Electroencephalogram-Based Anesthesia Indices Differently React to Modulations of Alpha-Oscillatory Activity.

BACKGROUND: The electroencephalographic (EEG) provides the anesthesiologist with information regarding the level of anesthesia. Processed EEG indices are available that reflect the level of anesthesia as a single number. Strong oscillatory EEG activity in the alpha-band may be associated with an adequate level of anesthesia and a lower incidence of cognitive sequelae. So far, we do not know how the processed indices would react to changes in the alpha-band activity. Hence, we modulated the alpha-oscillatory activity of intraoperative EEG to assess possible index changes. METHODS: We performed our analyses based on data from 2 studies. Intraoperative EEG was extracted, and we isolated the alpha-band activity by band-pass filtering (8-12 Hz). We added or subtracted this activity to the original EEG in different steps with different amplifications of the alpha signal. We then replayed these signals to the bispectral index (BIS), the Entropy Module (state entropy [SE]), the CONOX (qCON), and the SEDLine (patient state index [PSI]); and evaluated the alpha-band modulation's impact on the respective index. RESULTS: The indices behaved differently to the modulation. In general, indices decreased with stronger alpha-band activity, but the rate of change was different with SE showing the strongest change (9% per step) and PSI and BIS (<5% per step) showing the weakest change. A simple regression analysis revealed a decrease of 0.02 to 0.09 index points with increasing alpha amplification. CONCLUSIONS: While the alpha-band in the intraoperative EEG seems to carry information regarding the quality of anesthesia, changes in the alpha-band activity do neither strongly nor uniformly influence processed EEG indices. Hence, to assess alpha-oscillatory activity's strength, the user needs to focus on the raw EEG or its spectral representation also displayed on the monitoring systems.

Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase.

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.

The Strength of Alpha Oscillations in the Electroencephalogram Differently Affects Algorithms Used for Anesthesia Monitoring.

BACKGROUND: Intraoperative patient monitoring using the electroencephalogram (EEG) can help to adequately adjust the anesthetic level. Therefore, the processed EEG (pEEG) provides the anesthesiologist with the estimated anesthesia level. The commonly used approaches track the changes from a fast- and a low-amplitude EEG during wakefulness to a slow- and a high-amplitude EEG under general anesthesia. However, besides these changes, another EEG feature, a strong oscillatory activity in the alpha band (8-12 Hz), develops in the frontal EEG. Strong alpha-band activity during general anesthesia seems to reflect an appropriate anesthetic level for certain anesthetics, but the way the common pEEG approaches react to changes in the alpha-band activity is not well explained. Hence, we investigated the impact of an artificial alpha-band modulation on pEEG approaches used in anesthesia research. METHODS: We performed our analyses based on 30 seconds of simulated sedation (n = 25) EEG, simulated anesthesia (n = 25) EEG, and EEG episodes from 20 patients extracted from a steady state that showed a clearly identifiable alpha peak in the density spectral array (DSA) and a state entropy (GE Healthcare) around 50, indicative of adequate anesthesia. From these traces, we isolated the alpha activity by band-pass filtering (8-12 Hz) and added this alpha activity to or subtracted it from the signals in a stepwise manner. For each of the original and modified signals, the following pEEG values were calculated: (1) spectral edge frequency (SEF95), (2) beta ratio, (3) spectral entropy (SpEntr), (4) approximate entropy (ApEn), and (5) permutation entropy (PeEn). RESULTS: The pEEG approaches showed different reactions to the alpha-band modification that depended on the data set and the amplification step. The beta ratio and PeEn decreased with increasing alpha activity for all data sets, indicating a deepening of anesthesia. The other pEEG approaches behaved nonuniformly. SEF95, SpEntr, and ApEn decreased with increasing alpha for the simulated anesthesia data (arousal) but decreased for simulated sedation. For the patient EEG, ApEn indicated an arousal, and SEF95 and SpEntr showed a nonuniform change. CONCLUSIONS: Changes in the alpha-band activity lead to different reactions for different pEEG approaches. Hence, the presence of strong oscillatory alpha activity that reflects an adequate level of anesthesia may be interpreted differently, by an either increasing (arousal) or decreasing (deepening) pEEG value. This could complicate anesthesia navigation and prevent the adjustment to an adequate, alpha-dominant anesthesia level, when titrating by the pEEG values.

Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases.

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic "hot spot" amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology.

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of ~100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex.

Improving enzyme functional annotation by integrating in vitro and in silico approaches: The example of histidinol phosphate phosphatases.

Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species.

Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis.

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades. However, their evolution has remained unknown to date. Here, we analyzed the evolutionary history of the HolPase from γ-Proteobacteria (HisB-N). It has been argued that HisB-N and its closest homologue d-glycero-d-manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB) have emerged from the same promiscuous ancestral phosphatase. GmhB variants catalyze the hydrolysis of the anomeric d-glycero-d-manno-heptose-1,7-bisphosphate (αHBP or ßHBP) with a strong preference for one anomer (αGmhB or ßGmhB). We found that HisB-N from Escherichia coli shows promiscuous activity for ßHBP but not αHBP, while ßGmhB from Crassaminicella sp. shows promiscuous activity for HolP. Accordingly, a combined phylogenetic tree of αGmhBs, ßGmhBs, and HisB-N sequences revealed that HisB-Ns form a compact subcluster derived from ßGmhBs. Ancestral sequence reconstruction and in vitro analysis revealed a promiscuous HolPase activity in the resurrected enzymes prior to functional divergence of the successors. The following increase in catalytic efficiency of the HolP turnover is reflected in the shape and electrostatics of the active site predicted by AlphaFold. An analysis of the phylogenetic tree led to a revised evolutionary model that proposes the horizontal gene transfer of a promiscuous ßGmhB from δ- to γ-Proteobacteria where it evolved to the modern HisB-N.

Sexual Dysfunctions in Parkinson's Disease and Their Influence on Partnership-Data of the PRISM Study.

BACKGROUND: Sexual dysfunctions (SD) are common but underreported in Parkinson's disease (PD) and have negative impacts on the quality of life (QoL) and partnership. METHODS: We analyzed the data set from the PRISM study for demographics of SD and their influence on quality of life and partnership. RESULTS: 449/861 (52.1%) PD patients reported SD, with male patients being affected more often and having a longer course of disease. The most common SD in men was erectile dysfunction (ED) (n = 152), while women's most frequent complaints were orgasm dysfunction (n = 84) and reduced libido (n = 81). Hypersexual SDs were reported significantly more often by men. Spousal caregivers of patients reporting inability to relax and enjoy sex and reduced libido indicated a negative influence on the relationship in general. Negative effects on the sexual relationship were reported significantly more often for patients with ED, difficulties with sexual arousal, inability to relax and enjoy sex, and reduced libido. Hypersexual dysfunctions showed no effect on the relationship. CONCLUSION: SD is a common but underreported problem in the treatment of patients with PD. Due to the negative influence on the relationship and QoL of patients and caregivers, SD should be assessed routinely.

In Silico Identification and Experimental Validation of Distal Activity-Enhancing Mutations in Tryptophan Synthase.

Allostery is a central mechanism for the regulation of multi-enzyme complexes. The mechanistic basis that drives allosteric regulation is poorly understood but harbors key information for enzyme engineering. In the present study, we focus on the tryptophan synthase complex that is composed of TrpA and TrpB subunits, which allosterically activate each other. Specifically, we develop a rational approach for identifying key amino acid residues of TrpB distal from the active site. Those residues are predicted to be crucial for shifting the inefficient conformational ensemble of the isolated TrpB to a productive ensemble through intra-subunit allosteric effects. The experimental validation of the conformationally driven TrpB design demonstrates its superior stand-alone activity in the absence of TrpA, comparable to those enhancements obtained after multiple rounds of experimental laboratory evolution. Our work evidences that the current challenge of distal active site prediction for enhanced function in computational enzyme design has become within reach.