Properties and action pattern of the recombinant mannuronan C-5-epimerase AlgE2

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca²⁺-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of β- -mannuronic acid residues (M) to - -guluronic acid residues (G) in alginate. AlgE2 has been produced by fermentation with a recombinant strain of Escherichia coli, isolated and partially purified. Epimerization with AlgE2 increased the content of G-residues in different alginates from starting values of 0–45% up to approximately 70%. The new G-residues were mainly present in short blocks. Although G-residues may be introduced next to pre-existing G-residues, AlgE2 was not able to epimerize strictly alternating MG-structures. The epimerization with AlgE2 was greatly affected by the concentration of Ca²⁺. The type of alginate used as substrate affected the reaction rate and the reaction pattern especially at low Ca²⁺ concentration. AlgE2 appears to act by a preferred attack mechanism where the enzyme associates with different sequences in the alginate depending on the concentration of Ca²⁺. During epimerization, AlgE2 occasionally causes cleavage of the alginate chain. The observed frequency corresponds to 1–3 breaks per 1,000 M-units epimerized.     

Focus: Rare Disease: Psychosocial Functioning in Siblings of Children With Rare Disorders Compared to Controls

Siblings of children with chronic disorders are at increased risk of psychosocial problems. The risk may be exacerbated when the chronic disorder is rare and limited medical knowledge is available, due to more uncertainty and feelings of isolation. We examined mental health, parent-child communication, child-parent relationship quality, and social support among 100 children aged 8 to 16 years (M age 11.5 years, SD = 2.2; 50.0% boys, 50.0% girls). Fifty-six were siblings of children with rare disorders, and 44 were controls. The siblings of children with rare disorders (herein, siblings) were recruited from a resource centre for rare disorders and comprised siblings of children with a range of rare disorders including neuromuscular disorders and rare chromosomal disorders with intellectual disability. Controls were recruited from schools. Self-reported child mental health was significantly poorer for siblings compared to controls (effect size difference d = 0.75). Parent-reported child mental health was not significantly different between the groups (d = -0.06 to 0.16). Most child-parent relationships (anxiety/avoidance; mothers/fathers) were significantly poorer for siblings compared to controls (d = 0.47 to 0.91). There was no difference between groups in anxious relation with mother. Parent-child communication was significantly poorer for siblings compared to controls (d = -0.87 to -0.75). Social support was significantly poorer for siblings compared to controls (d = 0.61). We conclude that siblings of children with rare disorders display more psychosocial problems than controls. Interventions are indicated to prevent further maladjustment for siblings.     

Uracil-DNA glycosylase of Thermoplasma acidophilum directs long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and ATP/ADP, into short-patch repair

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5'-acting AP endonuclease and a 5'-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3'-acting AP lyase, followed by a 3'-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ～15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.     

hTERT mRNA dendritic cell vaccination: complete response in a pancreatic cancer patient associated with response against several hTERT epitopes

Immunotherapy targeting the hTERT subunit of telomerase has been shown to induce robust immune responses in cancer patients after vaccination with single hTERT peptides. Vaccination with dendritic cells (DCs) transfected with hTERT mRNA has the potential to induce strong immune responses to multiple hTERT epitopes and is therefore an attractive approach to more potent immunotherapy. Blood samples from such patients provide an opportunity for identification of new, in vivo processed T-cell epitopes that may be clinically relevant. A 62-year-old female patient underwent radical surgery for a pancreatic adenocarcinoma. After relapse, she obtained stable disease on gemcitabine treatment. Due to severe neutropenia, the chemotherapy was terminated. The patient has subsequently been treated with autologous DCs loaded with hTERT mRNA for 3 years. Immunomonitoring was performed at regular intervals following start of vaccination and clinical outcome measured by CT and PET/CT evaluation. The patient developed an immune response against several hTERT-derived Th and CTL epitopes. She presently shows no evidence of active disease based on PET/CT scans. No serious adverse events were experienced and the patient continues to receive regular booster injections. We here provide evidence for the induction of hTERT-specific immune responses following vaccination of a pancreas cancer patient with DCs loaded with hTERT mRNA. These responses are associated with complete remission. A thorough analysis of this patient immune response has provided a unique opportunity to identify novel epitopes, associated with clinical effects. These will be included in future hTERT vaccines.     

Towards more efficient longline fisheries: fish feeding behaviour,bait characteristics and development of alternative baits

Better knowledge of food search behaviour in fish is essential for studies that aim to improve longline fishing, particularly through bait development. This review provides an overview of our understanding of how fish detect and locate sources of food odour, focusing on the stimuli and sensory modalities involved, and on factors that affect feeding activity. Studies that identify feeding attractants and efforts to develop alternative longline baits are presented. The review reveals that such studies are few in number, and that to date there are no alternatives to traditional baits in commercial longlining despite the growing demand for these resources, which are also used for human consumption. The chemical compounds that elicit food search behaviour differ from species to species, and species selectivity could be improved by incorporating specific feeding attractants in manufactured baits. The unique properties of chemical stimuli and odour dispersal form the basis for improving longline efficiency through the development of a long-lasting bait. Vision is important in prey capture, and manufactured baits can be made more visible than natural baits by increasing the contrast (e.g. via fluorescent or polarising coatings) and creating motion through buoyancy. Physical properties such as size, shape, texture and strength can also be manipulated in a manufactured bait to improve catch efficiency. Knowledge obtained from studies of various aspects of food search behaviour is of paramount importance for future research aimed at alternative bait development and improving longline fishing.     

Safety in numbers: multiple occurrences of highly similar homologs among Azotobacter vinelandii carbohydrate metabolism proteins probably confer adaptive benefits

Background

Gene duplication and horizontal gene transfer are common processes in bacterial and archaeal genomes, and are generally assumed to result in either diversification or loss of the redundant gene copies. However, a recent analysis of the genome of the soil bacterium Azotobacter vinelandii DJ revealed an abundance of highly similar homologs among carbohydrate metabolism genes. In many cases these multiple genes did not appear to be the result of recent duplications, or to function only as a means of stimulating expression by increasing gene dosage, as the homologs were located in varying functional genetic contexts. Based on these initial findings we here report in-depth bioinformatic analyses focusing specifically on highly similar intra-genome homologs, or synologs, among carbohydrate metabolism genes, as well as an analysis of the general occurrence of very similar synologs in prokaryotes.

Results

Approximately 900 bacterial and archaeal genomes were analysed for the occurrence of synologs, both in general and among carbohydrate metabolism genes specifically. This showed that large numbers of highly similar synologs among carbohydrate metabolism genes are very rare in bacterial and archaeal genomes, and that the A. vinelandii DJ genome contains an unusually large amount of such synologs. The majority of these synologs were found to be non-tandemly organized and localized in varying but metabolically relevant genomic contexts. The same observation was made for other genomes harbouring high levels of such synologs. It was also shown that highly similar synologs generally constitute a very small fraction of the protein-coding genes in prokaryotic genomes. The overall synolog fraction of the A. vinelandii DJ genome was well above the data set average, but not nearly as remarkable as the levels observed when only carbohydrate metabolism synologs were considered.

Conclusions

Large numbers of highly similar synologs are rare in bacterial and archaeal genomes, both in general and among carbohydrate metabolism genes. However, A. vinelandii and several other soil bacteria harbour large numbers of highly similar carbohydrate metabolism synologs which seem not to result from recent duplication or transfer events. These genes may confer adaptive benefits with respect to certain lifestyles and environmental factors, most likely due to increased regulatory flexibility and/or increased gene dosage.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-192) contains supplementary material, which is available to authorized users.     

Effects of the Workplace Health Promotion Activities Soccer and Zumba on Muscle Pain,Work Ability and Perceived Physical Exertion among Female Hospital Employees

Objectives

This 40-week workplace physical training RCT investigated the effect of soccer and Zumba, respectively, on muscle pain intensity and duration, work ability, and rating of perceived exertion (RPE) during work among female hospital employees.

Methods

107 hospital employees were cluster-randomized into two training groups, and a control group. The training was conducted outside working hours as two-three 1-h sessions per week for the first 12 weeks, and continued as one-two 1-h sessions per week for the last 28 weeks. Muscle pain intensity and duration, work ability, and RPE during work were measured at baseline and after 12 and 40 weeks.

Results

After 12 weeks, both the soccer (−1.9, 95% CI, −3.0, −0.8, P = 0.001) and the Zumba group (−1.3, 95% CI, −2.3, −0.3, P = 0.01) reduced the pain intensity (on a scale from 0 to 10) in the neck-shoulder region (eta squared = 0.109), whereas only the soccer group (−1.9, 95% CI, −3.2, −0.7, P = 0.002, eta squared = 0.092) showed a reduction after 40 weeks referencing the control group. After 40 weeks, both the soccer (-16.4 days, 95% CI, −29.6, −3.2, P<0.02) and the Zumba group (-16.6 days, 95% CI, −28.9, −4.2, P<0.01) reduced the pain duration during the past 3 months in the neck-shoulder region (eta squared = 0.077). No significant effects on intensity or duration of pain in the lower back, RPE during work or work ability were found.

Conclusions

The present study indicates that workplace initiated soccer and Zumba training improve neck-shoulder pain intensity as well as duration among female hospital employees.

Trial Registration

International Standard Randomized Controlled Trial Number Register ISRCTN 61986892.     

Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.