Open Access

Show Abstract ▼

Abstract: Purpose : The purpose of this study was to investigate the distribution of Ca, Fe and Zn using X-ray fluorescence in human RPE/Bruch’s membrane/choroid with and without early AMD. Methods : We used a set of unfixed frozen human retinal pigment epithelium (RPE)/choroid samples from young (n=1, female aged 34) and aged donors with (n=4, males, aged 73-78) and without (n=3, one female, aged 75-77) early AMD from Manchester Eye Tissue Repository. Using X-ray fluorescence microscopy (I18 Diamond light source, UK) with a 2 um beam, we obtained high-resolution Ca, Zn, Fe, sulphur, potassium, chloride and phosphorus maps covering areas up to 100 x 600 um2 of 30 um thick sections placed on quartz holders and scanned at room temperature Results : Calcification was observed in the 3 groups. In the 34-year old sample, sparse small Ca spherules (2x2 um2) at the RPE/Bruch’s membrane interface not colocalising with Zn or Fe. In aged samples, with and without AMD, calcified nodules within RPE cells, at RPE/Bruch’s membrane interface and within druse in AMD. Every calcified nodule colocalised with Zn. Quantification revealed two types (high Zn content, low Zn content). In aged donors without AMD, high-Zn calcified nodules with Ca concentration of 3083+1679 ppm (mean+SD) (maximum 12809+9311) and Zn 66+55 ppm (max 125+75). The average size was 27+15 um2. In aged samples with AMD, high-Zn calcified nodules with average Ca concentration of 4316+1723 ppm (max 13105+7563) and Zn 97+57 ppm (max 201+143). Size 26x10 um2. In the aged non-AMD group, the low zinc-calcification nodules contained an average Ca ppm of 915+259 (max 1475+380) and Zn 33+18 ppm (max 40+21). The average size 28x16 um2. In the AMD group, the low Zn calcified nodules average Ca ppm 1544+1450 (max 3526+5289) and average Zn ppm 47+23 (max 64+37). Average size 19x10 um2. Calcified plaques in Bruch’s membrane from aged donors with and without AMD. Some of these plaques colocalised with Zn and also Fe. Fe-loaded structures in the choriocapillaris underlying calcified nodules and plaques. Conclusions : Calcific nodules contain zinc in older eyes with and without AMD. Calcified nodules with lower amounts of Ca contained lower amounts of Zn, so the accumulation of Ca may occur in parallel to Zn. It is possible that iron-loaded structures in the choriocapillaris are macrophages.

Open Access

Show Abstract ▼

Abstract: Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)–rich protein aggregates [termed “Lewy bodies” (LBs)], is a well-established characteristic of Parkinson’s disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning–based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

Open Access

Show Abstract ▼

Abstract: The first experimental results from a new transmissive diagnostic instrument for synchrotron X-ray beamlines are presented. The instrument utilizes a single-crystal chemical-vapour-deposition diamond plate as the detector material, with graphitic wires embedded within the bulk diamond acting as electrodes. The resulting instrument is an all-carbon transmissive X-ray imaging detector. Within the instrument's transmissive aperture there is no surface metallization that could absorb X-rays, and no surface structures that could be damaged by exposure to synchrotron X-ray beams. The graphitic electrodes are fabricated in situ within the bulk diamond using a laser-writing technique. Two separate arrays of parallel graphitic wires are fabricated, running parallel to the diamond surface and perpendicular to each other, at two different depths within the diamond. One array of wires has a modulated bias voltage applied; the perpendicular array is a series of readout electrodes. X-rays passing through the detector generate charge carriers within the bulk diamond through photoionization, and these charge carriers travel to the nearest readout electrode under the influence of the modulated electrical bias. Each of the crossing points between perpendicular wires acts as an individual pixel. The simultaneous read-out of all pixels is achieved using a lock-in technique. The parallel wires within each array are separated by 50 µm, determining the pixel pitch. Readout is obtained at 100 Hz, and the resolution of the X-ray beam position measurement is 600 nm for a 180 µm size beam.

Show Abstract ▼

Abstract: We report on the alteration history of the olivine-phyric, highly depleted (HD) shergottite, Northwest Africa (NWA) 10416, paying particular attention to the origin of the aqueous alteration seen affecting the meteorite’s olivine megacrysts. The rock’s interior displays 1 mm, zoned, altered olivine megacrysts set in a groundmass of clinopyroxene, unzoned olivine, and interstitial plagioclase and maskelynite. Synchrotron micro X-ray diffraction (µ-XRD) and transmission electron microscopy (TEM) show that plagioclase and maskelynite have been partially replaced by kaolinite. The relict olivine megacryst cores display a unique concentric colouration for Martian meteorites, having central amber-coloured zones surrounded by a brown mantle zone, with the rims remaining clear and unaltered. This colouration is a result of fluid alteration and partial replacement, with hydration. TEM analysis revealed the ∼200 nm scale banded and largely amorphous nature of the alteration, but with some (∼ 20%) relict crystalline olivine patches. Although the coloured olivine zones show cation and anion site vacancies compared to stoichiometric olivine, a relict igneous compositional trend is preserved in the megacrysts, from Mg-rich altered cores (Mg# = 76) to unaltered stoichiometric rims (Fo53). Synchrotron Fe-K X-ray absorption near-edge structure (XANES) analysis revealed that the coloured zones of the megacryst have different Fe oxidation values. High ferric contents are present in the brown mantle zones (Fe3+/ΣFe ≤ 0.92) and the amber zones (Fe3+/ΣFe ≤ 0.30), whereas the clear rims are ferrous. This suggests alteration occurred in an oxidising environment and that the sharp contrast in colour of the megacryst (brown to clear) is a record of a relict fluid reaction front. In order to test the terrestrial or extraterrestrial origin of the alteration, olivine material from a shock-melt vein was analysed by TEM. The analysis revealed 0.952 nm curved d-spacing’s from clay alteration undisturbed by any shock effects, strongly suggesting a terrestrial origin. The d-spacing values most likely represent a collapsed saponite or vermiculite, showing that in some places olivine has been replaced by crystalline clay. Oxygen isotope analysis of bulk (Δ17O = 0.309 ± 0.009 (2σ) ‰) and amber-coloured megacryst material (= 0.271 ± 0.002 (2σ) ‰), are also consistent with terrestrial alteration. We propose a model in which, during the meteorite’s time in Northwest Africa, low-temperature, likely acidic, groundwater exploited fractures. The fluid altered the olivine megacrysts in a way that was controlled by the pre-existing, igneous compositional zonation, with Mg-rich olivine being more susceptible to alteration in this fluid environment. The plagioclase and maskelynite were also altered to a high degree. After the alteration event it is likely that NWA 10416 had a significant residence time in Northwest Africa, accounting for terrestrial calcite and the dehydration of some clay phases.

Show Abstract ▼

Abstract: The hallmark of neurodegenerative disease in humans, including Alzheimer’s, Amyotrophic Lateral Sclerosis and Parkinson’s, lies in our inability to efficiently restore damaged neurons. Contrary to widespread belief, humans can regenerate neurons within the Peripheral Nervous System (PNS) and some areas of the Central Nervous System (CNS), however the underlying mechanisms which drive or repress complete functional and structural neuronal regeneration remain elusive. To develop our understanding of this natural phenomenon one should focus on species which are capable of efficient neuronal regeneration following neurotrauma. The earthworm species Eisenia fetida can regenerate their Cerebral Ganglion (CG) - loosely defined as the brain, within a few weeks following surgical removal. The characterization of fundamental aspects of neuronal regeneration in the earthworm promises to provide an insight as to why humans have largely lost that capability. Here we present a detailed micro-dissection protocol that has been developed to excise the CG and study the progression of its regeneration. The Ventral Nerve Cord (VNC) of the worm is a tissue which connects the CG to the rest of the nervous system via the Circumpharyngeal Connectives (CC). Exploration of molecular dynamics through changes in the transcriptome were determined in the VNC and CC at 1 week and 5-week post-decerebration using an RNAseq approach (90 million reads/condition, 100bp Paired End). RNAseq established that specific groups of genes are up- or downregulated in the regenerating VNC. More than 500 significantly enriched biological processes were identified throughout the regeneration process, including vascular development, neurogenesis and extracellular matrix organization, as well as more than 100 significantly enriched molecular functions, including calcium ion binding, metal ion binding and metalloendopeptidase activity. Differential expression of transcripts was confirmed via qPCR. Examples of transcripts which have been validated at 1w post-decerebration, include catalase (~34-fold), superoxide dismutase 1 (sod-1) (~12-fold) and transcription factor jun-B (junb) (~3-fold). On the other hand, examples of transcripts which have been validated at 5w post-decerebration, include Glial Fibrillary Acidic Protein (GFAP) (~101-fold), adam19 (~17-fold) and Bone Morphogenetic Protein 1 (BMP1) (~3-fold). Moreover, the large number of differentially expressed metalloproteins (ADAMs, BMPs, MMPs, metallothionein) identified in the transcriptome, led to the hypothesis that metal trafficking could play a role in the course of regeneration. This was confirmed using a synchrotron-based approach, namely X-Ray Fluorescence (XRF) spectroscopy, where Zinc (Zn) and Iron (Fe) show a differential distribution pattern across different stages (at 1 week to 10-weeks post-decerebration) of regeneration. Lastly, various histological techniques were implemented to characterize/describe neuronal structures during the regenerating process, including immunohistochemistry and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL). Furthermore, numerous novel markers and biological processes have been identified which, to date, have not been linked to neuronal regeneration. In summary, the results suggest that the increase of axon growth promoting factors as well the decrease of growth inhibitory factors act in conjunction to ensure efficient neuronal regeneration in the earthworm.