Biochemical and biophysical characterization of a prephenate dehyodrogenase from the hyperthermophilic bacterium Aquifex aeolicus

Prephenate dehydrogenase is a key enzyme from the TyrA protein family responsible for catalyzing the NAD+ -dependent oxidative decarboxylation of prephenate to hydroxylphenylpyruvate, one of the terminal steps in the biosynthesis of tyrosine (Tyr). To gain structural and biophysical information on this protein, PD from the thermophilic bacterium Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography. The enzyme is susceptible to proteolysis at the N-terminal region of the protein and the exact site of cleavage was determined by mass spectrometry. Crystallography trials on several N-terminally truncated variants performed by our collaborators at the University of Toronto indicated that only the PD variant missing the first 19 amino acids (E19PD) yielded quality diffraction crystals. The biochemical and biophysical properties of the full-length PD were compared to E19PD also expressed recombinantly in E. coli . The enzyme functions as a cyclohexadienyl dehydrogenase, accepting prephenate (effectively) and L-arogenate (poorly) as substrates. Both forms of the enzyme are thermally stable and show maximal activity only at high temperatures, although E19PD is less stable but more active than the full-length protein. Low concentrations of the denaturant guanidinium hydrochloride (Gdn-HCl) activate the activity of E19PD, but at higher concentrations activity is lost concomitant with a multi-state pathway of denaturation which proceeds through unfolding of the dimer, oligomerization, then unfolding of monomers. Measurements of steady-state fluorescence intensity and its quenching by acrylamide in the presence of Gdn-HCl suggest that of the two tryptophan (Trp) residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried Trp is at the active site. These findings are in accordance with the crystal structure of E19PD. Site-directed mutagenesis and steady-state kinetic analyses of variant proteins were used to probe the roles of conserved residues. In accord with the crystal structure of the enzyme bound with NAD+ plus product and product analogues, His147 acted as a catalytic hydrogen bond acceptor while Ser216 was responsible for coordinating NAD+ and His147 to facilitate hydride transfer. Arg250 and His217 were responsible for binding prephenate in the active site. Additionally and most importantly, His217 in A. aeolicus PD and the homologous residue in E. coli CM-PD (His257) was shown to be critical for inhibition of activity by Tyr. Two assays were developed to assess Tyr binding to wild-type and variant enzymes. Our results are placed in context of crystal structures of PD bound with Tyr and indicate how TyrA proteins can accept hydroxyphenylpuruvate and Tyr in the active site of the enzyme

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Non-aqueous capillary electrophoresis for the analysis of acidic compounds using negative electrospray ionization mass spectrometry

Non-aqueous capillary electrophoresis (NACE) is an attractive CE mode, in which water solvent of the background electrolyte (BGE) is replaced by organic solvent or by a mixture of organic solvents. This substitution alters several parameters, such as the pKa, permittivity, viscosity, zeta potential, and conductivity, resulting in a modification of CE separation performance (i.e., selectivity and/or efficiency). In addition, the use of NACE is particularly well adapted to ESI-MS due to the high volatility of solvents and the low currents that are generated. Organic solvents reduce the number of side electrochemical reactions at the ESI tip, thereby allowing the stabilization of the ESI current and a decrease in background noise. All these features make NACE an interesting alternative to the aqueous capillary zone electrophoresis (CZE) mode, especially in combination with mass spectrometry (MS) detection. The aim of this work was to evaluate the use of NACE coupled to negative ESI-MS for the analysis of acidic compounds with two available CE-MS interfaces (sheath liquid and sheathless). First, NACE was compared to aqueous CZE for the analysis of several pharmaceutical acidic compounds (non-steroidal anti-inflammatory drugs, NSAIDs). Then, the separation performance and the sensitivity achieved by both interfaces were evaluated, as were the impact of the BGE and the sample composition. Finally, analyses of glucuronides in urine samples subjected to a minimal sample pre-treatment (“dilute-and-shoot”) were performed by NACE-ESI-MS, and the matrix effect was evaluated. A 20- to 100-fold improvement in sensitivity was achieved using the NACE mode in combination with the sheathless interface and no matrix effect was observed regardless of the interfaces

Capillary electrophoresis–electrospray ionization-mass spectrometry interfaces: Fundamental concepts and technical developments

Capillary electrophoresis (CE) hyphenated to electrospray ionization (ESI) mass spectrometry (MS) is a powerful tool for analyzing a wide variety of analytes in different matrices. The major issue with CE–ESIMS lies in finding a suitable and versatile interface to ensure the best CE and ESI operations. Thus, the development and improvement of CE–ESI-MS interfaces have been the subjects of much research. The first part of the present review focuses on the fundamental aspects of the three steps of the ESI process, i.e., spray formation, droplet evolution, and the production of gas-phase ions. In the second part of the review, the electrochemical reactions involved in the ESI and CE processes and their influences on the sensitivity and performance are discussed in detail. Then, the existing interfaces are divided into two major classes according to their operating flow rate (electrospray vs. nanospray regime). The particular characteristics of these two regimes are discussed by considering their practical impacts on ionization and the MS response. Finally, the current CE–ESI-MS interfaces are summarized, including their major advantages, drawbacks, and fields of application

In-spray supercharging of intact proteins by capillary electrophoresis–electrospray ionization–mass spectrometry using sheath liquid interface

Capillary electrophoresis (CE) coupled with electrospray ionization (ESI) mass spectrometry (MS) is a suitable technique for the analysis of intact proteins. The main configuration to realize this coupling is the sheath liquid interface, which is characterized by the addition of a make-up liquid providing the electric contact as well as the appropriate flow and solvent composition for optimal ionization and evaporation. One main advantage of this interface is that the composition of the sheath liquid can be tuned to modify the ionization without affecting CE selectivity and efficiency. In the case of protein ionization, this feature is particularly interesting to modulate their charge-state distribution (CSD), while keeping the separation performance unchanged. In this context, the current work evaluated the effect on proteins’ CSD of adding supercharging molecules to the sheath liquid. Several supercharging reagents were tested with different background electrolyte (BGE) and their impact was estimated for three model proteins (i.e., human insulin, human growth hormone, hemoglobin A0) exhibiting various properties in terms of ionization, conformation, and flexibility. Their influence on the global sensitivity for each protein was also assessed. Among the supercharging reagents tested, m-NBA and sulfolane led to supercharging effect whose magnitude depended on the proteins as well of the BGE pH. The sensitivity and separation performance remained globally unchanged for each protein and supercharging additive, while sulfolane led in some cases to a sensitivity improvement

Evaluation of a sheathless nanospray interface based on a porous tip sprayer for CE-ESI-MS coupling

The hyphenation of capillary electrophoresis (CE) with mass spectrometry (MS) is a powerful method to obtain high efficient, sensitive, and selective analyses. The successful coupling with electrospray ionization (ESI) source requires closed electric circuits for both the CE separation and the ESI processes. A wide range of interfaces has been proposed to satisfy this requirement. Among them, the new high sensitivity porous sprayer based on a porous tip achieves the electric connection by inserting the capillary outlet made of a porous material into an ESI needle filled with a conductive liquid and independently grounded. This device is compatible with the minute flow rates exhibited in CE and therefore makes possible the use of a nano-electrospray behavior. In this work, this interface was evaluated for hyphenating a CE with a single quadrupoleMS instrument for lowmolecular weight analytes. Investigations aimed at highlighting the most influent parameters thanks to a design of experiments, reaching the best performance in terms of sensitivity and stability. MS signal intensities of various pharmaceutical compounds (e.g. amphetamines, blockers) emphasized high sensitivity and efficiency, while repeatability, expressed as relative standard deviation of corrected heights and areas, was suitable for quantitative purposes (<5%)

Fast chiral separation of drugs using columns packed with sub-2 mum particles and ultra-high pressure

The use of columns packed with sub-2 mum particles in liquid chromatography with very high pressure conditions (known as UHPLC) was investigated for the fast enantioseparation of drugs. Two different procedures were evaluated and compared using amphetamine derivatives and beta-blockers as model compounds. In one case, cyclodextrins (CD) were directly added to the mobile phase and chiral separations were carried out in less than 5 min. However, this strategy suffered from several drawbacks linked to column lifetime and low chromatographic efficiencies. In the other case, the analysis of enantiomers was carried out after a derivatization procedure using two different reagents, 2,3,4-tri-O-acetyl-alpha-D-arabinopyranosyl isothiocyanate (AITC) and N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent). Separation of several amphetamine derivatives contained within the same sample was achieved in 2-5 min with high efficiency and selectivity. The proposed approach was also successfully applied to the enantiomeric purity determination of (+)-(S)-amphetamine and (+)-(S)-methamphetamine. Similar results were obtained with beta-blockers, and the separation of 10 enantiomers was carried out in less than 3 min, whereas the individual separation of several beta-blocker enantiomers was performed in 1 min or less. Chirality, 2010. (c) 2009 Wiley-Liss, Inc

Expression, Purification, and Characterization of Recombinant Purine Nucleoside Phosphorylase from <i>Escherichia coli</i>

Recombinant purine nucleoside phosphorylase (PNPase) from <i>Escherichia coli</i> was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate−liberating enzymes. The <i>E. coli</i> enzyme was overexpressed in <i>E. coli</i> by inserting the genomic fragment containing the <i>deoD</i> gene downstream of the isopropyl β−D−thiogalactoside−inducible promotor of pSE380 expression vector. The recombinant protein was purified to about 90% homogeneity and with a yield of about 9000 units of activity/L of culture, using an efficient one−column procedure. A continuous spectrophotometric assay coupling Pi release to the phosphorolysis of the nucleoside analogue 7−methylinosine (m7Ino) was recently described. Here, we report the steady−state kinetic parameters of the recombinant <i>E. coli</i> PNPase catalyzed reaction with m7Ino and Pi as substrates and compare these parameters with those of a bacterial PNPase commercially available for use in coupled assays. Under the assay conditions described, the recombinant <i>E. coli</i> protein is active at higher pH values and is stable up to a temperature of about 55&deg;C and following multiple freeze−thaw cycles. It is activated by high ionic strength but loses some activity following dialysis or concentration under pressure. Finally, a new procedure for the synthesis of m7Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase−coupled Pi assays more attractiv

Novel personalized treatment strategy for patients with chronic stroke with severe upper-extremity impairment: The first patient of the AVANCER trial.

BACKGROUND Around 25% of patients who have had a stroke suffer from severe upper-limb impairment and lack effective rehabilitation strategies. The AVANCER proof-of-concept clinical trial (NCT04448483) tackles this issue through an intensive and personalized-dosage cumulative intervention that combines multiple non-invasive neurotechnologies. METHODS The therapy consists of two sequential interventions, lasting until the patient shows no further motor improvement, for a minimum of 11 sessions each. The first phase involves a brain-computer interface governing an exoskeleton and multi-channel functional electrical stimulation enabling full upper-limb movements. The second phase adds anodal transcranial direct current stimulation of the motor cortex of the lesioned hemisphere. Clinical, electrophysiological, and neuroimaging examinations are performed before, between, and after the two interventions (T0, T1, and T2). This case report presents the results from the first patient of the study. FINDINGS The primary outcome (i.e., 4-point improvement in the Fugl-Meyer assessment of the upper extremity) was met in the first patient, with an increase from 6 to 11 points between T0 and T2. This improvement was paralleled by changes in motor-network structure and function. Resting-state and transcranial magnetic stimulation-evoked electroencephalography revealed brain functional changes, and magnetic resonance imaging (MRI) measures detected structural and task-related functional changes. CONCLUSIONS These first results are promising, pointing to feasibility, safety, and potential efficacy of this personalized approach acting synergistically on the nervous and musculoskeletal systems. Integrating multi-modal data may provide valuable insights into underlying mechanisms driving the improvements and providing predictive information regarding treatment response and outcomes. FUNDING This work was funded by the Wyss-Center for Bio and Neuro Engineering (WCP-030), the Defitech Foundation, PHRT-#2017-205, ERA-NET-NEURON (Discover), and SNSF (320030L_197899, NiBS-iCog)