Atomic force microscopy and modeling of natural elastic fibrillin polymers

A central issue in the understanding of Marfan syndrome deals with the functional architecture of fibrillin-containing microfibrils. Fibrillin-rich microfibrils are long extracellular matrix fibrillar components exhibiting a 50 nm periodic beaded-structure with a width of around 20–25 nm after rotary shadowing and a 10–12 nm diameter when observed in ultra-thin sections. They are composed of fibrillin monomers more or less associated with many other components which are, for the most part, poorly characterized up to date. They are known to be elastic but few data have been accumulated to understand their properties. Atomic force microscopy (AFM) allowed us to morphologically differentiate fibrillin-rich microfibrils from other fibrillar components and to investigate the thin structure of these beaded filaments in their native state. They showed, in AFM, a periodic beaded structure ranging from 50 to 60 nm and a width of about 40 nm. The different sizes of fibrillin-containing microfibrils previously observed after rotary shadowing and in ultra-thin sections was resolved with our technique and is revealed to be 10 nm in diameter. Each beaded microfibril appears to be composed of heterogeneous beads connected by 2–3 arms. An orientation of the microfibrils has been shown, and allows us to propose a complementary model of microfibrillar monomer association.     

medplot: A Web Application for Dynamic Summary and Analysis of Longitudinal Medical Data Based on R

In biomedical studies the patients are often evaluated numerous times and a large number of variables are recorded at each time-point. Data entry and manipulation of longitudinal data can be performed using spreadsheet programs, which usually include some data plotting and analysis capabilities and are straightforward to use, but are not designed for the analyses of complex longitudinal data. Specialized statistical software offers more flexibility and capabilities, but first time users with biomedical background often find its use difficult. We developed medplot, an interactive web application that simplifies the exploration and analysis of longitudinal data. The application can be used to summarize, visualize and analyze data by researchers that are not familiar with statistical programs and whose knowledge of statistics is limited. The summary tools produce publication-ready tables and graphs. The analysis tools include features that are seldom available in spreadsheet software, such as correction for multiple testing, repeated measurement analyses and flexible non-linear modeling of the association of the numerical variables with the outcome. medplot is freely available and open source, it has an intuitive graphical user interface (GUI), it is accessible via the Internet and can be used within a web browser, without the need for installing and maintaining programs locally on the user’s computer. This paper describes the application and gives detailed examples describing how to use the application on real data from a clinical study including patients with early Lyme borreliosis.     

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.     

A combined computational and experimental investigation of the [2Fe–2S] cluster in biotin synthase

Biotin synthase was the first example of what is now regarded as a distinctive enzyme class within the radical S-adenosylmethionine superfamily, the members of which use Fe/S clusters as the sulphur source in radical sulphur insertion reactions. The crystal structure showed that this enzyme contains a [2Fe–2S] cluster with a highly unusual arginine ligand, besides three normal cysteine ligands. However, the crystal structure is at such a low resolution that neither the exact coordination mode nor the role of this exceptional ligand has been elucidated yet, although it has been shown that it is not essential for enzyme activity. We have used quantum refinement of the crystal structure and combined quantum mechanical and molecular mechanical calculations to explore possible coordination modes and their influences on cluster properties. The investigations show that the protonation state of the arginine ligand has little influence on cluster geometry, so even a positively charged guanidinium moiety would be in close proximity to the iron atom. Nevertheless, the crystallised enzyme most probably contains a deprotonated (neutral) arginine coordinating via the NH group. Furthermore, the Fe···Fe distance seems to be independent of the coordination mode and is in perfect agreement with distances in other structurally characterised [2Fe–2S] clusters. The exceptionally large Fe···Fe distance found in the crystal structure could not be reproduced.     

The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.     

Comparison of the most demanding scenarios during different in-season training sessions and official matches in professional basketball players

The purpose of this study was to compare physical demands during the most demanding scenarios (MDS) of different training sessions and official matches in professional basketball players across playing positions. Thirteen professional basketball players were monitored over a 9-week competitive season using a local positioning system. Peak physical demands included total distance, distance covered at > 18 km·h^-1, distance and number of accelerations (≥ 2 m∙s^-2) and decelerations (≤ -2 m∙s^-2) over a 60-second epoch. Analysis of variance for repeated measures, Bonferroni post-hoc tests and standardised Cohen’s effect size (ES) were calculated. Overall, almost all physical demands during the MDS of training were lower (-6.2% to -35.4%) compared to official matches. The only variable that surpassed competition demands was distance covered at > 18 km·h^-1, which presented moderate (ES = 0.61, p = 0.01) and small (ES = 0.48, p > 0.05) increases during training sessions four and three days before a competition, respectively. Conversely, the two previous practices before match day presented trivial to very large decreases (ES = 0.09–2.66) in all physical demands. Furthermore, centres achieved the lowest peak value in total distance covered during matches, forwards completed the greatest peak distance at > 18 km·h^-1, and guards performed the greatest distance and number of high-intensity accelerations and decelerations. In conclusion, physical demands during the MDS of different training sessions across the microcycle failed to match or surpass peak values during official matches, which should be considered when prescribing a training process intended to optimise the MDS of match play.     

Characterization of Microbial Arsenate Reduction in the Anoxic Bottom Waters of Mono Lake,California

Dissimilatory reduction of arsenate (DAsR) occurs in the arsenic-rich, anoxic water column of Mono Lake, California, yet the microorganisms responsible for this observed in situ activity have not been identified. To gain insight as to which microorganisms mediate this phenomenon, as well as to some of the biogeochemical constraints on this activity, we conducted incubations of arsenate-enriched bottom water coupled with inhibition/amendment studies and Denaturing Gradient Gel Electrophoresis (DGGE) characterization techniques. DAsR was totally inhibited by filter-sterilization and by nitrate, partially inhibited (~50%) by selenate, but only slightly (~25%) inhibited by oxyanions that block sulfate-reduction (molybdate and tungstate). The apparent inhibition by nitrate, however, was not due to action as a preferred electron acceptor to arsenate. Rather, nitrate addition caused a rapid, microbial re-oxidation of arsenite to arsenate, which gave the overall appearance of no arsenate loss. A similar microbial oxidation of As(III) was also found with Fe(III), a fact that has implications for the recycling of As(V) in Mono Lake's anoxic bottom waters. DAsR could be slightly (10%) stimulated by substrate amendments of lactate, succinate, malate, or glucose, but not by acetate, suggesting that the DAsR microflora is not electron donor limited. DGGE analysis of amplified 16S rDNA gene fragments from incubated arsenate-enriched bottom waters revealed the presence of two bands that were not present in controls without added arsenate. The resolved sequences of these excised bands indicated the presence of members of the epsilon ( Sulfurospirillum ) and delta ( Desulfovibrio ) subgroups of the Proteobacteria , both of which have representative species that are capable of anaerobic growth using arsenate as their electron acceptor.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Sex-biased dispersal promotes adaptive parental effects

Background  

In heterogeneous environments, sex-biased dispersal could lead to environmental adaptive parental effects, with offspring selected to perform in the same way as the parent dispersing least, because this parent is more likely to be locally adapted. We investigate this hypothesis by simulating varying levels of sex-biased dispersal in a patchy environment. The relative advantage of a strategy involving pure maternal (or paternal) inheritance is then compared with a strategy involving classical biparental inheritance in plants and in animals.