The sporulation-specific small regulatory RNAs of Bacillus subtilis

Constantly changing environments in nature have led to bacteria evolving regulatory strategies that result in differential gene expression. A novel and understudied aspect of these networks are regulatory RNAs. The Gram-positive model organism Bacillus subtilis not only modulates gene expression to survive a variety of stresses, but also can form endospores to ensure its survival. Sporulation is an essential survival mechanism for many species, allowing them to enter a state of dormancy with resistance to various harsh conditions. This, in turn, ensures survival of not only the population, but also the species. The process of sporulation requires the controlled expression of approximately a quarter of the genes encoded by B. subtilis. Previous large-scale studies have identified that many transcripts do not encode proteins, but exhibited expression profiles similar to genes already known to be part of the sporulation network. Many of these transcripts were selected to likely function as small regulatory RNAs (sRNAs). This study has shown that many putative sRNAs are active during sporulation, three of which show specific phenotypes that alter germination capabilities in the presence of specific germinants. Cells lacking the necessary components to reverse this process are at a strong disadvantage. Detection of favorable growth conditions is key, but how is this conveyed during metabolic inactivity? Initial selection of putative sRNAs was done by in silico characterization. Prediction of transcriptional control and regulatory regions combined with tiling array profiling was used to select putative sRNAs for confirmation in vivo. Transcriptional fusion constructs were generated to confirm compartmental specific expression during sporulation. Spore specific sRNAs were further characterized with phenotypic studies, which suggested a role in endospore formation. This study explored some of the global analysis methods to identify sRNA targets. Whilst no targets for the four chosen sRNAs could be identified, this study produced the most comprehensive data set of proteins to be identified from a B. subtilis endospore

Pre-project monitoring of the Qwuloolt restoration in the Snohomish River Estuary

The Qwuloolt restoration site is approximately 150 hectares of former estuarine wetland in the Snohomish River system that will have tidal inundation returned via levee breach in late 2015. Qwuloolt is one of several large restoration projects planned for the Snohomish River estuary in the next decade for recovery of salmon and other biota, which together could restore several thousand acres and constitute one of the most significant restoration efforts in Puget Sound. In 2008 we began development and implementation of a comprehensive monitoring plan for Qwuloolt that evaluates a broad suite of abiotic and biotic attributes (e.g., land forms, hydrology, and chemistry; taxonomic composition of plant, invertebrate, fish, and bird assemblages). Four years of pre-breach data document clear contrasts between Qwuloolt and adjacent reference sites. Qwuloolt is subsided, hydrologically isolated, and its biota composed of relatively few species and dominated by nonnative, freshwater species. These results provide an invaluable foundation for scientifically rigorous post-breach evaluation of project performance, and contribute to estuary-wide understanding of cumulative effects of restoration and basic estuarine ecology of Puget Sound

Consequences of <i>in vitro</i> benzyl butyl phthalate exposure for blubber gene expression and insulin-induced Akt activation in juvenile grey seals

Plastic and plasticiser pollution of marine environments is a growing concern. Although phthalates, one group of plasticisers, are rapidly metabolised by mammals, they are found ubiquitously in humans and have been linked with metabolic disorders and altered adipose function. Phthalates may also present a threat to marine mammals, which need to rapidly accumulate and mobilise their large fat depots. High molecular weight (HMW) phthalates may be most problematic because they can accumulate in adipose. We used blubber explants from juvenile grey seals to examine the effects of overnight exposure to the HMW, adipogenic phthalate, benzyl butyl phthalate (BBzP) on expression of key adipose-specific genes and on phosphorylation of Akt in response to insulin. We found substantial differences in transcript abundance of Pparγ, Insig2, Fasn, Scd, Adipoq and Lep between moult stages, when animals were also experiencing differing mass changes, and between tissue depths, which likely reflect differences in blubber function. Akt abundance was higher in inner compared to outer blubber, consistent with greater metabolic activity in adipose closer to muscle than skin, and its phosphorylation was stimulated by insulin. Transcript abundance of Pparγ and Fasn (and Adipoq in some animals) were increased by short term (30 min) insulin exposure. In addition, overnight in vitro BBzP exposure altered insulin-induced changes in Pparγ (and Adipoq in some animals) transcript abundance, in a tissue depth and moult stage-specific manner. Basal or insulin-induced Akt phosphorylation was not changed. BBzP thus acted rapidly on the transcript abundance of key adipose genes in an Akt-independent manner. Our data suggest phthalate exposure could alter seal blubber development or function, although the whole animal consequences of these changes are not yet understood. Knowledge of typical phthalate exposures and toxicokinetics would help to contextualise these findings in terms of phthalate-induced metabolic disruption risk and consequences for marine mammal health

Signs of life:oxygen sensors confirm viability, measure oxygen consumption and provide rapid, effective contamination monitoring for field-based tissue culture

Understanding the ecology and evolution of wildlife and domesticated species requires knowledge of their physiological responses to environmental change and the constraints under which they operate. However, whole animal experiments are often limited in sample size and can be logistically and ethically challenging. Culture techniques represent a powerful approach, but are used infrequently in field research due to practical constraints. We used minimal tissue culture equipment in a remote field site for in vitro explant experiments using blubber from wild grey seals Halichoerus grypus. Assessing explant viability and detecting microbial contamination in remote field sites, where facilities are often small, unspecialised and more vulnerable to bacterial or fungal infection, present major challenges. We investigated whether oxygen‐sensitive planar optodes (OSPO) in closed system respirometry could be used to assess oxygen consumption by blubber explants from suckling and fasting wild seal pups as a proxy for viability. We also explored whether OSPOs could provide rapid information on whole animal relevant physiological metrics by determining whether explant oxygen consumption correlated with the nutritional state of the animal, blubber depth and other tissue metabolic properties, including glucose uptake, lactate production and lipolysis. Vials containing blubber explants consumed significant amounts of oxygen compared to controls, showing tissues were metabolically active. Oxygen consumption differed between nutritional states and blubber tissue depth. These differences were reflected in other tissue metabolic properties. Dissolved oxygen levels remained consistent over 24 hr in 94% of control vials containing only culture media. In 6% of control vials extremely rapid oxygen consumption preceded, by 2–3 days, colour changes in the phenol‐red containing media that indicate lactic acidosis from microbial metabolic activity. Oxygen use in control vials was, therefore, an effective monitoring system that provided vital early warning of media contamination, allowing stocks to be discarded, which prevented erroneous results and avoided waste of valuable field time and irreplaceable samples. OSPO are thus a useful tool for simultaneously assessing tissue oxygen consumption, investigating functional physiological differences and monitoring microbiological contamination in culture experiments, particularly in field laboratories studying live tissues from wildlife

Metagenomic profiling of ticks: Identification of novel rickettsial genomes and detection of tick-borne canine parvovirus

Background: Across the world, ticks act as vectors of human and animal pathogens. Ticks rely on bacterial endosymbionts, which often share close and complex evolutionary links with tick-borne pathogens. As the prevalence, diversity and virulence potential of tick-borne agents remain poorly understood, there is a pressing need for microbial surveillance of ticks as potential disease vectors. Methodology/Principal Findings: We developed a two-stage protocol that includes 16S-amplicon screening of pooled samples of hard ticks collected from dogs, sheep and camels in Palestine, followed by shotgun metagenomics on individual ticks to detect and characterise tick-borne pathogens and endosymbionts. Two ticks isolated from sheep yielded an abundance of reads from the genus Rickettsia, which were assembled into draft genomes. One of the resulting genomes was highly similar to Rickettsia massiliae strain MTU5. Analysis of signature genes showed that the other represents the first genome sequence of the potential pathogen Candidatus Rickettsia barbariae. Ticks from a dog and a sheep yielded draft genome sequences of Coxiella strains. A sheep tick yielded sequences from the sheep pathogen Anaplasma ovis, while Hyalomma ticks from camels yielded sequences belonging to Francisella-like endosymbionts. From the metagenome of a dog tick from Jericho, we generated a genome sequence of a canine parvovirus. Significance: Here, we have shown how a cost-effective two-stage protocol can be used to detect and characterise tick-borne pathogens and endosymbionts. In recovering genome sequences from an unexpected pathogen (canine parvovirus) and a previously unsequenced pathogen (Candidatus Rickettsia barbariae), we demonstrate the open-ended nature of metagenomics. We also provide evidence that ticks can carry canine parvovirus, raising the possibility that ticks might contribute to the spread of this troublesome virus

Predicting consequences of POP-induced disruption of blubber glucose uptake, mass gain rate and thyroid hormone levels for weaning mass in grey seal pups

Persistent organic pollutants (POPs) are endocrine disruptors that alter adipose tissue development, regulation and function. Top marine predators are particularly vulnerable because they possess large fat stores that accumulate POPs. However, links between endocrine or adipose tissue function disruption and whole animal energetics have rarely been investigated. We predicted the impact of alterations to blubber metabolic characteristics and circulating thyroid hormone (TH) levels associated with polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and organochlorine pesticides (OCPs) on suckling mass gain and weaning mass in wild grey seal pups. Glucose uptake by inner blubber was a strong predictor of whole animal mass gain rate, which in turn, resulted in heavier weaning mass. Weaning mass was predicted to increase by 3.7 ± 1.59 (sem) %, through increased mass gain rate, in the absence of the previously reported suppressive effect of dioxin-like PCB (DL-PCBs) on blubber glucose uptake. PBDEs were, conversely, associated with faster mass gain. Alleviation of this effect was predicted to reduce weaning mass by 6.02 ± 1.86% (sem). To better predict POPs effects on energy balance, it is crucial to determine if and how PBDEs promote mass gain in grey seal pups. Weaning mass was negatively related to total T3 (TT3) levels. A 20% (range = 9.3–31.7%) reduction in TT3 by DL-PCBs partially overcame the effect of DL-PCB -mediated reduction in blubber glucose uptake. Overall, DL-PCBs were thus predicted to reduce weaning mass by 1.86 ± 1.60%. Organohalogen impacts on whole-animal energy balance in grey seal pups appear to partially offset each other through opposing effects on different mechanisms. POP effects were generally minor, but the largest POP-induced reductions in weaning mass were predicted to occur in pups that were already small. Since weaning mass is positively related to first-year survival, POPs may disproportionately affect smaller individuals, and could continue to have population-level impacts even when levels are relatively low compared to historical values. Our findings show how in vitro experiments combined with measurements in vivo can help elucidate mechanisms that underpin energy balance regulation and help to quantify the magnitude of disruptive effects by contaminants and other stressors in wildlife

Towards the Understanding of Chinese Collaboration in Hospitality – The Opinions of Professionals

The increasingly competitive market in China has resulted in the struggle of hotels for competitive advantage, and even for survival. Among the possible tools to rise above this cut-throat environment is the concept of hotel collaboration. This paper moves towards the understanding of hotel collaboration in China based on the focus group discussions of 22 experienced practitioners in the Chinese hotel and related industries. The data identified several threats, opportunities, and insights into hotel collaboration of domestic with overseas hotels as well as predictions for the future of this practice in China. Based on specific cultural, political, and economic contexts of contemporary China, the results indicate that partial collaboration is likely to succeed in the hotel market of the country

Autophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.This research was funded by the National Health and Medical Research Council of Australia through Project grant 433614 (JKH), Program grant 487922 (JKH), a Senior Research Fellowship (JKH), and a Howard Florey Centenary Fellowship (HV). Operational Infrastructure Support was provided by the Victorian Government, Australia. Additional support was from Australian Research Council grant DP0346823 (GJL); NIH grant DK060322 (DYRS); and CDMRP, Department of Defense, USA W81XWH-10-1-0854 (KCE)

Climate Change Meets the Law of the Horse

The climate change policy debate has only recently turned its full attention to adaptation - how to address the impacts of climate change we have already begun to experience and that will likely increase over time. Legal scholars have in turn begun to explore how the many different fields of law will and should respond. During this nascent period, one overarching question has gone unexamined: how will the legal system as a whole organize around climate change adaptation? Will a new distinct field of climate change adaptation law and policy emerge, or will legal institutions simply work away at the problem through unrelated, duly self-contained fields, as in the famous Law of the Horse? This Article is the first to examine that question comprehensively, to move beyond thinking about the law and climate change adaptation to consider the law of climate change adaptation. Part I of the Article lays out our methodological premises and approach. Using a model we call Stationarity Assessment, Part I explores how legal fields are structured and sustained based on assumptions about the variability of natural, social, and economic conditions, and how disruptions to that regime of variability can lead to the emergence of new fields of law and policy. Case studies of environmental law and environmental justice demonstrate the model’s predictive power for the formation of new distinct legal regimes. Part II applies the Stationarity Assessment model to the topic of climate change adaptation, using a case study of a hypothetical coastal region and the potential for climate change impacts to disrupt relevant legal doctrines and institutions. We find that most fields of law appear capable of adapting effectively to climate change. In other words, without some active intervention, we expect the law and policy of climate change adaptation to follow the path of the Law of the Horse - a collection of fields independently adapting to climate change - rather than organically coalescing into a new distinct field. Part III explores why, notwithstanding this conclusion, it may still be desirable to seek a different trajectory. Focusing on the likelihood of systemic adaptation decisions with perverse, harmful results, we identify the potential benefits offered by intervening to shape a new and distinct field of climate change adaptation law and policy. Part IV then identifies the contours of such a field, exploring the distinct purposes of reducing vulnerability, ensuring resiliency, and safeguarding equity. These features provide the normative policy components for a law of climate change adaptation that would be more than just a Law of the Horse. This new field would not replace or supplant any existing field, however, as environmental law did with regard to nuisance law, and it would not be dominated by substantive doctrine. Rather, like the field of environmental justice, this new legal regime would serve as a holistic overlay across other fields to ensure more efficient, effective, and just climate change adaptation solutions

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy