Complexome profiling reveals association of PPR proteins with ribosomes in the mitochondria of plants

Mitochondrial transcripts are subject to a wealth of processing mechanisms including cis- and trans-splicing events, as well as base modifications (RNA editing). HUndreds of proteins are required for these processes in plant mitochondria, many of which belong to the pentatricopeptide repeat (PPR) protein superfamily. The structure, localization, and function of these proteins is only poorly Understood. Here we present evidence that several PPR proteins are boUnd to mitoribosomes in plants. A novel complexome profiling strategy in combination with chemical crosslinking has been employed to systematically define the protein constituents of the large and the small ribosomal subunits in the mitochondria of plants. We identified more than 80 ribosomal proteins, which include several PPR proteins and other non-conventional ribosomal proteins. These findings reveal a potential coupling of transcriptional and translational events in the mitochondria of plants. Furthermore, the data indicate an extremely high molecular mass of the “small” subunit, even exceeding that of the “large” subunit

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5–9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity

Triblock copolymers of styrene and sodium methacrylate as smart materials:synthesis and rheological characterization

Well-defined amphiphilic triblock poly(sodium methacrylate)-polystyrene-poly(sodium methacrylate) (PMAA-b-PS-b-PMAA) copolymers characterized by a different length of either the hydrophilic or the hydrophobic block have been synthesized by ATRP. In solution the micelle-like aggregates consist of a collapsed PS core surrounded by stretched charged PMAA chains. The micelles are kinetically 'frozen' and as a consequence the triblock copolymers do not show a significant surface activity. The hydrophilic block length has a major influence on the rheology, the shortest PMAA blocks yielding the strongest gels (at the same total weight concentration). The hydrophobic block length has only a minor influence until a certain threshold, below which the hydrophobic interactions are too weak resulting in weak gels. A mathematical model is used to describe the micelle radius and the results were in good agreement with the experimentally found radius in transmission electron microscopy. The influences of the ionic strength, pH and temperature on the rheology has also been investigated, showing the potential of these polymers as smart hydrogels. The change in conformation of the hydrophilic corona from the collapsed state to the stretched state by changing the pH was quantified with zeta-potential measurements. To the best of our knowledge, this is the first systematic investigation of this kind of triblock copolymers in terms of their rheological behavior in water.</p

Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials:Application and Interpretation

Transmission electron microscopy (TEM) provides direct structural information on nano-structured materials and is popular as a characterization tool in soft matter and supramolecular chemistry. However, technical aspects of sample preparation are overlooked and erroneous image interpretations are regularly encountered in the literature. There are three most commonly used TEM methods as we derived from literature: drying, staining and cryo-TEM, which are explained here with respect to their application, limitations and interpretation. Since soft matter chemistry relies on a lot of indirect evidence, the role of TEM for the correct evaluation of the nature of an assembly is very large. Mistakes in application and interpretation can therefore have enormous impact on the quality of present and future studies. We provide helpful background information of these three techniques, the information that can and cannot be derived from them and provide assistance in selecting the right technique for soft matter imaging. This essay warns against the use of drying and explains why. In general cryo-TEM is by far the best suited method and many mistakes and over-interpretations can be avoided by the use of this technique

Attentional bias retraining in cigarette smokers attempting smoking cessation (ARTS): study protocol for a double bline randomised controlled trial

YesSmokers attend preferentially to cigarettes and other smoking-related cues in the environment, in what is known as an attentional bias. There is evidence that attentional bias may contribute to craving and failure to stop smoking. Attentional retraining procedures have been used in laboratory studies to train smokers to reduce attentional bias, although these procedures have not been applied in smoking cessation programmes. This trial will examine the efficacy of multiple sessions of attentional retraining on attentional bias, craving, and abstinence in smokers attempting cessation. This is a double-blind randomised controlled trial. Adult smokers attending a 7-session weekly stop smoking clinic will be randomised to either a modified visual probe task with attentional retraining or placebo training. Training will start 1 week prior to quit day and be given weekly for 5 sessions. Both groups will receive 21 mg transdermal nicotine patches for 8–12 weeks and withdrawal-orientated behavioural support for 7 sessions. Primary outcome measures are the change in attentional bias reaction time and urge to smoke on the Mood and Physical Symptoms Scale at 4 weeks post-quit. Secondary outcome measures include differences in withdrawal, time to first lapse and prolonged abstinence at 4 weeks post-quit, which will be biochemically validated at each clinic visit. Follow-up will take place at 8 weeks, 3 months and 6 months post-quit. This is the first randomised controlled trial of attentional retraining in smokers attempting cessation. This trial could provide proof of principle for a treatment aimed at a fundamental cause of addiction.National Institute for Health Research (NIHR) Doctoral Research Fellowship (DRF) awarded to RB (DRF-2009-02-15

Cross-sectional and longitudinal associations between different exercise types and food cravings in free-living healthy young adults

Introduction: An increase in energy intake due to alterations in hedonic appetite sensations may, at least in part, contribute to lower-than-expected weight loss in exercise interventions. The aim of this study was to examine cross-sectional and longitudinal associations between habitual exercise participation and food cravings in free-living young adults. Methods: A total of 417 adults (49% male, 28 ± 4 years) reported frequency and duration of walking, aerobic exercise, resistance exercise and other exercise at baseline and every 3 months over a 12-month period. Food cravings were assessed via the Control of Eating Questionnaire at baseline and 12-month follow-up. Results: Cross-sectional analyses revealed more frequent cravings for chocolate and a greater difficulty to resist food cravings in women compared to men (p < 0.01). Only with resistance exercise significant sex by exercise interaction effects were observed with favorable responses in men but not in women. Significant main effects were shown for walking and aerobic exercise with exercisers reporting more frequent food cravings for chocolate and fruits and greater difficulty to resist eating compared to non-exercisers (p < 0.05). Longitudinal analyses revealed significant interaction effects for other exercise (p < 0.05) with favorable results in men but not women. Furthermore, significant main effects were observed for aerobic exercise, resistance exercise and total exercise with an increase in exercise being associated with a reduced difficulty to resist food cravings (p < 0.05). Discussion: The association between exercise participation and hedonic appetite sensations varies by exercise type and sex. Even though exercise was associated with more frequent and greater difficulty to food cravings in the cross-sectional analyses, which may be attributed to greater energy demands, longitudinal results indicate beneficial effects of increased exercise on appetite control, particularly in men

The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus

The cyanobacterial NADPH: plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the similar to 450 kDa NDH-1L complex, that is involved in respiration and CET at high CO2 concentration, and not detectable in the NDH-1MS and NDH-1MS' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a Delta ndhP::Km mutant

Molecular Packaging of Biocatalysts Using a Robust Protein Cage

In this paper, we report on the discovery of a novel, robust protein cage (encapsulin) that we could use for packaging various biocatalysts. We have elucidated the structure of the stable encapsulin by electron microscopy and X-ray diffraction. Furthermore, we developed an effective expression system for the encapsulin and a facile protocol for preparing encapsulated enzymes. By packaging and testing various enzymes (varying in size, oligomeric structure, and cofactor type) we demonstrate that, through encapsulation, the enzymes become significantly more stable. We also provide evidence that the pores of the encapsulin, through a size-exclusion effect, can modulate the substrate acceptance profile of an encapsulated enzyme

Molecular packaging of biocatalysts using a robust protein cage

Bacterial microcompartments serve as metabolic modules (pseudo-organelles) that help to increase the local concentration of enzymes, facilitate substrate transfer between a series of connected reactions and sequester potentially toxic substrates or products. Encapsulation of enzyme(s) might be beneficial for biocatalytic approaches due to efficient transfer of intermediates, protection of packaged proteins against proteolysis, extended lifetime of the enzyme and protection against thermal inactivation. Please click Additional Files below to see the full abstract