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Abstract: Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen–antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to ∼10 000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.

Open Access

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Abstract: Background: Despite their efficacy in the treatment of chronic inflammation, the prolonged application of therapeutic glucocorticoids (GCs) is limited by significant systemic side effects including glucocorticoid-induced osteoporosis (GIOP). 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional enzyme that primarily activates GCs in vivo, regulating tissue-specific exposure to active GC. We aimed to determine the contribution of 11β-HSD1 to GIOP. Methods: Wild type (WT) and 11β-HSD1 knockout (KO) mice were treated with corticosterone (100 μg/ml, 0.66% ethanol) or vehicle (0.66% ethanol) in drinking water over 4 weeks (six animals per group). Bone parameters were assessed by micro-CT, sub-micron absorption tomography and serum markers of bone metabolism. Osteoblast and osteoclast gene expression was assessed by quantitative RT-PCR. Results: Wild type mice receiving corticosterone developed marked trabecular bone loss with reduced bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N). Histomorphometric analysis revealed a dramatic reduction in osteoblast numbers. This was matched by a significant reduction in the serum marker of osteoblast bone formation P1NP and gene expression of the osteoblast markers Alp and Bglap. In contrast, 11β-HSD1 KO mice receiving corticosterone demonstrated almost complete protection from trabecular bone loss, with partial protection from the decrease in osteoblast numbers and markers of bone formation relative to WT counterparts receiving corticosterone. Conclusions: This study demonstrates that 11β-HSD1 plays a critical role in GIOP, mediating GC suppression of anabolic bone formation and reduced bone volume secondary to a decrease in osteoblast numbers. This raises the intriguing possibility that therapeutic inhibitors of 11β-HSD1 may be effective in preventing GIOP in patients receiving therapeutic steroids.

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Abstract: The local and systemic dissemination of implant-related derivatives into the body is of great interest to human health. Biological exposures of metal micro/nano-particles, complexes and soluble ions, generated as a consequence of in-service degradation of metallic prosthetics, can result in severe adverse tissues reactions. However, individual reactions are highly variable and are not easily predicted, due to in part a lack of understanding of the properties of the metalstimuli which dictates cellular interactions and toxicity. However, interrogating ultra-dilute metals within fragile, hydrated biological tissues without influencing the native physical and/or chemical composition is challenging. Therefore, a drive for more advanced characterisation techniques of the exogenous metallic particles is required. Here, synchrotron-based X-ray fluorescence spectroscopy (XRF) and X-ray absorption near edge structure (XANES) were deployed to investigate the quantitative spatial distribution and local chemistry of implant-related metal particles within soft tissues. Analytical and experimental steps were outlined and improved when using XRF for greater accuracy in quantification. This included the implications of the matrix composition, the effects of saturation and how the instability in the flux and beam profile affected the measurement. Two post-analytical algorithms were generated to reduce the effects of image artefacts observed frequently in micro-focus XRF images. These methodological advances will have significant positive impact on quantitative XRF. Specifically, the quantitative XRF imaging displayed within this thesis, combined in a multi-institution research effort, influenced the future use of specific metallic implants. The chemistry of exogenous implant-related particles was also investigated, for the first time, using a XANES mapping approach. This enabled twodimensional chemical imaging at a high spatial resolution, allowing superior chemical discrimination of small, isolated, heterogenous features. This method highlighted the unreported variability in metal chemistry associated with orthopaedic implants, which may alter the perceived toxicity of implant derivatives. The results shown within this thesis outline the advantages of using a XANES mapping approach over conventional spectroscopy methods and should be considered when interrogating chemical species of metals in biological environments. Finally, a novel approach to co-locative analysis between exogenous metal particles and the underlying cellular content was developed using a lanthanide-antibody conjugate with XRF. This allowed simultaneous imaging of the native elemental distributions and biological epitopes. The development of this imaging modality is promising for understanding spatial relationships between such components. Collectively, advances within these interrogation methods applied to implant- related products will ultimately help our understanding of associated immunological responses and will generate more appropriate biological exposure models. Additionally, the advantages reported from the development of these techniques can be applied to an array of samples types in many areas of science.

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Abstract: Synchrotron radiation X-ray fluorescence microscopy is frequently used to investigate the spatial distribution of elements within a wide range of samples. Interrogation of heterogeneous samples that contain large concentration ranges has the potential to produce image artefacts due to the profile of the X-ray beam. The presence of these artefacts and the distribution of flux within the beam profile can significantly affect qualitative and quantitative analyses. Two distinct correction methods have been generated by referencing the beam profile itself or by employing an adaptive-thresholding procedure. Both methods significantly improve qualitative imaging by removing the artefacts without compromising the low-intensity features. The beam-profile correction method improves quantitative results but requires accurate two-dimensional characterization of the X-ray beam profile.

Open Access

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Abstract: Corrosion products generated in artificial pits of zirconium were characterized in–situ by synchrotron X-ray diffraction and X-ray absorption near edge structure (XANES) in physiological saline, with and without addition of 4% albumin and/or 0.1% H2O2. Zr metal fragments and tetragonal ZrO2 particles were detected in aggregated black corrosion products away from the corrosion front. At the corrosion front, a ZrOCl2⋅8H2O salt layer of a few hundreds of microns thickness was formed. Coarsened ZrOCl2⋅8H2O crystallites were found farther out into the solution. The Zr solution species were confirmed to be in a tetravalent state by XANES. TEM imaging of the corrosion products revealed heterogeneity of the morphology of the Zr metal fragments and confirmed their size to be less than a few microns. The formation and speciation of Zr corrosion products were found not affected by the presence of H2O2 and/or albumin in physiological saline. Furthermore, bulk Zr electrochemistry identified that the presence of H2O2 and/or albumin did not affect passive current densities and pitting potentials of the bulk Zr surface. Therefore, it is concluded that the pitting susceptibility and pit chemistry of Zr in physiological saline were unaffected by the presence of H2O2, albumin or their combinations.