associations of ABI and faPWV with a composite measure of prevalent CVD. METHODS We evaluated ABI and faPWV in 4,330 older-aged (75.3±5.0 years) adults using an oscillometric screening device. Associations between ABI and faPWV with CVD risk factors and CVD were determined using mixed-model linear- and logistic-regression. RESULTS ABI and faPWV were associated with age, HDL, and smoking. ABI was associated with sex, TC, diabetes. faPWV was associated with SBP. Both ABI and faPWV were inversely associated with CVD. Low ABI (≤0.9 vs. >0.9) and low faPWV (≤9.94 vs. >9.94) increased the odds of CVD by 2.41-fold (95% CI:1.85,3.17) and 1.46-fold (95% CI:1.23,1.74), respectively. The inverse association between faPWV and CVD was independent of ABI and CVD risk factors. CONCLUSION ABI and faPWV, measures of lower-limb atherosclerosis and arteriosclerosis, are independently associated with CVD risk factors and prevalent CVD. Assessment of faPWV may confer additional risk information beyond ABI

Toxin Neutralization Using Alternative Binding Proteins.

Animal toxins present a major threat to human health worldwide, predominantly through snakebite envenomings, which are responsible for over 100,000 deaths each year. To date, the only available treatment against snakebite envenoming is plasma-derived antivenom. However, despite being key to limiting morbidity and mortality among snakebite victims, current antivenoms suffer from several drawbacks, such as immunogenicity and high cost of production. Consequently, avenues for improving envenoming therapy, such as the discovery of toxin-sequestering monoclonal antibodies against medically important target toxins through phage display selection, are being explored. However, alternative binding protein scaffolds that exhibit certain advantages compared to the well-known immunoglobulin G scaffold, including high stability under harsh conditions and low cost of production, may pose as possible low-cost alternatives to antibody-based therapeutics. There is now a plethora of alternative binding protein scaffolds, ranging from antibody derivatives (e.g., nanobodies), through rationally designed derivatives of other human proteins (e.g., DARPins), to derivatives of non-human proteins (e.g., affibodies), all exhibiting different biochemical and pharmacokinetic profiles. Undeniably, the high level of engineerability and potentially low cost of production, associated with many alternative protein scaffolds, present an exciting possibility for the future of snakebite therapeutics and merit thorough investigation. In this review, a comprehensive overview of the different types of binding protein scaffolds is provided together with a discussion on their relevance as potential modalities for use as next-generation antivenoms

Vibration-modified torsional potentials and vibration-torsion (“vibtor”) levels in the m-fluorotoluene cation

Zero-kinetic-energy (ZEKE) spectra are presented for m-fluorotoluene, employing different low-lying (≤350 cm−1) intermediate torsional and vibration-torsional (“vibtor”) levels of the S1 state. The adiabatic ionization energy (AIE) is found to be 71 997 ± 5 cm−1 (8.9265 ± 0.0006 eV). It is found that the activity in the ZEKE spectra varies greatly for different levels and is consistent with the assignments of the S1 levels deduced in the recent fluorescence study of Stewart et al. [J. Chem. Phys. 150, 174303 (2019)]. For cation torsional levels, the most intense band corresponds to changes in the torsional quantum number, in line with the known change in the phase of the torsional potential upon ionization. This leads to the observation of an unprecedented number of torsions and vibtor levels, with the pronounced vibtor activity involving out-of-plane vibrations. Interactions between levels involving torsions are discussed, with evidence presented, for the first time it is believed, for modification of a torsional potential induced by a vibration. Also, we discuss the possibility of distortion of the methyl group leading to a change from G6 molecular symmetry to Cs point group symmetry

Longitudinal Synaptic Loss in Primary Tauopathies: An In Vivo [11 C]UCB-J Positron Emission Tomography Study

BACKGROUND: Synaptic loss is characteristic of many neurodegenerative diseases; it occurs early and is strongly related to functional deficits. OBJECTIVE: In this longitudinal observational study, we determine the rate at which synaptic density is reduced in the primary tauopathies of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), and we test the relationship with disease progression. METHODS: Our cross-sectional cohort included 32 participants with probable PSP and 16 with probable CBD (all amyloid-negative corticobasal syndrome), recruited from tertiary care centers in the United Kingdom, and 33 sex- and age-matched healthy control subjects. Synaptic density was estimated by positron emission tomography imaging with the radioligand [11 C]UCB-J that binds synaptic vesicle 2A. Clinical severity and cognition were assessed by the PSP Rating Scale and the Addenbrooke's cognitive examination. Regional [11 C]UCB-J nondisplaceable binding potential was estimated in Hammersmith Atlas regions of interest. Twenty-two participants with PSP/CBD had a follow-up [11 C]UCB-J positron emission tomography scan after 1 year. We calculated the annualized change in [11 C]UCB-J nondisplaceable binding potential and correlated this with the change in clinical severity. RESULTS: We found significant annual synaptic loss within the frontal lobe (-3.5%, P = 0.03) and the right caudate (-3.9%, P = 0.046). The degree of longitudinal synaptic loss within the frontal lobe correlated with the rate of change in the PSP Rating Scale (R = 0.47, P = 0.03) and cognition (Addenbrooke's Cognitive Examination-Revised, R = -0.62, P = 0.003). CONCLUSIONS: We provide in vivo evidence for rapid progressive synaptic loss, correlating with clinical progression in primary tauopathies. Synaptic loss may be an important therapeutic target and outcome variable for early-phase clinical trials of disease-modifying treatments. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society