Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria

A negative feedback control of kaiC expression by KaiC protein has been proposed to generate a basic oscillation of the circadian clock in the cyanobacterium Synechococcus sp. PCC 7942. KaiC has two P loops or Walker's motif As, that are potential ATP-/GTP-binding motifs and DXXG motifs conserved in various GTP-binding proteins. Herein, we demonstrate that in vitro KaiC binds ATP and, with lower affinity, GTP. Point mutation by site-directed mutagenesis of P loop 1 completely nullified the circadian rhythm of kaiBC expression and markedly reduced ATP-binding activity. Moreover, KaiC can be autophosphorylated in vitro. These results suggest that the nucleotide-binding activity of KaiC plays important roles in the generation of circadian oscillation in cyanobacteria.

Detecting KaiC Phosphorylation Rhythms of the Cyanobacterial Circadian Oscillator In Vitro and In Vivo

The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins: KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period, and these states can be analyzed simply by SDS-PAGE. The purity of the proteins is critical for obtaining robust oscillation. Contaminating proteases will destroy oscillation by degradation of Kai proteins, and ATPases will attenuate robustness by consumption of ATP. Here, we provide a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro. In addition, we present a protocol that facilitates analysis of phosphorylation states of KaiC and other phosphorylated proteins from in vivo samples.

Robustness and Coherence of a Three-Protein Circadian Oscillator: Landscape and Flux Perspectives

Three-protein circadian oscillations in cyanobacteria sustain for weeks. To understand how cellular oscillations function robustly in stochastic fluctuating environments, we used a stochastic model to uncover two natures of circadian oscillation: the potential landscape related to steady-state probability distribution of protein concentrations; and the corresponding flux related to speed of concentration changes which drive the oscillations. The barrier height of escaping from the oscillation attractor on the landscape provides a quantitative measure of the robustness and coherence for oscillations against intrinsic and external fluctuations. The difference between the locations of the zero total driving force and the extremal of the potential provides a possible experimental probe and quantification of the force from curl flux. These results, correlated with experiments, can help in the design of robust oscillatory networks.

Recommendations for a trans-European dietary assessment method in children between 4 and 14 years

Background/Objectives: The main objective of European Food Consumption Validation (EFCOVAL)-child Project is to define and evaluate a trans-European methodology for undertaking national representative dietary surveys among children in the age group of 4–14 years. In the process of identifying the best dietary assessment methodologies, experts were brought together at a workshop. The paper presents the discussion of the best available method and the final recommendations for a trans-European dietary assessment method among 4- to 14-year-old children.
Subjects/Methods: The starting point was to investigate whether the method (two non-consecutive 24-h dietary recalls (24-HDRs)) suggested for the adults in European Food Consumption Survey Method (EFCOSUM) would be usable for children in
the age group between 4 and 14 years. However, all available dietary assessment methods were included in the discussion to ensure that the final recommendation would be based on the best evidence. Six criteria were defined and used as additional
guidance in the process.
Results: The literature does not give a clear recommendation on the dietary assessment methods that are most suitable for children in the age group of 4–14 years. Nevertheless, on the basis of the literature, the recommendations were separated for preschoolers (4–6 years) and schoolchildren (7–14 years).
Conclusion: For preschoolers, two non-consecutive days of a structured food record are recommended, using a (for children adapted) picture booklet and household measures for portion-size estimation. For schoolchildren, repeated 24-HDRs are recommended, using a picture booklet and household measures for portion-size estimation. In addition, the child should bring a booklet to register what is eaten out of home. One parent should assist the schoolchild at the 24-HDR interview, and therefore face-to-face interviews are required.

Feasibility of repeated 24-h dietary recalls combined with a food-recording booklet, using EPIC-Soft, among preschoolers

Background/Objectives: This study evaluates the feasibility among preschoolers of the 224-h dietary recalls (24-HDRs) method combined with a food-recording booklet (FRB), using EPIC-Soft pc-program for the 24-HDR (the software developed to conduct 24-HDRs in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study).
Subjects/Methods: A total of 20 and 25 (4- to 5-year-old) children were recruited, as a convenience sample, through worksites or day or healthcare in Denmark and Spain, respectively. One parent (or both parents together) completed two face-to-face 24- HDR, combined with an optional use of a FRB. Feasibility was evaluated by evaluation questionnaires completed by parents and interviewers.
Results: The face-to-face interviews were primarily conducted with the mothers. The FRB was used by 90% of the participants, and proxy persons, other than the parent, were also involved; involvement of proxy persons seems necessary in a majority of the recalls in both the countries.
Conclusions: The results suggest that 224-HDR with one parent combined with a FRB is feasible for registering preschoolers’ diet. An FRB and/or information from proxy persons, other than the parent, is needed for a majority of the parents. In future studies, it may be beneficial to develop the FRB more like a structured food record (FR), which might, in principle, change the method to a one-day FR method from more than a 24-HDR method. It is recommended then to further investigate the use of EPIC-Soft as a data-entry tool.