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The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V . parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella . Our results showed that two subpopulations (P1 and P2) of V . parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:Δ lldD ) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:Δ lldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Clostridium perfringens epsilon toxin (Etx) is a pore forming toxin that causes enterotoxaemia in ruminants and may be a cause of multiple sclerosis in humans. To date, most in vitro studies of Etx have used the Madin-Darby canine kidney (MDCK) cell line. However, studies using Chinese hamster ovary (CHO) cells engineered to express the putative Etx receptor, myelin and lymphocyte protein (MAL), suggest that amino acids important for Etx activity differ between species. In this study, we investigated the role of amino acids Y42, Y43 and H162, previously identified as important in Etx activity towards MDCK cells, in Etx activity towards CHO-human MAL (CHO-hMAL) cells, human red blood cells (hRBCs) and synthetic bilayers using site-directed mutants of Etx. We show that in CHO-hMAL cells Y42 is critical for Etx binding and not Y43 as in MDCK cells, indicating that surface exposed tyrosine residues in the receptor binding domain of Etx impact efficiency of cell binding to MAL-expressing cells in a species-specific manner. We also show that Etx mutant H162A was unable to lyse CHO-hMAL cells, lysed hRBCs, whilst it was able to form pores in synthetic bilayers, providing evidence of the complexity of Etx pore formation in different lipid environments.

A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin–Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine. Genetic toxoids avoid MAL for reduced host toxicity Cells expressing myelin and lymphocyte protein (MAL), the putative receptor for Clostridium perfringens’ epsilon toxin, can be sensitive to otherwise attenuated mutants of the toxin. Here, the team led by Richard Titball at United Kingdom’s University of Exeter found that a previous variant exhibits differential toxic effects when cells express sheep or human MAL. To circumvent this, Titball’s team applied site-directed mutagenesis of the receptor binding site to develop a new variant with enhanced reduction in toxicity towards MAL-expressing cells and able to induce high levels of neutralising antibodies upon immunisation of sheep. These findings suggests that testing genetic toxoids in cells expressing MAL from the target species might be relevant for enterotoxaemia vaccine development and warrant further studies into the role of MAL in epsilon toxin-mediated pathogenesis.

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that 11168H forms persister cells at a frequency of 10 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

ABSTRACTHere, we present the genome sequence of Staphylococcus aureus Ex1, isolated in 2015 from a patient with spinal osteomyelitis at the Royal Devon and Exeter Hospital in the United Kingdom. The availability of the Ex1 genome sequence provides a resource for studying the basis for spinal infection and horizontal gene transfer in S. aureus.

Clostridium perfringens epsilon toxin (Etx) is a pore forming toxin that causes enterotoxaemia in ruminants and may be a cause of multiple sclerosis in humans. To date, most in vitro studies of Etx have used the Madin-Darby canine kidney (MDCK) cell line. However, studies using Chinese hamster ovary (CHO) cells engineered to express the putative Etx receptor, myelin and lymphocyte protein (MAL), suggest that amino acids important for Etx activity differ between species. In this study, we investigated the role of amino acids Y42, Y43 and H162, previously identified as important in Etx activity towards MDCK cells, in Etx activity towards CHO-human MAL (CHO-hMAL) cells, human red blood cells (hRBCs) and synthetic bilayers using site-directed mutants of Etx. We show that in CHO-hMAL cells Y42 is critical for Etx binding and not Y43 as in MDCK cells, indicating that surface exposed tyrosine residues in the receptor binding domain of Etx impact efficiency of cell binding to MAL-expressing cells in a species-specific manner. We also show that Etx mutant H162A was unable to lyse CHO-hMAL cells, lysed hRBCs, whilst it was able to form pores in synthetic bilayers, providing evidence of the complexity of Etx pore formation in different lipid environments.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two key subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. The two subpopulations displayed different abilities for revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) has also revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. The proteins that were significantly up and down regulated between the different VBNC populations were determined and significantly regulated proteins were assigned into 23 functional groups, the majority being included in metabolism functional categories. A lactate dehydrogenase (lldD) protein was significantly upregulated in all subpopulations of VBNC cells and is responsible for converting lactate to pyruvate. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene causes the cells to enter the VBNC state significantly faster than the wild-type, and exogenously adding lactate to VBNC cells aided resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase plays a putative key role in regulating the VBNC state. Competing Interest Statement The authors have declared no competing interest.

We describe Global Atmosphere 6.0 and Global Land 6.0 (GA6.0/GL6.0): the latest science configurations of the Met Office Unified Model and JULES (Joint UK Land Environment Simulator) land surface model developed for use across all timescales. Global Atmosphere 6.0 includes the ENDGame (Even Newer Dynamics for General atmospheric modelling of the environment) dynamical core, which significantly increases mid-latitude variability improving a known model bias. Alongside developments of the model's physical parametrisations, ENDGame also increases variability in the tropics, which leads to an improved representation of tropical cyclones and other tropical phenomena. Further developments of the atmospheric and land surface parametrisations improve other aspects of model performance, including the forecasting of surface weather phenomena. We also describe GA6.1/GL6.1, which includes a small number of long-standing differences from our main trunk configurations that we continue to require for operational global weather prediction. Since July 2014, GA6.1/GL6.1 has been used by the Met Office for operational global numerical weather prediction, whilst GA6.0/GL6.0 was implemented in its remaining global prediction systems over the following year.

A globally complete, high temporal resolution and multiple-variable approach is employed to analyse the diurnal cycle of Earth's outgoing energy flows. This is made possible via the use of Met Office model output for September 2010 that is assessed alongside regional satellite observations throughout. Principal component analysis applied to the long-wave component of modelled outgoing radiation reveals dominant diurnal patterns related to land surface heating and convective cloud development, respectively explaining 68.5 and 16.0 % of the variance at the global scale. The total variance explained by these first two patterns is markedly less than previous regional estimates from observations, and this analysis suggests that around half of the difference relates to the lack of global coverage in the observations. The first pattern is strongly and simultaneously coupled to the land surface temperature diurnal variations. The second pattern is strongly coupled to the cloud water content and height diurnal variations, but lags the cloud variations by several hours. We suggest that the mechanism controlling the delay is a moistening of the upper troposphere due to the evaporation of anvil cloud. The short-wave component of modelled outgoing radiation, analysed in terms of albedo, exhibits a very dominant pattern explaining 88.4 % of the variance that is related to the angle of incoming solar radiation, and a second pattern explaining 6.7 % of the variance that is related to compensating effects from convective cloud development and marine stratocumulus cloud dissipation. Similar patterns are found in regional satellite observations, but with slightly different timings due to known model biases. The first pattern is controlled by changes in surface and cloud albedo, and Rayleigh and aerosol scattering. The second pattern is strongly coupled to the diurnal variations in both cloud water content and height in convective regions but only cloud water content in marine stratocumulus regions, with substantially shorter lag times compared with the long-wave counterpart. This indicates that the short-wave radiation response to diurnal cloud development and dissipation is more rapid, which is found to be robust in the regional satellite observations. These global, diurnal radiation patterns and their coupling with other geophysical variables demonstrate the process-level understanding that can be gained using this approach and highlight a need for global, diurnal observing systems for Earth outgoing radiation in the future.