Establishing an untargeted lipidomics workflow for cellular analysis: insights into endothelial cell function in anaphylaxis

BackgroundRheumatoid arthritis (RA) is a kind of autoimmune disease characterized with chronic aggressive arthritis, presence of abnormal antibodies and persistent synovitis[1]. However, the pathogenesis of RA remained unclear by now....

Botulinum toxin type A activates protective autophagy by modulating endoplasmic reticulum stress in hypoxia/reoxygenation-treated endothelial cells

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

Positive correlations between volatile organic compounds (VOCs) and defense chemicals indicate signaling of defense status. Such aposematic signaling has been hypothesized to be widespread in plants, however, it has up to now only been shown for visual signals. Correlations between identical compounds in different plant tissues, on the other hand, can be informative about the (co-)regulation of their biosynthesis or emission. Here I use Brassica rapa to investigate 1) correlations between identical metabolites (volatiles, glucosinolates) in leaf and flower tissue, and 2) correlations between volatiles and glucosinolates in the same plant organs (flowers and leaves). Whereas the amounts of many glucosinolates were positively correlated in leaves and flower tissue, identical leaf and floral VOCs showed no such correlations, indicating independent regulation of emission. None of the leaf or flower volatiles showed positive correlations with the two major glucosinolates (gluconapin, glucobrassicanapin) or the sum of all glucosinolates in either leaves or flowers. Some VOCs, however, showed positive correlations with minor glucosinolates which, however, represented less than one percent of the total amounts of glucosinolates. Some leaf monoterpenes showed negative associations with gluconapin. The lack of consistent positive correlations between VOCs and major defense compounds suggests that plants do not chemically signal their defense status. This could be adaptive as it may avoid eavesdropping by specialist herbivores to locate their host plants. Negative correlations likely indicate chemical trade-offs in the synthesis of secondary metabolites.

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

To evaluate a novel measurement method based on X-ray images for accurately determining the relationship between pedicle screws and the medial wall of the pedicle in thoracic and lumbar spine surgery. A total of 650 pedicle screws from 126 patients who underwent thoracic or lumbar internal fixation surgery were included. Postoperative X-ray images were used to calculate the distance between the medial edge of the screw threads and the medial wall of the pedicle ("b-x") based on our measurement method. This distance was also measured using postoperative CT ("Y"). The agreement of "b-x" and "Y" was assessed using Spearman's correlation analysis, intraclass correlation coefficient (ICC) and Bland-Altman plot analysis. The cutoff value of "b-x" and its accuracy in predicting "Y" value was evaluated using the receiver operating characteristic (ROC) curve. Of the 650 screws, 59 (9.08%) showed medial wall breaching on CT. A strong positive correlation (r = 0.82) and strong agreement (ICC = 0.855) were shown between "b-x" and "Y" in Spearman's correlation and ICC analysis, respectively. Bland-Altman plot analysis indicated most values (96.2%) fell within the 95% confidence interval. The cutoff value for predicting medial wall breaching ("Y" < 0) was determined as "b-x" < 0.97mm, with sensitivity of 89.83% and specificity of 88.49%. This novel measurement method, based on X-ray images, accurately determines the relationship between the pedicle screws and medial wall of the pedicle. It offers the advantages of high efficiency and low cost, facilitating timely detection and correction of screws that breach the medial pedicle wall during surgery.

Chronic inflammation in diabetes mellitus (DM) is the leading cause of non-healing wounds. Chemokine CC motif ligand 4 (CCL4) is enhanced in the circulation and in the wounds of DM patients. This study aimed to investigate the effect of endogenous CCL4 inhibition on diabetic wound healing. Endothelial progenitor cells (EPCs) and human dermal microvascular endothelial cells (HDMECs) were used. Mice were injected with streptozotocin to generate hyperglycemia. An enhanced CCL4 level as well as decreased tube formation and migration abilities were observed in high-glucose-treated HDMECs and in EPCs from type 2 DM patients. CCL4 inhibition by siRNA restored the damaged cell function by upregulating the Akt/endothelial nitric oxide synthase/vascular endothelial growth factor/stromal cell-derived factor-1α pathways. Wild-type diabetic mice had delayed wound repair, whereas the CCL4-knockout diabetic mice showed an accelerated rate of wound closure. In a Matrigel plug assay, CCL4-knockout diabetic mice showed higher blood vessel and hemoglobin levels. Higher CD31 and Ki67 expression in the wound area and Matrigel plugs was detected in the CCL4-knockout diabetic mice. CCL4-knockout mice had upregulated angiogenic factors and downregulated inflammatory factors. This study might provide the theoretical basis for CCL4 inhibition as a therapeutic option for clinical diabetic wound treatment.

Inhibition of Adipocyte-Derived FABP4 Reduces Adipocyte Inflammation, Improves Angiogenesis, and Facilitates Wound Healing in Metabolic Dysfunctions

Linear Relationship Between Lymphocyte Counts in Peripheral Blood and Buffy Coat During Extracorporeal Photopheresis in Patients with Graft-Versus-Host Disease

CXCR6+CD8+T cells have been implicated in the pathogenesis of various liver and autoimmune diseases. However, their involvement in primary biliary cholangitis (PBC) has not been elucidated. We used immunohistochemistry and flow cytometry to quantify CXCR6+CD8+T cells in hepatic tissue and peripheral blood samples obtained from CXCR6+CD8+T cells obtained from PBC patients. Then, we performed comprehensive statistical analyses to access the correlation between the abundance of these cells and clinical as well as pathological data across different stages of PBC. Our research revealed that CXCR6+ cell frequencies in CD3+CD8+T cells from PBC patients significantly exceeded that of healthy controls (HCs) (2.24 vs. 0.61%, p < 0.01). A similar pattern emerged for hepatic CXCR6+CD8+T cell counts, which were notably higher in the PBC cohort compared to HCs. Our cohort consisted of 118 PBC patients, categorized into 62 early-stage (E-PBC) and 56 late-stage (L-PBC) cases. Notably, significant disparities existed between these groups in terms of liver enzyme and lipid profile levels (p < 0.05), with no notable differences observed in gender, age, blood counts, cholesterol levels, or autoantibodies (p > 0.05). Intriguingly, the quantity of hepatic CXCR6+CD8+T cells per high power field (HPF) was significantly elevated in both E-PBC and L-PBC patients as opposed to normal liver samples, indicating a substantial increase in these cells across all stages of PBC (p = 0.000). Spearman's rank correlation analysis showed a positive correlation between CXCR6+CD8+T cell counts and serum levels of Alkaline Phosphatase (AKP) and Gamma-Glutamyl Transferase (GGT), ANA, IgG and IgM, while revealing a negligible correlation with Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST). Subsequent findings indicated significant variances in CXCR6+ cell numbers not only among different PBC stages but also across various degrees of inflammation and fibrosis (p ≤ 0.007). In a follow-up study post-Ursodeoxycholic Acid (UDCA) treatment, stark differences were identified in biochemical and immunohistochemical profiles between responder (31 patients) and non-responder (33 patients) groups (p < 0.05). A Wilcoxon rank-sum test further demonstrated a significant difference in the level of hepatic CXCR6+CD8+T cells between these two response groups (p = 0.002). CXCR6+CD8+T cells play a vital role in the pathogenesis of PBC, exhibiting correlations with the extent of inflammation, staging of liver fibrosis, and response to pharmacological interventions in PBC patients.

ObjectiveAcute myocardial infarction (AMI) is typically caused by an abrupt blockage of a coronary artery, leading to necrosis of the affected heart tissue. Immediately, distinct leukocyte cell populations infiltrate the injured heart mediating self‐repair and regeneration of the injured tissue as well as pathological autoimmune responses. The latter of which may subsequently lead to ventricular remodeling and heart failure. Hence, regulating this immune response may prove beneficial as an adjunct therapy in the management of AMI. B lymphocytes act as important sensors of pathogens and self‐antigens and promote both protective and autoimmune effects. We explored the role of subtypes of maturating B lymphocytes in the setting of ischemic injury.MethodsWe quantified the number of mature B cells in the arterial blood of 31 AMI patients (23% women, age 66±15 years). Myocardial ischemia was determined by coronary angiography. Additionally, 10 healthy individuals (30% women, age 59±7 years) were included. Absolute cell counts were assessed in whole blood, drawn from the arterial port prior to coronary intervention, by multi‐color FACS analysis. Mature CD19+ B cells were further subdivided in CD19+/IgD+/CD27− naïve B cells, CD19+/IgD+/CD27−/CD38++/CD24++ transitional B cells, CD19+/IgD−/CD27++ plasma cells, and CD19+/IgD+/CD27+ memory B cells. The total number of cells was normalized to the white blood cell count of the whole blood analysis. Linear regression analysis was performed to associate total B cell counts with troponin I and creatine kinase (CK) plasma levels in MI patients. The multivariable regression model comprised sex, age, current smoking, symptom duration, leukocyte count, and CRP‐levels. Risk assessment for AMI patients was done using the GRACE score, which was calculated using http://www.gracescore.org. The variables that constitute the GRACE score are age, heart rate, systolic blood pressure, and serum creatinine.ResultsThe study population included 14 patients with ST‐segment elevation MI (STEMI) (64±14 years) and 17 patients with non‐ST‐segment elevation MI (NSTEMI) (68±15 years). Mean left ventricular ejection fraction was 53% (40–60%) in STEMI and 57% (38–63%) in NSTEMI patients. The number of CD19+ B cells was significantly increased in STEMI patients compared to controls (31.9±6.2 ×104 vs. 13.6±3.3 ×104 cells/mL; p=0.026) and NSTEMI group (31.9±6.2 ×104 vs. 15.9±2.1 cells/mL; p=0.016). Results were associated with plasma troponin I and CK and revealed a strong relation of the CD19+ B cells count with these markers of the severity of infarction (table 1). Further, investigation of B cell subpopulations revealed a higher number of cells in STEMI patients compared to controls and to NSTEMI group (table 1). Noteworthy, only naïve B cells and memory cells show a correlation to troponin I (Pearson r=0.770; p=0.0005 and r=0.717; p=0.0058, respectively) and CK (Pearson r=0.827; p=0.0005 and r=0.717; p=0.0058, respectively). Finally, the number of memory cells correlates to the 6 months probability of death from admission, which was calculated from the GRACE Score (Pearson r=0.609; p=0.0466).ConclusionOur data indicate a potential relation of elevated B cell counts with adverse outcomes in patients with AMI. Further, the strong association of naïve and memory B cells with markers of myocardial damage suggests a prevalent role for these subsets. How B cells contribute to the injured heart itself is an area of future investigations.Support or Funding InformationThis study is funded by the German Centre for Cardiovascular Research (DZHK) Partner Site Greifswald, Greifswald, Germany.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phyllanthus emblica L. is a medicinal and edible plant renowned for its potent antioxidant and anti-inflammatory activity. However, the digestive stability, bioaccessibility, and intestinal protective effects of its polyphenolic components remain unclear. This study aimed to evaluate systematically the in vitro digestion behavior, antioxidant capacity, and anti-colitis potential of P. emblica extract (PE). The bioaccessibility of total phenolics in PE was 92.03%, indicating high digestive stability, whereas the bioaccessibility of flavonoids was 42.69%, indicating partial degradation during digestion. Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) identified 3-O-galloylmucic acid and gallic acid as the dominant and stable phenolics throughout all digestive phases. Phyllanthus emblica L. retained strong antioxidant capacity across the oral, gastric, and intestinal stages. In a zebrafish model of trinitrobenzenesulfonic acid (TNBS)-induced inflammatory bowel disease, PE significantly ameliorated intestinal injury, reduced gut lumen expansion, inhibited neutrophil recruitment, and restored goblet cell numbers. Gene expression analysis showed a dose-dependent downregulation of pro-inflammatory cytokines (tnf-α, il-1β, il-6) (P < 0.05). Phyllanthus emblica L. extract exhibited high digestive stability and polyphenol bioaccessibility while maintaining antioxidant activity. Its pronounced anti-inflammatory and mucosal-protective effects in vivo provided evidence supporting the potential application of P. emblica as a functional food ingredient for intestinal health and inflammatory bowel disease prevention. © 2025 Society of Chemical Industry.

Purpose: To investigate the effect of niclosamide on the radiosensitivity of MCF-7 cancer stem cells (CSC) and to explore the molecular mechanism of radiosensitization. Material and methods: Cancer stem cells were obtained from MCF-7 mammary adenocarcinoma monolayer culture (2D) and propagated as mammospheres (3D) in serum-free DMEM/F12 supplemented with EGF, bFGF, B 27, heparin, and insulin in ultra-low attachment plates. The single cell suspensions were irradiated with 1, 2, 4, 6, and 8 Gy using 60Co source at a dose rate of 0.54 Gy/min. Controls were sham irradiated. The size of the CD44+/CD24-/low CSC fraction was measured using flow cytometry at the 7th and 14th days after irradiation. The radiosensitizing effect of niclosamide on the 2D- and 3D-cell cultures was assessed by the number of cells at the 7th and 14th days after irradiation with doses of 2 or 4 Gy. Niclosamide was added to the cell cultures in a dose of 2 µM 1 h before irradiation. Media with niclosamide were changed to corresponding media without niclosamide 24 h post-irradiation. Cell survival was evaluated by hemocytometer cell counting using trypan blue. The size of the CSC fraction was measured at the 7th day after the irradiation with or without niclosamide. To investigate the effect of niclosamide on DNA damage the level of histone γH2AX was measured by flow cytometry at 60 min after irradiation. Results: Culturing adherent MCF-7 cells line under non-adherent mammosphere-forming conditions led to significantly increase in CSCs proportion. CD44+/CD24–/low cell fraction in 2D-culture was 0.2 ± 0.1 %, and it was 3.2 ± 0.6 % in 3D-culture (the third-generation spheres). Subpopulation of CSCs increased with the increase in irradiation dose. At day 14 after irradiation with the dose of 8 Gy, CD44+/CD24–/low cell fraction in 2D-culture was 2.2 ± 0.6 %, and it was 12.0 ± 0.9 % in 3D-culture. Cells grown in mammosphere-culture were found to have a higher survival rate after irradiation at doses of 2 and 4 Gy than cells grown in adherent conditions. At day 7 after irradiation with 2 and 4 Gy the 3D-culture cells counts decreased less than the 2D-culture cells counts (92 ± 13 % and 61 ± 10 % versus 58 ± 4 % and 37 ± 3 % compared to non-irradiated control). Moreover, cells grown in mammosphere-culture showed a higher renewal capacity. At day 14 after irradiation with 2 and 4 Gy the number of 3D-culture cells was 66 ± 11 % and 30 ± 7 % while the number of adherent cells was 43 ± 2 % and 7 ± 1 % compared to non-irradiated control. It was found that niclosomide has radiosensitizing effect on cells grown in mammospheres. Pretreatment with niclosamide 1 h prior to irradiation resulted in decrease in cell survival. At day 7 after irradiation at 2 and 4 Gy in the presence of niclosamide, the numbers of 3D-culture cells were reduced by 13 % and 7 % compared to their numbers after irradiation with appropriate doses in the absence of niclosamide. Furthermore, niclosamide was shown to reduce the percentage of CSCs. At day 7 after irradiation with 2 and 4 Gy, 3D-culture showed elevated percentages of CD44+/CD24−/low cells: CSC fraction in mammospheres were 4.6 ± 0.3 % and 6.1 ± 0.5 %, respectively, and after pretreatment by niclosamide and irradiation the sizes of the CSC fractions were 2.5 ± 0.1 % and 4.0 ± 0.3 %, respectively. Radiosensitizing effect of niclosamide lasted for 2 weeks. At day 14 after irradiation at 2 and 4 Gy in combination with niclosamide the numbers of 3D-culture cells were reduced by 13 % and 9 % compared to cell numbers after irradiation with appropriate doses without of niclosamide. Radiosensitization of cells grown in mammospheres might be related with increase in DNA double-strand breaks (DSB) prodused by niclosomide. At 1 h after 2 and 4 Gy irradiation cells grown in mammospheres shown 2.4-fold and 4.8-fold increase in DNA DSB, as indicated by γH2AX levels, compared to baseline. The combined treatment of niclosamide and irradiation with 2 and 4 Gy resulted in 3.4-fold and 5.5-fold increase in the levels of histone γ-H2AX, respectively. Conclusion: Niclosamide increased the radiosensetivity not only of the bulk cells of mammary adenocarcinoma line MCF-7 but also of CSCs. The radiosensitizing effect of niclosamide was associated with increase in the level of DNA damage.

Lentiviral vectors (LV) are emerging tools for genetic therapies and novel cancer treatments. While effective, LV-based therapies have extremely large costs associated with their manufacturing and delivery. LV technology descends from human immunodeficiency virus (HIV), whose lipid envelope has been previously measured and shown to have a direct impact on its transduction efficiency. We developed a rapid, robust, and sensitive untargeted lipidomics pipeline to analyze novel LV biotherapeutic products and demonstrate its utility on HEK 293T packaging cells and concentrated culture media containing LV. The impact of 48 hours of LV production on the lipidome of HEK 293T cells was measured and compared to the expression of vesicular stomatitis virus G protein (VSV G) over the same timeframe. 151 lipids were identified in HEK 293T packaging cells, 84 of which had fold changes with FDR-corrected P < 0.05 compared to HEK 293T treated with media. It was found that fold changes with FDR-adjusted P < 0.05 after VSV G expression and LV production were highly correlated (R2 = 0.89). Concentrating LV in culture media led to the identification of 102 lipids, half of which were determined to be unique LV virion lipids after subtracting the media lipidome. Our approach can be readily used to study the lipid dynamics of large-scale LV production and be rapidly translated into targeted methods to quantify individual lipid components or applied to other viral vector platforms.

The objective of our work was to investigate the influence of Fasudil, a Rho inhibitor on the number and function of the late endothelial progenitor cells in peripheral blood of chronic obstructive pulmonary diseases (COPD) patients with pulmonary artery hypertension. Eighty COPD patients with pulmonary artery hypertension were selected and divided into two groups: the treatment group and the control group, which had 40 patients respectively. The control group received routine treatment, including oxygen uptake, anti-infection and phlegm dissolving. The treatment group received the Fasudil in addition to the routine treatment. The changes on the number and function of the late endothelial progenitor cells in peripheral blood of the patients before and after the treatment were compared between the two groups. The changes on the pulmonary artery pressure were also compared. The number of the late endothelial progenitor cells in peripheral blood of the treatment group increased and the function was enhanced. The pulmonary artery pressure was reduced. The difference before and after the treatment and with the control group was statistically significant (p<0.05). The changes on the number and function of the late endothelial progenitor cells in peripheral blood and the pulmonary artery pressure before and after the treatment of the control group were not statistically significant (p>0.05). The Rho-kinase inhibitor Fasudil increased the number and enhanced the function of the late endothelial progenitor cells in peripheral blood of COPD patients with pulmonary artery hypertension.