Immunomagnetic Separation Research Articles

Characterization of Sugammadex-Related Isomeric Cyclodextrin Impurities Using Cyclic Ion Mobility High-Resolution Mass Spectrometry.

Cyclic ion mobility-mass spectrometry (cIM-MS) is a powerful technique for separating and identifying isomeric mixtures of compounds. When coupled with chromatography, cIM-MS creates a multidimensional separation system, with high resolving power and peak capacity. In this study, we report the cyclic ion mobility separation and high-resolution mass spectrometry identification of four regioisomers of a Sugammadex-related impurity, abbreviated as Di-OH-SGM. Separation using multipass cyclic ion mobility was achieved by selecting the [M + 2Na]2+ ion, while other adducts, such as [M + Na]+, [M + 2H]2+, [M + H + Na]2+, and [M - 2H]2- did not yield isomer separation. Two methods were developed for ion mobility separation of the isomers: a conventional multipass method and a slicing method. Isomer assignment was based on the characteristic fragment ions. The collision cross section values (cTWCCSN2) of the resolved cyclodextrin isomers were also determined. Ion mobility separation of structurally different fragment ions was demonstrated. Additionally, by coupling cIM-MS with reversed-phase liquid chromatography (HPLC-cIM-MS), two-dimensional separation of the isomers was achieved. The isomers, separated using HPLC-cIM-MS, were identified with the same approach as with cIM-MS alone, and their elution order provided insights into their relative hydrophobicity.

A refined method for high-purity isolation of uterine glandular epithelial cells in mouse.

The uterine endometrium consists of luminal epithelium, glandular epithelium, and stromal cells, with uterine glands playing a pivotal role in pregnancy success among mammals. Uterine glands secrete essential factors that regulate embryo development and implantation; however, their cellular biology remains poorly understood. This study presents a refined method for isolating three distinct endometrial cell types with high purity, with a specific emphasis on glandular epithelial cells. The method combines mechanical dissociation, enzymatic digestion, and immunomagnetic separation. The isolated glandular epithelial cells were maintained in culture and exhibited proliferation in response to steroid hormones. Furthermore, estrogen responsiveness was abrogated by Estrogen Receptor 1 (Esr1) knockdown mediated by siRNA. Here, we present an efficient and reproducible method for isolating uterine glandular epithelial cells with high purity, enabling their in vitro maintenance, hormone responsiveness assessment, and functional gene knockdown. These findings establish a robust platform for advancing our understanding of uterine gland biology, facilitating detailed investigations into molecular mechanisms underlying glandular function and their critical roles in establishing pregnancy success. Future research could explore the contribution of these isolated cells to endometrial receptivity and embryo implantation.

Compositional analysis of traditional liquid gold with separation of compounds containing heavy atoms in ion mobility-mass spectrometry.

"Liquid gold" has been traditionally used for over a century to decorate ceramicware, but its chemical composition has not been thoroughly investigated. One of the keys to successfully characterizing liquid gold, which is a complex mixture, is to distinguish Au-containing products from other chemicals. In this paper, we propose a separation based on the difference in collision cross section, of which chemicals with heavy atoms are relatively smaller than those without in ion mobility-mass spectrometry (IM-MS). Chemicals containing a single Au atom (and Pt atom) were successfully separated from other species in the two-dimensional distribution map for IM-MS. By a detailed analysis of the spectra obtained by IM-MS/MS with collision-induced dissociation before and after IM separation, we found that liquid gold (gold resinate) was a mixture of a series of (1) Au reacted with α-pinene-related units and (2) Au reacted with abietic acid units. α-Pinene and abietic acid are the main components of turpentine and rosin, the raw materials of liquid gold as reported previously (Anal. Sci. 2024, 40, 133-139). All Au-containing species contain sulfur atoms. Species of Au reacted with α-pinene-related units with different degrees of unsaturation and oxidation have also been identified. Liquid gold, a complex mixture of chemicals containing Au, has been successfully analyzed compositionally.

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and understanding epigenomic regulation. Neutrophils, as a crucial leukocyte type in immune responses, undergo substantial chromatin architectural changes during differentiation and activation, which significantly impact the gene expression necessary for their functions. ATAC-seq has been instrumental in uncovering key transcription factors in neutrophil maturation, revealing pathogen-specific epigenomic signatures, and identifying therapeutic targets for autoimmune diseases. However, neutrophils' sensitivity to the external milieu complicates high-quality ATAC-seq data production. Here, we propose a scalable protocol for preparing ATAC-seq libraries from rodent bone marrow-derived neutrophils, featuring improved immunomagnetic separation to ensure optimal cell viability and high-quality libraries. The vital elements impacting the library quality and optimization principles for methodological extension are discussed in detail. This protocol will support the researchers who are willing to study the chromatin architecture and epigenomic reprogramming of neutrophils, advancing studies in basic and clinical immunology.

Association Between Glycemic Control and Complications With Concentration of Urinary Exfoliated Proximal Tubule Kidney Cells in People With Diabetes Mellitus.

Background: Emerging evidence suggests cell exfoliation could be operating under the control of cell metabolism. It is unclear if there are associations between the concentration of exfoliated kidney proximal tubule cells (PTCs) in urine with glycemic control and complications. Our study is aimed at exploring this. Methods: Urine samples were collected from 122 adult study participants and stored at -80°C. Exfoliated PTCs were extracted from thawed urine using a validated specific immunomagnetic separation method based on anti-CD13 and anti-SGLT-2 antibodies. The number of PTCs was assessed using brightfield microscopy. Study participants were grouped into those with no diabetes mellitus (DM) and those with DM. Individuals with DM were further subgrouped into those with and without retinopathy. Adjusted Poisson regression analysis was conducted for the DM cohort, investigating associations between demographic, clinical, and biochemical parameters with mean urinary exfoliated PTCs. Results: The adjusted Poisson regression analysis noted sex to have a significant association with mean number of urinary exfoliated PTCs, with a lower incidence rate in males compared to females (incidence rate ratio (IRR) 0.56, 95% CI 0.35-0.89, p = 0.014). Each 1% increase in glycated haemoglobin (HbA1c) was associated with an increase of 1.03 times in mean exfoliated PTCs (IRR 1.03, 95% CI 1.01-1.04, p = 0.007), and DM patients with retinopathy had an increase of 1.68 times in mean exfoliated PTCs compared to those without retinopathy (IRR 1.68, 95% CI 1.07-2.62, p = 0.024). No significant associations were observed with albuminuria or estimated glomerular filtration rate (eGFR). Conclusions: Our results indicate increased shedding of PTCs into the urinary tract in patients with poorer glycemic control, particularly those with diabetic retinopathy and in females.

Rounded Turn SLIM Design for High-Resolution Ion Mobility Mass Spectrometry Analysis of Small Molecules.

Various rounded turn designs in Structures for Lossless Ion Manipulation (SLIM) were explored via ion trajectory simulations. The optimized design was integrated into a SLIM ion mobility (IM) system coupled with a time-of-flight (TOF) mass spectrometer (MS) for further experimental investigation. The SLIM-TOF IM-MS system was assessed for IM resolution and ion transmission efficiency across a wide m/z range using various RF frequencies and buffer gas combinations. High ion transmission efficiency and high resolution ion mobility (HRIM) separation were achieved for Agilent tune mix ions through a ∼12.8 m serpentine separation path in both nitrogen and helium. In helium, ion transmission for low m/z ions was enhanced at higher RF trapping frequency, enabling the detection of ions with m/z below 50 and all 17 amino acids from a standard mixture. Lossless ion transmission was observed for glycine (m/z 76) in both passthrough and HRIM modes. HRIM resolution was benchmarked using L-isoleucine, L-leucine, and various other isobaric and isomeric metabolites with m/z values of 60-89. This work demonstrates a rounded turn SLIM design that enables HRIM measurements for small molecule analytes, with a particular focus on metabolomics, where IM offers a means to enhance the speed, robustness, and specificity of analytical workflows.

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.

Design of buffer property for the new enrichment method of circulating tumor cell based on immunomagnetic-negative separation

Metastasis is a significant contributor to cancer-related mortality and a critical issue in cancer. Monitoring the changes in circulating tumor cells (CTCs) with metastatic potential is a valuable prognostic and predictive biomarker. CTCs are a rare population in the peripheral blood of patients with cancer. The enrichment process is extremely important for the isolation of clinically significant CTC subpopulations, which can then be used for further analysis. The present study postulates that the buffer serves as an essential field for immunomagnetic separation, thereby enhancing the efficacy of CTC enrichment in peripheral blood. This, in turn, facilitates CTC detection. Here, we describe the design of buffers for developing a novel immunomagnetic-negative separation method for CTC enrichment. During the design process, the buffer properties of the floating and cell coatings had a synergistic effect on the efficiency of cell enrichment in blood samples. The efficacy of the method was evaluated using peripheral blood samples from patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The developed method enriched clinically relevant CTC subpopulations that expressed the epithelial-mesenchymal transition (EMT)-related molecule vimentin and/or the cancer immune checkpoint marker programmed death ligand 1 (PD-L1). Furthermore, it was applicable as a part of the enrichment process in a TelomeScan® (OBP-401)-based CTC detection assay with high sensitivity and specificity. From the perspective of methodological approaches, the design of buffer properties can be useful for developing a highly versatile enrichment method for handling CTC heterogeneity.

Synthetic γ‐AApeptides as Targeting Ligands for Immunomagnetic separation of Alzheimer’s disease (AD) Biomarkers

Synthetic γ‐AApeptides as Targeting Ligands for Immunomagnetic separation of Alzheimer’s disease (AD) Biomarkers

Development of a Platform for High-Resolution Ion Mobility Separations Coupled with Messenger Tagging Infrared Spectroscopy for High-Precision Structural Characterizations.

The ability to uniquely identify a compound requires highly precise and orthogonal measurements. Here we describe a newly developed analytical platform that integrates high resolution ion mobility and cryogenic vibrational ion spectroscopy for high-precision structural characterizations. This platform allows for the temporal separation of isomeric/isobaric ions and provides a highly sensitive description of the ion's adopted geometry in the gas phase. The combination of these orthogonal structural measurements yields precise descriptors that can be used to resolve between and confidently identify highly similar ions. The unique benefits of our instrument, which integrates a structures for lossless ion manipulations ion mobility (SLIM IM) device with messenger tagging infrared spectroscopy, include the ability to perform high-resolution ion mobility separations and to record the IR spectra of all ions simultaneously. The SLIM IM device, with its 13 m separation path length, allows for multipass experiments to be performed for increased resolution as needed. It is integrated with an Agilent qTOF MS where the collision cell was replaced with a cryogenically held (30 K) TW-SLIM module. The cryo-SLIM is operated in a novel manner that allows ions to be streamed through the device and collisionally cooled to a temperature where they can form noncovalently bound N2 complexes that are maintained as they exit the device and are detected by the TOF mass analyzer. The instrument can be operated in two modes: IMS+IR where the IR spectra for mobility-selected ions can be recorded and IR-only mode where the IR spectra for all mass-resolved ions can be recorded. In IR-only mode, IR spectra (400 cm-1 spectral range) can be recorded in as short as 2 s for high throughput measurements. This work details the construction of the instrument and modes of operation. It provides initial benchmarking of CCS and IR measurements to demonstrate the utility of this instrument for targeted and untargeted approaches.

SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

Here, we developed and demonstrated a novel integrative system—Silica Nanorods (SNA) substrate cell capture combined with Supramolecular Nanoparticle (SMNP) delivery mediated CBE base editing (SNA·SMNP·CBE)—achieving the synchronization of CD34+HSPCs cell capture and gene editing for β-hemoglobinopathies. First, in vitro study shows it enables efficient and precise modification of BCL11A promoter in CD34+HSPCs, yielding the highly editing efficiency of 50.4 %, thus making an alternative strategy to conventional immunomagnetic cell separation and electroporation transfection system mediated CBE editing (IMS·EP·CBE). Then, we transplanted the edited human CD34+HSPCs into severe combined immunodeficiency (SCID) mice by using intraosseous injection strategy. When compared with conventional IMS·EP·CBE methods, our results showed that significantly higher human HBG expression in the bone marrow and peripheral blood of recipient mice, and long-term engraftment, evidenced from similar gene expression profiles to naïve CD34+HSPCs at 14 weeks. Conclusively, our integrative system—SNA·SMNP·CBE·intraosseous injection—offers an appealing novel way for the unique potential of gene therapy in the clinic application for β-hemoglobinopathies patients.

A rapid and sensitive sandwich assay for the detection of Alicyclobacillus acidoterrestris in apple juice based on magnetosome immunomagnetic separation combined with quantum dots immunoassay technology

The contamination of apple juice by Alicyclobacillus acidoterrestris (A. acidoterrestris) can cause significant economic losses. Therefore, developing a rapid and sensitive method for detecting A. acidoterrestris is necessary. To address this issue, this study prepared magnetosome–monoclonal antibodies immunomagnetic microspheres (IMMs) as capture probes to enrich A. acidoterrestris (IMMs-Aa) and monoclonal antibodies-quantum dots (mAb-CdTeQDs) as detection probes for detecting A. acidoterrestris. The obtained IMMs-Aa-mAb-CdTeQDs “sandwich” structure was used to detect A. acidoterrestris based on the fluorescence intensity. The strategy presented a good linear correlation (y = 1048× + 1891, R2 = 0.995) between the fluorescence intensity and the concentration of A. acidoterrestris (101–105 CFU/mL) with a low detection limit of 25.4 CFU/mL within 50 min. The recovery rate of this strategy in spiked apple juice ranged from 88.16 % to 107.03 %. Thus, this study established an efficient and highly sensitive magnetic capture-enrichment-separation-detection method for A. acidoterrestris.

Studying Structural Details in Complex Samples: II. High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Coupled to High Resolution Tandem Mass Spectrometry (MS/MS).

The elucidation of structural motifs in extremely complex mixtures is very difficult since the standard methods for structural elucidation are not capable to provide significant information on a single molecule. The best method for the analysis of complex mixtures is ultrahigh resolution mass spectrometry, but the utilization of this method alone does not provide significant information about structural details. Here, a combination with a separation method is necessary. While chromatography is a well-established technique, it has some disadvantages in regard to the separation of complex mixtures, as often no separation of individual isomers is possible. Therefore, here the combination of an ion mobility separation with ultrahigh resolution mass spectrometry is evaluated. As a sample matrix, crude oil is used because it is an excellent matrix to develop new analytical techniques on complex samples. Crude oil is the most complex natural sample known, but only little information is available on the structural identity or functionalities due to a high number of structural isomers or isobars. A lab-built APPI/APLI-FAIMS source was revised to optimize ion transmission and used to follow up on the ion mobility of crude oil constituents after photoionization. An MS/MS approach using collision-induced dissociation (CID) was used to elucidate structural motifs of the transmitted isomers.

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.

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 a concomitant decrease in mitochondrial respiration rates. The results uncovered the increased mitochondrial unfolded protein response. This protective, redox-sensitive activity is inherent in Tregs when contrasted with homologous, spCD25-treated, conventional T cells. Although 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.

Sample Type and Processing Plant Differences in the Proportion of Enterohemorrhagic Escherichia coli O157 and Non-O157 Serogroups in Feces and on Hides of Cull Dairy Cattle at Slaughter.

The study was conducted to determine the proportion and concentration of enterohemorrhagic Escherichia coli (EHEC) O157 and six non-O157 (O26, O45, O103, O111, O121, and O145) serogroups and identify seasonal and processing plant differences in feces and on hides of cull dairy cattle processed in commercial slaughterhouses in the United States. Approximately 60 rectal and 60 hide-on samples from matched carcasses were collected in each of three processing plants, in two periods; summer of 2017 and spring of 2018. Samples before enrichment were spiral plated to quantify EHEC, and postenriched samples underwent culture methods that included immuno-magnetic separation, plating on selective media, and PCR assays for identification and serogroup confirmation of putative isolates. An isolate was considered EHEC O157 positive if it harbored serogroup-specific (rfbE), Shiga toxin (stx1 and/or stx2), and intimin (eae) genes and EHEC non-O157 positive if at least one of the non-O157 serogroup-specific, stx1 and/or stx2, and eae genes was identified. Generalized linear mixed models were fitted to estimate overall proportion of positives for EHEC O157 and non-O157 EHEC serogroups, as well as seasonal and processing plant differences in fecal and hide-on proportion of positives. The fecal EHEC proportion at the sample level was 1.8% (95% CI = 0.0-92.2%) and 4.2% (95% CI = 0.0-100.0%) for EHEC O157 and EHEC non-O157, respectively. Hide sample level proportion of positives was 3.0% (95% CI = 0.0-99.9%) for EHEC O157 and 1.6% (95% CI = 0.0-100.0%) for EHEC non-O157. The proportion of EHEC O157 and non-O157 significantly differed by processing plant and sample type (hide vs. feces), but not by season. The association between proportion of EHEC serogroups in feces with the proportion on hides collected from matched cattle was 7.8% (95% CI = 0.6-53.3%) and 3.8% (95% CI = 0.3-30.8%) for EHEC O157 and non-O157, respectively. Taken together, our findings provide evidence of a low proportion of EHEC serogroups in the feces and on hides of cull dairy cattle and that their proportion varies across processing plants.

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.

DiaTracer enables spectrum-centric analysis of diaPASEF proteomics data.

Data-independent acquisition (DIA) has become a widely used strategy for peptide and protein quantification in mass spectrometry-based proteomics studies. The integration of ion mobility separation into DIA analysis, such as the diaPASEF technology available on Bruker's timsTOF platform, further improves the quantification accuracy and protein depth achievable using DIA. We introduce diaTracer, a new spectrum-centric computational tool optimized for diaPASEF data. diaTracer performs three-dimensional (m/z, retention time, ion mobility) peak tracing and feature detection to generate precursor-resolved "pseudo-MS/MS" spectra, facilitating direct ("spectral-library free") peptide identification and quantification from diaPASEF data. diaTracer is available as a stand-alone tool and is fully integrated into the widely used FragPipe computational platform. We demonstrate the performance of diaTracer and FragPipe using diaPASEF data from triple-negative breast cancer (TNBC), cerebrospinal fluid (CSF), and plasma samples, data from phosphoproteomics and HLA immunopeptidomics experiments, and low-input data from a spatial proteomics study. We also show that diaTracer enables unrestricted identification of post-translational modifications from diaPASEF data using open/mass-offset searches.

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.