Michael J. Knight

Expression, purification, and reconstitution of a diatom silicon transporter

The synthesis and manipulation of silicon materials on the nanoscale are core themes in nanotechnology research. Inspiration is increasingly being taken from the natural world because the biological mineralization of silicon results in precisely controlled, complex silica structures with dimensions from the millimeter to the nanometer. One fascinating example of silicon biomineralization occurs in the diatoms, unicellular algae that sheath themselves in an ornate silica-based cell wall. To harvest silicon from the environment, diatoms have developed a unique family of integral membrane proteins that bind to a soluble form of silica, silicic acid, and transport it across the cell membrane to the cell interior. These are the first proteins shown to directly interact with silicon, but the current understanding of these specific silicon transport proteins is limited by the lack of in vitro studies of structure and function. We report here the recombinant expression, purification, and reconstitution of a silicon transporter from the model diatom Thalassiosira pseudonana. After using GFP fusions to optimize expression and purification protocols, a His(10)-tagged construct was expressed in Saccharomyces cerevisiae, solubilized in the detergent Fos-choline-12, and purified by affinity chromatography. Size-exclusion chromatography and particle sizing by dynamic light scattering showed that the protein was purified as a homotetramer, although nonspecific oligomerization occurred at high protein concentrations. Circular dichroism measurements confirmed sequence-based predictions that silicon transporters are α-helical membrane proteins. Silicic acid transport could be established in reconstituted proteoliposomes, and silicon uptake was found to be dependent upon an applied sodium gradient. Transport data across different substrate concentrations were best fit to the sigmoidal Hill equation, with a K(0.5) of 19.4 ± 1.3 μM and a cooperativity coefficient of 1.6. Sodium binding was noncooperative with a K(m)(app) of 1.7 ± 1.0 mM, suggesting a transport silicic acid:Na(+) stoichiometry of 2:1. These results provide the basis for a full understanding of both silicon transport in the diatom and protein-silicon interactions in general.

Magnetic Resonance Imaging to Detect Early Molecular and Cellular Changes in Alzheimer's Disease

Recent pharmaceutical trials have demonstrated that slowing or reversing pathology in Alzheimers disease is likely to be possible only in the earliest stages of disease, perhaps even before significant symptoms develop. Pathology in Alzheimers disease accumulates for well over a decade before symptoms are detected giving a large potential window of opportunity for intervention. It is therefore important that imaging techniques detect subtle changes in brain tissue before significant macroscopic brain atrophy. Current diagnostic techniques often do not permit early diagnosis or are too expensive for routine clinical use. Magnetic Resonance Imaging (MRI) is the most versatile, affordable, and powerful imaging modality currently available, being able to deliver detailed analyses of anatomy, tissue volumes, and tissue state. In this mini-review, we consider how MRI might detect patients at risk of future dementia in the early stages of pathological change when symptoms are mild. We consider the contributions made by the various modalities of MRI (structural, diffusion, perfusion, relaxometry) in identifying not just atrophy (a late-stage AD symptom) but more subtle changes reflective of early dementia pathology. The sensitivity of MRI not just to gross anatomy but to the underlying “health” at the cellular (and even molecular) scales, makes it very well suited to this task.

Direct evidence of the molecular basis for biological silicon transport

Diatoms are an important group of eukaryotic algae with a curious evolutionary innovation: they sheath themselves in a cell wall made largely of silica. The cellular machinery responsible for silicification includes a family of membrane permeases that recognize and actively transport the soluble precursor of biosilica, silicic acid. However, the molecular basis of silicic acid transport remains obscure. Here, we identify experimentally tractable diatom silicic acid transporter (SIT) homologues and study their structure and function in vitro, enabled by the development of a new fluorescence method for studying substrate transport kinetics. We show that recombinant SITs are Na+/silicic acid symporters with a 1:1 protein: substrate stoichiometry and KM for silicic acid of 20 μM. Protein mutagenesis supports the long-standing hypothesis that four conserved GXQ amino acid motifs are important in SIT function. This marks a step towards a detailed understanding of silicon transport with implications for biogeochemistry and bioinspired materials.

The yeast enzyme Eht1 is an octanoyl-CoA:ethanol acyltransferase that also functions as a thioesterase

Fatty acid ethyl esters are secondary metabolites that are produced during microbial fermentation, in fruiting plants and in higher organisms during ethanol stress. In particular, volatile medium‐chain fatty acid ethyl esters are important flavour compounds that impart desirable fruit aromas to fermented beverages, including beer and wine. The biochemical synthesis of medium‐chain fatty acid ethyl esters is poorly understood but likely involves acyl‐CoA:ethanol O‐acyltransferases. Here, we characterize the enzyme ethanol hexanoyl transferase 1 (Eht1) from the brewers yeast Saccharomyces cerevisiae. Full‐length Eht1 was successfully overexpressed from a recombinant yeast plasmid and purified at the milligram scale after detergent solubilization of sedimenting membranes. Recombinant Eht1 was functional as an acyltransferase and, unexpectedly, was optimally active toward octanoyl‐CoA, with kcat = 0.28 ± 0.02/s and KM = 1.9 ± 0.6 μm. Eht1 was also revealed to be active as a thioesterase but was not able to hydrolyse p‐nitrophenyl acyl esters, in contrast to the findings of a previous study. Low‐resolution structural data and site‐directed mutagenesis provide experimental support for a predicted α/β‐hydrolase domain featuring a Ser–Asp–His catalytic triad. The S. cerevisiae gene YBR177C/EHT1 should thus be reannotated as coding for an octanoyl‐CoA:ethanol acyltransferase that can also function as a thioesterase.

Anisotropy of spin-echo T 2 relaxation by magnetic resonance imaging in the human brain in vivo

BACKGROUND: The use of T2 relaxation contrast, as measured by MRI, is particularly commonplace in non-invasive assessment of the brain. However, the mechanisms and uses of T2 relaxation in the brain are still not fully understood. The hypothesis that T2 relaxation may show anisotropy in the human brain was studied at 3 T. T2 anisotropy refers to the variation of T2 in ordered structures with respect to the direction of the applied magnetic field. METHODS: Using a 3 T clinical MRI scanner, we made quantitative multi-contrast spin-echo T2 and diffusion tensor imaging (DTI) measurements in healthy volunteers, repeating the measurements with the subject’s head oriented differently relative to the applied field, for the measurement of possible spin-echo T2 anisotropy. RESULTS: We report T2 relaxation anisotropy measurements and present a means for visualising it according to the principal orientation of ordered structures in the brain parenchyma. We introduce a parameter for the model-free description of T2 anisotropy, namely the T2 “fractional anisotropy”, similar to that used to describe anisotropy of translational diffusion. This parameterisation enables the overall level of anisotropy in T2 across a chosen region or tissue to be calculated. Anisotropic T2 relaxation was observed in both gray and white matter, though to a greater extent in the latter, with a strong relationship with the anisotropy of translational diffusion. This is evidenced by making repeat measurements with the subject’s head tilted to different angles relative to the applied magnetic field, by which means we observed the T2 at the same anatomical site to change. CONCLUSIONS: Relaxation anisotropy has a significant effect on T2 in the brain parenchyma. It has the potential to offer non-invasive access to tissue microstructure not available by other imaging modalities, and may be sensitive to pathology or noxious factors not detected by other means.

Diffusion-mediated nuclear spin phase decoherence in cylindrically porous materials

Graphical abstract

Magnetic Resonance Relaxation Anisotropy: Physical Principles and Uses in Microstructure Imaging

Magnetic resonance imaging (MRI) provides an excellent means of studying tissue microstructure noninvasively since the microscopic tissue environment is imprinted on the MRI signal even at macroscopic voxel level. Mesoscopic variations in magnetic field, created by microstructure, influence the transverse relaxation time (T2) in an orientation-dependent fashion (T2 is anisotropic). However, predicting the effects of microstructure upon MRI observables is challenging and requires theoretical insight. We provide a formalism for calculating the effects upon T2 of tissue microstructure, using a model of cylindrical magnetic field perturbers. In a cohort of clinically healthy adults, we show that the angular information in spin-echo T2 is consistent with this model. We show that T2 in brain white matter of nondemented volunteers follows a U-shaped trajectory with age, passing its minimum at an age of ∼30 but that this depends on the particular white matter tract. The anisotropy of T2 also interacts with age and declines with increasing age. Late-myelinating white matter is more susceptible to age-related change than early-myelinating white matter, consistent with the retrogenesis hypothesis. T2 mapping may therefore be incorporated into microstructural imaging.

Magnetic resonance scanning and image segmentation procedure at 3 T for volumetry of human hippocampal subfields

BACKGROUND: Recent evidence suggests that dementia affects hippocampal substructures differentially and thus identifying anatomical details of this structure is potentially clinically important. Visualising details of human hippocampal substructures in vivo is challenging by imaging due to the small size of the medial temporal lobe structure. METHODS: MRI data were acquired with a 3 T MR scanner using a 2D multi-echo spin echo pulse sequence at such a spatial resolution to reveal hippocampal subfield boundaries. These images were used to develop a manual segmentation procedure for the hippocampal subfields based on image contrast within the structures and/or geometric constraints by anatomical landmarks. RESULTS: The T2-images were used to devise a segmentation protocol for Cornu Ammonis (CA) CA1, CA2, CA3, dentate gyrus, subiculum and lumped Stratum Lacunosum + Stratum Moleculare + Stratum Radiatum. The segmentation protocol was applied to MRI data from healthy young and aged controls as well as a small cohort of mild cognitive impairment (MCI) subjects. The reported subfield volumes showed high levels of inter-rater reliability arguing for potential as a tool in documenting subfield volumetry in clinical research. CONCLUSIONS: A comprehensive and robust MRI protocol which allows the labelling of six separate hippocampal subfields from images acquired with a 3 T clinical scanner is presented to promote hippocampal subfield volumetry of clinical cohorts.

Timing the ischaemic stroke by 1H-MRI: improved accuracy using absolute relaxation times over signal intensities.

One in four ischaemic stroke patients are ineligible for thrombolytic treatment due to unknown onset time. Quantification of absolute MR relaxation times and signal intensities are potential methods for estimating stroke duration. We compared the accuracy of these approaches and determined whether changes in relaxation times and signal intensities identify the same ischaemic tissue as diffusion MRI. Seven Wistar rats underwent permanent middle cerebral artery occlusion to induce focal ischaemia and were scanned at six time points. The trace of the diffusion tensor (DAV), T1&rgr; and T2 were acquired at 4.7 T. Results show relaxation times, and signal intensities of the MR relaxation parameters increase linearly with ischaemia duration (P<0.001). Using T1&rgr; and T2 relaxation times, an estimate of 4.5 h after occlusion has an uncertainty of ±12 and ±35 min, respectively, compared with over 50 min for signal intensities. In addition, we present a pixel-by-pixel method that simultaneously estimates stroke onset time and identifies potentially irreversible ischaemic tissue using absolute relaxation times. This method demonstrates signal intensity changes during ischaemia display an ambiguous pattern and highlights the possibility that diffusion MRI overestimates the true extent of irreversible ischaemia. In conclusion, quantification of absolute relaxation times at a single time point enables a more accurate estimation of stroke duration than signal intensities and provides more information about tissue status in ischaemia.

Cerebral White Matter Maturation Patterns in Preterm Infants: An MRI T2 Relaxation Anisotropy and Diffusion Tensor Imaging Study: Multiparameteric MRI of White Matter in Preterm Brain

Preterm birth is associated with worse neurodevelopmental outcome, but brain maturation in preterm infants is poorly characterized with standard methods. We evaluated white matter (WM) of infant brains at term‐equivalent age, as a function of gestational age at birth, using multimodal magnetic resonance imaging (MRI).