ORCID Profile
0000-0002-7078-5443
Current Organisations
NYU Langone Medical Center
,
La Trobe University
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Genome Structure and Regulation | Biochemistry and Cell Biology | Structural Biology (incl. Macromolecular Modelling) | Cell Development, Proliferation and Death
Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Biological Sciences |
Publisher: Radiation Research Society
Date: 07-2015
DOI: 10.1667/RR13798.1
Publisher: Elsevier BV
Date: 08-2017
Publisher: Oxford University Press (OUP)
Date: 28-03-2011
DOI: 10.1093/NAR/GKR175
Publisher: American Chemical Society (ACS)
Date: 13-01-2015
DOI: 10.1021/JZ5019702
Abstract: Application of single-molecule fluorescence detection has led to the development of light microscopy techniques that make it possible to study fluorescent s les at spatial resolutions significantly improved upon the diffraction limit of light. The biological and materials science applications of these "super-resolution" microscopy methods are vast, causing current demand for them to be high. However, implementation, execution, and interpretation of these techniques, particularly involving biological s les, require a broad interdisciplinary skillset, not often found in a single laboratory. Those already used to interdisciplinary work as well as navigating communication and collaboration between more pure forms of physics, chemistry, and biology are well-positioned to spearhead such efforts. In this Perspective, we describe various aspects of single-molecule super-resolution imaging, discussing, in particular, the role that physical chemistry has so far played in its development and establishment. We also highlight a selection of some of the remarkable recent research achievements in this vibrant field.
Publisher: Springer Science and Business Media LLC
Date: 18-12-2018
DOI: 10.1038/S41467-018-07799-2
Abstract: DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1. We also show that BRCA2 directly interacts with RNase H2, mediates its localization to DSBs in the S/G2 cell-cycle phase, and controls DNA:RNA hybrid levels at DSBs. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.
Publisher: Public Library of Science (PLoS)
Date: 28-12-2020
DOI: 10.1371/JOURNAL.PGEN.1009256
Abstract: Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to s le preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize in idual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways.
Publisher: Public Library of Science (PLoS)
Date: 24-02-2015
Publisher: Elsevier BV
Date: 09-2014
Publisher: Oxford University Press (OUP)
Date: 07-2016
Publisher: Wiley
Date: 19-09-2013
Abstract: A technique capable of detecting and monitoring nucleic acid concentration offers potential in diagnosing cancer and further developing an understanding of the biochemistry of disease. The application of Fourier transform infrared (FTIR) spectroscopy has previously been hindered by the supposed non-Beer-Lambert absorption behavior of DNA in intact cells making elucidation of the DNA bands difficult. We use known composition DNA/hemoglobin standards to successfully estimate the DNA content in avian erythrocyte nuclei (44.2%) and intact erythrocytes (12.8%). Furthermore we demonstrate that the absorption of cellular DNA does follow the Beer-Lambert Law and highlights the role of conformation and hydration in FTIR spectroscopy of biological s les.
Publisher: Elsevier BV
Date: 05-2018
DOI: 10.1016/J.CELREP.2018.04.093
Abstract: The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.
Publisher: Springer Science and Business Media LLC
Date: 08-01-2021
DOI: 10.1038/S41598-020-80505-9
Abstract: The role of central orexin in the sympathetic control of interscapular brown adipose tissue (iBAT) thermogenesis has been established in rodents. Stimulatory doses of caffeine activate orexin positive neurons in the lateral hypothalamus, a region of the brain implicated in stimulating BAT thermogenesis. This study tests the hypothesis that central administration of caffeine is sufficient to activate BAT. Low doses of caffeine administered either systemically (intravenous [IV] 10 mg/kg) and centrally (intracerebroventricular [ICV] 5–10 μg) increases BAT thermogenesis, in anaesthetised (1.5 g/kg urethane, IV) free breathing male rats. Cardiovascular function was monitored via an indwelling intra-arterial cannula and exhibited no response to the caffeine. Core temperature did not significantly differ after administration of caffeine via either route of administration. Caffeine administered both IV and ICV increased neuronal activity, as measured by c-Fos-immunoreactivity within subregions of the hypothalamic area, previously implicated in regulating BAT thermogenesis. Significantly, there appears to be no neural anxiety response to the low dose of caffeine as indicated by no change in activity in the basolateral amygdala. Having measured the physiological correlate of thermogenesis (heat production) we have not measured indirect molecular correlates of BAT activation. Nevertheless, our results demonstrate that caffeine, at stimulatory doses, acting via the central nervous system can increase thermogenesis, without adverse cardio-dynamic impact.
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3AN00316G
Abstract: The application of FTIR spectroscopy to disease diagnosis requires a thorough knowledge of the spectroscopy associated with the cell cycle to discern disease markers from normal cellular events. We have applied synchrotron FTIR spectroscopy to monitor cells at different phases of the cell cycle namely G1, S and G2 phases. By applying Principal component analysis (PCA) from three independent trials we show clustering on a 2-dimensional scores plots (PC1 versus PC2) from cell spectra only two hours apart within the cell cycle. The corresponding PCA Loadings Plots indicate the clustering is primarily based on changes to the overall concentration of nucleic acids, proteins and lipids. During the first ten hours post mitosis, cells are observed to increase in protein and decrease in both lipid and nucleic acid concentration. During the synthesis phase, (beginning 9-11 hours post-mitosis) the PCA Loadings Plots show the accumulation of lipids within the cell as well the duplication of the genome as evidenced by strong DNA contributions. In the 4-6 hours following the synthesis phase, the cells once again accumulate protein while the relative nucleic acid and lipid concentrations decrease. These results, in comparison to previous studies on dehydrated cells, show previously unresolvable biochemical information as well as highlighting the advantages of FTIR spectroscopy applied to single living cells.
Publisher: Proceedings of the National Academy of Sciences
Date: 11-03-2021
Abstract: DNA resection is an initial, decisive step in the homologous recombination repair pathway of DNA double-strand breaks (DSBs). However, the in idual roles and cross-talk of key proteins in this process remain unclear. To resolve the spatiotemporal dynamics of this intricate process, we applied multicolor single-molecule localization microscopy along with robust analytical approaches. Our data reveal the dynamic actions and interactions of MRE11, BRCA1, and CtIP both as a multimer and in idually the recruitment and spatial exclusion of 53BP1 the role of BLM helicase alongside EXO1 and DNA2 nucleases and the inhibitory mechanisms of MRE11 exo-/endonuclease inhibition. Together, our findings describe important aspects of DNA DSB repair and highlight key differences between repair of clustered damage and sporadic endogenous breaks.
Publisher: Springer US
Date: 26-08-2020
Publisher: The Royal Society
Date: 06-08-2014
Abstract: The role that DNA conformation plays in the biochemistry of cells has been the subject of intensive research since DNA polymorphism was discovered. B-DNA has long been considered the native form of DNA in cells although alternative conformations of DNA are thought to occur transiently and along short tracts. Here, we report the first direct observation of a fully reversible en masse conformational transition between B- and A-DNA within live bacterial cells using Fourier transform infrared (FTIR) spectroscopy. This biospectroscopic technique allows for non-invasive and reagent-free examination of the holistic biochemistry of s les. For this reason, we have been able to observe the previously unknown conformational transition in all four species of bacteria investigated. Detection of this transition is evidence of a previously unexplored biological significance for A-DNA and highlights the need for new research into the role that A-DNA plays as a cellular defence mechanism and in stabilizing the DNA conformation. Such studies are pivotal in understanding the role of A-DNA in the evolutionary pathway of nucleic acids. Furthermore, this discovery demonstrates the exquisite capabilities of FTIR spectroscopy and opens the door for further investigations of cell biochemistry with this under-used technique.
Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/CH13499
Abstract: The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss s le preparation and labelling methods.
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/CH19571_CO
Abstract: The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live s les. Super-resolution optical fluctuation imaging (SOFI) is well suited to live s les while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.
Publisher: Springer Science and Business Media LLC
Date: 11-2017
DOI: 10.1038/S41598-017-14922-8
Abstract: We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF = 0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.
Publisher: Springer Science and Business Media LLC
Date: 24-09-2018
DOI: 10.1038/S41467-018-06435-3
Abstract: Homologous recombination (HR) is a crucial pathway for the repair of DNA double-strand breaks. BRCA1/2 breast cancer proteins are key players in HR via their mediation of RAD51 nucleofilament formation and function however, their in idual roles and crosstalk in vivo are unknown. Here we use super-resolution (SR) imaging to map the spatiotemporal kinetics of HR proteins, revealing the interdependent relationships that govern the dynamic interplay and progression of repair events. We show that initial single-stranded DNA/RAD51 nucleofilament formation is mediated by RAD52 or, in the absence of RAD52, by BRCA2. In contrast, only BRCA2 can orchestrate later RAD51 recombinase activity during homology search and resolution. Furthermore, we establish that upstream BRCA1 activity is critical for BRCA2 function. Our analyses reveal the underlying epistatic landscape of RAD51 functional dependence on RAD52, BRCA1, and BRCA2 during HR and explain the phenotypic similarity of diseases associated with mutations in these proteins.
Publisher: IOP Publishing
Date: 16-08-2022
Abstract: Correlative imaging methods can provide greater information for investigations of cellular ultra-structure, with separate analysis methods complementing each other's strengths and covering for deficiencies. Here we present a method for correlative applications of super resolution and atomic force microscopies, optimising the s le preparation for correlative imaging of the cellular cytoskeleton in COS-7 cells. This optimisation determined the order of permeabilisation and fixation, the concentration of Triton X-100 surfactant used and time required for sufficient removal of the cellular membrane while maintaining the microtubule network. Correlative SMLM/AFM imaging revealed the different information that can be obtained through each microscopy. The widths of microtubules and microtubule clusters were determined from both AFM height measurements and Gaussian fitting of SMLM intensity cross sections, these were then compared to determine the orientation of microtubules within larger microtubule bundles. The ordering of microtubules at intersections was determined from the AFM height profiles as each microtubule crosses the other. The combination of both microtubule diameter measurements enabled greater information on their structure to be found than either measurement could in idually.
Publisher: Springer Science and Business Media LLC
Date: 21-09-2016
DOI: 10.1038/SREP33493
Abstract: Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and d STORM super-resolution microscopy to quantify protein interactions with the MT network and with in idual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development.
Publisher: Springer Science and Business Media LLC
Date: 21-01-2015
DOI: 10.1038/SREP07924
Publisher: Springer Science and Business Media LLC
Date: 09-09-2020
DOI: 10.1186/S13024-020-00386-4
Abstract: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H 2 O 2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.
Publisher: IOP Publishing
Date: 05-05-2021
Abstract: Super-resolution microscopy (SRM) comprises a suite of techniques well-suited to probing the nanoscale landscape of genomic function and dysfunction. Offering the specificity and sensitivity that has made conventional fluorescence microscopy a cornerstone technique of biological research, SRM allows for spatial resolutions as good as 10 nanometers. Moreover, single molecule localization microscopies (SMLMs) enable examination of in idual molecular targets and nanofoci allowing for the characterization of subpopulations within a single cell. This review describes how key advances in both SRM techniques and s le preparation have enabled unprecedented insights into DNA structure and function, and highlights many of these new discoveries. Ongoing development and application of these novel, highly interdisciplinary SRM assays will continue to expand the toolbox available for research into the nanoscale genomic landscape.
Publisher: American Chemical Society (ACS)
Date: 23-10-2015
DOI: 10.1021/ACSCHEMBIO.5B00754
Abstract: 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 s le, 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 s les. 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.
Publisher: Springer Science and Business Media LLC
Date: 14-05-2019
Start Date: 01-2020
End Date: 12-2024
Amount: $424,636.00
Funder: Australian Research Council
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