Implementation of Modern Therapeutic Drug Monitoring and Lipidomics Approaches in Clinical Practice: A Case Study with Colistin Treatment.

Nowadays, lipidomics plays a crucial role in the investigation of novel biomarkers of various diseases. Its implementation into the field of clinical analysis led to the identification of specific lipids and/or significant changes in their plasma levels in patients suffering from cancer, Alzheimer's disease, sepsis, and many other diseases and pathological conditions. Profiling of lipids and determination of their plasma concentrations could also be helpful in the case of drug therapy management, especially in combination with therapeutic drug monitoring (TDM). Here, for the first time, a combined approach based on the TDM of colistin, a last-resort antibiotic, and lipidomic profiling is presented in a case study of a critically ill male patient suffering from Pseudomonas aeruginosa-induced pneumonia. Implementation of innovative analytical approaches for TDM (online combination of capillary electrophoresis with tandem mass spectrometry, CZE-MS/MS) and lipidomics (liquid chromatography-tandem mass spectrometry, LC-MS/MS) was demonstrated. The CZE-MS/MS strategy confirmed the chosen colistin drug dosing regimen, leading to stable colistin concentrations in plasma samples. The determined colistin concentrations in plasma samples reached the required minimal inhibitory concentration of 1 µg/mL. The complex lipidomics approach led to monitoring 545 lipids in collected patient plasma samples during and after the therapy. Some changes in specific individual lipids were in good agreement with previous lipidomics studies dealing with sepsis. The presented case study represents a good starting point for identifying particular individual lipids that could correlate with antimicrobial and inflammation therapeutic management.

A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4'-Nitroacetophenone.

Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4'-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5-8.2% and 2.3-7.4%, accuracy was in the range of 93.3-109.7% and 94.7-110.1%, limits of detection (LODs) were in the range of 0.15-9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer's disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

Quantitative analysis of therapeutic peptides by CZE using multiple sample injection in hydrodynamically closed separation system.

Therapeutic peptides have emerged as an innovative and promising class of therapeutic compounds in modern medicine. Synthetic peptide analogs triptorelin and lanreotide are known for their pronounced clinical versatility and potency. In this study, we present the development and validation of novel methods based on capillary zone electrophoresis performed in hydrodynamically closed system (HCS) and paired with ultraviolet detection and repeated injection sample introduction. To the best of our knowledge, we developed the first capillary electrophoresis-based method for the determination of lanreotide, and concurrently, the first HCS method for the determination of triptorelin. Maximal separation efficiency and signal intensity were achieved using background electrolytes composed of 50 mM formic acid with the addition of 0.05% (v/v) methyl-hydroxyethyl cellulose. The proposed methods exhibit favorable performance characteristics, namely, calibration curve (r2 exceeding 0.99), low limits of detection (0.25 µg/mL in a water matrix and 0.5 µg/mL in synthetic urine), acceptable precision (relative standard deviation ranging from 2.2% to 9.6% for intraday repeatability and between 5.2% and 14.9% for interday reproducibility), and accuracy (relative errors falling within the 91.1%-107.8% range). The method for triptorelin determination was then used for its quantification in a commercially available drug dosage form (powder for injection) and in spiked synthetic urine samples. The developed methods were also evaluated according to the novel blue applicability grade index, revealing their superior applicability. The results collectively point out the potential of the proposed methods for both quality control and clinical investigations.

Development and Validation of a Capillary Zone Electrophoresis-Tandem Mass Spectrometry Method for Simultaneous Quantification of Eight ß-Lactam Antibiotics and Two ß-Lactamase Inhibitors in Plasma Samples.

Monitoring plasma concentrations of ß-lactam antibiotics is crucial, particularly in critically ill patients, where variations in concentrations can lead to treatment failure or adverse events. Standardized antimicrobial regimens may not be effective for all patients, especially in special groups with altered physiological parameters. Pharmacokinetic/pharmacodynamic (PK/PD) studies highlight the time-dependent antibacterial activity of these antibiotics, emphasizing the need for personalized dosing. Therapeutic drug monitoring (TDM) is essential, requiring rapid and accurate analytical methods for precise determination of drugs in biological material (typically plasma or serum). This study presents a novel capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) method designed for the simultaneous quantification of five penicillin antibiotics, two cephalosporins, one carbapenem, and two ß-lactamase inhibitors in a single run. The method involves a simple sample pretreatment-precipitation with organic solvent-and has a run time of 20 min. Optimization of CZE separation conditions revealed that 20 mM ammonium hydrogen carbonate (NH4HCO3) serves as the optimal background electrolyte (BGE). Positive electrospray ionization (ESI) mode, with isopropyl alcohol (IP)/10 mM ammonium formate water solution (50/50, v/v) as the sheath liquid, was identified as the optimal condition for MS detection. Method validation according to the Food and Drug Administration (FDA) guideline for development of bioanalytical methods demonstrated satisfactory selectivity, linearity, recovery, robustness, and stability. The method's practicality was evaluated using the Blue Applicability Grade Index (BAGI), yielding a score of 77.5. Moreover, the greenness of the proposed method was evaluated by two commonly used metric tools-Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI). The developed CZE-MS/MS method offers a practical and reliable approach for quantifying a broad spectrum of ß-lactam antibiotics in plasma. Its ability to simultaneously quantify multiple analytes in a single run, coupled with a straightforward sample pretreatment, positions it as a valuable and prospective tool for TDM in critically ill patients.

Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies.

BACKGROUND: Accumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer's disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood. METHODS: We used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation. RESULTS: We showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism. CONCLUSION: Our results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Derivatization of carboxylic groups prior to their LC analysis - A review.

Carboxylic acids (CAs) represent a large group of important molecules participating in various biologically significant processes. Analytical study of these compounds is typically performed by liquid chromatography (LC) combined with various types of detection. However, their analysis is often accompanied by a wide variety of problems depending on used separation system or detection method. The dominant ones are: i) poor chromatographic behavior of the CAs in reversed-phase LC; ii) absence of a chromophore (or fluorophore); iii) weak ionization in mass spectrometry (MS). To overcome these problems, targeted chemical modification, and derivatization, come into play. Therefore, derivatization still plays an important and, in many cases, irreplaceable role in sample preparation, and new derivatization methods of CAs are constantly being developed. The most commonly used type of reaction for CAs derivatization is amidation. In recent years, an increased interest in the isotopic labeling derivatization method has been observed. In this review, we comprehensively summarize the possibilities and actual trends in the derivatization of CAs that have been published over the past decade.

An Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method with Online Solid-Phase Extraction Sample Preparation for the High-Throughput and Sensitive Determination of Ostarine in Human Urine.

Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase extraction (online-SPE) in conjunction with ultra-high-performance liquid chromatography and tandem mass spectrometry (triple quadrupole). This automated 2D separation approach is characterized by minimum manual steps in the sample preparation (only dilute-and-shoot), reflecting high sample throughput and the reliability of analytical data. It provides favorable performance parameters, including a limit of detection of 0.5 pg/mL, high accuracy (relative error = 1.6-7.5%), precision (relative standard deviation = 0.8-4.5%), and sensitivity. Additionally, it demonstrates excellent linearity (r2 = 0.9999) in the calibration range of 0.05 to 25 ng/mL and robustness, with no carryover effects observed. This comparative study revealed a two-decadic-order-lower LOD of the SPE-UHPLC-MS/MS method to the corresponding UHPLC-MS/MS method and the lowest one in the group of currently published LC-MS methods. The World Anti-Doping Agency screening and confirmation criteria were met through the analysis of spiked urine samples from ten healthy volunteers. Accordingly, the proposed method is suitable for routine use in antidoping laboratories.

Recent Analytical Methodologies in Lipid Analysis.

Lipids represent a large group of biomolecules that are responsible for various functions in organisms. Diseases such as diabetes, chronic inflammation, neurological disorders, or neurodegenerative and cardiovascular diseases can be caused by lipid imbalance. Due to the different stereochemical properties and composition of fatty acyl groups of molecules in most lipid classes, quantification of lipids and development of lipidomic analytical techniques are problematic. Identification of different lipid species from complex matrices is difficult, and therefore individual analytical steps, which include extraction, separation, and detection of lipids, must be chosen properly. This review critically documents recent strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation published in the last five years (2019 to 2023). The advantages and disadvantages of various extraction methods are covered. The instrumental analysis step comprises methods for lipid identification and quantification. Mass spectrometry (MS) is the most used technique in lipid analysis, which can be performed by direct infusion MS approach or in combination with suitable separation techniques such as liquid chromatography or gas chromatography. Special attention is also given to the correct evaluation and interpretation of the data obtained from the lipid analyses. Only accurate, precise, robust and reliable analytical strategies are able to bring complex and useful lipidomic information, which may contribute to clarification of some diseases at the molecular level, and may be used as putative biomarkers and/or therapeutic targets.

A simple UHPLC-MS/MS method for determination of SET2, a selective antagonist of TRPV2 receptor, in rat plasma samples.

Targeting the transient receptor potential vanilloid 2 channels (TRPV2) in order to alleviate or reverse the course of several diseases including multiple cancers, cardiovascular, immunological, or neurological disorders have been a matter of focus for several years now. SET2, a selective TRPV2 inhibitor, represents an innovative molecule which came into recognition in 2019 and seems to be a promising therapeutic modality in cancer and cardiac diseases. Drug discovery and bioanalysis in clinical environment demands simple, excellent, highly reliable, fast, sensitive, and selective analytical approaches which enable unambiguous identification and quantification of demanded molecule. Here, a targeted ultra-high-performance liquid chromatography - tandem mass spectrometry with electrospray ionization was developed for the quantification of SET2 in plasma samples. The developed method enabled analysis of approx. 15 samples within one hour. Simplicity of the whole analytical procedure can be emphasized by a very simple sample pretreatment based only on the protein precipitation with organic acid (here, 2 M tricholoroacetic acid). The validation procedure was characterized by promising validation parameters and excellent sensitivity what was documented by the limit of detection value at pg.mL-1 concentration level. Analytical validation reported intra- and interday accuracy < 15 % for all quality control samples concentration levels. Similarly, excellent level of intra- (0.1 - 4.8 %) and interday (0.5 - 3.3 %) precision for the tested quality control samples was obtained. The applicability of the developed method was proven by quantifying SET2 concentration levels in plasma samples obtained from Wistar rats that were administered this drug intraperitoneally at a dose of 25 mg/kg. We expect that our new analytical method represents a very attractive tool that could be easily implemented in pharmacokinetics studies and/or therapeutic drug monitoring. Moreover, its applicability was confirmed by the new practicability evaluation metric tool.

Capillary electrophoresis in the analysis of therapeutic peptides-A review.

Therapeutic peptides are a growing class of innovative drugs with high efficiency and a low risk of adverse effects. These biomolecules fall within the molecular mass range between that of small molecules and proteins. However, their inherent instability and potential for degradation underscore the importance of reliable and effective analytical methods for pharmaceutical quality control, therapeutic drug monitoring, and compliance testing. Liquid chromatography-mass spectrometry (LC-MS) has long time been the "gold standard" conventional method for peptide analysis, but capillary electrophoresis (CE) is increasingly being recognized as a complementary and, in some cases, superior, highly efficient, green, and cost-effective alternative technique. CE can separate peptides composed of different amino acids owing to differences in their net charge and size, determining their migration behavior in an electric field. This review provides a comprehensive overview of therapeutic peptides that have been used in the clinical environment for the last 25 years. It describes the properties, classification, current trends in development, and clinical use of therapeutic peptides. From the analytical point of view, it discusses the challenges associated with the analysis of therapeutic peptides in pharmaceutical and biological matrices, as well as the evaluation of CE as a whole and the comparison with LC methods. The article also highlights the use of microchip electrophoresis, nonaqueous CE, and nonconventional hydrodynamically closed CE systems and their applications. Overall, the article emphasizes the importance of developing new CE-based analytical methods to ensure the high quality, safety, and efficacy of therapeutic peptides in clinical practice.

A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples.

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis - tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis - mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

Determination of short-chain fatty acids as putative biomarkers of cancer diseases by modern analytical strategies and tools: a review.

Short-chain fatty acids (SCFAs) are the main metabolites produced by bacterial fermentation of non-digestible carbohydrates in the gastrointestinal tract. They can be seen as the major flow of carbon from the diet, through the microbiome to the host. SCFAs have been reported as important molecules responsible for the regulation of intestinal homeostasis. Moreover, these molecules have a significant impact on the immune system and are able to affect inflammation, cardiovascular diseases, diabetes type II, or oncological diseases. For this purpose, SCFAs could be used as putative biomarkers of various diseases, including cancer. A potential diagnostic value may be offered by analyzing SCFAs with the use of advanced analytical approaches such as gas chromatography (GC), liquid chromatography (LC), or capillary electrophoresis (CE) coupled with mass spectrometry (MS). The presented review summarizes the importance of analyzing SCFAs from clinical and analytical perspective. Current advances in the analysis of SCFAs focused on sample pretreatment, separation strategy, and detection methods are highlighted. Additionally, it also shows potential areas for the development of future diagnostic tools in oncology and other varieties of diseases based on targeted metabolite profiling.

An Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Determination of 4 ß-Lactam Antibiotics, Tazobactam, and Linezolid in Human Plasma Samples.

BACKGROUND: Optimization of antimicrobial therapy is a challenge in critically ill patients who develop extreme interindividual and intraindividual pharmacokinetic variability. Therapeutic drug monitoring is a valuable tool for maximizing the effect of a drug and minimizing its adverse and unwanted effects. The aim of the current work was to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine multiple antibiotics in clinical plasma samples from critically ill patients; low sample volume and rapid processing of samples were considered the main criteria. METHODS: A separation method based on an online combination of UHPLC-MS/MS was developed for the simultaneous determination of 4 ß-lactam antibiotics (cefepime, meropenem, cefotaxime, and piperacillin), tazobactam, and linezolid in human plasma samples. The volume of plasma sample used for analysis was 20 µL. The developed method was validated according to Food and Drug Administration guidelines. RESULTS: The chromatographic run time was 8 minutes. Calibration curves were linear for concentration ranges of 0.1-100 mcg/mL (r 2 > 0.99) for tazobactam, meropenem, cefotaxime, linezolid, and piperacillin and 1-100 mcg/mL (r 2 > 0.99) for cefepime. The intraday and interday accuracy of the method ranged from 92.4% to 110.7% and 93.6% to 113.3%, respectively. The intraday and interday precision values were ≤17.3% and ≤17.4%, respectively. No interfering and carryover analytes were observed. CONCLUSIONS: The developed UHPLC-MS/MS method is an appropriate and practical tool for therapeutic drug monitoring of the selected antibiotics. Owing to its rapidity, requirement of low sample volume, and high selectivity, sensitivity, and reliability, it can be effectively implemented in routine clinical laboratory tests for critically ill patients.

Analog of kynurenic acid decreases tau pathology by modulating astrogliosis in rat model for tauopathy.

Kynurenines have immunomodulatory and neuroactive properties and can influence the central nervous system. Previous studies showed the involvement of the kynurenines in the pathogenesis and progression of neurodegenerative disease. In neurodegenerative disorders, including tauopathies, the tryptophan metabolism is shifted toward neurotoxic agents and the reduction of neuroprotectant products. Astrocyte-derived kynurenic acid serves as a neuroprotectant. However, systemic administration of kynurenic acid is not effective because of low permeability across the blood-brain barrier (BBB). We used a kynurenic acid analog with similar biological activity but higher brain permeability to overcome BBB limitations. In the present study, we used amide derivate of kynurenic acid N-(2-N, N-dimethylaminoethyl)- 4-oxo-1 H-quinoline-2-carboxamid (KYNA-1). We administered KYNA-1 for three months to tau transgenic rats SHR-24 and analyzed the effect on tau pathology and activation of glial cells. Primary glial cell cultures were applied to identify the mechanism of the KYNA-1 effect. KYNA-1 was not toxic to rats after chronic three-month administration. When chronically administered, KYNA-1 reduced hyperphosphorylation of insoluble tau in the brain of transgenic rats. Noteworthily, the plasma total tau was also reduced. We determined that the effect of KYNA-1 on tau pathology was induced through the modulation of glial activation. KYNA-1 inhibited LPS induced activation of astrocytes and induced transformation of microglia to M2 phenotype. We identified that the administration of KYNA-1 reduced tau hyperphosphorylation and neuroinflammation. KYNA-1 may serve as a promising treatment for tauopathies.

Capillary zone electrophoresis in combination with UV detection for simultaneous determination of tramadol and paracetamol in pharmaceutical and biological samples.

The aim of the present study is the development and validation of a simple method based on capillary zone electrophoresis coupled with UV detection for simultaneous determination of tramadol and paracetamol in pharmaceutical and biological samples. The background electrolyte was composed of 50 mM ammonium carbonate, which is a type of a non-conventional electrolyte system. The developed method was characterized by suitable validation parameters, such as linearity (coefficient of determination r2 0,995), selectivity or the limit of detection at the level of 0.25 - 0.5 μg/ml. Acceptable values of accuracy and precision were obtained, which were in good agreement with the recommended validation guidelines for analysis of pharmaceutical and biological samples. Detection was performed at a wavelength of 200 nm. The developed method was successfully applied to determine tramadol and paracetamol in various dosage forms and in urine biological samples. Achieved results indicate a potential of the method to be integrated in the common quality control processes of drugs and/or in bioanalysis.

Interaction kinetics reveal distinct properties of conformational ensembles of three-repeat and four-repeat tau proteins.

Tau protein is an intrinsically disordered protein. Its physiological state is best described as a conformational ensemble (CE) of metastable structures interconverting on the local and molecular scale. The monoclonal antibody DC39C recognizes a linear C-terminal tau epitope, and as the tau interaction partner, its binding parameters report about tau CE. Association kinetics of DC39C binding, together with crosslinking mass spectrometry, show differences in the accessibility of the C terminus in CEs of tau isoforms. Furthermore, removal of the C terminus accelerated the aggregation kinetics of three-repeat tau proteins. Our results suggest a novel mechanism of splicing-driven regulation of the tau C-terminal domain with consequences on the specific roles of tau isoforms in microtubule assembly and pathological aggregation.

Amino acids in inflammatory bowel diseases: Modern diagnostic tools and methodologies.

Amino acids are crucial building blocks of living organisms. Together with their derivatives, they participate in many intracellular processes to act as hormones, neuromodulators, and neurotransmitters. For several decades amino acids have been studied for their potential as markers of various diseases, including inflammatory bowel diseases. Subsequent improvements in sample pretreatment, separation, and detection methods have enabled the specific and very sensitive determination of these molecules in multicomponent matrices-biological fluids and tissues. The information obtained from targeted amino acid analysis (biomarker-based analytical strategy) can be further used for early diagnostics, to monitor the course of the disease or compliance of the patients. This review will provide an insight into current knowledge about inflammatory bowel diseases, the role of proteinogenic amino acids in intestinal inflammation and modern analytical techniques used in its diagnosis and disease activity monitoring. Current advances in the analysis of amino acids focused on sample pretreatment, separation strategy, or detection methods are highlighted, and their potential in clinical laboratories is discussed. In addition, the latest clinical data obtained from the metabolomic profiling of patients suffering from inflammatory bowel diseases are summarized with a focus on proteinogenic amino acids.

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern.

BACKGROUND: The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. METHODS: We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. FINDINGS: AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. INTERPRETATION: The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. FUNDING: The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

Comparison of 1D a 2D ITP-MS performance parameters and application possibilities: Ultratrace determination of B vitamins in human urine.

The possibility to investigate analytes at ultra-low concentration levels still remains a hot topic in bioanalysis. In this area, various preconcentration techniques are an integral part of analytical procedures. When applying electromigration separation techniques, an isotachophoresis has been advantageously employed many times for this purpose. To solve current biomedical tasks effectively, an advanced two-dimensional isotachophoretic instrument (in a hydrodynamically closed separation system with an enhanced sample load capacity) hyphenated with mass spectrometry (ITP-ITP-MS) has been proposed by Foret and coworkers. As a continuation, this work represents the first study dealing with a full validation of an ITP-ITP-MS method. In order to see the benefits of an online ITP sample pretreatment (preconcentration and clean-up) on the performance parameters, the developed 2D ITP-MS method was compared with a corresponding 1D ITP-MS method. Application potentialities of the compared methods were demonstrated via a determination of two B vitamins, namely thiamine and pyridoxine, in human urine samples. The developed 2D ITP-MS method showed its enhanced effectivity and usefulness for a routine biomedical use (here, a reliable screening of trace B vitamins in human urine without an offline sample preparation).

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy.

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized-GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.