Dynamic Control of the DNA Replication Initiation Protein DnaA by Soj/ParA

SummaryRegulation of DNA replication and segregation is essential for all cells. Orthologs of the plasmid partitioning genes parA, parB, and parS are present in bacterial genomes throughout the prokaryotic evolutionary tree and are required for accurate chromosome segregation. However, the mechanism(s) by which parABS genes ensure proper DNA segregation have remained unclear. Here we report that the ParA ortholog in B. subtilis (Soj) controls the activity of the DNA replication initiator protein DnaA. Subcellular localization of several Soj mutants indicates that Soj acts as a spatially regulated molecular switch, capable of either inhibiting or activating DnaA. We show that the classical effect of Soj inhibiting sporulation is an indirect consequence of its action on DnaA through activation of the Sda DNA replication checkpoint. These results suggest that the pleiotropy manifested by chromosomal parABS mutations could be the indirect effects of a primary activity regulating DNA replication initiation

Scoping the impact of tidal and wave energy extraction on suspended sediment concentrations and underwater light climate

The depth to which sunlight penetrates below the sea surface is one of the key factors determining the species composition and productivity of marine ecosystems. The effects range from the rate and fate of primary production, through the performance of visual predators such as fish, the potential for refuge from predators by migrating to depth, to the scope for seabed stabilisation by algal mats. Light penetration depends partly on spectral absorption by seawater and dissolved substances, but mainly on the scattering caused by suspended particulate material (SPM). Some of this SPM may be of biological origin, but in coastal waters the majority is mineral material originating ultimately from seabed disturbance and land erosion, the latter being deposited in the sea by rivers and aerial processes. SPM is maintained in the water column or deposited on the seabed depending on combinations of hydrodynamic processes including baroclinic (density-driven) or barotropic (mainly tidal and wind driven) currents, and wave action (Ward et al. 1984; Huettel et al. 1996). Since tidal and wave energy extraction must alter these hydrodynamic properties at some scales depending on the nature of the extraction process, we can expect some kind of impact on the concentration of the SPM. If these are large enough, we may have to consider the extent to which these may impact the underwater light environment and the local or regional ecology. Whilst several coupled hydrodynamic-sediment models exist to predict SPM distributions in aquatic systems, their skill level in open coastal and offshore marine waters is acknowledged to be relatively low. This is largely because the processes are not well understood and the formulations are largely based on empirical relationships rather than fundamental physical principles. The models are also highly demanding in terms of calibration data and computational resources. Hence their utility for predicting relatively subtle effects arising from changes in flow or wave environments due to energy extraction devices seems rather low. Here, we summarise the key mathematical functions describing the processes involved in sediment suspension, and propose a lightweight one-dimensional (vertical) model which can be used to scope the effects of changes in flow and wave energy on SPM

Modelling the sensitivity of suspended sediment profiles to tidal current and wave conditions

Seawater turbidity due to suspended particulate material (SPM) is an important property of a marine ecosystem, determining the underwater light environment and many aspects of biological production and ecology. SPM concentrations are largely determined by patterns of sediment resuspension from the seabed due to shear stress caused by waves and currents. Hence planning for the construction of large scale offshore structures which will alter regional hydrodynamics needs to consider the consequences for SPM concentrations. Here we develop a one-dimensional (vertical) model of SPM dynamics which can be used to scope the effects of changes in wave and tidal current properties at a site. We implement the model for a number of sites off the east coast of Scotland where we have extensive data sets to enable numerical parameter optimisation. The model performs well at simulating fluctuations in turbidity varying from flood-ebb tidal cycles, spring-neap cycles, storm wave events, and an annual cycle of SPM concentration which is attributed to seasonal consolidation of seabed sediments. Sensitivity analysis shows that, for the range of seabed sediment types in the study (water depth 16 – 50 m; mud content 0.006 – 0.380 proportion by weight), relatively large (50%) attenuations of tidal current speed are required to produce changes in water column turbidity which would be detectable by observations given the variability in measurements. The model has potential for application to map the large scale sensitivity of turbidity distributions to the installation of wave and tidal energy extraction arrays

Modelling wave-current interactions off the east coast of Scotland

Densely populated coastal areas of the North Sea are particularly vulnerable to severe wave conditions, which overtop or damage sea-defences leading to dangerous flooding. Around the shallow southern North Sea, where the coastal margin is low-lying and population density is high, oceanographic modelling has helped to develop forecasting systems to predict flood risk. However coastal areas of the deeper northern North Sea are also subject to regular storm damage but there has been little or no effort to develop coastal wave models for these waters. Here we present a high spatial resolution model of northeast Scottish coastal waters, simulating waves and the effect of tidal currents on wave propagation, driven by global ocean tides, far-field wave conditions, and local air pressure and wind stress. We show that the wave- current interactions and wave-wave interactions are particularly important for simulating the wave conditions close to the coast at various locations. The model can simulate the extreme conditions experienced when high (spring) tides are combined with sea-level surges and large Atlantic swell. Such a combination of extremes represents a high risk for damaging conditions along the Scottish coast

Modelling sea level surges in the Firth of Clyde, a fjordic embayment in south-west Scotland

Storm surges are an abnormal enhancement of the water level in response to weather perturbations. They have the capacity to cause damaging flooding of coastal regions, expecially when they coincide with astronomical high spring tides. Some areas of the UK have suffered particularly damaging surge events, and the Firth of Clyde is a region with high risk due to its location and morphology. Here we use a three-dimensional high spatial resolution hydrodynamic model to simulate the local bathymetric and morphological enhancement of surge in the Clyde, and disaggregate the effects of far-field atmospheric pressure distribution and local scale wind forcing of surges. A climatological analysis, based on 30 years of data from Millport tide gauges is also discussed. The results suggest that floods are not only caused by extreme surge events, but also by the coupling of spring high tides with moderate surges. Water level is also enhanced by a funnelling effect due to the bathymetry and the morphology of fjordic sealochs and the River Clyde estuary. In a world of rising sea level, studying the propagation and the climatology of surges and high water events is fundamental. In addition, high-resolution hydrodynamic models are essential to forecast extreme events and prevents the loss of lives, or to plan coastal defences solutions

Student experiences and perceptions of compulsory research projects: a veterinary perspective

Background Although research underpins clinical work, many students training to be clinicians are not inherently interested in developing research skills. Aim To characterise and understand veterinary student experiences and perceptions of compulsory research projects. Methods This was an explanatory sequential mixed-methods study, with a questionnaire survey of an entire cohort informing purposive selection for focus group discussions. Student views were triangulated with staff questionnaire data. Results About a third of the cohort felt that the project had not been worthwhile or had not fostered useful skills. Focus group data analysis identified fragility of motivation and lack of clear schemata for the research process as key themes. Students were easily demotivated by typical research challenges and lack of schemata contributed to a poor understanding of the rationale for the project, encouraging highly extrinsic forms of motivation. Triangulation with staff questionnaire data indicated that staff understood students’ challenges, but were more likely than students to consider it to be a valuable learning experience. Conclusions Findings support ongoing curriculum development and emphasise that, to optimise motivation, engagement and learning, students training to be clinicians need a clear rationale for research, based on development of critical inquiry skills as a core clinical competency

The APC network regulates the removal of mutated cells from colonic crypts

Self-renewal is essential for multicellular organisms but carries the risk of somatic mutations that can lead to cancer, which is particularly critical for rapidly renewing tissues in a highly mutagenic environment such as the intestinal epithelium. Using computational modeling and in vivo experimentation, we have analyzed how adenomatous polyposis coli (APC) mutations and β-catenin aberrations affect the maintenance of mutant cells in colonic crypts. The increasing abundance of APC along the crypt axis forms a gradient of cellular adhesion that causes more proliferative cells to accelerate their movement toward the top of the crypt, where they are shed into the lumen. Thus, the normal crypt can efficiently eliminate β-catenin mutant cells, whereas APC mutations favor retention. Together, the molecular design of the APC/β-catenin signaling network integrates cell proliferation and migration dynamics to translate intracellular signal processing and protein gradients along the crypt into intercellular interactions and whole-crypt physiological or pathological behavior

DNA replication initiation in Bacillus subtilis: Structural and functional characterization of the essential DnaA-DnaD interaction

© 2018 The Author(s). The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation

In situ delivery of nanoparticles formulated with micron-sized crystals protects from murine melanoma.

INTRODUCTION Intratumoral injections of novel therapeutics can activate tumor antigen-specific T cells for locoregional tumor control and may even induce durable systemic protection (against distant metastases) via recirculating T cells. Here we explored the possibility of a universal immunotherapy that promotes T-cell responses in situ and beyond, upon intratumoral injection of nanoparticles formulated with micron-sized crystals. METHODS Cucumber mosaic virus-like particles containing a tetanus toxin peptide (CuMVTT) were formulated with microcrystalline tyrosine (MCT) adjuvant and injected directly in B16F10 melanoma tumors. To further enhance immunogenicity, we loaded the nanoparticles with a TLR7/8 ligand and incorporated a universal tetanus toxin T-helper cell peptide. We assessed therapeutic efficacy and induction of local and systemic immune responses, including RNA sequencing, providing broad insight into the tumor microenvironment and correlates of protection. RESULTS MCT crystals were successfully decorated with CuMVTT nanoparticles. This 'immune-enhancer' formed immunogenic depots in injected tumors, enhanced polyfunctional CD8+ and CD4+ T cells, and inhibited B16F10 tumor growth locally and systemically. Local inflammation and immune responses were associated with upregulation of genes involved in complement activation and collagen formation. CONCLUSIONS Our new immune-enhancer turned immunologically cold tumors into hot ones and inhibited local and distant tumor growth. This type of immunotherapy does not require the identification of (patient-individual) relevant tumor antigens. It is well tolerated, non-infectious, and affordable, and can readily be upscaled for future clinical testing and broad application in melanoma and likely other solid tumors

On the protection of extrasolar Earth-like planets around K/M stars against galactic cosmic rays

Previous studies have shown that extrasolar Earth-like planets in close-in habitable zones around M-stars are weakly protected against galactic cosmic rays (GCRs), leading to a strongly increased particle flux to the top of the planetary atmosphere. Two main effects were held responsible for the weak shielding of such an exoplanet: (a) For a close-in planet, the planetary magnetic moment is strongly reduced by tidal locking. Therefore, such a close-in extrasolar planet is not protected by an extended magnetosphere. (b) The small orbital distance of the planet exposes it to a much denser stellar wind than that prevailing at larger orbital distances. This dense stellar wind leads to additional compression of the magnetosphere, which can further reduce the shielding efficiency against GCRs. In this work, we analyse and compare the effect of (a) and (b), showing that the stellar wind variation with orbital distance has little influence on the cosmic ray shielding. Instead, the weak shielding of M star planets can be attributed to their small magnetic moment. We further analyse how the planetary mass and composition influence the planetary magnetic moment, and thus modify the cosmic ray shielding efficiency. We show that more massive planets are not necessarily better protected against galactic cosmic rays, but that the planetary bulk composition can play an important role.Comment: 7 figure