Homologous Recombination under the Single-Molecule Fluorescence Microscope

Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein–protein and protein–DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination

Single Molecule Investigations of Holliday Junction Binding Protein RuvA

DNA breaks are inevitable as they mainly occur due to cells’ own reactive oxygen species (ROS). While DNA breaks can be single-stranded or double-stranded, the double-stranded DNA (dsDNA) breaks are more dangerous. If such damage is not repaired, it can lead to genetic instability and serious health issues including cancers. One way dsDNA breaks can be repaired is via a process called homologous recombination (HR), which involves several DNA-binding proteins. Therefore, to have a better insight into the repair mechanism and origin of repair defects, we need a better understanding of how these proteins interact with DNA itself and DNA intermediates of the HR process such as Holliday junctions (HJs). The HJ is a four-way branched structure formed between two homologous DNA molecules during exchange of nucleotide sequences, which is a central intermediate of the DNA repair via the HR process. The HJs are eventually resolved into regular dsDNA molecules by a set of proteins called HJ resolvases. Therefore, knowledge of the binding interaction of these proteins and HJ can provide critical insights into the origin of diseases and potential treatments. Although the HR process has been the subject of intensive study for more than three decades, the complex and dynamic nature of protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA, which is difficult to capture by routine biochemical or structural biology methods. One remedy for this problem is the employment of single molecule techniques such as single-molecule fluorescence microscopy and optical tweezers. These tools provide unique ways of probing these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. However, for single molecule fluorescence microscopy experiments we needed a single molecule total internal reflection fluorescence microscope which we custom built. Using single-molecule fluorescence resonance energy transfer (smFRET) and ensemble analyses, we recently investigated the binding interaction between the HJ and RuvA – a prokaryotic protein that recognizes the HJ and initiates its resolution by forming a resolvase protein complex called RuvABC. Using the HJ labeled with a donor and acceptor fluorophores to enable smFRET, we show that RuvA stably binds to a specific conformation of the HJ, halting its conformational dynamics. Further, the FRET experiments in different ionic environments created by Mg2+ ions suggest that RuvA binds to the HJ via electrostatic interaction. These insights led us to a follow up study looking at the mechanical stability of the RuvA-HJ complex. We have recently developed an optical tweezers-based single-molecule manipulation assay to detect the formation of protein-HJ complexes, which we implemented to study the RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (Funfold) of the HJ by ~2-fold, demonstrating that the RuvA protein stabilizes the junction. Further, the analysis of F-X curves. To our surprise, we also observed that RuvA provides stabilization that permits refolding of the HJ at a force higher than the unfolding force of the HJ without protein. This observation suggests that RuvA stays bound to the DNA construct even after unfolding of the HJ motif, may serve as a nucleation site for HJ refolding, and reduces the energy required for HJ refolding. Together, using high-resolution single-molecule studies we have revealed several molecular insights of the binding interaction between aforementioned proteins and HJ furthering our understating of their roles in the critical HR process. The better the HR process is understood the more likely the scientific community will be able to develop ways of modulating this process for the treatment of recombination related diseases

A web-based biodiversity toolkit as a conservation management tool for natural fragments in an urban context

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol)The collection of biological information has a long history, motivated by a variety of reasons and in more recent years is largely being driven for research and academic purposes. As a result biological information is often linked to a specific species or ecosystem management and is discipline specific, not relating to general management actions at a specific conservation site. The biological data that exists is often not consolidated in a central place to allow for effective management of conservation sites. Different databases and formats are often used to cover biological, infrastructural, heritage and management information. Biological information has traditionally not influenced real-time site-specific conservation management, with long term data sets being used to draw conclusions before they can influence management actions. In order to overcome this problem of scattered and unfocused data a biodiversity database related to specific site management was developed. This study focuses on the development of this database and its links to the management of spatially defined sites. Included in the solution of scattered data are the applications of information management tools which interpret data and convert it into management actions, both in terms of long term trends and immediate real- time management actions as the information is received and processed

A web-based biodiversity toolkit as a conservation management tool for natural fragments in an urban context

>Magister Scientiae - MScThe collection of biological information has a long history, motivated by a variety of reasons and in more recent years is largely being driven for research and academic purposes. As a result biological information is often linked to a specific species or ecosystem management and is discipline specific, not relating to general management actions at a specific conservation site. The biological data that exists is often not consolidated in a central place to allow for effective management of conservation sites. Different databases and formats are often used to cover biological, infrastructural, heritage and management information. Biological information has traditionally not influenced real-time site-specific conservation management, with long term data sets being used to draw conclusions before they can influence management actions. In order to overcome this problem of scattered and unfocused data a biodiversity database related to specific site management was developed. This study focuses on the development of this database and its links to the management of spatially defined sites. Included in the solution of scattered data are the applications of information management tools which interpret data and convert it into management actions, both in terms of long term trends and immediate real- time management actions as the information is received and processed. Information systems are always difficult to describe in words as much of the layout and information is visual and hence difficult to convey I just the text of this document. A breakdown of the resultant information system is outlined in detail in the conclusion section. During the development of a Biodiversity Database it was found that management tools had to be developed to integrated data with management. Furthermore it was found that human error was a significant factor in poor data quality; as a result an observer training programme was developed

Renal neuroendocrine control of desiccation and cold tolerance by Drosophila suzukii

Background: Neuropeptides are central to the regulation of physiological, and behavioural processes in insects, directly impacting cold and desiccation survival. However, little is known about the control mechanisms governing these responses in D. suzukii. The close phylogenetic relationship of D. suzukii with D. melanogaster allows, through genomic and functional studies, an insight into the mechanisms directing stress tolerance in D. suzukii. Results: Capa, Leucokinin, DH44 and DH31 neuropeptides demonstrate a high level of conservation between D. suzukii and D. melanogaster with respect to peptide sequences, neuronal expression, receptor localisation, and diuretic function in the Malpighian tubules. Despite D. suzukii’s ability to populate cold environments, they proved sensitive to both cold and desiccation. Furthermore, in D. suzukii, Capa acts as a desiccation-and cold stress-responsive gene, while DH44 gene expression is increased only after desiccation exposure, and the LK gene after nonlethal cold stress recovery. Conclusion: This study provides a comparative investigation into stress tolerance mediation by neuroendocrine signalling in two Drosophila species, providing evidence that similar signalling pathways control fluid secretion in the Malpighian tubules. Identifying processes governing specific environmental stresses affecting D. suzukii could lead to the development of targeted integrated management strategies to control insect pest populations

What's in a name? - A short history of coordination chemistry from then to now

This article traces the development of coordination chemistry and shows how progress in the science has been paralleled by the development of a vocabulary and nomenclature to describe new concepts, structural features and compound types

The percutaneous toxicokinetics of sulphur mustard in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant

This is the peer reviewed version of the following article: Charlotte A. Hall, et al, 'The percutaneous toxicokinetics of Sulphur mustard in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant', Journal of Applied Toxicology, July 2017, which has been published in final form at DOI: 10.1002/jat.3453. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Copyright © 2017 John Wiley & Sons, Ltd.This study used a damaged skin, porcine model to evaluate the in vivo efficacy of WoundStat™ for decontamination of superficial (non-haemorrhaging), sulphur mustard-contaminated wounds. The dorsal skin of 12 female pigs was subjected to controlled physical damage and exposed to 10 μL 14C–radiolabelled sulphur mustard (14C–SM). Animals were randomly assigned to either a control or a treatment group. In the latter, WoundStat™ was applied 30 s post exposure and left in situ for 1 h. Skin lesion progression and decontaminant efficacy were quantified over 6 h using a range of biophysical measurements. Skin, blood and organ samples were taken post mortem for histopathological assessment, 14C–SM distribution and toxicokinetic analyses. Application of SM to damaged skin without decontamination was rapidly followed by advanced signs of toxicity, including ulceration and decreased blood flow at the exposure site in all animals. WoundStat™ prevented ulceration and improved blood flow at the exposure site in all decontaminated animals (n = 6). Furthermore, significantly smaller quantities of 14C–SM were detected in the blood (45% reduction), and recovered from skin (70% reduction) and skin surface swabs (99% reduction) at 6 h post-challenge. Overall, the distribution of 14C–SM in the internal organs was similar for both groups, with the greatest concentration in the kidneys, followed by the liver and small intestine. WoundStat™ significantly reduced the amount of 14C–SM recovered from the liver, a key organ for SM metabolism and detoxification. This study demonstrates that WoundStat™ is a suitable product for reducing the ingress and toxicity of a chemical warfare agent.Peer reviewedFinal Accepted Versio

Serotonin controlling feeding and satiety

Serotonin has been implicated in the control of satiety for almost four decades. Historically, the insight that the appetite suppressant effect of fenfluramine is linked to serotonin has stimulated interest in and research into the role of this neurotransmitter in satiety. Various rodent models, including transgenic models, have been developed to identify the involved 5-HT receptor subtypes. This approach also required the availability of receptor ligands of different selectivity, and behavioural techniques had to be developed simultaneously which allow differentiating between unspecific pharmacological effects of these ligands and ‘true’ satiation and satiety. Currently, 5-HT1B, 5-HT2C and 5-HT6 receptors have been identified to mediate serotonergic satiety in different ways. The recently approved anti-obesity drug lorcaserin is a 5-HT2C receptor agonist. In brain, both hypothalamic (arcuate nucleus, paraventricular nucleus) and extrahypothalamic sites (parabrachial nucleus, nucleus of the solitary tract) have been identified to mediate the serotonergic control of satiety. Serotonin interacts within the hypothalamus with endogenous orexigenic (Neuropeptide Y/Agouti related protein) and anorectic (α-melanocyte stimulating hormone) peptides. In the nucleus of the solitary tract serotonin integrates peripheral satiety signals. Here, the 5-HT3, but possibly also the 5-HT2C receptor play a role. It has been found that 5-HT acts in concert with such peripheral signals as cholecystokinin and leptin. Despite the recent advances of our knowledge, many of the complex interactions between 5-HT and other satiety factors are not fully understood yet. Further progress in research will also advance the development of new serotonergic anti-obesity drugs

Importance of patient bed pathways and length of stay differences in predicting COVID-19 hospital bed occupancy in England.

Background: Predicting bed occupancy for hospitalised patients with COVID-19 requires understanding of length of stay (LoS) in particular bed types. LoS can vary depending on the patient’s “bed pathway” - the sequence of transfers of individual patients between bed types during a hospital stay. In this study, we characterise these pathways, and their impact on predicted hospital bed occupancy. Methods: We obtained data from University College Hospital (UCH) and the ISARIC4C COVID-19 Clinical Information Network (CO-CIN) on hospitalised patients with COVID-19 who required care in general ward or critical care (CC) beds to determine possible bed pathways and LoS. We developed a discrete-time model to examine the implications of using either bed pathways or only average LoS by bed type to forecast bed occupancy. We compared model-predicted bed occupancy to publicly available bed occupancy data on COVID-19 in England between March and August 2020. Results: In both the UCH and CO-CIN datasets, 82% of hospitalised patients with COVID-19 only received care in general ward beds. We identified four other bed pathways, present in both datasets: “Ward, CC, Ward”, “Ward, CC”, “CC” and “CC, Ward”. Mean LoS varied by bed type, pathway, and dataset, between 1.78 and 13.53 days. For UCH, we found that using bed pathways improved the accuracy of bed occupancy predictions, while only using an average LoS for each bed type underestimated true bed occupancy. However, using the CO-CIN LoS dataset we were not able to replicate past data on bed occupancy in England, suggesting regional LoS heterogeneities. Conclusions: We identified five bed pathways, with substantial variation in LoS by bed type, pathway, and geography. This might be caused by local differences in patient characteristics, clinical care strategies, or resource availability, and suggests that national LoS averages may not be appropriate for local forecasts of bed occupancy for COVID-19. Trial registration: The ISARIC WHO CCP-UK study ISRCTN66726260 was retrospectively registered on 21/04/2020 and designated an Urgent Public Health Research Study by NIHR.</p