Caught on the hop: In multistep electron transfer (ET) reactions through peptides, aliphatic amino acids can also act as relay stations. With cysteine, the reaction occurs as a proton-coupled electron transfer (PCET) with water used as a mediator for the proton transfer (see picture).

Lipoteichoic acids (LTA), cell wall components of gram-positive bacteria, have been reported to induce various inflammatory mediators and to play a key role in gram-positive-microbe-mediated septic shock. In a large number of these studies, investigators used commercially available LTA purified from a variety of gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, and Streptococcus sanguis. We report here that, although these commercially available LTA could be readily shown to stimulate production of nitric oxide (NO) in RAW 264.7 mouse macrophages, the activity was dramatically inhibited by polymyxin B, a relatively specific inhibitor of endotoxin biological activity. One-step purification of the commercially available S. aureus LTA using hydrophobic interaction chromatography resulted in two well-separated peak fractions, one highly enriched for LTA and a second highly enriched for endotoxin. The LTA-enriched fractions did not induce production of NO in RAW 264.7 macrophages, although they caused a dose-dependent induction of NO in the presence of low concentrations of gamma interferon (IFN-␥) (which by itself induced little NO), regardless of the presence of polymyxin B. In contrast, the endotoxin-enriched fractions by themselves inhibited in high levels of NO in RAW 264.7 macrophages but activity was almost completely inhibited in the presence of polymyxin B. Consistent with these findings, our data also indicate that commercial LTA preparations from S. aureus, B. subtilis, and S. sanguis were not able to induce NO from lipopolysaccharidehyporesponsive C3H/HeJ mouse peritoneal macrophages, but in the presence of IFN-␥, these LTA preparations were able to induce relatively high levels of NO from C3H/HeJ macrophages. These results indicate that commercially available LTA can contain contaminating and potentially significant levels of endotoxin that can be expected to contribute to the putative macrophage-stimulating effects of LTA as assessed by NO production. The fact that the purified LTA, by itself, was not able to induce significant levels of NO secretion in RAW 264.7 macrophages supports the conclusion that caution in attributing high-level biological activity to this microbial cell wall constituent should be exercised.

Each cell of higher organism adults is derived from a fertilized egg through a series of divisions, during which mutations can occur. Both the rate and timing of mutations can have profound impacts on both the individual and the population, because mutations that occur at early cell divisions will affect more tissues and are more likely to be transferred to the next generation. Using large-scale multigeneration screening experiments for recessive lethal or nearly lethal mutations of Drosophila melanogaster and recently developed statistical analysis, we show for male D. melanogaster that (i) mutation rates (for recessive lethal or nearly lethal) are highly variable during germ cell development; (ii) first cell cleavage has the highest mutation rate, which drops substantially in the second cleavage or the next few cleavages; (iii) the intermediate stages, after a few cleavages to right before spermatogenesis, have at least an order of magnitude smaller mutation rate; and (iv) spermatogenesis also harbors a fairly high mutation rate. Because germ-line lineage shares some (early) cell divisions with somatic cell lineage, the first conclusion is readily extended to a somatic cell lineage. It is conceivable that the first conclusion is true for most (if not all) higher organisms, whereas the other three conclusions are widely applicable, although the extent may differ from species to species. Therefore, conclusions or analyses that are based on equal mutation rates during development should be taken with caution. Furthermore, the statistical approach developed can be adopted for studying other organisms, including the human germ-line or somatic mutational patterns.within-host coalescent | mutation cluster | likelihood

Long-range electron transfer (ET) through proteins is a fundamental reaction in living organisms, playing a role in energy-conversion processes like photosynthesis [1a] and respiration [1b] as well as in enzymatic reactions. [1c] The mechanism of these ET processes is still a matter of controversy. Two cases are discussed, a bridge-mediated superexchange (single-step reaction) and a hopping mechanism (stepwise reaction). [2] Today, the hopping mechanism, where each step follows the Marcus rule, [3] is favored for long-range ET through peptides. [4] Nevertheless, for special conformations like a helices with their intramolecular hydrogen bonds, experiments on long-range ET have been discussed as single-step reactions. [5] However, quantum chemical calculations favor a multistep reaction also in a helices. [6] For the polyproline II helix (PPII helix), which does not have hydrogen bonds, a mechanistic transition between a singlestep and a stepwise reaction was observed when the number of the proline units between the electron donor and the electron acceptor is larger than 4. [7] Quantum chemical calculations explain the effect of peptide conformations on the distance influence of ET by a pathway model. [8] Recently it was shown that the rate also depends upon the direction of the ET process, demonstrating the important impact of dipole moments. [9] Another parameter influencing ET is the charge of ions bound as cofactors to the systems. [10] Such a Coulomb effect should also be of importance in peptides with unprotected amino or carboxylate groups that exist as ions under biological conditions. Herein we present our new results, which demonstrate that ET in peptides is indeed influenced by their charged termini.The investigations were carried out with our recently developed model peptides 1 in which ET occurs between the radical cation of a dialkoxyphenyl group (electron acceptor at the C-terminal end) and tyrosine as the electron donor at the N-terminal end. Halfway between the donor and the acceptor we introduced an amino acid with a side chain X. [11] These three amino acids were separated by sequences of three prolines (Scheme 1). If X is a side chain that can be oxidized by the electron acceptor, this central amino acid acts as a stepping stone for a hopping process (relay amino acid). [11] The structure of peptides 1 (in CH 3 CN/H 2 O 3:1) was characterized by their CD spectra as PPII helices. [12] Interestingly, the CD spectra remained unchanged when the temperature was varied between 20 8C and 80 8C, which supports the conformational stability of the peptides. These findings are in agreement with experimental and theoretical data from other polyproline systems. [13,14] In a PPII helix each of the triproline spacers corresponds to a distance of about 10 . [13,14] The triproline units thus separate the donor, the acceptor, and the relay amino acids, and act as a medium for the ET process. The 13 C NMR spectra [15,16] indicate that approximately 80 % of the proline peptide bonds adopt a trans conformat...

Conventional transcranial direct current stimulation (tDCS) targeting the left dorsolateral prefrontal cortex (DLPFC) could improve arousal in disorders of consciousness (DOC). However, the comparative effectiveness of anodal stimulation of the left DLPFC and the electrophysiological effect of tDCS are yet to be determined. In this randomized sham-controlled design, patients were separated into three groups (left/right anodal tDCS, sham). Data on the clinical assessments and EEG were collected at baseline and after 2 weeks of tDCS. The outcome at 3-month follow-up was evaluated using the Glasgow Outcome Scale-Extended. Results showed that sessions of the left tDCS facilitated the excitability of the prefrontal cortex, whereas only one patient had a positive outcome. Targeting the right DLPFC was less effective, merely leading to activation of the stimulation site, with no effect on the state of arousal. Moreover, sham stimulation had minimal or no effect on any of the outcomes. These results provide evidence for a hemispheric asymmetry of tDCS effects in patients with DOC. Left anodal tDCS might be more effective for modulating cortical excitability compared to tDCS on the right DLPFC. However, future studies with large sample sizes are needed to confirm these findings. This trial is registered with NCT03809936.