Immunomagnetic Separation Research Articles

The concept of the liquid biopsy in the treatment of malignant eye tumours

The liquid biopsy is acutting-edge technique that involves analysing non-solid biological tissues, primarily blood but also ocular fluids, for the presence of cancer cells or fragments of tumour DNA. Unlike traditional biopsies, liquid biopsies are usually minimally invasive and can be performed more frequently, enabling continuous monitoring of disease progression and treatment efficacy. This article (and the associated series of articles) outlines the key developments in liquid biopsy, which include the analysis of circulating tumor DNA (ctDNA), circulating tumor cells (CTC) and exosomal RNA and protein biomarkers. Techniques, such as digital droplet PCR and next-generation sequencing (NGS) have made it possible to detect even very low levels of ctDNA, which is crucial for early cancer detection and monitoring minimal residual disease. The detection of rare CTCs is enhanced by techniques, such as microfluidic devices and immunomagnetic separation. Multiomic approaches, whereby exosomal RNA, protein and ctDNA analyses are combined, help to create amore comprehensive picture of tumour biology, including insights into tumour heterogeneity, potentially leading to better diagnostic and prognostic tools and helping to predict treatment response and resistance. The challenges of liquid biopsy application, which will be described in the following article, include (a)standardization, (b)cost and accessibility, (c)validation and clinical utility. However, the liquid biopsy represents apromising frontier in the application of precision ocular oncology, with ongoing research likely to expand its applications and improve its effectiveness in the coming years.

Effect of mitochondrial oxidative stress on Regulatory T Cell manufacturing for clinical application in transplantation: results from a pilot study

The manufacturing process of Regulatory T (Treg) cells for clinical application begins with the positive selection of CD25+ cells using superparamagnetic iron-oxide nanoparticle (SPION)-conjugated anti-CD25 antibodies (spCD25) and immunomagnetic cell separation technology. Our findings revealed that the interaction of spCD25 with its cell target induced the internalization of the complex spCD25-Interleukin-2 Receptor. Accumulation of intracellular spCD25 triggered oxidative stress, causing delayed Treg expansion and temporary reduction in suppressor activity. This activation delay hindered the efficient generation of clinically competent cells. During this early phase, Treg cells exhibited elevated mitochondrial superoxide and lipid peroxidation levels, with concomitant decrease on mitochondrial respiration rates. The results uncovered the increased mitochondrial unfolded protein response (mitoUPR). This protective, redox-sensitive activity is inherent of Tregs when contrasted with homologous, spCD25-treated, conventional T cells. While the temporary effects of spCD25 on clinically competent cells did not impede their use in a safety/feasibility pilot study with kidney transplant recipients*, it is reasonable to anticipate a potential reduction in their therapeutic efficacy. The mechanistic understanding of the adverse effects triggered by spCD25 is crucial for improving the manufacturing process of clinically competent Treg cells, a pivotal step in the successful implementation of immune cell therapy in transplantation.*Clinical trial registration number NCT03284242 at ClinicalTrials.gov.

Development of an automated processing platform based on luciferase fused TPP17 to detect specific Treponema pallidum antibody in clinical serum with high sensitivity and rapidity

Syphilis (T. pallidum) is a systemic and sexually transmitted disease. Over 80 % of syphilis infections are asymptomatic, and some patients tend to conceal high-risk sex histories. Therefore, fast and effective screening of syphilis is crucial to control its spread. In this study, a methodology, combining a luciferase immuno-magnetic separation system (LIMS) with an automated magnetic-processing machine, has been designed to detect syphilis-specific antibodies against TPP17 of T. pallidum. Protein A/G-immobilized magnetic particles captured the antibodies in serum and then were specifically labeled with a chimeric protein of TPP17 with a luciferase hGluc. The magnetic separation was automatically executed. It takes only 25 min from samples to results for 32 samples once. Further, for clinical samples, antibody-negative serum samples from pregnant women were unexpectedly found to have higher bioluminescence than regular sera. Innovatively, two cutoff values were determined. ROC (receiver operating characteristic) analyses showed both the sensitivity and the specificity of 100 % for the regular sera, and the sensitivity and the specificity of 96.5 % and 100 % for the sera during pregnancy. The methodology was 32 times more sensitive than ELISA. The automated platform can potentially be commercialized and would be of great significance in controlling the spread of syphilis.

The kisspeptin effects on the thymic regulatory cell compositions (Th17, Treg, iNKT) <i>in vitro</i>

The peptide hormone kisspeptin, produced by neurons of the hypothalamus anterior zone, is a key regulator of the gonadostat formation and reproductive function, and has immunoregulatory activity. During pregnancy, kisspeptin is produced by the placenta, presents in the peripheral blood and affects on immune cells that express specific receptors for the hormone, including thymus cells. However, the kisspeptin effects on thymopoiesis during pregnancy have not been studied. The purpose of the work was to study the kisspeptin effect on the thymic regulatory cell composition in vitro. Thymocytes were cultured for 72 hours with kisspeptin at a concentration (9.6 pM) characterizing its maximum level in the peripheral blood during pregnancy, in the presence of CD3/CD28-activating particles and further assessed the regulatory cell composition and Ki-67 and Bcl-2 expression by flow cytometry. The percentage of T regulatory lymphocytes (Treg) was assessed as the percentage of CD4+CD25+FoxP3+ cells; T helper cells producing interleukin17 (Th17), as a percentage of CD4+IL-17A+RORγt+ cells; invariant T lymphocytes with natural killer functions(iNKT), as a percentage of CD3hiVa24Ja18+ cells in thymocyte culture. Dexamethasone (10-6M) was added to induce apoptosis. The effect of kisspeptin-primed thymic plasmacytoid dendritic cells (pDCs) on the regulatory cell composition (Th17, Treg, iNKT) in thymocyte culture was assessed. Thymic pDCs isolated by immunomagnetic separation were cultured for 24 hours with kisspeptin, and then intact autologous thymocytes were added in a ratio of 1:10 and cultured for another 72 hours. The thymocyte incubation of with kisspeptin did not affect the Tregs, iNKT and Th17 percentage in vitro, as well as the expression of Ki-67 and Bcl-2 in these cells. Under dexamethasone influence, the Bcl-2+Th17 percentage in thymocyte cultures with kisspeptin was increased. The Th17 percentage was increased in cultures of kisspeptin-primed pDCs with thymocytes, while the Treg and iNKT number did not change. It can be concluded that kisspeptin has regulatory effects on the Th17 percentage in thymocyte culture in vitro, mediating its effects by influencing thymic pDCs. And kisspeptin acted directly on thymocytes, increases the resistance of thymic Th17 to dexamethasone-induced apoptosis. The obtained results expand our understanding of the hormonal regulation of regulatory cell balance during pregnancy.

Characteristics of the proinflammatory response of monocytes/macrophages in patients with metabolic syndrome, exemplified by the production of IL-6 and MCP-1

Metabolic syndrome (MS) is one of the most common socially significant diseases. Around 1.9 billion people suffer from this disease, which places an enormous burden on healthcare systems around the world. This is particularly true in connection with concomitant diseases. With the progression of MS, disorders in the function of the immune system occur in the body, including those associated with mitochondrial dysfunction, leading to the development of chronic inflammation. In particular, there is an increase in the number of circulating monocytes actively recruited to inflamed adipose tissue, where there is non-specific proinflammatory activation of innate immune cells, which adopt an M1-like phenotype and become less sensitive to anti-inflammatory stimuli. This ultimately leads to a decrease in the functional activity of monocytes/macrophages and their immunoplasticity. The subject of the study was the venous blood of patients and the CD14+ monocytes/macrophages obtained from it by immunomagnetic separation. In our work, we focused on the search for significant relationships between markers of chronic inflammation and the formation of immune tolerance of monocytes/macrophages in patients with metabolic syndrome. Biochemical parameters, basal and LPS-stimulated production of proinflammatory cytokines (IL-6 and MCP-1) were investigated by culture of monocytes/macrophages in patients with metabolic syndrome. The evaluation of biochemical parameters in blood samples from MS patients and healthy donors revealed that the levels of ALAT, AST, GGT, alkaline phosphatase, uric acid, C-reactive protein, glucose and insulin were significantly higher in MS patients than in healthy donors. The levels of P-amylase and high-density lipoprotein were significantly lower than in the control group. Within the experimental model, repeated stimulation showed a decrease in cytokine production in response to LPS compared to the first stimulation on day 7. It was also found that the response to the primary stimulus was higher in cells from patients with a body mass index (BMI) 40 kg/m2, which could indirectly indicate the presence of a phenotype associated with chronic inflammation and consequently with reduced plasticity of the monocyte/macrophage immune response.

Immunomagnetic separation for the enrichment of trace enrofloxacin and analysis of degradation byproducts in water

ABSTRACT The presence of trace antibiotics in water can lead to the development of drug-resistant bacterial strains, posing risks to ecosystems and human health. Immunomagnetic separation (IMS) using magnetic nanoparticles (MNPs) is an effective technique for targeted enrichment. This study established and optimized a separation system for the immunomagnetic microsphere enrichment of enrofloxacin (ENR) antibiotics, achieving efficient enrichment and isolation of ENR. To address potential elution degradation, an analysis of ENR degradation pathways and toxicity assessment of degradation products was performed. The study manifested the successful conjugation of antibodies to magnetic microspheres, leading to a 97.68% separation efficiency for ENR in water through IMS. Specifically, 1 mg of MNP@Ab could specifically bind to 1.5 ng of ENR at 37 °C for 30 min, and the elution rate exceeded 83%. No degradation products of ENR were detected during the enrichment and isolation procedures. Nevertheless, extending the elution time to 1 h disclosed three major degradation pathways with higher toxicity risks than ENR based on ecological risk assessment. To strictly control the elution temperature and elution time, the increase in temperature and time will heighten the risk of degradation products. This study presents an efficient strategy for water treatment and environmental protection.

Investigation of Feedlot-level Use of a Direct-fed Microbial on Fecal Shedding of E. coli O157:H7

Our objectives were to determine whether the feedlot-level use of a direct-fed microbial (DFM; Lactobacillus animalis LA51 and Propionibacterium freudenreichii PF24; Bovamine Defend®, 2 × 109 CFU/g) was associated with fecal prevalence and concentration of E. coli O157:H7, and determine pen- and feedlot-level risk factors associated with fecal E. coli O157:H7 prevalence in cattle pens from commercial feedlot operations. Twenty commercial feedlots in Nebraska, ten that included DFM (DFM) and ten that did not (no-DFM), were sampled during the summer of 2017. In each sampling month, 22 pen-floor fecal samples were collected from three pens in each feedlot. Samples were subjected to cultural and molecular procedures for the detection of E. coli O157:H7 (immunomagnetic separation, plating on selective media, followed by PCR confirmation) and spiral plating for quantification. A total of 1,320 samples from 180 pens of finishing cattle belonging to 20 feedlots, which were sampled three times throughout a 12-week period, were processed and tested. Across all feedlots and sampling months, the mean within-pen prevalence was 13.5% (95% CI = 2.6–47.4%). The association between DFM status and the within-pen prevalence of E. coli O157:H7 depended significantly (p < 0.05) on the sampling month. The second sampling month between late July and mid-August corresponded to the highest within-pen prevalence estimates reported in this study, with no-DFM pens having a higher prevalence than DFM pens. After accounting for the DFM status, and based on multivariable analyses, sampling month, average pen body weight, and weather conditions were significantly associated with the within-pen fecal prevalence of E. coli O157:H7. Collectively, these findings demonstrate that the use of a DFM containing Lactobacillus animalis LA51 and Propionibacterium freudenreichii PF26 in feedlots showed potential in reducing fecal E. coli O157:H7 prevalence in cattle during times when prevalence peaks.

Identification of dystrophin Dp71dΔ71-associated proteins in PC12 cells by quantitative proteomics

Dystrophin Dp71 is essential for the development of the nervous system. Its alteration is associated with intellectual disability. Different Dp71 isoforms are generated by alternative splicing; however, their functions have not been fully described. Here, we identified Dp71dΔ71-associated proteins to understand the complex functions. PC12 cells, stably transfected with pTRE2pur-Myc/Dp71dΔ71 or pTRE2pur-Myc empty vector (EV), were analyzed by immunoprecipitation followed with quantitative proteomics with data-independent acquisition and ion mobility separation. We used the Top3 method to quantify absolutely every protein detected. A total of 106 proteins were quantified with Progenesis QI software and the database UP000002494. Seven new proteins associated with Dp71dΔ71 were selected with at least 2-fold quantity between immunoprecipitated proteins of PC12-Myc/Dp71dΔ71 versus PC12-EV cells. These results revealed new proteins that interact with Dp71dΔ71, including β-Tubulin, S-adenosylmethionine synthase isoform type-2, adapter molecule crk, helicase with zinc finger 2, WD repeat domain 93, cyclin-L2 and myosin-10, which are related to cell migration and/or cell growth. The results lay the foundation for future research on the relationship between these proteins and Dp71 isoforms.

Metal Polycation Adduction to Lipids Enables Superior Ion Mobility Separations with Ultrafast Ozone-Induced Dissociation.

Specific lipid isomers are functionally critical, but their structural rigidity and usually minute geometry differences make separating them harder than other biomolecules. Such separations by ion mobility spectrometry (IMS) were recently enabled by new high-definition methods using dynamic electric fields, but major resolution gains are needed. Another problem of identifying many isomers with no unique fragments in ergodic collision-induced dissociation (CID) was partly addressed by the direct ozone-induced dissociation (OzID) that localizes the double bonds, but a low reaction efficiency has limited the sensitivity, dynamic range, throughput, and compatibility with other tools. Typically lipids are analyzed by MS as singly charged protonated, deprotonated, or ammoniated ions. Here, we explore the differential IMS (FAIMS) separations with OzID for exemplary lipids cationized by polyvalent metals. These multiply charged adducts have much greater FAIMS compensation voltages (UC) than the 1+ ions, with up to 10-fold resolution gain enabling baseline isomer separations even at a moderate resolving power of the SelexION stage. Concomitantly OzID speeds up by many orders of magnitude, producing a high yield of diagnostic fragments already in 1 ms. These capabilities can be ported to the superior high-definition FAIMS and high-pressure OzID systems to take lipidomic analyses to the next level.

Ultrasensitive Analysis of Escherichia coli O157:H7 Based on Immunomagnetic Separation and Labeled Surface-Enhanced Raman Scattering with Minimized False Positive Identifications.

It is a big challenge to monitor pathogens in food with high selectivity. In this study, we reported an ultrasensitive method for Escherichia coli O157:H7 detection based on immunomagnetic separation and labeled surface-enhanced Raman scattering (SERS). The bacterium was identified by heterogeneous recognition elements, monoclonal antibody (mAb), and aptamer. E. coli O157:H7 was separated and enriched by magnetic nanoparticles modified by mAb, and then a plasmonic nanostructure functionalized by aptamers with embedded Raman tags and interior gaps was utilized for further discrimination and detection. The selectivity was enhanced by two binding sites. The higher Raman enhancement was obtained by strong local electromagnetic field oscillation in the gap and the firm embedment of 4-mercaptopyridine (4-Mpy). Optimum experiments created that SERS signals of 4-Mpy at 1010 cm-1 had a good linearity with E. coli O157:H7 at a large range of 10 to 107 CFU/mL with a limit of detection of 2 CFU/mL. This method has great potential for on-site food pathogenic bacterial detection.

Impaired tolerance of monocyte-macrophages to lipopolysaccharide in patients with coronary atherosclerosis

Atherosclerosis is a chronic disease in which lipids, cells and various proteins accumulate in the walls of the arteries, forming atherosclerotic plaques. The growth of plaques leads narrowing of the lumen of the blood vessels. Atherosclerosis is accompanied by local inflammation, while the number of hematogenous macrophages are derived from monocytes increases in the vascular wall. The reasons why the inflammatory reaction cannot be completed and becomes chronic are not clear. To resolve inflammation and protect tissues from high concentrations of cytokines that can cause apoptosis, there is a mechanism of immune tolerance of innate immunity. Lipopolysaccharide (LPS) tolerance of monocyte-macrophages is a phenomenon in which cells reduce their sensitivity to repeated exposure to LPS. This condition is characterized by a decrease in the ability of macrophages to produce proinflammatory cytokines and promotes resolution of inflammation. We hypothesized that in atherosclerosis, tolerance violations in monocyte-macrophages are possible. The study included patients who were admitted to the department of cardiac surgery, Moscow Regional Research and Clinical Institute (MONIKI). Patients were divided into patients with coronary atherosclerosis (CAD) with detected stenosis in 2 or more arteries and healthy controls without stenosis in the arteries according to the results of coronary angiography. In the present study, we examined the ability of macrophages from 13 patients with CAD and 11 patients without CAD to develop tolerance to LPS. To do this, we isolated CD14+ monocytes from the blood by positive selection using immunomagnetic separation and subjected them to two sequential LPS stimulations, immediately after cell isolation and after 6 days of culture. The secretion of cytokines TNFα, IL-1b, IL-6, IL-10, IL-8, and CCL2 was measured in cell culture supernatants using by ELISA. Our results showed impaired macrophage tolerance for CCL2 secretion and improved tolerance for IL-8 secretion in macrophages from patients with CAD compared with patients without. Since IL-8 and CCL2 are chemoattractants for other immune cells, it can be assumed that the observed impairment of macrophage tolerance to LPS in atherosclerosis increases the infiltration of other monocytes into the inflammatory site, contributing to the chronicity of inflammation.

Revisiting Dissolved Organic Matter Analysis Using High-Resolution Trapped Ion Mobility and FT-ICR Mass Spectrometry.

The molecular level characterization of complex mixtures remains an analytical challenge. We have shown that the integration of complementary, high-resolution, gas-phase separations allows for chemical formula level isomeric content description. In the current work, we revisited the current challenges associated with the analysis of dissolved organic matter using high-resolution trapped ion mobility separation (TIMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In particular, we evaluated the separation capabilities provided by TIMS-MS compared to MS alone, the use of ICR complementary data acquisition (DAQ) systems and transient processing strategies, ICR cell geometries (e.g., Infinity cell vs harmonized cell), and magnetic field strengths (7 T vs 9.4 T vs 21 T) for the case of a Harney River DOM sample. Results showed that the external high-performance DAQ enables direct representation of mass spectra in absorption mode FT (aFT), doubling the MS resolution compared to the default magnitude mode FT (mFT). Changes between half- vs full-apodization result in greater MS signal/noise vs superior MS resolving power (RP); in the case of DOM analysis, a 45% increase in assigned formulas is observed when employing the DAQ half (Kaiser-type)-apodization window and aFT when compared to the default instrument mFT. Results showed the advantages of reprocessing 2D-TIMS-FT-ICR MS data with higher RP and magnetic field chemical formulas generated list acquired (e.g., 21 T led to a 24% increase in isomers reported) or the implementation of alternative strategies.

Utilizing ESI-MS/MS and ion mobility spectrometry for structural characterization and isomer differentiation of 1, 2-unsaturated (1-4) linked disaccharide derivatives and their 2-C-functionalized analogues.

The 1, 2-unsaturated derivatives of (1-4) linked disaccharides serve as versatile building blocks for synthesizing biologically active compounds. Distinguishing between four pairs of stereoisomers in mixtures presents a challenging task. In this study, disaccharide derivatives are analyzed as alkali metal adducts using electrospray ionization tandem mass spectrometry (ESI-MS/MS), both as individual compounds and in mixtures by ion mobility mass spectrometry (IMS). Electrospray ionization (ESI) in combination with tandem mass spectrometry (MS/MS) in positive mode was employed to differentiate lithium adduct ions of hexa-acetyl/hexa-benzyl-D-lactals, and hexa-acetyl/hexa-benzyl-D-maltals, along with their corresponding 2-C-branched malonates. The high resolving power of trapped ion mobility spectrometry (TIMS) with the imeX™ functionality rapidly identified different metal adducts (Li, Na, Cs) as individual isomers and separated mixtures of stereoisomers. The measured collisional cross section (CCS) values were analyzed in relation to predicted CCS values. MS/MS spectra of the [M + Li]+ ions of glycal disaccharide analogues exhibited typical cross-ring and glycosidic bond cleavages. Collision-induced dissociation (CID) spectra provided insights into their fragmentation behavior, allowing differentiation of (1-4) linked disaccharides. TIMS technology delivered adjustable ion mobility resolution for suitable separation of the four sets of stereo isomeric compounds. However, accurately predicting CCS values to differentiate between respective isomeric pairs using the SigmaCCS program for sodium adducts is only partially achievable. ESI CID spectra of [M + Li]+ adduct ions for individual glycal disaccharide analogues facilitate the discrimination between alpha and beta (1-4) linked unsaturated disaccharides and their 2-C-branched analogues. Through optimized experimental conditions, complete baseline ion mobility separation of stereoisomer pairs of the Cs adducts is achieved.

Introducing Ion Mobility Mass Spectrometry in Brain Glycosaminoglycomics: Application to Chondroitin/Dermatan Sulfate Octasaccharide Domains.

Glycosaminoglycans (GAGs) are sulfated linear O-glycan chains abundantly expressed in the extracellular matrix (ECM). Among GAGs, chondroitin sulfate (CS) and dermatan sulfate (DS) play important roles at the brain level, where the distribution and location of the sulfates within the CS/DS chains are responsible for numerous biological events. The diversity of the neural hybrid CS/DS expressed in the brain and the need to elucidate their structure gave rise to considerable efforts toward the development of analytical methods able to discover novel regularly and irregularly sulfated domains. In this context, we report here the introduction of ion mobility separation (IMS) mass spectrometry (MS) in brain glycosaminoglycomics. Based on IMS MS and tandem MS (MS/MS) by collision-induced dissociation (CID), we have developed a powerful approach for the screening and structural analysis of neural CS/DS and optimized and validated the method for the structural analysis of octasaccharides that were released from brain proteoglycans by β-elimination and pooled after chain depolymerization using chondroitin AC lyase. The IMS MS data revealed the separation of CS/DS octamers into mobility families based on the amount of sulfation. Among the discovered oversulfated domains, of major biological importance is the pentasulfated-[4,5-Δ-GlcAGalNAc(IdoAGalNAc)3], for which the (-) nanoESI IMS CID MS/MS analysis disclosed through the presence of distinct drift times, the incidence of two isomers. Moreover, the generated fragment ions revealed an uncommon trisulfated motif and an uncommon pentasulfated motif. Hence, using IMS MS and CID MS/MS, novel brain-related CS/DS domains of atypical sulfation patterns were discovered and structurally characterized in detail.

Ion confinement and separation using asymmetric electrodynamic fields in structures for lossless ion manipulations.

TW-SLIM ion mobility separations have demonstrated exceptional resolution by leveraging long paths with minimal loss. All previously reported experiments have used electrode surfaces which are mirrored to generate symmetrically opposing electric fields for ion confinement. However, work with other planar ion optics indicates this may be unnecessary. This study explores conditions under which separations may be obtained using a SLIM with asymmetric electric fields. The asymmetric field configuration was defined by applying a uniform DC potential to all electrodes of the top PCB of a standard TW-SLIM board pair, with no electrode placement modifications. This configuration was simulated in SIMION to assess transmission through the SLIM. A benchtop TW-SLIM instrument outfitted with a Faraday plate detector was modified likewise, so the top PCB had a uniform DC potential applied to all electrodes, while the bottom board was operated normally. Simulations show full ion transmission for four different m/z ion populations over a range of DC biases applied to the "pusher" board. Likewise, the modified benchtop instrument is capable of transmitting, separating, and cycling ions with minimal losses. The effect of pusher strength on separation quality is explored, and comparisons between the standard and modified SLIM are made with respect to resolving the +2 and +3 charge states of neurotensin ions. A functional IMS instrument using asymmetric confining fields demonstrates additional field modifications may be a means to achieve additional functionality with limited interruption of the analysis. A TW-SLIM PCB specifically designed as a pusher board would benefit from minimized manufacturing cost, simplifying assembly, reducing drive electronics, and improved field consistency.

Identifying and Quantifying Relative Concentrations of Epimers in Mixtures via Cyclic Ion Mobility Mass Spectrometry: Dexamethasone and Betamethasone as a Case Study.

Epimers can show different biological activities and different pharmacological behaviors; therefore, their separation and analysis are crucial in the drug development process. Due to their similar chemical and physical properties, separation of epimers is challenging. This study demonstrates the application of cyclic ion mobility-mass spectrometry to separate, identify, and quantify dexamethasone and betamethasone in a binary mixture. Cyclic IMS separation of the isolated protonated dimer resulted in three peaks: dexamethasone homodimer, betamethasone homodimer, and their heterodimer. Besides providing improved separation over the protonated monomer, the presence of a heterodimer peak provides additional confirmation of an isomeric mixture. We identified the dexamethasone and betamethasone homodimer peaks by infusing pure solutions of each epimer and measuring each pure homodimer's arrival time. The measured peak areas indicated that the heterodimer is formed at twice the rate of each homodimer and that dexamethasone and betamethasone contribute equally to the heterodimer signal. Using this observation, we could accurately calculate the relative concentrations of each epimer by adding half of the heterodimer peak area to each homodimer peak area. These findings enable the identification and quantification of dexamethasone and betamethasone based on the arrival time distributions of their protonated dimers. This is the first demonstration of accurate relative quantification of epimers by separating charged dimers in the gas phase.

Assessing the additivity of mass distribution-based isotopic shifts in high-resolution cyclic ion mobility separations

Recent advancements in high-resolution ion mobility spectrometry-mass spectrometry (IMS-MS) have enabled the separation of isotopologues and isotopomers based on their mass distribution-based isotopic shifts (i.e., changes in center of mass and moments of inertia). To better understand the fundamental nature of these isotopic shifts, we investigated whether they were additive in nature by introducing varying isotopic substitutions (e.g., 13C, 2H/D, and 81Br) through either hydrogen deuterium exchange or permethylation. From there, we measured the relative arrival times between light and heavy isotopologues with high-resolution cyclic ion mobility separations. Globally, we observed that the isotopic shifts were approximately additive in nature regardless of the molecule system or specific isomer studied. Furthermore, we discovered that additivity occurs in the isotopic shifts irrespective of the absolute shift, potentially indicating this observation may be more global in nature. We believe that our findings will serve to better understand the fundamental nature of mass distribution-based isotopic shifts and will inform theoretical ion mobility calculations in the future.

Association of Telomere Length in T Lymphocytes, B Lymphocytes, NK Cells and Monocytes with Different Forms of Age-Related Macular Degeneration.

Age plays a primary role in the development of age-related macular degeneration (AMD). Telomere length (TL) is one of the most relevant biomarkers of aging. In our study, we aimed to determine the association of TL with T lymphocytes, B lymphocytes, NK cells or monocytes with different forms of AMD. Our study included 62 patients with AMD: geographic atrophy (GA), neovascular AMD (NVAMD) with and without macular atrophy and 22 healthy controls. Each leukocyte subtype was isolated from peripheral blood by immunomagnetic separation, and the DNA was purified. The TL in the genomic DNA was determined using qPCR by amplifying the telomere region with specific oligonucleotide primers and normalizing to the control gene. Statistical analysis was performed using R version 4.5.1. We observed a statistically significant increase in TL in the T cells between the control and NVAMD groups but not for the GA group. The B cells and monocytes showed a significant decrease in TL in all AMD groups. The TL in the NK cells did not decrease in any of the AMD groups. The TL in the monocytes had the strongest association with AMD. It reflects a person's "telomeric status" and may become a diagnostic hallmark of these degenerative processes.

Sequencing Sialic Acid Positioning in Gangliosides by High-Resolution Cyclic Ion Mobility Separations Coupled with Multiple Collision-Induced Dissociation-Based Tandem Mass Spectrometry Strategies.

Gangliosides, a diverse class of glycosphingolipids, are highly abundant in neural tissue and have been implicated in numerous aging-related diseases. Their characterization with methods such as liquid chromatography-tandem mass spectrometry is often precluded by their structural complexity, isomeric heterogeneity, and lack of commercially available authentic standards. In this work, we coupled high-resolution cyclic ion mobility spectrometry with multiple collision-induced dissociation-based tandem mass spectrometry strategies to sequence the sialic acid positions in various ganglioside isomers. Initially, as a proof-of-concept demonstration, we were able to characterize the sialic acid positions in several GD1 and GT1 species. From there, we extended our approach to identify the location of N-glycolylneuraminic acid (NeuGc) residues in previously uncharacterized GD1 and GQ1 isomers. Our results highlight the potential of this presented methodology for the de novo characterization of gangliosides within complex biological matrices without the need for authentic standards.

Effect of statin administration on the inflammatory response of monocytes in patients with atherosclerosis

Currently, statins are the main preparations of anti-atherosclerotic therapy due to a number of effects that reduce the progression of atherosclerosis, including anti-inflammatory effectiveness. The purpose of this study was to evaluate the inflammatory response of monocytes in patients with severe atherosclerosis during therapy with hydrophilic and lipophilic statins, as well as in patients with atherosclerosis not receiving lipid-lowering therapy. A total of 60 patients with severe atherosclerosis of the coronary arteries were included in the study in three groups: 1) receiving atovastatin therapy for at least 12 months before inclusion in the study, n = 20; 2) receiving rosuvastatin therapy for at least 12 months before inclusion in the study, n = 20; and 3) those who had not received statin therapy within a year before inclusion in the study, n = 20. The primary culture of monocytes from study participants was obtained by gradient centrifugation followed by immunomagnetic separation of CD14+ monocytes. The isolated cells were cultured for 7 days without stimulation and with pro-inflammatory stimulation using lipopolysaccharide (LPS). The level of basal, LPS-stimulated and re-stimulated secretion of TNFα and IL-1β was determined by enzyme immunoassay. Basal secretion of TNFα and IL-1β in patients receiving statins was lower than in patients who did not receive statins for a year; the secretion of both cytokines was significantly lower in the rosuvastatin group. LPS-stimulated TNFα secretion was significantly lower in the groups of patients receiving statins; IL-1β secretion was significantly lower in the atorvastatin and rosuvastatin groups compared to the group without statins. Re-stimulated IL-1β secretion did not differ significantly between groups; re-stimulated TNFα secretion was significantly lower in the rosuvastatin group compared to the atorvastatin and non-statin groups. Thus, the results of the study demonstrate the anti-inflammatory effectiveness of rosuvastatin, expressed in a decrease in the secretion of pro-inflammatory cytokines by cultured monocytes/macrophages of patients with severe coronary atherosclerosis.