Nutrient homeostasis within the plant circadian network

Circadian clocks have evolved to enhance adaptive physiology in the predictable, fluctuating environment caused by the rotation of the planet. Nutrient acquisition is central to plant growth performance and the nutrient demands of a plant change according to the time of day. Therefore, major aspects of nutrient homeostasis, including carbon assimilation and mineral uptake, are under circadian control. It is also emerging that there is feedback of nutritional status to the circadian clock to integrate these processes. This review will highlight recent insights into the role of the circadian clock in regulating plant nutrition as well as discuss the role for nutrients in affecting circadian function

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue- or cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®-compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap-compatible split luciferase assay (ETSLA) system to investigate tissue-specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest

An NgAgo tool for genome editing: did CRISPR/Cas9 just find a competitor?

AbstractWhile CRISPR/Cas9-mediated genome editing technology has been experiencing a rapid transformation during the past few years, a recent report on NgAgo-mediated single-stranded DNA-guided genome editing may offer an attractive alternative for genome manipulation. While it's too early to predict whether NgAgo will be able to compete with or be superior to CRISPR/Cas9, the scientific community is anxiously waiting for further optimization and broader applications of the NgAgo genome editing technology

Sucrose and Ethylene Signaling Interact to Modulate the Circadian Clock.

Circadian clocks drive rhythmic physiology and metabolism to optimize plant growth and performance under daily environmental fluctuations caused by the rotation of the planet. Photosynthesis is a key metabolic process that must be appropriately timed to the light-dark cycle. The circadian clock contributes to the regulation of photosynthesis, and in turn the daily accumulation of sugars from photosynthesis also feeds back to regulate the circadian oscillator. We have previously shown that GIGANTEA (GI) is required to sustain Suc-dependent circadian rhythms in darkness. The mechanism by which Suc affects the circadian oscillator in a GI-dependent manner was unknown. Here, we identify that Suc sustains rhythms in the dark by stabilizing GI protein, dependent on the F-box protein ZEITLUPE, and implicate CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a negative regulator of ethylene signaling. Our identification of a role for CTR1 in the response to Suc prompted a reinvestigation of the effects of ethylene on the circadian oscillator. We demonstrate that ethylene shortens the circadian period, conditional on the effects of Suc and requiring GI These findings reveal that Suc affects the stability of circadian oscillator proteins and can mask the effects of ethylene on the circadian system, identifying novel molecular pathways for input of sugar to the Arabidopsis (Arabidopsis thaliana) circadian network.This research was supported by the BBSRC (BB/H006826/1 and BB/L021188/1

Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies

Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses

Superoxide is promoted by sucrose and affects amplitude of circadian rhythms in the evening

Plants must coordinate photosynthetic metabolism with the daily environment and adapt rhythmic physiology and development to match carbon availability. Circadian clocks drive biological rhythms which adjust to environmental cues. Products of photosynthetic metabolism, including sugars and reactive oxygen species (ROS), are closely associated with the plant circadian clock, and sugars have been shown to provide metabolic feedback to the circadian oscillator. Here, we report a comprehensive sugar-regulated transcriptome of Arabidopsis and identify genes associated with redox and ROS processes as a prominent feature of the transcriptional response. We show that sucrose increases levels of superoxide (O2–), which is required for transcriptional and growth responses to sugar. We identify circadian rhythms of O2–-regulated transcripts which are phased around dusk and find that O2– is required for sucrose to promote expression of TIMING OF CAB1 (TOC1) in the evening. Our data reveal a role for O2– as a metabolic signal affecting transcriptional control of the circadian oscillator in Arabidopsis

Revision total hip arthroplasty using the Zweymuller femoral stem

Background: A variety of femoral stem designs have been reported to be successful in revision total hip arthroplasty without consensus as to optimal design. We evaluated the clinical and radiographic outcomes in a consecutive series of femoral revisions using a wedge-shape, tapered-stem design at medium and long-term follow-up. Materials and methods: We performed a retrospective review of clinical and radiographic outcomes of twenty-eight consecutive femoral revisions arthroplasties, which were done using the Zweymuller femoral stem. Results: The mean follow-up was 7.4 years (range 2-15 years). No stem re-revision was necessary. All stems were judged to be stable by radiographic criteria at the most recent follow-up. The final mean Harris hip score was 90. There was no difference in Harris hip scores, implant stability, or radiological appearance (distal cortical hypertrophy or proximal stress shielding) of the implants between medium-term (mean 5.7 years) and long-term (mean 12.4 years) follow-up. Conclusions: We found the Zweymuller femoral stem design to be durable for revision hip arthroplasty when there is an intact metaphyseal-diaphyseal junction for adequate press-fit stability at surgery. © Springer-Verlag 2008

Organ specificity in the plant circadian system is explained by different light inputs to the shoot and root clocks

Circadian clocks allow the temporal compartmentalisation of biological processes. In Arabidopsis circadian rhythms display organ specificity but the underlying molecular causes have not been identified. We investigated the mechanisms responsible for the similarities and differences between the clocks of mature shoots and roots in constant conditions and in light:dark cycles. We developed an imaging system to monitor clock gene expression in shoots and light- or dark-grown roots, modified a recent mathematical model of the Arabidopsis clock and used this to simulate our new data. We showed that the shoot and root circadian clocks have different rhythmic properties (period and amplitude) and respond differently to light quality. The root clock was entrained by direct exposure to low-intensity light, even in antiphase to the illumination of shoots. Differences between the clocks were more pronounced in conditions where light is present than in constant darkness, and persisted in the presence of sucrose. We simulated the data successfully by modifying those parameters of a clock model that are related to light inputs. We conclude that differences and similarities between the shoot and root clocks can largely be explained by organ-specific light inputs. This provides mechanistic insight into the developing field of organ-specific clocks

Quantifying the Risk of Localised Animal Movement Bans for Foot-and-Mouth Disease

The maintenance of disease-free status from Foot-and-Mouth Disease is of significant socio-economic importance to countries such as the UK. The imposition of bans on the movement of susceptible livestock following the discovery of an outbreak is deemed necessary to prevent the spread of what is a highly contagious disease, but has a significant economic impact on the agricultural community in itself. Here we consider the risk of applying movement restrictions only in localised zones around outbreaks in order to help evaluate how quickly nation-wide restrictions could be lifted after notification. We show, with reference to the 2001 and 2007 UK outbreaks, that it would be practical to implement such a policy provided the basic reproduction ratio of known infected premises can be estimated. It is ultimately up to policy makers and stakeholders to determine the acceptable level of risk, involving a cost benefit analysis of the potential outcomes, but quantifying the risk of spread from different sized zones is a prerequisite for this. The approach outlined is relevant to the determination of control zones and vaccination policies and has the potential to be applied to future outbreaks of other diseases

Latent class evaluation of the performance of serological tests for exposure to Brucella spp. in cattle, sheep, and goats in Tanzania

Background: Brucellosis is a neglected zoonosis endemic in many countries, including regions of sub-Saharan Africa. Evaluated diagnostic tools for the detection of exposure to Brucella spp. are important for disease surveillance and guiding prevention and control activities. Methods and findings: Bayesian latent class analysis was used to evaluate performance of the Rose Bengal plate test (RBT) and a competitive ELISA (cELISA) in detecting Brucella spp. exposure at the individual animal-level for cattle, sheep, and goats in Tanzania. Median posterior estimates of RBT sensitivity were: 0.779 (95% Bayesian credibility interval (BCI): 0.570–0.894), 0.893 (0.636–0.989), and 0.807 (0.575–0.966), and for cELISA were: 0.623 (0.443–0.790), 0.409 (0.241–0.644), and 0.561 (0.376–0.713), for cattle, sheep, and goats, respectively. Sensitivity BCIs were wide, with the widest for cELISA in sheep. RBT and cELISA median posterior estimates of specificity were high across species models: RBT ranged between 0.989 (0.980–0.998) and 0.995 (0.985–0.999), and cELISA between 0.984 (0.974–0.995) and 0.996 (0.988–1). Each species model generated seroprevalence estimates for two livestock subpopulations, pastoralist and non-pastoralist. Pastoralist seroprevalence estimates were: 0.063 (0.045–0.090), 0.033 (0.018–0.049), and 0.051 (0.034–0.076), for cattle, sheep, and goats, respectively. Non-pastoralist seroprevalence estimates were below 0.01 for all species models. Series and parallel diagnostic approaches were evaluated. Parallel outperformed a series approach. Median posterior estimates for parallel testing were ≥0.920 (0.760–0.986) for sensitivity and ≥0.973 (0.955–0.992) for specificity, for all species models. Conclusions: Our findings indicate that Brucella spp. surveillance in Tanzania using RBT and cELISA in parallel at the animal-level would give high test performance. There is a need to evaluate strategies for implementing parallel testing at the herd- and flock-level. Our findings can assist in generating robust Brucella spp. exposure estimates for livestock in Tanzania and wider sub-Saharan Africa. The adoption of locally evaluated robust diagnostic tests in setting-specific surveillance is an important step towards brucellosis prevention and control