Modifications of Protein-Bound Substrates by Trans-Acting Enzymes in Natural Products Biosynthesis.

Enzymatic modifications of small molecules are a common phenomenon in natural product biosynthesis, leading to the production of diverse bioactive compounds. In polyketide biosynthesis, modifications commonly take place after the completion of the polyketide backbone assembly by the polyketide synthases and the mature products are released from the acyl-carrier protein (ACP). However, exceptions to this rule appear to be widespread, as on-line hydroxylation, methyl transfer, and cyclization during polyketide assembly process are common, particularly in trans-AT PKS systems. Many of these modifications are catalyzed by specific domains within the modular PKS systems. However, several of the on-line modifications are catalyzed by stand-alone proteins. Those include the on-line Baeyer-Villiger oxidation, α-hydroxylation, halogenation, epoxidation, and methyl esterification during polyketide assembly, dehydrogenation of ACP-bound short fatty acids by acyl-CoA dehydrogenase-like enzymes, and glycosylation of ACP-bound intermediates by discrete glycosyltransferase enzymes. This review article highlights some of these trans-acting proteins that catalyze enzymatic modifications of ACP-bound small molecules in natural product biosynthesis.

Recent Advances in On-line Mass Spectrometry Toolbox for Mechanistic Studies of Organic Electrochemical Reactions.

Electrochemical reactions are very complex and involve a variety of physicochemical processes. Accurate and systematic monitoring of intermediate process changes during the reaction is essential for understanding the mechanism of electrochemical reactions and is the basis for rational design of new electrochemical reactions. On-line electrochemical analysis based on mass spectrometry (MS) has become an important tool for studying electrochemical reactions. This technique is based on different ionization and sampling means and realizes on-line analysis of electrochemical reactions by establishing electrochemistry-MS (EC-MS) coupling devices. In particular, it provides key evidence for elucidating the reaction mechanism by capturing and identifying the reactive reaction intermediates. This review will categorize various EC-MS devices and the organic electrochemical reaction systems they study, highlighting the latest research progress in recent years. It will also analyze the properties of various devices and look forward to the future development of EC-MS.

On-line targeted metabolomics for real-time monitoring of relevant compounds in fermentation processes.

Fermentation monitoring is a powerful tool for bioprocess development and optimization. On-line metabolomics is a technology that is starting to gain attention as a bioprocess monitoring tool, allowing the direct measurement of many compounds in the fermentation broth at a very high time resolution. In this work, targeted on-line metabolomics was used to monitor 40 metabolites of interest during three Escherichia coli succinate production fermentation experiments every 5 min with a triple quadrupole mass spectrometer. This allowed capturing high-time resolution biological data that can provide critical information for process optimization. For nine of these metabolites, simple univariate regression models were used to model compound concentration from their on-line mass spectrometry peak area. These on-line metabolomics univariate models performed comparably to vibrational spectroscopy multivariate partial least squares regressions models reported in the literature, which typically are much more complex and time consuming to build. In conclusion, this work shows how on-line metabolomics can be used to directly monitor many bioprocess compounds of interest and obtain rich biological and bioprocess data.

Quantification of [99Tc]TcO4- in urine by means of anion-exchange chromatography-aerosol desolvation nebulization-inductively coupled plasma-mass spectrometry.

To sensitively determine 99Tc, a new method for internal quantification of its most common and stable species, [99Tc]Tc O 4 - , was developed. Anion-exchange chromatography (IC) was coupled to inductively coupled plasma-mass spectrometry (ICP-MS) and equipped with an aerosol desolvation system to provide enhanced detection power. Due to a lack of commercial Tc standards, an isotope dilution-like approach using a Ru spike and called isobaric dilution analysis (IBDA) was used for internal quantification of 99Tc. This approach required knowledge of the sensitivities of 99Ru and 99Tc in ICP-MS. The latter was determined using an in-house prepared standard manufactured from decayed medical 99mTc-generator eluates. This standard was cleaned and preconcentrated using extraction chromatography with TEVA resin and quantified via total reflection X-ray fluorescence (TXRF) analysis. IC coupled to ICP-MS enabled to separate, detect and quantify [99Tc]Tc O 4 - as most stable Tc species in complex environments, which was demonstrated in a proof of concept. We quantified this species in untreated and undiluted raw urine collected from a patient, who previously underwent scintigraphy with a 99mTc-tracer, and determined a concentration of 19.6 ± 0.5 ng L-1. The developed method has a high utility to characterize a range of Tc-based radiopharmaceuticals, to determine concentrations, purity, and degradation products in complex samples without the need to assess activity parameters of 99(m)Tc.

When the WRENCH turns a few heads: Expectation and semantic relatedness in inattentional blindness.

Semantic relatedness and expectation were investigated in inattentional blindness-failure to perceive an unexpected object in plain sight when attention is engaged elsewhere. Participants named primary-task pictures and ignored distractor pictures. Four trials preceded a 'critical' trial where an unexpected six-letter-word appeared at fixation, simultaneously with the pictures. In Experiment 1, we found robust effects for both in-lab and on-line-Zoom methodology. More participants reported the unexpected word semantically-related to the primary-task pictures than a semantically-unrelated word. In Experiment 2, expectations were violated, by changing the semantic category of the primary-task pictures. More participants reported the unexpected word semantically-related to the unexpected picture category than a semantically-unrelated word. When attentional resources are consumed by a task, a violation to task expectations is not enough to reorient attention to an unexpected word. Attention reorients to what is meaningful to the task, and what is meaningful is updated in light of unexpected information.

Synthesis and absolute configuration of cyclic synthetic cathinones derived from α-tetralone.

The emergence of new synthetic cathinones continues to be a matter of public health concern. In fact, already known products (drugs) are being rapidly replaced by new structurally related alternatives, whereby modifications in the basic cathinone structure are used by manufacturers to circumvent the legislation. On the other hand, some derivatives of synthetic cathinones represent important pharmaceuticals with antidepressant properties. In the search for pharmaceutically relevant analogs, the main goal of the present study was to design and characterize novel cyclic α-tetralone-based derivatives of synthetic cathinones. We synthesized a series of derivatives and verified their chemical structure. Subsequently, chiral separation has been accomplished by high-performance liquid chromatography (HPLC) equipped with a circular dichroism (CD) detector, which directly provided CD spectra of the enantiomers of the analyzed substances at 252 nm. Using density functional theory calculations, we have obtained stable conformers of selected enantiomers in solution and their relative abundances, which we used to simulate their spectra. The experimental and calculated data have been used to assign the absolute configuration of six as-yet unknown synthetic cathinones.

Reaction monitoring via benchtop nuclear magnetic resonance spectroscopy: A practical comparison of on-line stopped-flow and continuous-flow sampling methods.

The ability for nuclear magnetic resonance (NMR) spectroscopy to provide quantitative, structurally rich information makes this spectroscopic technique an attractive reaction monitoring tool. The practicality of NMR for this type of analysis has only increased in the recent years with the influx of commercially available benchtop NMR instruments and compatible flow systems. In this study, we aim to compare 19F NMR reaction profiles acquired under both on-line continuous-flow and stopped-flow sampling methods, with modern benchtop NMR instrumentation, and two reaction systems: a homogeneous imination reaction and a biphasic activation of a carboxylic acid to acyl fluoride. Reaction trends with higher data density can be acquired with on-line continuous-flow analyses, and this work highlights that representative reaction trends can be acquired without any correction when monitoring resonances with a shorter spin-lattice relaxation time (T1), and with the used flow conditions. On-line stopped-flow analyses resulted in representative reaction trends in all cases, including the monitoring of resonances with a long T1, without the need of any correction factors. The benefit of easier data analysis, however, comes with the cost of time, as the fresh reaction solution must be flowed into the NMR system, halted, and time must be provided for spins to become polarized in the instrument's external magnetic field prior to spectral measurement. Results for one of the reactions were additionally compared with the use of a high-field NMR.

Gas Phase Transformations in Carbon-11 Chemistry.

The short-lived positron-emitter carbon-11 (t1/2 = 20.4 min; ß+, 99.8%) is prominent for labeling tracers for use in biomedical research with positron emission tomography (PET). Carbon-11 is produced for this purpose with a cyclotron, nowadays almost exclusively by the 14N(p,α)11C nuclear reaction, either on nitrogen containing a low concentration of oxygen (0.1-0.5%) or hydrogen (~5%) to produce [11C]carbon dioxide or [11C]methane, respectively. These primary radioactive products can be produced in high yields and with high molar activities. However, only [11C]carbon dioxide has some utility for directly labeling PET tracers. Primary products are required to be converted rapidly and efficiently into secondary labeling synthons to provide versatile radiochemistry for labeling diverse tracer chemotypes at molecular positions of choice. This review surveys known gas phase transformations of carbon-11 and summarizes the important roles that many of these transformations now play for producing a broad range of labeling synthons in carbon-11 chemistry.

Development of an automatic measurement system using atmospheric pressure photoionization ultrahigh-resolution mass spectrometry and application for on-line analysis of particulate matter.

On-line chemical characterization of atmospheric particulate matter (PM) with soft ionization technique and ultrahigh-resolution Mass Spectrometry (UHRMS) provides molecular information of organic constituents in real time. Here we describe the development and application of an automatic measurement system that incorporates PM2.5 sampling, thermal desorption, atmospheric pressure photoionization, and UHRMS analysis. Molecular formulas of detected organic compounds were deducted from the accurate (±10 ppm) molecular weights obtained at a mass resolution of 100,000, allowing the identification of small organic compounds in PM2.5. Detection efficiencies of 28 standard compounds were determined and we found a high sensitivity and selectivity towards organic amines with limits of detection below 10 pg. As a proof of principle, PM2.5 samples collected off-line in winter in the urban area of Beijing were analyzed using the Ionization Module and HRMS of the system. The automatic system was then applied to conduct on-line measurements during the summer time at a time resolution of 2 hr. The detected organic compounds comprised mainly CHON and CHN compounds below 350 m/z. Pronounced seasonal variations in elemental composition were observed with shorter carbon backbones and higher O/C ratios in summer than that in winter. This result is consistent with stronger photochemical reactions and thus a higher oxidation state of organics in summer. Diurnal variation in signal intensity of each formula provides crucial information to reveal its source and formation pathway. In summary, the automatic measurement system serves as an important tool for the on-line characterization and identification of organic species in PM2.5.

Corporate Social Responsibility and Directors' and Officers' Liability Risk: The Moderating Effect of Risk Environment and Growth Potential.

Theoretical arguments regarding the effect of corporate social responsibility (CSR) on firm liability risk are abundant; however, empirical evidence about this relationship is scarce. We investigate the relationship between CSR and the personal liability risk of a firm's directors and officers. We argue that companies with better CSR performance represent a better underwriting risk for directors' and officers' (D&O) insurance providers and, therefore, have a lower cost of insurance. Our results show that firms with better CSR performance are more likely to purchase D&O insurance and have a lower premium-to-coverage ratio, known as the insurance rate-on-line. We also show that this risk-reduction effect is stronger for firms that operate in a high-risk environment and have higher sales growth. These results provide evidence that CSR can be used as a risk management tool to mitigate liability risk and suggest which firms benefit most from this effect.

[The on-line turnover of biologically active additives. Part I. Legal regulation of on-line market of imported biologically active additives in Russia].

The interest of population to biologically active additives drastically increased both due to consequences of the COVID-19 corona-virus infection and dynamic development of online trade and active propaganda of healthy lifestyle through mass media. Such production contains in its compound substances that are necessary for maintaining normal vital activity of organism and increasing human immunity. In other cases it can be used as additional and auxiliary agent of treatment of various diseases. In Russia, biologically active additives can be sold through three channels: pharmacies, on-line market and network marketing. In Russia, the results of monitoring of on-line market in 2021-2022 testifies that purchasing of such biologically active additives at on-line platforms turned out to be the riskiest action for human life and health. The biologically active additives were sold at on-line market with serious violations that confirms their danger to health and in some cases for human life. The study demonstrated that nowadays there is no clear-cut legal regulation of turn-over of imported biologically active additives at on-line market. The article presents detailed analysis of normative legal field that regulates in Russia on-line trade of imported biologically active additives and relevant conclusions and recommendations.

[The application of artificial intellect in health care: prospects and challenges for science and clinical medicine].

The paper analyses publications data related to issues of application of AI and machine learning systems in medical science and practice. The particular attention is paid to key points of AI application in health care: diagnostics, telemedicine, development of new medications, medical rehabilitation and management decision-making process. Despite broad perspectives of applying the given systems in clinical practice and pharmaceutical industry, there are a number of such unsolved problems as ensuring information security, risk of making erroneous decisions and necessity to change existing normative legal base of health care.

Sensitive Analysis of Recombinant Human Erythropoietin Glycopeptides by On-Line Phenylboronic Acid Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry.

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N24 and N38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CE-MS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest.

The role of EPID in vivo dosimetry in the risk management of stereotactic lung treatments.

BACKGROUND AND OBJECTIVE: In this work we report our experience with the use of in vivo dosimetry (IVD) in the risk management of stereotactic lung treatments. METHODS: A commercial software based on the electronic portal imaging device (EPID) signal was used to reconstruct the actual planning target volume (PTV) dose of stereotactic lung treatments. The study was designed in two phases: i) in the observational phase, the IVD results of 41 consecutive patients were reviewed and out-of-tolerance cases were studied for root cause analysis; ii) in the active phase, the IVD results of 52 patients were analyzed and corrective actions were taken when needed. Moreover, proactive preventions were further introduced to reduce the risk of future failures. The error occurrence rate was analyzed to evaluate the effectiveness of proactive actions. RESULTS: A total of 330 fractions were analyzed. In the first phase, 13 errors were identified. In the active phase, 12 errors were detected, 5 of which needed corrective actions; in 4 patients the actions taken corrected the error. Several preventions and barriers were introduced to reduce the risk of future failures: the planning checklist was updated, the procedure for vacuum pillows was improved, and use of the respiratory compression belt was optimized. A decrease in the failure rate was observed, showing the effectiveness of procedural adjustment. CONCLUSION: The use of IVD allowed the quality of lung stereotactic body radiation therapy (SBRT) treatments to be improved. Patient-specific and procedural corrective actions were successfully taken as part of risk management, leading to an overall improvement in the dosimetric accuracy.

Methodology for fast development of digital solutions in integrated continuous downstream processing.

The methodology for production of biologics is going through a paradigm shift from batch-wise operation to continuous production. Lot of efforts are focused on integration, intensification, and continuous operation for decreased foot-print, material, equipment, and increased productivity and product quality. These integrated continuous processes with on-line analytics become complex processes, which requires automation, monitoring, and control of the operation, even unmanned or remote, which means bioprocesses with high level of automation or even autonomous capabilities. The development of these digital solutions becomes an important part of the process development and needs to be assessed early in the development chain. This work discusses a platform that allows fast development, advanced studies, and validation of digital solutions for integrated continuous downstream processes. It uses an open, flexible, and extendable real-time supervisory controller, called Orbit, developed in Python. Orbit makes it possible to communicate with a set of different physical setups and on the same time perform real-time execution. Integrated continuous processing often implies parallel operation of several setups and network of Orbit controllers makes it possible to synchronize complex process system. Data handling, storage, and analysis are important properties for handling heterogeneous and asynchronous data generated in complex downstream systems. Digital twin applications, such as advanced model-based and plant-wide monitoring and control, are exemplified using computational extensions in Orbit, exploiting data and models. Examples of novel digital solutions in integrated downstream processes are automatic operation parameter optimization, Kalman filter monitoring, and model-based batch-to-batch control.

Development of setups for real-time analysis of the effluent of a microreactor by mass spectrometry.

Monitoring a synthesis reaction in real time could allow not only the detection of the intermediates involved in the synthesis, to better understand its mechanisms, but also the impurities. Spectroscopic methods could be performed but are not so performant when analyzing complex mixtures and could require specific properties for the detection of the molecules of interest, the presence of a chromophore moiety for example. Mass spectrometry (MS) may overcome these limitations and is able to reach the accuracy and sensitivity required to efficiently detect, quantify, identify, and characterize the reagents and species produced during the synthesis. This is why the hyphenation of a microreactor with MS has already allowed synthesis processes to be monitored, but most of the time it targets a specific reaction or compounds and involves solvents compatible with MS. In this study, a universal setup for the hyphenation of a microreactor with MS and based on two valves has been developed. This two-valve setup has proven itself for the analysis of molecules of different nature and hydrophilicity, soluble in a large number of solvents even in non-MS-compatible ones. The developed setup evidenced a good repeatability and a linear response for the detection of the studied compounds. In addition, the dilution step included in the two-valve setup allows the MS monitoring of compounds initially synthesized at different concentrations. Finally, it was successfully used to study an amination reaction allowing the detection of the reaction products in 4 min with good repeatability as RSD values of MS signals were lower than 17%.

Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV.

In this paper, we demonstrate the coupling of synchrotron small angle X-ray scattering (SAXS) to asymmetrical flow-field flow fractionation (AF4) for protein characterization. To the best of our knowledge, this is the first time AF4 is successfully coupled to a synchrotron for on-line measurements on proteins. This coupling has potentially high impact, as it opens the possibility to characterize individual constituents of sensitive and/or complex samples, not suited for separation using other techniques, and for low electron density samples where high X-ray flux is required, e.g., biomolecules and biologics. AF4 fractionates complex samples in native or close to native environment, with low shear forces and system surface area. Many orders of magnitude in size can be fractionated in one measurement, without having to reconfigure the experimental setup. We report AF4 fractionations with correlated UV and statistically adequate SAXS data of bovine serum albumin and a monoclonal antibody and evaluate SAXS data recorded for the two protein systems.

Development of a capillary electrophoretic method for determination of ketorolac enantiomers in human plasma using cationic ß-cyclodextrin derivative as a chiral selector.

A novel approach for the separation of ketorolac enantiomers by capillary electrophoresis is presented. A cationic ß-cyclodextrin derivative based on imidazole was synthesized and used as a chiral selector in the background electrolyte. The influence of pH and ionic strength of background electrolyte, as well as cationic ß-cyclodextrin derivative concentration on the resolution of ketorolac enantiomers, was investigated. The highest value of the resolution for ketorolac enantiomers was 1.46 when the background electrolyte consisted of 25 mM NaH2 PO4 (pH 6.4) with 1 mM 1-butyl-3-ß-cyclodextrinimidazolium tosylate. Additionally, the possibilities of cationic derivatives for the separation of ketoprofen enantiomers were shown (peak resolution 1.06). The two-step preconcentration mode was developed to reduce the limit of detection of individual enantiomers. The proposed approach was successfully applied to determine ketorolac enantiomers in tablet "Ketorol express" and human plasma. The calibration range of ketorolac enantiomers for plasma samples was 0.25-2.50 µg/ml with coefficients of determination ≥ 0.99. The relative standard deviation both of the peak area and migration time was less than 15%, as well as the accuracy ranged from 90.1% to 110.2% for both analytes. The limits of detection were 44 and 55 ng/ml for R- and S-ketorolac. The quantity of ketorolac in plasma was verified with high-performance liquid chromatography.

Sensitivity enhancement of capillary electrophoresis-frontal analysis-based method for characterization of drug-protein interactions using on-line sample preconcentration.

Capillary electrophoresis-frontal analysis is one of the most frequently used approaches for the study of plasma protein-drug interactions as a substantial part of new drug development. However, the capillary electrophoresis-frontal analysis typically combined with ultraviolet-visible detection suffers from insufficient concentration sensitivity, particularly for substances with limited solubility and low molar absorption coefficient. The sensitivity problem has been solved in this work by its combination with an on-line sample preconcentration. According to the knowledge of the authors this combination has never been used to characterize plasma protein-drug binding. It resulted in a fully automated and versatile methodology for the characterization of binding interactions. Further, the validated method minimalizes the experimental errors due to a reduction in the manipulation of samples. Moreover, employing an on-line preconcentration strategy with capillary electrophoresis-frontal analysis using human serum albumin-salicylic acid as a model system improves the drug concentration sensitivity 17-fold compared to the conventional method. The value of binding constant (1.51 ± 0.63) · 104 L/mol obtained by this new capillary electrophoresis-frontal analysis modification is in agreement with the value (1.13 ± 0.28) ·104 L/mol estimated by a conventional variant of capillary electrophoresis-frontal analysis without the preconcentration step, as well as with literature data obtained using different techniques.

Comparison of 2 medium cutoff dialyzers versus on-line hemodiafiltration regarding depurative ability and albumin loss: An uncontrolled clinical research.

BACKGROUND: Hemodialysis (HD) techniques that best remove molecules in the middle to high molecular weight range are on-line hemodiafiltration (OL-HDF) and HD with medium cut-off (MCO) membranes. The aim of this study was to compare efficacy and safety of OL-HDF with FxCordiax HDF 800™, with HD with 2 MCO dialyzers: Theranova 500® and the new Elisio 21HX™ dialyzer. METHODS: Fourteen patients following treatment with OL-HDF using FxCordiax HDF 800™ were randomized to receive a consecutive 1-week HD treatment with Theranova 500® and Elisio 21HX™.The reduction rate (RR) of differently sized molecules was compared, as well as the variation rate in molecules smaller than 1000, detected by nuclear magnetic resonance based chemometrics (metabolomics). Albumin loss in dialysate was quantified. RESULTS: Lower RRs were found for molecules around 20 000 with Elisio 21HX™ compared to OL- HDF (RR prolactin 58.5% versus 66.7%, p = 0.034; RR Kappa light chain 63.1% versus 71.8%, p = 0.010). Albumin loss per session was higher with Theranova 500® than with OL-HDF and with Elisio 21HX™ (2249.9 ± 714.1 mg, 815.2 ± 474.0 mg, 442.9 ± 135.9 mg, p < 0.001, respectively). Metabolomic studies suggested, by semi-quantitative analysis, a greater depurative capacity of OL-HDF, followed by Elisio 21HX™, and then Theranova 500®. CONCLUSIONS: In this study, HD with Theranova 500® has proven to be very similar in efficacy to OL-HDF, although with a significantly higher albumin loss. HD with Elisio 21HX™ resulted in lower removal of molecules around 20 000 compared to OL-HDF, with no significant difference compared to Theranova 500®, and with less albumin loss than Theranova 500®.