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The reuse of the same chromatography resin to purify multiple products is of high economic and ecologic interest in the pharmaceutical industry. An effective cleaning program to ensure no product carryover from one product to the subsequent is key to enable a multi-product use strategy. The methods utilised to assess product carryover during resin lifetime studies for a given product are not specific enough to detect carryover to support multi-product use. Here the proposed use of liquid chromatography coupled to mass spectrometry (LC-MS) is the analytical method of choice due to its high sensitivity and selectivity. A proof-of-concept study using protein A resin and two distinct IgG product streams produced by CHO cell lines was conducted. The experimental design was developed to determine any carryover from previous products after a multiple product Protein A resin reuse process, using LC-MS and capillary electrophoresis (CE). The capability of LC-MS coupled with bioinformatic analysis to detect carryover where CE with UV detection was not able to detect any carryover was demonstrated. The findings from this approach suggest that the acceptance of chromatographic resin reuse for multiple products will be dependent on the development of analytical/bioinformatics tools such as those proposed in this work. [Display omitted] • A multiple product Protein A resin reuse process was designed. • Capillary electrophoresis with UV detection was not able to detect any carryover. • LC-MS coupled with bioinformatic analysis did detect carryover. • These data provide leverage for chromatographic resin reuse for biopharmaceuticals. [ABSTRACT FROM AUTHOR]

Background: Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal (GI) emergency in preterm neonates. Untargeted metabolomics may allow the identification of biomarkers involved in NEC pathophysiology. Methods: We conducted a prospective study including preterm infants born at < 34 gestational weeks (GWs) whose urine was longitudinally collected at birth (< 48 h, T0) and at 14 (T1) and 28 days (T2). Neonates were followed for their development of NEC, spontaneous intestinal perforation (SIP), or other GI conditions and compared to those of matched healthy controls. Urine samples were investigated by untargeted metabolomic analysis based on mass-spectrometry. Results: Thirty-five patients with NEC, 5 patients with SIP, 14 patients with other GI diseases and 113 controls were enrolled and selected for metabolomic analysis on the basis of their clinical characteristics and available samples. Considering urine samples at T0, the one-class classification approach was able to correctly classify 16/20 subjects (80%) who developed NEC, 3/3 (100%) who developed SIP and 5/7 subjects (71.4%) with other GI pathologies as not belonging to the control group. Neonates with surgical NEC had higher N-acetylaspartic acid, butyrylcarnitine and propionylcarnitine levels than did those with medical NEC. Considering the time evolution of the urinary metabolome, the NEC and control groups showed differences independently of the time point. Conclusions: The urinary metabolome is closely associated with the underlying GI disease from birth. Urinary metabolic features characterize NEC patients from healthy controls until 28 days of life. The early urinary metabolome has the potential to predict surgical NEC. Future studies are needed to validate our results. [ABSTRACT FROM AUTHOR]

Introduction: The release of pro-inflammatory cytokines in critically ill patients with sepsis leads to endothelial dysfunction resulting in cardiocirculatory insufficiency. Their extracorporeal elimination using the cytokine adsorber CytoSorb® (CS) (adsorption of especially hydrophobic molecules < 60 kDa) might be promising, but data about the adsorption capacity as well as a potential harmful adsorption of anti-inflammatory cytokines are missing so far. Methods: The prospective Cyto-SOLVE-study included 15 patients with sepsis or other hyperinflammatory conditions (interleukin 6 > 500 pg/ml), continuous kidney replacement therapy, and the application of CS. Various cytokines and chemokines were measured pre- and post-CS as well as in patients' blood at predefined timepoints. Significant changes in the concentrations were detected with the Wilcoxon test with associated samples. Clearance of the adsorber (ml/min) was calculated with: b l o o d f l o w ∗ c o n c e n t r a t i o n p r e - p o s t c o n c e n t r a t i o n pre . Results: Most of the inflammatory mediators showed a high initial extracorporeal clearance of 70–100 ml/min after CS installation, which dropped quickly to 10-30 ml/min after 6 h of treatment. No difference in clearance was observed between pro- and anti-inflammatory cytokines. Despite extracorporeal adsorption, a significant (p < 0.05) decrease in the blood concentration after 6 h was only observed for the pro-inflammatory cytokines tumor necrosis factorα (TNF-α) (median 284 vs. 230 pg/ml), vascular endothelial growth factor (VEGF) (median 294 vs. 252 pg/ml), macrophage inflammatory protein 1a (MIP-1a) (median 11.1 vs. 9.0 pg/ml), and regulated upon activation, normal T cell expressed and secreted (RANTES) (median 811 vs. 487 pg/ml) as well as the anti-inflammatory cytokines interleukin 4 (median 9.3 vs. 6.4 pg/ml), interleukin 10 (median 88 vs. 56 pg/ml), and platelet-derived growth factor (PDGF) (median 177 vs. 104 pg/ml). A significant (p < 0.05) decrease in patients' blood after 12 h was only detected for interleukin 10. Conclusions: CS can adsorb pro- as well as anti-inflammatory mediators with no relevant difference regarding the adsorption rate. A fast saturation of the adsorber resulted in a rapid decrease of the clearance. The potential clinical benefit or harm of this unspecific cytokine adsorption needs to be evaluated in the future. Trial Registration: ClinicalTrials.gov NCT04913298, registration date June 4, 2021. [ABSTRACT FROM AUTHOR]

Background: Data suggest that metabolites, other than blood phenylalanine (Phe), better and independently predict clinical outcomes in patients with phenylketonuria (PKU). Methods: To find new biomarkers, we compared the results of untargeted lipidomics and metabolomics in treated adult PKU patients to those of matched controls. Samples (lipidomics in EDTA-plasma (22 PKU and 22 controls) and metabolomics in serum (35 PKU and 20 controls)) were analyzed using ultra-high-performance liquid chromatography and high-resolution mass spectrometry. Data were subjected to multivariate (PCA, OPLS-DA) and univariate (Mann–Whitney U test, p < 0.05) analyses. Results: Levels of 33 (of 20,443) lipid features and 56 (of 5885) metabolite features differed statistically between PKU patients and controls. For lipidomics, findings include higher glycerolipids, glycerophospholipids, and sphingolipids species. Significantly lower values were found for sterols and glycerophospholipids species. Seven features had unknown identities. Total triglyceride content was higher. Higher Phe and Phe catabolites, tryptophan derivatives, pantothenic acid, and dipeptides were observed for metabolomics. Ornithine levels were lower. Twenty-six metabolite features were not annotated. Conclusions: This study provides insight into the metabolic phenotype of PKU patients. Additional studies are required to establish whether the observed changes result from PKU itself, diet, and/or an unknown reason. [ABSTRACT FROM AUTHOR]

Abstract Background Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal (GI) emergency in preterm neonates. Untargeted metabolomics may allow the identification of biomarkers involved in NEC pathophysiology. Methods We conducted a prospective study including preterm infants born at

The majority of patients with resected stage II‐IIIA non‐small cell lung cancer (NSCLC) are treated with platinum‐based adjuvant chemotherapy (ACT) in a one‐size‐fits‐all approach. However, a significant number of patients do not derive clinical benefit, and no predictive patient selection biomarker is currently available. Using mass spectrometry‐based proteomics, we have profiled tumour resection material of 2 independent, multi‐centre cohorts of in total 67 patients with NSCLC who underwent ACT. Unsupervised cluster analysis of both cohorts revealed a poor response/survival sub‐cluster composed of ~ 25% of the patients, that displayed a strong epithelial‐mesenchymal transition signature and stromal phenotype. Beyond this stromal sub‐population, we identified and validated platinum response prediction biomarker candidates involved in pathways relevant to the mechanism of action of platinum drugs, such as DNA damage repair, as well as less anticipated processes such as those related to the regulation of actin cytoskeleton. Integration with pre‐clinical proteomics data supported a role for several of these candidate proteins in platinum response prediction. Validation of one of the candidates (HMGB1) in a third independent patient cohort using immunohistochemistry highlights the potential of translating these proteomics results to clinical practice.

The aim of this work was the investigation of novel organocatalysts for the Michael addition of diethyl malonate to β-nitrostyrene. The methodology of the study included NMR titration, reaction monitoring by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS), product characterization by MALDI, IR spectroscopy, scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), and elemental analysis. As a result, evidence of supramolecular interactions between two pairs of components of the reaction was found. In addition to the supramolecular complexes, an unusual reaction, i.e., the Michael addition of NH-bispidines to β-nitrostyrene, was found, which led to previously unknown oligomers of β-nitrostyrene. A new mechanism for the catalytic action of NH-bispidine was proposed, which involved catalysis not by the initial organocatalyst but rather by its adduct with β-nitrostyrene. Thus, in this reaction, N-benzylbispidine acted as an initiator, and the real catalyst was the betaine formed during the initiation stage. [ABSTRACT FROM AUTHOR]

The majority of patients with resected stage II‐IIIA non‐small cell lung cancer (NSCLC) are treated with platinum‐based adjuvant chemotherapy (ACT) in a one‐size‐fits‐all approach. However, a significant number of patients do not derive clinical benefit, and no predictive patient selection biomarker is currently available. Using mass spectrometry‐based proteomics, we have profiled tumour resection material of 2 independent, multi‐centre cohorts of in total 67 patients with NSCLC who underwent ACT. Unsupervised cluster analysis of both cohorts revealed a poor response/survival sub‐cluster composed of ~ 25% of the patients, that displayed a strong epithelial‐mesenchymal transition signature and stromal phenotype. Beyond this stromal sub‐population, we identified and validated platinum response prediction biomarker candidates involved in pathways relevant to the mechanism of action of platinum drugs, such as DNA damage repair, as well as less anticipated processes such as those related to the regulation of actin cytoskeleton. Integration with pre‐clinical proteomics data supported a role for several of these candidate proteins in platinum response prediction. Validation of one of the candidates (HMGB1) in a third independent patient cohort using immunohistochemistry highlights the potential of translating these proteomics results to clinical practice. [ABSTRACT FROM AUTHOR]

Introduction: Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. Methods: In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting highabundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Results and discussion: Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies. [ABSTRACT FROM AUTHOR]

Background: Lipids play key roles in numerous biological processes, including energy storage, cell membrane structure, signaling, immune responses, and homeostasis, making lipidomics a vital branch of metabolomics that analyzes and characterizes a wide range of lipid classes. Addressing the complex etiology, age-related risk, progression, inflammation, and research overlap in conditions like Alzheimer's Disease, Parkinson's Disease, Cardiovascular Diseases, and Cancer poses significant challenges in the quest for effective therapeutic targets, improved diagnostic markers, and advanced treatments. Mass spectrometry is an indispensable tool in clinical lipidomics, delivering quantitative and structural lipid data, and its integration with technologies like Liquid Chromatography (LC), Magnetic Resonance Imaging (MRI), and few emerging Matrix-Assisted Laser Desorption Ionization- Imaging Mass Spectrometry (MALDI-IMS) along with its incorporation into Tissue Microarray (TMA) represents current advances. These innovations enhance lipidomics assessment, bolster accuracy, and offer insights into lipid subcellular localization, dynamics, and functional roles in disease contexts. Aim of the review: The review article summarizes recent advancements in lipidomic methodologies from 2019 to 2023 for diagnosing major neurodegenerative diseases, Alzheimer's and Parkinson's, serious non-communicable cardiovascular diseases and cancer, emphasizing the role of lipid level variations, and highlighting the potential of lipidomics data integration with genomics and proteomics to improve disease understanding and innovative prognostic, diagnostic and therapeutic strategies. Key scientific concepts of review: Clinical lipidomic studies are a promising approach to track and analyze lipid profiles, revealing their crucial roles in various diseases. This lipid-focused research provides insights into disease mechanisms, biomarker identification, and potential therapeutic targets, advancing our understanding and management of conditions such as Alzheimer's Disease, Parkinson's Disease, Cardiovascular Diseases, and specific cancers. Lipidome analysis methodology in major diseases and discovery of therapeutics and biomarkers [ABSTRACT FROM AUTHOR]

For the first time, the distribution of rare earth elements (REE) has been studied in detail for a number of coal facilities (30 deposits, 650 samples of coal and 210 samples of carbonaceous rocks). The ubiquitous presence of elevated concentrations of REE in coals has been noted. The REE mineral cluster in coals includes the association: ash content of coals – SiO2 – K2O – Al2o3 – TiO2 – Sc – Y – Dy – Ho – Er – Tm – Yb – Lu, and the association La – Ce – Pr – Nd – Sm – Eu – Gd – Tb. The presence of these elements of the mineral part of the coals is preferably in the composition of phosphate minerals – monazite and apatite (according to electron microscopy with microanalysis, the correlation of REE with P2o5). The content of individual REE in humic acids isolated from coals and fractions of coals of different densities has been studied. The specific role of organic matter(s) in the concentration of REE, their presence in the humus component of S and in low-ash coals is shown. Selective accumulation (fractionation) of heavy REE by organic matter has been experimentally established for the first time. Two genetic types of REE mineralization have been identified in coals: mainly terrigenous (hydrogenic) and tufogenic. The increased concentrations of REE in coals are due to the influence of the petrofund. The deposits were ranked according to the degree of prospects for REE based on an assessment of the resource potential of associated REE in the coals of the studied brown coal deposits. REE raw materials (lanthanides in coal ash) differ significantly from traditional types of rare earths ores by an incomparably large relative amount of heavy REE (on average 3–4 times), sometimes reaching 46% of the total REE content. Thus, coal ash is a unique non–traditional source of heavy lanthanides – more rare, valuable and expensive. The coals of the studied deposits should be considered as associated raw materials for rare earths.

Quantum chemistry calculations were performed to elucidate the reaction sites and degradation routes of dexamethasone. Flow Injection Analysis Electrospray Fourier‐Transform Ion Cyclotron Resonance Mass Spectrometry (FIA‐ESI‐FTCIR‐MS) was used to identify dexamethasone and its byproducts in Mexico City's wastewater, guided by results from ab‐initio calculations on the bond energy analysis and thus propose possible degradation routes. Products of hydroxylation/reduction, decarbonylation/formylation and defluorination reactions were identified. The results of this study contribute to the identification of the degradation products of drugs commonly used in the treatment of COVID‐19 in urban areas. Through wastewater analysis and computationally assisted Mass‐Spectrometry techniques, an alternative approach is presented to identify byproducts in complex matrices when analytical standards are unavailable or in case of limited understanding on the stability of the degradation byproducts in aqueous medium. [ABSTRACT FROM AUTHOR]

Friction and wear of polyoxymethylene upon friction against polyoxymethylene and polyetheretherketone have been analyzed. It is shown that wear exponentially increases upon growth of contact pressure and sliding velocity in the homogeneous friction pair polyoxymethylene–polyoxymethylene. In the heterogeneous pair polyetheretherketone–polyoxymethylene the wear of POM is small and exhibits no registered dependence on sliding velocity and contact pressure. Mass-spectrometric analysis shows that the macromolecular decomposition products are not detected for polyetheretherketone–polyoxymethylene pair. However, for thermodynamically compatible polyoxymethylene–polyoxymethylene pair their formation, accompanied by the growth of friction force, is detected already at the levels of friction power as low as 10–2 MPa m/s. These differences have been interpreted to result from interpenetration of polyoxymethylene macromolecules across the interface and their rupture in the shear field in homogeneous polyoxymethylene–polyoxymethylene friction pair and its absence in polyetheretherketone–polyoxymethylene pair. Thermally activated interpenetration of macromolecules for polyoxymethylene–polyoxymethylene pair and its absence in polyetheretherketone–polyoxymethylene pair has been visualized by means of molecular dynamics simulations. The experimental approach and the results of its application will be useful in detailed studies of molecular level friction mechanisms of friction and wear of industrially used polymers and their composites. [ABSTRACT FROM AUTHOR]

In a balanced diet, regular fish consumption provides positive outcomes for human health. On the other hand, the seafood supply chain faces a significant food safety risk due to the presence of potentially toxic elements (PTEs). In the present study, to assess the risk for Italian consumers, the concentrations of five PTEs, namely lead, chromium, cadmium, mercury, nickel, and aluminum, were determined in the three most consumed preserved fish in Italy: tuna (Thunnus albacares, Katsuwonus pelamis), mackerel (Scomber scombrus) and anchovy (Engraulis encrasicolus). Samples were collected from the national market, and the instrumental analysis was performed by inductively coupled plasma mass spectrometry (ICP-MS). The analyzed PTEs were found in all the species that were investigated. However, after considering the target hazard quotient (THQ) and the hazard index (HI), it was observed that the three fish preserves did not pose any risk of chronic toxicity for the average consumer, even at the highest concentrations detected. However, for significant consumers, mercury detected in tuna samples represented almost 90% of the tolerable weekly intake (TWI) reported by the European Food Safety Authority (EFSA), representing a matter of concern for consumers, particularly regarding developmental neurotoxicity, whose HI exceeded 111%. The acute toxicity of nickel was also considered for significant consumers at the highest concentration detected, and the margin of exposure (MOE) calculated was above 7000, much higher than the value of 30 indicated by EFSA. Due to the lack of data on non-professional carcinogenicity or human intake through foods with low cancer risk, this toxicity was not considered in the analysis of PTEs. [ABSTRACT FROM AUTHOR]

In the context of the Collaborative Materials Exercise CMX-7 organized by the Nuclear Forensics International Technical Working Group (ITWG), four sample of uranium materials were analysed by IFIN-HH, Romania. The presented work is focused on presenting the analytical process of adapting a SQ ICP-MS such that to be used for determining fingerprints of the nuclear materials of interest. The "in-house" protocol developed made possible the determination of the uranium isotopic ratio of the samples without the use of chemical separation and also to determine the concentration of the trace elements. The age determination calculated based on the 230Th/234U chronometer. [ABSTRACT FROM AUTHOR]

Due to the segmented functions and complexity of the human brain, the characterization of molecular profiles within specific areas such as brain structures and biofluids is essential to unveil the molecular basis for structure specialization as well as the molecular imbalance associated with neurodegenerative and psychiatric diseases. Much of our knowledge about brain functionality derives from neurophysiological, anatomical, and transcriptomic approaches. More recently, laser capture and imaging proteomics, technological and computational developments in LC-MS/MS, as well as antibody/aptamer-based platforms have allowed the generation of novel cellular, spatial, and posttranslational dimensions as well as innovative facets in biomarker validation and druggable target identification. Proteomics is a powerful toolbox to functionally characterize, quantify, and localize the extensive protein catalog of the human brain across physiological and pathological states. Brain function depends on multi-dimensional protein homeostasis, and its elucidation will help us to characterize biological pathways that are essential to properly maintain cognitive functions. In addition, comprehensive human brain pathological proteomes may be the basis in computational drug-repositioning methods as a strategy for unveiling potential new therapies in neurodegenerative and psychiatric disorders. [ABSTRACT FROM AUTHOR]

The analysis of the ultraviolet induced transformation of the thermal decomposition spectra of the monomer of the submicrometer layers of polymethylmethacrylate and its composite with fullerene C60 for different fullerene concentrations and irradiation doses has been carried out. Formation of the new decomposition stages in the spectra has been interpreted by the binding between C60 and side ester groups of polymethylmethacrylate. [ABSTRACT FROM AUTHOR]

The formation of decomposition products of macromolecules was found upon friction of polyoxymethylene against polyoxymethylene in the vacuum of mass-spectrometer. Their composition is shown to be different from that of the products of thermal decomposition of polyoxymethylene. It is shown that the formation of decomposition products is accompanied by the transition of the friction force from low to high level. The effect has been interpreted by the mechanical rupture of macromolecules upon friction. Quantitative parameters of the process have been discussed. [ABSTRACT FROM AUTHOR]

The misfolding of the mammalian prion protein from its α‐helix rich cellular isoform to its β‐sheet rich infectious isoform is associated with several neurodegenerative diseases. The determination of the structural mechanism by which misfolding commences, still remains an unsolved problem. In the current study, native‐state hydrogen exchange coupled with mass spectrometry has revealed that the N state of the mouse prion protein (moPrP) at pH 4 is in dynamic equilibrium with multiple partially unfolded forms (PUFs) capable of initiating misfolding. Mutation of three evolutionarily conserved aromatic residues, Tyr168, Phe174, and Tyr217 present at the interface of the β2‐α2 loop and the C‐terminal end of α3 in the structured C‐terminal domain of moPrP significantly destabilize the native state (N) of the protein. They also reduce the free energy differences between the N state and two PUFs identified as PUF1 and PUF2**. It is shown that PUF2** in which the β2‐α2 loop and the C‐terminal end of α3 are disordered, has the same stability as the previously identified PUF2*, but to have a very different structure. Misfolding can commence from both PUF1 and PUF2**, as it can from PUF2*. Hence, misfolding can commence and proceed in multiple ways from structurally distinct precursor conformations. The increased extents to which PUF1 and PUF2** are populated at equilibrium in the case of the mutant variants, greatly accelerate their misfolding. The results suggest that the three aromatic residues may have been evolutionarily selected to impede the misfolding of moPrP. [ABSTRACT FROM AUTHOR]

IntroductionBloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine.MethodsIn this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose.Results and discussionAmong all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

Abstract Background Chromatographic peakpicking continues to represent a significant bottleneck in automated LC–MS workflows. Uncontrolled false discovery rates and the lack of manually-calibrated quality metrics require researchers to visually evaluate individual peaks, requiring large amounts of time and breaking replicability. This problem is exacerbated in noisy environmental datasets and for novel separation methods such as hydrophilic interaction columns in metabolomics, creating a demand for a simple, intuitive, and robust metric of peak quality. Results Here, we manually labeled four HILIC oceanographic particulate metabolite datasets to assess the performance of individual peak quality metrics. We used these datasets to construct a predictive model calibrated to the likelihood that visual inspection by an MS expert would include a given mass feature in the downstream analysis. We implemented two novel peak quality metrics, a custom signal-to-noise metric and a test of similarity to a bell curve, both calculated from the raw data in the extracted ion chromatogram, and found that these outperformed existing measurements of peak quality. A simple logistic regression model built on two metrics reduced the fraction of false positives in the analysis from 70–80% down to 1–5% and showed minimal overfitting when applied to novel datasets. We then explored the implications of this quality thresholding on the conclusions obtained by the downstream analysis and found that while only 10% of the variance in the dataset could be explained by depth in the default output from the peakpicker, approximately 40% of the variance was explained when restricted to high-quality peaks alone. Conclusions We conclude that the poor performance of peakpicking algorithms significantly reduces the power of both univariate and multivariate statistical analyses to detect environmental differences. We demonstrate that simple models built on intuitive metrics and derived from the raw data are more robust and can outperform more complex models when applied to new data. Finally, we show that in properly curated datasets, depth is a major driver of variability in the marine microbial metabolome and identify several interesting metabolite trends for future investigation.

Background: Data suggest that metabolites, other than blood phenylalanine (Phe), better and independently predict clinical outcomes in patients with phenylketonuria (PKU). Methods: To find new biomarkers, we compared the results of untargeted lipidomics and metabolomics in treated adult PKU patients to those of matched controls. Samples (lipidomics in EDTA-plasma (22 PKU and 22 controls) and metabolomics in serum (35 PKU and 20 controls)) were analyzed using ultra-high-performance liquid chromatography and high-resolution mass spectrometry. Data were subjected to multivariate (PCA, OPLS-DA) and univariate (Mann–Whitney U test, p < 0.05) analyses. Results: Levels of 33 (of 20,443) lipid features and 56 (of 5885) metabolite features differed statistically between PKU patients and controls. For lipidomics, findings include higher glycerolipids, glycerophospholipids, and sphingolipids species. Significantly lower values were found for sterols and glycerophospholipids species. Seven features had unknown identities. Total triglyceride content was higher. Higher Phe and Phe catabolites, tryptophan derivatives, pantothenic acid, and dipeptides were observed for metabolomics. Ornithine levels were lower. Twenty-six metabolite features were not annotated. Conclusions: This study provides insight into the metabolic phenotype of PKU patients. Additional studies are required to establish whether the observed changes result from PKU itself, diet, and/or an unknown reason.

The experimental study of the pharmacokinetic characteristics of a new pyrrole derivative of carnosine, pyrrolylcarnosine, was carried out. Based on the results of the experiment, the basic pharmacokinetic parameters of the drug are expected. The tissues and organs bioavailability was studied. The tropism of pyrrolylcarnosine to the organs of elimination and the ability of pyrrolylcarnosine to penetrate into the heart muscle tissue were shown.

Trialkylphosphates are of considerable interest from the military-chemical point of view as precursors of highly toxic organophosphorus toxicants, possible products of their decontamination, complexing agents in processes of reprocessing of used nuclear fuel, etc. Meanwhile, the development of ways to identify these substances is still in its infancy. In addition, trialkylphosphates are an interesting subject for the search of a solution to the analytical problem of establishing the structure of O-alkyl fragments in molecules of M(O) (OR)n esters of multibasic acids, where M is the central atom (central core), n is the basicity of the acid, R are alkyl radicals with an unknown and in general case different number of carbon atoms in each of them. No general solution to this problem could be found in the book sources. The aim of this work is to develop an algorithm for in-depth identification of trialkyl phosphates, which includes establishing the belonging of the investigated substance to this class, as well as a general and reliable method for determining the number of carbon atoms in each of the O-alkyl radicals of the investigated substances. On the basis of the revealed regularities of the decay of positive molecular ions of trialkyl phosphates, as well as negative ions of resonant capture of electrons of the same compound obtained under conditions of electron ionization, an algorithm for in-depth identification of trialkyl phosphates is proposed, which includes establishing the belonging of the investigated substance to this class, as well as a general and reliable method determining the number of carbon atoms in each of the O-alkyl radicals of the substance molecule.

Results of tests of six Actinobacillus pleuropneumoniae isolates recovered from the diseased pigs kept in animal holdings located in the Russian Federation for their biological properties (biochemical, proteomic, antigenic and pathogenic ones) are presented in the paper. Proteomic properties were determined with mass-spectrometry using Autof MS 1000 mass-spectrometer (Autobio Diagnostics Co., Ltd, China): protein profiles were plotted and the peaks characteristic for each isolate were identified. Mass-spectra of tested Actinobacillus isolates and reference Actinobacillus pleuropneumoniae DSM 13472 strain were found to be in the m/z range of 2,000–12,000 Da. The following peaks (m/z) were common for all Actinobacillus pleuropneumoniae isolates and the strain: 2,541 ± 2; 4,267 ± 2; 5,085 ± 2; 6,450 ± 2; 7,207 ± 4; 9,408 ± 3; 11,820 ± 6. Therewith, the highest intensity (100%) was reported for the peak at 5,085 ± 2, that was supposed to be a specific feature of Actinobacillus pleuropneumoniae. All isolates were confirmed to belong to Actinobacillus pleuropneumoniae species and to 2, 5 and 9 serotypes by real-time polymerase chain reaction using species-specific and serotype-specific primers. Actinobacillus рleuropneumoniae isolates were tested for their pathogenic properties by experimental infection of white mice and 2.5–3 month-old piglets. All tested isolates were pathogenic for both white mice and piglets. Isolate No. 4 belonging to serotype 5 was found to be the most virulent out of tested isolates. Thus, LD50 was 4.19 lg microbial cells for white mice and 5.49 lg microbial cells for piglets that was consistent to the data of other authors carried out tests of actinobaccilli isolated in the Russian Federation for their pathogenicity. The isolates were deposited to the FGBI “ARRIAH” Collection of Microorganism Strains.

BackgroundCalcineurin inhibitors, including tacrolimus, remain a cornerstone of immunosuppressive therapy after kidney transplantation. However, the therapeutic window is narrow, and nephrotoxic side effects occur with overdose, while the risk of alloimmunization and graft rejection increases with underdose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows quantification of tacrolimus in biological samples from patients. This study investigates the feasibility of quantifying tacrolimus in scalp hair from kidney transplant (KT) recipients and correlates hair tacrolimus concentrations with tacrolimus dosage and blood trough levels. The aim was to provide proof-of-principle for hair tacrolimus drug monitoring in KT recipients.MethodSingle-center prospective study between September 9, 2021 and December 4, 2021, including KT recipients under tacrolimus. Minors, patients with active skin or hair diseases, and patients with scalp hair shorter than 4 cm were excluded from participation. Scalp hair was collected from the posterior vertex of patients, cut into segments, and analyzed for tacrolimus by LC-MS/MS. Patients filled out a questionnaire on hair treatments and washing habits. In parallel, tacrolimus trough levels were measured in whole blood and correlated with hair tacrolimus concentrations.ResultsIn total, 39 consenting KT recipients were included, and hair samples were collected at 53 visits. Tacrolimus was detected in 98% of hair samples from patients exposed to the drug. Tacrolimus hair levels and whole blood trough levels were correlated with a beta coefficient of 0.42 (95% CI: −0.22–1.1, p = n.s.). Age and dark hair affected hair tacrolimus measurements, while different tacrolimus formulations (immediate release vs. extended release), hair washes, and permanent coloring did not. Longitudinal measurements in a subgroup of patients indicate that long-term measurement of hair tacrolimus levels is feasible.ConclusionMeasuring tacrolimus in hair is a potentially reliable method to monitor drug exposure in KT patients. Rapid wash-in effects and consistent concentrations over time indicate that tacrolimus is incorporated into the hair matrix, allowing temporal resolution in the analysis of recent exposure and exposure history. This method provides a simple and low-risk alternative to regular blood sampling, sparing patients from frequent hospital visits through the self-collection of hair samples.

Background: Chromatographic peakpicking continues to represent a significant bottleneck in automated LC–MS workflows. Uncontrolled false discovery rates and the lack of manually-calibrated quality metrics require researchers to visually evaluate individual peaks, requiring large amounts of time and breaking replicability. This problem is exacerbated in noisy environmental datasets and for novel separation methods such as hydrophilic interaction columns in metabolomics, creating a demand for a simple, intuitive, and robust metric of peak quality. Results: Here, we manually labeled four HILIC oceanographic particulate metabolite datasets to assess the performance of individual peak quality metrics. We used these datasets to construct a predictive model calibrated to the likelihood that visual inspection by an MS expert would include a given mass feature in the downstream analysis. We implemented two novel peak quality metrics, a custom signal-to-noise metric and a test of similarity to a bell curve, both calculated from the raw data in the extracted ion chromatogram, and found that these outperformed existing measurements of peak quality. A simple logistic regression model built on two metrics reduced the fraction of false positives in the analysis from 70–80% down to 1–5% and showed minimal overfitting when applied to novel datasets. We then explored the implications of this quality thresholding on the conclusions obtained by the downstream analysis and found that while only 10% of the variance in the dataset could be explained by depth in the default output from the peakpicker, approximately 40% of the variance was explained when restricted to high-quality peaks alone. Conclusions: We conclude that the poor performance of peakpicking algorithms significantly reduces the power of both univariate and multivariate statistical analyses to detect environmental differences. We demonstrate that simple models built on intuitive metrics and derived from the raw data are more robust and can outperform more complex models when applied to new data. Finally, we show that in properly curated datasets, depth is a major driver of variability in the marine microbial metabolome and identify several interesting metabolite trends for future investigation. [ABSTRACT FROM AUTHOR]

The brain extracellular matrix (ECM) is a highly glycosylated environment and plays important roles in many processes including cell communication, growth factor binding, and scaffolding. The formation of structures such as perineuronal nets (PNNs) is critical in neuroprotection and neural plasticity, and the formation of molecular networks is dependent in part on glycans. The ECM is also implicated in the neuropathophysiology of disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Schizophrenia (SZ). As such, it is of interest to understand both the proteomic and glycomic makeup of healthy and diseased brain ECM. Further, there is a growing need for site‐specific glycoproteomic information. Over the past decade, sample preparation, mass spectrometry, and bioinformatic methods have been developed and refined to provide comprehensive information about the glycoproteome. Core ECM molecules including versican, hyaluronan and proteoglycan link proteins, and tenascin are dysregulated in AD, PD, and SZ. Glycomic changes such as differential sialylation, sulfation, and branching are also associated with neurodegeneration. A more thorough understanding of the ECM and its proteomic, glycomic, and glycoproteomic changes in brain diseases may provide pathways to new therapeutic options. [ABSTRACT FROM AUTHOR]

The spread of SARS-CoV-2 variants of concern (VOCs) is of great importance since genetic changes may increase transmissibility, disease severity and reduce vaccine effectiveness. Moreover, these changes may lead to failure of diagnostic measures. Therefore, variant-specific diagnostic methods are essential. To date, genetic sequencing is the gold-standard method to discriminate between variants. However, it is time-consuming (taking several days) and expensive. Therefore, the development of rapid diagnostic methods for SARS-CoV-2 in accordance with its genetic modification is of great importance. In this study we introduce a Mass Spectrometry (MS)-based methodology for the diagnosis of SARS-CoV-2 in propagated in cell-culture. This methodology enables the universal identification of SARS-CoV-2, as well as variant-specific discrimination. The universal identification of SARS-CoV-2 is based on conserved markers shared by all variants, while the identification of specific variants relies on variant-specific markers. Determining a specific set of peptides for a given variant consists of a multistep procedure, starting with an in-silico search for variant-specific tryptic peptides, followed by a tryptic digest of a cell-cultured SARS-CoV-2 variant, and identification of these markers by HR-LC-MS/MS analysis. As a proof of concept, this approach was demonstrated for four representative VOCs compared to the wild-type Wuhan reference strain. For each variant, at least two unique markers, derived mainly from the spike (S) and nucleocapsid (N) viral proteins, were identified. This methodology is specific, rapid, easy to perform and inexpensive. Therefore, it can be applied as a diagnostic tool for pathogenic variants. [ABSTRACT FROM AUTHOR]

Abstract Background The respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000–5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B. Methods We used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function. Results Proteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1–4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B. Conclusions These results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1–4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis.

Protein sequencing algorithms process data from a variety of instruments that has been generated under diverse experimental conditions. Currently there is no way to predict the accuracy of an algorithm for a given data set. Most of the published algorithms and associated software has been evaluated on limited number of experimental data sets. However, these performance evaluations do not cover the complete search space the algorithmand the software might encounter in real-world. To this end, we present a database of simulated spectra that can be used to benchmark any spectra to peptide search engine. We demonstrate the usability of this database by bench marking two popular peptide sequencing engines. We show wide variation in the accuracy of peptide deductions and a complete quality profile of a given algorithm can be useful for practitioners and algorithm developers. All benchmarking data is available at https://users.cs.fiu.edu/~fsaeed/Benchmark.html.

One of the human proteome puzzles is an imbalance between the theoretically calculated and experimentally measured amounts of proteoforms. Considering the possibility of combinations of different post-translational modifications (PTMs), the quantity of possible proteoforms is huge. An estimation gives more than a million different proteoforms in each cell type. But, it seems that there is strict control over the production and maintenance of PTMs. Although the potential complexity of proteoforms due to PTMs is tremendous, available information indicates that only a small part of it is being implemented. As a result, a protein could have many proteoforms according to the number of modification sites, but because of different systems of personal regulation, the profile of PTMs for a given protein in each organism is slightly different.

Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the Plasmodium falciparum-exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria-causing parasite, PfEMP1. We generated independent TurboID fusions of two proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein–protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

Daratumumab is an anti‐CD38 antibody that is increasingly incorporated in induction regimens for treating patients with newly diagnosed multiple myeloma (NDMM). Previous reports have demonstrated a lower yield of hematopoietic stem cells (HSCs) after induction with daratumumab; however, none of them reported a failure to collect an adequate number of HSCs. We describe a case of adequate HSC mobilization failure in a patient who inadvertently received excessive doses of daratumumab and was confirmed by higher‐than‐expected circulating levels of daratumumab by mass spectrometry. Eventual clearance of circulating daratumumab was associated with the successful mobilization and harvesting of HSCs. [ABSTRACT FROM AUTHOR]

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults. Tissue reorganization at the site of the epileptogenic focus is accompanied by changes in the expression patterns of protein molecules. The study of mRNA and its corresponding proteins is crucial for understanding the pathogenesis of the disease. Protein expression profiles do not always directly correlate with the levels of their transcripts; therefore, it is protein profiling that is no less important for understanding the molecular mechanisms and biological processes of TLE. The study and annotation of proteins that are statistically significantly different in patients with TLE is an approach to search for biomarkers of this disease, various stages of its development, as well as a method for searching for specific targets for the development of a further therapeutic strategy. When writing a systematic review, the following aggregators of scientific journals were used: MDPI, PubMed, ScienceDirect, Springer, and Web of Science. Scientific articles were searched using the following keywords: "proteomic", "mass-spectrometry", "protein expression", "temporal lobe epilepsy", and "biomarkers". Publications from 2003 to the present have been analyzed. Studies of brain tissues, experimental models of epilepsy, as well as biological fluids, were analyzed. For each of the groups, aberrantly expressed proteins found in various studies were isolated. Most of the studies omitted important characteristics of the studied patients, such as: duration of illness, type and response to therapy, gender, etc. Proteins that overlap across different tissue types and different studies have been highlighted: DPYSL, SYT1, STMN1, APOE, NME1, and others. The most common biological processes for them were the positive regulation of neurofibrillary tangle assembly, the regulation of amyloid fibril formation, lipoprotein catabolic process, the positive regulation of vesicle fusion, the positive regulation of oxidative stress-induced intrinsic apoptotic signaling pathway, removal of superoxide radicals, axon extension, and the regulation of actin filament depolymerization. MS-based proteomic profiling for a relevant study must accept a number of limitations, the most important of which is the need to compare different types of neurological and, in particular, epileptic disorders. Such a criterion could increase the specificity of the search work and, in the future, lead to the discovery of biomarkers for a particular disease. [ABSTRACT FROM AUTHOR]

Fucose is a signaling carbohydrate that is attached at the end of glycan processing. It is involved in a range of processes, such as the selectin‐dependent leukocyte adhesion or pathogen‐receptor interactions. Mass‐spectrometric techniques, which are commonly used to determine the structure of glycans, frequently show fucose‐containing chimeric fragments that obfuscate the analysis. The rearrangement leading to these fragments—often referred to as fucose migration—has been known for more than 25 years, but the chemical identity of the rearrangement product remains unclear. In this work, we combine ion‐mobility spectrometry, radical‐directed dissociation mass spectrometry, cryogenic IR spectroscopy of ions, and density‐functional theory calculations to deduce the product of the rearrangement in the model trisaccharides Lewis x and blood group H2. The structural search yields the fucose moiety attached to the galactose with an α(1→6) glycosidic bond as the most likely product. [ABSTRACT FROM AUTHOR]

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common inherited mitochondrial metabolic disease of fatty acid β-oxidation, especially in newborns. MCADD is clinically diagnosed using Newborn Bloodspot Screening (NBS) and genetic testing. Still, these methods have limitations, such as false negatives or positives in NBS and the variants of uncertain significance in genetic testing. Thus, complementary diagnostic approaches for MCADD are needed. Recently, untargeted metabolomics has been proposed as a diagnostic approach for inherited metabolic diseases (IMDs) due to its ability to detect a wide range of metabolic alterations. We performed an untargeted metabolic profiling of dried blood spots (DBS) from MCADD newborns (n = 14) and healthy controls (n = 14) to discover potential metabolic biomarkers/pathways associated with MCADD. Extracted metabolites from DBS samples were analyzed using UPLC-QToF-MS for untargeted metabolomics analyses. Multivariate and univariate analyses were used to analyze the metabolomics data, and pathway and biomarker analyses were also performed on the significantly identified endogenous metabolites. The MCADD newborns had 1034 significantly dysregulated metabolites compared to healthy newborns (moderated t-test, no correction, p-value ≤ 0.05, FC 1.5). A total of 23 endogenous metabolites were up-regulated, while 84 endogenous metabolites were down-regulated. Pathway analyses showed phenylalanine, tyrosine, and tryptophan biosynthesis as the most affected pathways. Potential metabolic biomarkers for MCADD were PGP (a21:0/PG/F1alpha) and glutathione, with an area under the curve (AUC) of 0.949 and 0.898, respectively. PGP (a21:0/PG/F1alpha) was the first oxidized lipid in the top 15 biomarker list affected by MCADD. Additionally, glutathione was chosen to indicate oxidative stress events that could happen during fatty acid oxidation defects. Our findings suggest that MCADD newborns may have oxidative stress events as signs of the disease. However, further validations of these biomarkers are needed in future studies to ensure their accuracy and reliability as complementary markers with established MCADD markers for clinical diagnosis. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with an average age-of-onset of ∼60 years and is usually fatal within 2–5 years of diagnosis. Mouse models based upon single gene mutations do not recapitulate all ALS pathological features. Environmental insults may also contribute to ALS, and β-N-methylamino-L-alanine (BMAA) is an environmental toxin linked with an increased risk of developing ALS. BMAA, along with cycasin, are hypothesized to be the cause of the Guam-ALS epicenter of the 1950s. We developed a multihit model based on low expression of a dominant familial ALS TDP-43 mutation (Q331K) and chronic low-dose BMAA exposure. Our two-hit mouse model displayed a motor phenotype absent from either lesion alone. By LC/MS analysis, free BMAA was confirmed at trace levels in brain, and were as high as 405 ng/mL (free) and 208 ng/mL (protein-bound) in liver. Elevated BMAA levels in liver were associated with dysregulation of the unfolded protein response (UPR) pathway. Our data represent initial steps towards an ALS mouse model resulting from combined genetic and environmental insult. [ABSTRACT FROM AUTHOR]

Introduction. Highly glycosylated proteins are the most abundant class of modern biopharmaceuticals. A majority of such therapeuticals produced by Russian biopharmaceutical companies is biosimilars. The foundation of biosimilar manufacturing is analytical assessment of structure equivalence to an original molecule. Fc-fusions present a challenge due to their structural properties. The only biosimilar of this kind registered in Russia is etanercept – a fusion of tumor necrosis factor receptor α and Fc-fragment of IgG1. Existing approaches widely used in protein analysis do not allow accurate and reliable description of glycoylation of these proteins. Development of new approaches and principles of such analysis is necessary, as the changes in biosimilar’s molecular structure can seriously affect its efficacy and safety.Aim. Development of double proteolysis approaches for glycopeptide mapping of Fc-fusion protein etanercept using Arg-C protease.Materials and methods. Etanercept was subjected to enzymatic hydrolysis using trypsin in combination with Arg-C or Asp-N. The resulting peptides were analyzed using HPLC-MS system Xevo G2-XS QTOF (Waters Corporation, USA). The conformation of glycan structure was performed via analysis of fragment spectra of glycopeptides, acquired with high collision energy mode (MS E ). UNIFI (version 1.8) with biopharmasuetical assessment setting (Waters Corporation, USA) was used to analyze the peptide maps.Results and discussion. It was found that using the combination of trypsin with protease Arg-C leads to reliable results Using the developed approach we successfully determined the majority of O-glycosylation sites and types of O-glycans. It was shown that for an effective O-glycopeptide maping N-deglycosylation stage is required. Most abundant N-glycan structures were identified for each of three N-glycosylation sites (N149, N171, N317). It was determined, that the combination of trypsin with Arg-C allows identification of three-antenna forms despite the presence of O-glycosylation site on the analyzed peptide. General N-glycosylation profile shows comparability of results for both approaches.Conclusion. As a result of this research we developed glycopeptide mapping approaches in which a combination of proteases is used. Using these methods sites of N- and O-glycosilation and glycofoms of etanercept were accurately and reproducibly determined. Developed procedures can be applied to other types of Fc-fusion proteins, making it of broader appeal and benefit to the overall biopharmaceutical industry. These approaches provide comprehensive information useful for structure-function studies and of potential value for product comparability measurements and possibly even future manufacturing control strategies.

The use of gas chromatography and mass spectrometry in the laboratory diagnostics of syphilis is described. The data of the indication of syphilitic lesions on the basis of identification of molecular markers are presented in this paper.

Cystic fibrosis is an important medical and social problem. Despite significant advances in modern science and practice, bacterial complications are still among the main causes of patient mortality. One of the crucial bacterial pathogens in cystic fibrosis is Pseudomonas aeruginosa, which can lead to rapidly decreased pulmonary function. At the same time, the lungs are not the only site for microbial colonization. The oral cavity can also serve as a reservoir for initial entry and adaptation of microbial strains, which, however, were only sparsely investigated. A clinical case of isolated heterogeneous microbial population of P. aeruginosa from various loci of the oral cavity and sputum of a patient with cystic fibrosis is described. A proteomic mass-spectrum analysis obtained from eleven isolates as well as their mutual comparison with the control strain of P. aeruginosa ATCC 27853 visualized with CCI matrix was performed. As a result, a high level of heterogeneity of the studied population was revealed, while high CCI Score values were obtained only for individual pairs of isolates. It was shown that the bacterial strains isolated from the patient sputum turned out to be more homogeneous in protein profiles than cultures detected from various oral sites. At the same time, in case of isolated several morphotypes of P. aeruginosa from single oral site, no closely related strains were found. Thus, it is shown that the oral cavity is more important viewed from microbial dissociation in cystic fibrosis, which may be due to more diverse microecological conditions. Representatives of the microbiological community can recolonize the tracheobronchial tree through microaspiration, thereby contributing to the maintenance of infectious inflammation in the lower respiratory tract and partially accounting for ineffectiveness of nebulized antibacterial therapy. At the same time, clones adapted to the lung tissue upon coughing up sputum are able to colonize oral sites. The data obtained actualize the question of the need for a comprehensive microbiological approach while examining patients to increase effectiveness of eradication measures in cystic fibrosis.

BACKGROUND: Male hypogonadism is associated with type 2 diabetes mellitus (T2DM), therefore, it is of interest to study its frequency. The clinical symptoms of hypogonadism are not specific, and laboratory diagnostics is the basis for its detection. The optimal method for this diagnostics is isotope dilution liquid chromatography/tandem mass spectrometry, which was used in our study. AIMS: Assessment of the incidence and aggravating factors of male hypogonadism in type 2 diabetesMATERIALS AND METHODS: A full-design, cross-sectional, screening, single-center, non-interventional study included men with T2DM, who were he was treated in Endocrinology Research Centre, Moscow. The study was conducted from October 2021 to January 2022. Medical history assessment, physical examination with determination of body mass index (BMI), measurement estimation of total testosterone by isotope dilution liquid chromatography/tandem mass spectrometry, glycated hemoglobin (HbA1c) and lipid profiles were performed. The groups were compared using the Mann-Whitney U-test for quantitative indicators and χ² with Yates’ correction for qualitative ones. Differences were considered statistically significant with p0,05.RESULTS: Hypogonadism was detected in 355 (70.3%) men with T2DM. Patients with hypogonadism had statistically significantly higher BMI, worse glycemic control, lower HDL levels, and higher triglycerides than eugonadal men. An additional comparative analysis among non-obese individuals showed the presence of statistically significant differences in the level of HbA1c (higher in hypogonadal men) and HDL (lower in hypogonadal men). An analysis of hypogonadal patients depending on the presence of obesity showed statistically significant differences between groups in the level of total testosterone (lower in obese men) and triglycerides (higher in obese men).CONCLUSIONS: The prevalence of male hypogonadism in type 2 diabetes was 70,3%. Its development was associated with obesity and poor glycemic control.

Untargeted metabolomics is a powerful tool for measuring and understanding complex biological chemistries. However, employment, bioinformatics and downstream analysis of mass spectrometry (MS) data can be daunting for inexperienced users. Numerous open-source and free-to-use data processing and analysis tools exist for various untargeted MS approaches, including liquid chromatography (LC), but choosing the 'correct' pipeline isn't straight-forward. This tutorial, in conjunction with a user-friendly online guide presents a workflow for connecting these tools to process, analyse and annotate various untargeted MS datasets. The workflow is intended to guide exploratory analysis in order to inform decision-making regarding costly and time-consuming downstream targeted MS approaches. We provide practical advice concerning experimental design, organisation of data and downstream analysis, and offer details on sharing and storing valuable MS data for posterity. The workflow is editable and modular, allowing flexibility for updated/changing methodologies and increased clarity and detail as user participation becomes more common. Hence, the authors welcome contributions and improvements to the workflow via the online repository. We believe that this workflow will streamline and condense complex mass-spectrometry approaches into easier, more manageable, analyses thereby generating opportunities for researchers previously discouraged by inaccessible and overly complicated software. [ABSTRACT FROM AUTHOR]