DETECT schools study protocol: A prospective observational cohort surveillance study investigating the impact of COVID-19 in Western Australian schools

Introduction: Amidst the evolving COVID-19 pandemic, understanding the transmission dynamics of the SARS-CoV-2 virus is key to providing peace of mind for the community and informing policy-making decisions. While available data suggest that school-aged children are not significant spreaders of SARS-CoV-2, the possibility of transmission in schools remains an ongoing concern, especially among an aging teaching workforce. Even in low-prevalence settings, communities must balance the potential risk of transmission with the need for students\u27 ongoing education. Through the roll out of high-throughput school-based SARS-CoV-2 testing, enhanced follow-up for individuals exposed to COVID-19 and wellbeing surveys, this study investigates the dynamics of SARS-CoV-2 transmission and the current psychosocial wellbeing impacts of the pandemic in school communities. Methods: The DETECT Schools Study is a prospective observational cohort surveillance study in 79 schools across Western Australia (WA), Australia. To investigate the incidence, transmission and impact of SARS-CoV-2 in schools, the study comprises three “modules”: Module 1) Spot-testing in schools to screen for asymptomatic SARS-CoV-2; Module 2) Enhanced surveillance of close contacts following the identification of any COVID-19 case to determine the secondary attack rate of SARS-CoV-2 in a school setting; and Module 3) Survey monitoring of school staff, students and their parents to assess psycho-social wellbeing following the first wave of the COVID-19 pandemic in WA. Clinical Trial Registration: Trial registration number: ACTRN1262000092297

Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy

The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail about the molecular structure of DNA has been derived primarily from in vitro experiments on extracted or synthetic DNA. Here, we report that a B- to A-like DNA conformational change can occur in the nuclei of intact cells in response to dehydration. This transition is reversible upon rehydration in air-dried cells. By systematically monitoring the dehydration and rehydration of single and double-stranded DNA, RNA, extracted nuclei and three types of eukaryotic cells including chicken erythrocytes, mammalian lymphocytes and cancerous rodent fibroblasts using Fourier transform infrared (FTIR) spectroscopy, we unequivocally assign the important DNA conformation marker bands within these cells. We also demonstrate that by applying FTIR spectroscopy to hydrated samples, the DNA bands become sharper and more intense. This is anticipated to provide a methodology enabling differentiation of cancerous from non-cancerous cells based on the increased DNA content inherent to dysplastic and neoplastic tissue

Pulse pressure and age at menopause

BACKGROUND: The objective of this study was to study the association of early age at menopause with pulse pressure (PP), a marker of arterial stiffness, and PP change. METHODS: The effect of natural menopause was studied in 2484 women from the Atherosclerosis Risk in Communities (ARIC) Study who had not used hormone replacement therapy and who had not had a hysterectomy. The cross-sectional association of age with PP was evaluated in the entire cohort. The cross-sectional association of recalled age at menopause was evaluated in the 1688 women who were postmenopausal at baseline. PP change over 6 years was assessed in relation to menopausal age separately in women who were postmenopausal at baseline and in those whose menopause occurred during the 6-year interval. RESULTS: Chronological age was strongly and positively associated with PP in cross-sectional analyses, but not independently associated with PP change. While menopausal age was not associated cross-sectionally with PP, early age at menopause (age<45) was significantly and independently associated with a slightly larger increase in PP (8.4, 95% CI 7.0–9.8) than later menopause (6.5, 95% CI 5.8;7.2). However, among normotensive women the difference was not statistically significant (p = 0.07, 6.1 vs 4.7). CONCLUSIONS: Early age at menopause may be related to a greater increase in arterial stiffness, but the effect appears to be small and further evidence is needed

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

New infrared and fluorescence techniques for the elucidation of cellular ultrastructure

The underlying philosophy of this doctoral thesis is that application of advanced experimental techniques to address open research questions while synchronously working to benchmark and augment the instrumentation and methodologies, produces an enhanced appreciation and understanding of both the experiments undertaken and the resultant data. This approach guided and informed the research carried out during the thesis which sought to investigate the constituents, concentrations, structures and functions of biological samples using two state-of-the-art techniques: Fourier transform infrared (FTIR) spectroscopy and super resolution single molecule localisation microscopy (SMLM). Significant contributions to a diverse range of research areas as well as improvements to experimental methodologies were achieved demonstrating the soundness of this approach and the efficacy of both FTIR spectroscopy and SMLM. New methodologies for benchtop and synchrotron source (S-)FTIR spectroscopy of live and hydrated samples were developed and applied to detect a previously unknown en masse reversible B- to A-DNA conformational transition in single cells. This was in counterpoint to the long-standing assumption that the majority of DNA exists persistently in the B-form. Subsequent detection of the B-A-B transition in functional prokaryotes during desiccation and subsequent rehydration demonstrated a possible role for the A-form helix as a defence mechanism against various forms of damage and as key in the evolution of nucleic acids. Successful detection of DNA conformation in cells using S-FTIR was also contrary to previous FTIR spectroscopic work that hypothesised that absorption of infrared light by compacted DNA did not obey Beer-Lambert’s Law and, as a result, dictated that DNA absorptions from cell samples could not be regarded quantitatively. FTIR spectra of standard mixtures of protein and DNA were used to produce multivariate regression models, which were then used to accurately estimate the DNA content of simple cells. As well as debunking the “Dark DNA” hypothesis which had hampered FTIR biospectroscopic research for over a decade, these experiments clearly demonstrated improved detection sensitivity in hydrated cell work. This motivated application of the developed live-cell S-FTIR spectroscopy methodologies to investigate the changing biochemistry of cells as they progressed through the cell cycle. Multivariate cluster analysis of spectra collected from COS-7 cells over the course of the cell cycle allowed differentiation of cells only two hours apart within the same phase. Furthermore, through analysis of the associated causes for variance, it was demonstrated that the spectroscopic detection of subtle changes in relative DNA, protein and lipid concentrations was possible. This level of sensitivity had previously been unreachable with FTIR spectra of dehydrated or fixed cells and is now envisioned as stimulating further live-cell spectroscopic research and allowing detection of previously unknown biochemistry. Concurrent to this work, the first home-built SMLM setup in Australia was established to provide a complementary technique for the FTIR spectroscopic work being undertaken. As a very new technique, development and application of SMLM involved design and construction of the necessary instrumentation from scratch as well as extensive optimisation of sample preparation protocols for imaging of microtubules, actin and mitochondria. To achieve this various parameters involved in cell fixation, data acquisition and image rendering were investigated systematically and the numerous encountered causes of image artefacts were characterised. Artefacts were shown to arise from all aspects of the experiment: hardware, software, and particularly from the sample. Importantly, it was shown that a commonly employed permeabilisation step in the preparation of microtubules, compromises biological relevance in pursuit of a ‘better’ image. Application of the protocols developed for labelling of microtubules enabled identification of a novel microtubule bundling response in cells expressing a Rabies protein. A number of mutants of the protein were investigated and it was found that the extent of microtubule bundling correlated with both the pathogenicity of virus in previous live mouse experiments and the antagonism to the innate interferon response. These results demonstrate a previously unknown interaction between the viral protein, the host cell immune response, and the cytoskeletal architecture, and have inspired much future research aimed at elucidating the mechanisms at play. SMLM was also applied to the imaging of fluorescently labelled 100 and 20 nm silica nanospheres and the exemplary spatial resolution achievable using the set-up was convincingly demonstrated. These nanospheres were also established as a proof-of-principle material for correlative electron microscopy-SMLM. Finally, SMLM was successfully used to image extracted double stranded DNA exhibiting the potential for future use of this technique to reveal sub-diffraction chromatin and DNA structure. This research underscores the potential of FTIR spectroscopy and SMLM both independently and for future complementary use, as well as highlights the importance of ongoing technique augmentation in parallel with the application of experimental methods to novel research endeavours.Awards: Vice-Chancellor’s Commendation for Doctoral Thesis Excellence in [2014]

New infrared and fluorescence techniques for the elucidation of cellular ultrastructure

The underlying philosophy of this doctoral thesis is that application of advanced experimental techniques to address open research questions while synchronously working to benchmark and augment the instrumentation and methodologies, produces an enhanced appreciation and understanding of both the experiments undertaken and the resultant data. This approach guided and informed the research carried out during the thesis which sought to investigate the constituents, concentrations, structures and functions of biological samples using two state-of-the-art techniques: Fourier transform infrared (FTIR) spectroscopy and super resolution single molecule localisation microscopy (SMLM). Significant contributions to a diverse range of research areas as well as improvements to experimental methodologies were achieved demonstrating the soundness of this approach and the efficacy of both FTIR spectroscopy and SMLM. New methodologies for benchtop and synchrotron source (S-)FTIR spectroscopy of live and hydrated samples were developed and applied to detect a previously unknown en masse reversible B- to A-DNA conformational transition in single cells. This was in counterpoint to the long-standing assumption that the majority of DNA exists persistently in the B-form. Subsequent detection of the B-A-B transition in functional prokaryotes during desiccation and subsequent rehydration demonstrated a possible role for the A-form helix as a defence mechanism against various forms of damage and as key in the evolution of nucleic acids. Successful detection of DNA conformation in cells using S-FTIR was also contrary to previous FTIR spectroscopic work that hypothesised that absorption of infrared light by compacted DNA did not obey Beer-Lambert’s Law and, as a result, dictated that DNA absorptions from cell samples could not be regarded quantitatively. FTIR spectra of standard mixtures of protein and DNA were used to produce multivariate regression models, which were then used to accurately estimate the DNA content of simple cells. As well as debunking the “Dark DNA” hypothesis which had hampered FTIR biospectroscopic research for over a decade, these experiments clearly demonstrated improved detection sensitivity in hydrated cell work. This motivated application of the developed live-cell S-FTIR spectroscopy methodologies to investigate the changing biochemistry of cells as they progressed through the cell cycle. Multivariate cluster analysis of spectra collected from COS-7 cells over the course of the cell cycle allowed differentiation of cells only two hours apart within the same phase. Furthermore, through analysis of the associated causes for variance, it was demonstrated that the spectroscopic detection of subtle changes in relative DNA, protein and lipid concentrations was possible. This level of sensitivity had previously been unreachable with FTIR spectra of dehydrated or fixed cells and is now envisioned as stimulating further live-cell spectroscopic research and allowing detection of previously unknown biochemistry. Concurrent to this work, the first home-built SMLM setup in Australia was established to provide a complementary technique for the FTIR spectroscopic work being undertaken. As a very new technique, development and application of SMLM involved design and construction of the necessary instrumentation from scratch as well as extensive optimisation of sample preparation protocols for imaging of microtubules, actin and mitochondria. To achieve this various parameters involved in cell fixation, data acquisition and image rendering were investigated systematically and the numerous encountered causes of image artefacts were characterised. Artefacts were shown to arise from all aspects of the experiment: hardware, software, and particularly from the sample. Importantly, it was shown that a commonly employed permeabilisation step in the preparation of microtubules, compromises biological relevance in pursuit of a ‘better’ image. Application of the protocols developed for labelling of microtubules enabled identification of a novel microtubule bundling response in cells expressing a Rabies protein. A number of mutants of the protein were investigated and it was found that the extent of microtubule bundling correlated with both the pathogenicity of virus in previous live mouse experiments and the antagonism to the innate interferon response. These results demonstrate a previously unknown interaction between the viral protein, the host cell immune response, and the cytoskeletal architecture, and have inspired much future research aimed at elucidating the mechanisms at play. SMLM was also applied to the imaging of fluorescently labelled 100 and 20 nm silica nanospheres and the exemplary spatial resolution achievable using the set-up was convincingly demonstrated. These nanospheres were also established as a proof-of-principle material for correlative electron microscopy-SMLM. Finally, SMLM was successfully used to image extracted double stranded DNA exhibiting the potential for future use of this technique to reveal sub-diffraction chromatin and DNA structure. This research underscores the potential of FTIR spectroscopy and SMLM both independently and for future complementary use, as well as highlights the importance of ongoing technique augmentation in parallel with the application of experimental methods to novel research endeavours.<div><br></div><div> Awards: Vice-Chancellor’s Commendation for Doctoral Thesis Excellence in 2014.</div

Lipid Droplet Motility Increases Following Viral Immune Stimulation

Lipid droplets (LDs) have traditionally been thought of as solely lipid storage compartments for cells; however, in the last decade, they have emerged as critical organelles in health and disease. LDs are highly dynamic within cells, and their movement is critical in organelle–organelle interactions. Their dynamics are known to change during cellular stress or nutrient deprivation; however, their movement during pathogen infections, especially at very early timepoints, is under-researched. This study aimed to track LD dynamics in vitro, in an astrocytic model of infection. Cells were either stimulated with a dsRNA viral mimic, poly I:C, or infected with the RNA virus, Zika virus. Individual LDs within infected cells were analysed to determine displacement and speed, and average LD characteristics for multiple individual cells calculated. Both LD displacement and mean speed were significantly enhanced in stimulated cells over a time course of infection with an increase seen as early as 2 h post-infection. With the emerging role for LDs during innate host responses, understanding their dynamics is critical to elucidate how these organelles influence the outcome of viral infection

Correlative Synchrotron Fourier Transform Infrared Spectroscopy and Single Molecule Super Resolution Microscopy for the Detection of Composition and Ultrastructure Alterations in Single Cells

Single molecule localization microscopy (SMLM) and synchrotron Fourier transform infrared (S-FTIR) spectroscopy are two techniques capable of elucidating unique and valuable biological detail. SMLM provides images of the structures and distributions of targeted biomolecules at spatial resolutions up to an order of magnitude better than the diffraction limit, whereas IR spectroscopy objectively measures the holistic biochemistry of an entire sample, thereby revealing any variations in overall composition. Both tools are currently applied extensively to detect cellular response to disease, chemical treatment, and environmental change. Here, these two techniques have been applied correlatively at the single cell level to probe the biochemistry of common fixation methods and have detected various fixation-induced losses of biomolecular composition and cellular ultrastructure. Furthermore, by extensive honing and optimizing of fixation protocols, many fixation artifacts previously considered pervasive and regularly identified using IR spectroscopy and fluorescence techniques have been avoided. Both paraformaldehyde and two-step glutaraldehyde fixation were identified as best preserving biochemistry for both SMLM and IR studies while other glutaraldehyde and methanol fixation protocols were demonstrated to cause significant biochemical changes and higher variability between samples. Moreover, the potential complementarity of the two techniques was strikingly demonstrated in the correlated detection of biochemical changes as well as in the detection of fixation-induced damage that was only revealed by one of the two techniques