Biochemical and spectroscopic characterization of the bacterial phytochrome of Pseudomonas aeruginosa

Phytochromes are photochromic biliproteins found in plants as well as in some cyanotrophic, photoautotrophic and heterotrophic bacteria. In many bacteria, their function is largely unknown. Here we describe the biochemical and spectroscopic characterization of recombinant bacterial phytochrome from the opportunistic pathogen Pseudomonas aeruginosa (PaBphP). The recombinant protein displays all the characteristic features of a bonafide phytochrome. In contrast with cyanobacteria and plants, the chromophore of this bacterial phytochrome is biliverdin IXalpha, which is produced by the heme oxygenase BphO in P. aeruginosa. This chromophore was shown to be covalently attached via its A-ring endo-vinyl group to a cysteine residue outside the defined bilin lyase domain of plant and cyanobacterial phytochromes. Site-directed mutagenesis identified Cys12 and His247 as being important for chromophore binding and photoreversibility, respectively. PaBphP is synthesized in the dark in the red-light-absorbing Pr form and immediately converted into a far-red-light-absorbing Pfr-enriched form. It shows the characteristic red/far-red-light-induced photoreversibility of phytochromes. A chromophore analog that lacks the C15/16 double bond was used to show that this photoreversibility is due to a 15Z/15E isomerization of the biliverdin chromophore. Autophosphorylation of PaBphP was demonstrated, confirming its role as a sensor kinase of a bacterial two-component signaling system.     

Prospective Analysis of the Association of a Common Variant of FTO (rs9939609) with Adiposity in Children: Results of the IDEFICS Study

Objectives

We investigated cross-sectionally and longitudinally the relationship between FTO rs9939609 and obesity-related characteristics in the European children of the IDEFICS project and the interaction of this variant with a lifestyle intervention.

Population and Methods

A cohort of 16224 children (2–9 years) was recruited into a population-based survey (T0) from eight European countries. A second survey (T1) reassessed the children two years later. A random sample of 4405 children was extracted for genetic studies. 3168 children were re-examined two years later. Half of them underwent a lifestyle intervention program. The FTO rs9939609 was genotyped. Weight, height, waist circumference, triceps and subscapular skinfolds were measured at T0 and T1.

Results

At T0, the risk A allele of rs9939609 was significantly associated with higher values of body mass index (BMI), waist circumference and skinfolds (age, sex, and country-adjusted p-values: all p<0.001) and with a statistically significant increased risk of overweight/obesity.Over the two year follow-up, no interaction between genotype and intervention was observed. The A allele was associated to a significantly higher increase in all the anthropometric variables examined at T0 independently from the study group (intervention versus control) (p-values: all p<0.002, adjusted for age, sex, country, intervention/control study group, T0 values, and individual time interval between T0 and T1). Over the two-year follow–up, 210 new cases of overweight/obesity occurred. A statistically significant higher incidence of overweight/obesity was associated to the A allele [OR_A = 1.95, 95% CI = (1.29; 2.97)].

Conclusions

We confirmed the association between the FTO rs9939609 and body mass and overweight/obesity risk in European children. The main finding of the study is that the A allele carriers present higher increase of body mass and central adiposity over time and higher risk of developing overweight/obesity during growth, independently from intervention measures.     

Using Hidden Markov Models to Improve Quantifying Physical Activity in Accelerometer Data – A Simulation Study

Introduction

The use of accelerometers to objectively measure physical activity (PA) has become the most preferred method of choice in recent years. Traditionally, cutpoints are used to assign impulse counts recorded by the devices to sedentary and activity ranges. Here, hidden Markov models (HMM) are used to improve the cutpoint method to achieve a more accurate identification of the sequence of modes of PA.

Methods

1,000 days of labeled accelerometer data have been simulated. For the simulated data the actual sedentary behavior and activity range of each count is known. The cutpoint method is compared with HMMs based on the Poisson distribution (HMM[Pois]), the generalized Poisson distribution (HMM[GenPois]) and the Gaussian distribution (HMM[Gauss]) with regard to misclassification rate (MCR), bout detection, detection of the number of activities performed during the day and runtime.

Results

The cutpoint method had a misclassification rate (MCR) of 11% followed by HMM[Pois] with 8%, HMM[GenPois] with 3% and HMM[Gauss] having the best MCR with less than 2%. HMM[Gauss] detected the correct number of bouts in 12.8% of the days, HMM[GenPois] in 16.1%, HMM[Pois] and the cutpoint method in none. HMM[GenPois] identified the correct number of activities in 61.3% of the days, whereas HMM[Gauss] only in 26.8%. HMM[Pois] did not identify the correct number at all and seemed to overestimate the number of activities. Runtime varied between 0.01 seconds (cutpoint), 2.0 minutes (HMM[Gauss]) and 14.2 minutes (HMM[GenPois]).

Conclusions

Using simulated data, HMM-based methods were superior in activity classification when compared to the traditional cutpoint method and seem to be appropriate to model accelerometer data. Of the HMM-based methods, HMM[Gauss] seemed to be the most appropriate choice to assess real-life accelerometer data.     

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku^−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.     

Using graphical chain models to analyze differences in structural correlates of undernutrition in Benin and Bangladesh

Undernutrition among children is one of the most important health problems in developing countries. In order to understand the complex pathways affecting undernutrition which is crucial for policy interventions, one needs to explicitly model the dependence chain of immediate, intermediate, and underlying factors affecting undernutrition. Graphical chain models are used here to investigate the determinants of undernutrition in Benin and Bangladesh. While the dependence chain affecting undernutrition contains many common elements, the influence of demographic, cultural, and socioeconomic factors seems to have stronger direct and indirect influences in Benin than in Bangladesh, where many socioeconomic and gender related factors have a more direct influence on undernutrition.