Creatine and creatinine quantification in olympic athletes: dried blood spot analysis pilot study

Capillary dried blood spot (DBS) samples facilitate field-based collection without venipuncture. This pilot study aims to evaluate the viability of creatine (Cr) and creatinine (Crt) quantification using fresh capillary serum (Cr_S/Crt_S) and DBS samples (Cr_DBS/Crt_DBS), using Flow Injection Analysis Mass Spectrometry (FIA – MS). Nine Olympic Athletes provided a capillary blood sample to assess Cr_S/Crt_S and Cr_DBS/Crt_DBS quantified by FIA – MS. No difference between Crt_S (mean ± SD: 813.6 ± 102.4 μmol/L) and Crt_DBS (812.4 ± 108.1 μmol/L) was observed with acceptable variance [SEM 88.7; CV 10.7%; ICC 0.57 (CI 95% 0.06 – 0.84)] and agreement [very strong (Spearman: r = 0.77; p < 0.01) or strong (Pearson: r = 0.56; p = 0.04); Bland Altman: lower (-193) and upper (+196) limits of agreement]. Cr_S (mean ± SD: 691.8 ± 165.2 μmol/L) was significantly different to Cr_DBS (2911 ± 571.4 μmol/L) with unacceptable variance [SEM 171.6; CV 27%; ICC 0.002 (CI 95% -0.02 – 0.07)] and ‘weak’ agreement [Spearman: r = 0.21, p = 0.47 and Pearson: r = 0.06, p = 0.84; Bland Altman lower (-3367) and upper (-1072) limits of agreement]. Crt quantification is viable using both Crt_S and Crt_DBS (but not for Cr and Cr_S/Cr_DBS), with the DBS tissue handling technique offering several methodological and practice facing advantages. Future work should expand upon the sample size, explore sport/discipline relevant analytes across a full competitive season, including key training, recovery and performance blocks of their periodized performance plan.     

Directed evolution of a highly active Yersinia mollaretii phytase

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073?U/mg which is ～10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ～8,400 clones) a phytase variant with ～20% improved thermostability (58°C for 20?min; residual activity wild type ～34%; variant ～53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993?U/mg).     

A Simple and Efficient DNA Isolation Method for Salvia officinalis

We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4?M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6?μg DNA g^?1 of dry leaf tissue, and the absorbance ratios 260/280 and 260/230?nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.     

Monitoring the transport of recombinant Candida rugosa lipase by a green fluorescent protein-lipase fusion

In a batch cultivation of Pichia pastoris expressing Candida rugosa lipase 1 (CRL1), secretion of 200 microg lipase ml(-1) of culture was achieved in sorbitol-based medium. However, a large amount of recombinant protein was retained intracellularly throughout the fermentation, pointing to the transport step as a major bottleneck. Therefore a translational fusion with the green fluorescent protein (GFP) was constructed that was expressed and transported similarly to the native lipase and retained catalytic activity. This analytical tool enables a rapid monitoring of product localization and amount, based on GFP-associated fluorescence.     

Selective excitation of native fluctuations during thermal unfolding simulations: horse heart cytochrome c as a case study

The effect of temperature on the activation of native fluctuation motions during molecular dynamics unfolding simulations of horse heart cytochrome c has been studied. Essential dynamics analysis has been used to analyze the preferred directions of motion along the unfolding trajectories obtained by high temperature simulations. The results of this study have evidenced a clear correlation between the directions of the deformation motions that occur in the first stage of the unfolding process and few specific essential motions characterizing the 300 K dynamics of the protein. In particular, one of those collective motions, involved in the fluctuation of a loop region, is specifically excited in the thermal denaturation process, becoming progressively dominant during the first 500 ps of the unfolding simulations. As further evidence, the essential dynamics sampling performed along this collective motion has shown a tendency of the protein to promptly unfold. According to these results, the mechanism of thermal induced denaturation process involves the selective excitation of one or few specific equilibrium collective motions.     

Endogenous Stress Caused by Faulty Oxidation Reactions Fosters Evolution of 2,4-Dinitrotoluene-Degrading Bacteria

Environmental strain Burkholderia sp. DNT mineralizes the xenobiotic compound 2,4-dinitrotoluene (DNT) owing to the catabolic dnt genes borne by plasmid DNT, but the process fails to promote significant growth. To investigate this lack of physiological return of such an otherwise complete metabolic route, cells were exposed to DNT under various growth conditions and the endogenous formation of reactive oxygen species (ROS) monitored in single bacteria. These tests revealed the buildup of a strong oxidative stress in the population exposed to DNT. By either curing the DNT plasmid or by overproducing the second activity of the biodegradation route (DntB) we could trace a large share of ROS production to the first reaction of the route, which is executed by the multicomponent dioxygenase encoded by the dntA gene cluster. Naphthalene, the ancestral substrate of the dioxygenase from which DntA has evolved, also caused significant ROS formation. That both the old and the new substrate brought about a considerable cellular stress was indicative of a still-evolving DntA enzyme which is neither optimal any longer for naphthalene nor entirely advantageous yet for growth of the host strain on DNT. We could associate endogenous production of ROS with likely error-prone repair mechanisms of DNA damage, and the ensuing stress-induced mutagenesis in cells exposed to DNT. It is thus plausible that the evolutionary roadmap for biodegradation of xenobiotic compounds like DNT was largely elicited by mutagenic oxidative stress caused by faulty reactions of precursor enzymes with novel but structurally related substrates-to-be.