Concentration In Cells Research Articles

Automated H2O2 monitoring during photo-Fenton processes using an Arduino self-assembled automatic system

A cheap and easy to use Arduino self-assembled automatic system was employed to continuously monitor the hydrogen peroxide consumption during the photo-Fenton degradation of caffeine, selected as model target compound. The automatic system made it possible to measure the H2O2 concentration in the reaction cell via a colorimetric reaction and to take samples for HPLC analysis minimising the operator manual intervention and exposure to UV radiation. The obtained results were compared in terms of LOD and LOQ with H2O2 measurements manually performed using UV-Vis spectrophotometry, evidencing better analytical performance when using the automatic system; LOD and LOQ were respectively 0.032mM and 0.106mM for the automatic system against 0.064mM and 0.213mM for UV-Vis spectrophotometry. Furthermore, the photo-Fenton treatment was optimised by means of a Design of Experiments (DoE) investigating the effect of added H2O2 concentration, iron concentration and caffeine initial concentration on system performances. The use of the automatic device for such monitoring provided several advantages: automation (with consequent reduction of the workload), measurement increased precision, reduced reagents consumption and waste production in agreement with the principles of Green Analytical Chemistry.

Contact Lens that Enabled Early Diagnosis for Diseases Induced by Oxidative Stress and Potassium Ion (K+) in Ocular Tissues

The present study used sensor technology to design a lens that could replace a docto.r It consists of an oxidative stress sensor, a (K+) ion sensor and a pressure sensor placed on a Lotrafilcon A silicone hydrogel lens for early diagnosis, as well as a recording and display device that the user can use on their own, recording 24 hours a day and alerting when needed. Additionally, power will be provided to the sensors for data transmission via an external wireless power transfer device. The oxidative stress sensor detects oxidative stress in the structures of the eye and indicates that the patient may have an abnormal condition like diabetic retinopathy, glaucoma and cataracts. Likewise, the (K +) ion sensor detects the (K +) ion concentration in the ocular cells and detects abnormal conditions where the concentration is elevated. The pressure sensor allows the intraocular pressure of patients diagnosed with glaucoma to be kept under control. This way, diseases can be diagnosed early, and continuous monitoring and control of the disease will be ensured. This will prevent the user from having to make frequent visits to the hospital, while also reducing the workload.

Vitamin D triggers hCAP18/LL-37 production: Implications for LL-37-induced human osteoblast cytotoxicity

The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3–4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 μM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.

Translational research of boron neutron capture therapy for spinal cord gliomas using rat model

Boron neutron capture therapy (BNCT) is a type of targeted particle radiation therapy with potential applications at the cellular level. Spinal cord gliomas (SCGs) present a substantial challenge owing to their poor prognosis and the lack of effective postoperative treatments. This study evaluated the efficacy of BNCT in a rat SCGs model employing the Basso, Beattie, and Bresnahan (BBB) scale to assess postoperative locomotor activity. We confirmed the presence of adequate in vitro boron concentrations in F98 rat glioma and 9L rat gliosarcoma cells exposed to boronophenylalanine (BPA) and in vivo tumor boron concentration 2.5 h after intravenous BPA administration. In vivo neutron irradiation significantly enhanced survival in the BNCT group when compared with that in the untreated group, with a minimal BBB scale reduction in all sham-operated groups. These findings highlight the potential of BNCT as a promising treatment option for SCGs.

Carrier-Free Self-Assembly Nano-Sonosensitizers for Sonodynamic-Amplified Cuproptosis-Ferroptosis in Glioblastoma Therapy.

Cuproptosis is a newly discovered form of programmed cell death significantly depending on the transport efficacy of copper (Cu) ionophores. However, existing Cu ionophores, primarily small molecules with a short blood half-life, face challenges in transporting enough amounts of Cu ions into tumor cells. This work describes the construction of carrier-free nanoparticles (Ce6@Cu NPs), which self-assembled by the coordination of Cu2+ with the sonosensitizer chlorin e6 (Ce6), facilitating sonodynamic-triggered combination of cuproptosis and ferroptosis. Ce6@Cu NPs internalized by U87MG cells induce a sonodynamic effect and glutathione (GSH) depletion capability, promoting lipid peroxidation and eventually inducing ferroptosis. Furthermore, Cu+ concentration in tumor cells significantly increases as Cu2+ reacts with reductive GSH, resulting in the downregulation of ferredoxin-1 and lipoyl synthase. This induces the oligomerization of lipoylated dihydrolipoamide S-acetyltransferase, causing proteotoxic stress and irreversible cuproptosis. Ce6@Cu NPs possess a satisfactory ability to penetrate the blood-brain barrier, resulting in significant accumulation in orthotopic U87MG-Luc glioblastoma. The sonodynamic-triggered combination of ferroptosis and cuproptosis in the tumor by Ce6@Cu NPs is evidenced both in vitro and in vivo with minimal side effects. This work represents a promising tumor therapeutic strategy combining ferroptosis and cuproptosis, potentially inspiring further research in developing logical and effective cancer therapies based on cuproptosis.

Natural anti-neuroinflammatory inhibitors in vitro and in vivo from Aglaia odorata

Fifty compounds including seven undescribed (1, 13, 18–20, 30, 31) and forty-three known (2–12, 14–17, 21–29, 32–50) ones were isolated from the extract of the twigs and leaves of Aglaia odorata with anti-neuroinflammatory activities. Their structures were determined by a combination of spectral analysis and calculated spectra (ECD and NMR). Among them, compounds 13–25 were found to possess tertiary amide bonds, with compounds 16, 17, and 19–21 existing detectable cis/trans mixtures in 1H NMR spectrum measured in CDCl3. Specifically, the analysis of the cis–trans isomerization equilibrium of tertiary amides in compounds 19–24 was conducted using NMR spectroscopy and quantum chemical calculations. Bioactivity evaluation showed that the cyclopenta[b]benzofuran derivatives (2–6, 8, 10, 12) could inhibit nitric oxide production at the nanomolar concentration (IC50 values ranging from 2 to 100 nM) in lipopolysaccharide-induced BV-2 cells, which were 413–20670 times greater than that of the positive drug (minocycline, IC50 = 41.34 μM). The cyclopenta[bc]benzopyran derivatives (13–16), diterpenoids (30–35), lignan (40), and flavonoids (45, 47, 49, 50) also demonstrated significant inhibitory activities with IC50 values ranging from 1.74 to 38.44 μM. Furthermore, the in vivo anti-neuroinflammatory effect of rocaglaol (12) was evaluated via immunofluorescence, qRT-PCR, and western blot assays in the LPS-treated mice model. The results showed that rocaglaol (12) attenuated the activation of microglia and decreased the mRNA expression of iNOS, TNF-α, IL-1β, and IL-6 in the cortex and hippocampus of mice. The mechanistic study suggested that rocaglaol might inhibit the activation of the NF-κB signaling pathway to relieve the neuroinflammatory response.

A 50-gene biomarker identifies estrogen receptor-modulating chemicals in a microarray compendium

High throughput transcriptomics (HTTr) profiling has the potential to rapidly and comprehensively identify molecular targets of environmental chemicals that can be linked to adverse outcomes. We describe here the construction and characterization of a 50-gene expression biomarker designed to identify estrogen receptor (ER) active chemicals in HTTr datasets. Using microarray comparisons, the genes in the biomarker were identified as those that exhibited consistent directional changes when ER was activated (4 ER agonists; 4 ESR1 gene constitutively active mutants) and opposite directional changes when ER was suppressed (4 antagonist treatments; 4 ESR1 knockdown experiments). The biomarker was evaluated as a predictive tool using the Running Fisher algorithm by comparison to annotated gene expression microarray datasets including those evaluating the transcriptional effects of hormones and chemicals in MCF-7 cells. Depending on the reference dataset used, the biomarker had a predictive accuracy for activation of up to 96%. To demonstrate applicability for HTTr data analysis, the biomarker was used to identify ER activators in a set of 15 chemicals that are considered potential bisphenol A (BPA) alternatives examined at up to 10 concentrations in MCF-7 cells and analyzed by full-genome TempO-Seq. Using benchmark dose (BMD) modeling, the biomarker genes stratified the ER potency of BPA alternatives consistent with previous studies. These results demonstrate that the ER biomarker can be used to accurately identify ER activators in transcript profile data derived from MCF-7 cells.

Populus euphratica R2R3-MYB transcription factor RAX2 binds ANN1 promoter to increase cadmium enrichment in Arabidopsis

The expression of R2R3-MYB transcription factor PeRAX2 increased transiently upon CdCl2 exposure (100 μM, 48h) in leaves and roots of Populus euphratica. We observed that overexpression of PeRAX2 increased Cd2+ concentration in Arabidopsis root cells and Cd2+ amount in whole plant, which was due to the increased Cd2+ influx into root tips. However, the Cd2+ influx facilitated by PeRAX2 overexpression was substantially reduced by LaCl3 (an inhibitor of Ca2+-channels), suggesting that PeRAX2 could promote the Cd2+ entering through PM Ca2+-permeable channels (CaPCs) in the roots. It is noting that the expression of annexin1 (AtANN1), which mediates the influx of divalent cations through the PM calcium channels, was upregulated by Cd2+ in PeRAX2-transgenic Arabidopsis. Bioinformatic analysis revealed that the AtANN1 promoter (AtANN1-pro) contains four cis-elements for MYB binding. The PeRAX2 interaction with AtANN1-pro was validated by LUC reporter assay, EMSA, and Y1H assay. Our data showed that PeRAX2 binds to the AtANN1 promoter region to regulate gene transcription and that AtANN1 mediates the Cd2+ entry through CaPCs in the PM, leading to a Cd2+ enrichment in transgenic plants. The PeRAX2-stimulated Cd2+ enrichment consequently resulted in high H2O2 production in root cells of transgenic plants. The expression of AtSOD and AtPOD and activities of CAT, SOD, POD increased in the transgenic lines under Cd2+ stress. However, the Cd2+-upregulated expression and activity of antioxidative enzymes were less pronounced in the PeRAX2-overexpressed lines, compared to the wildtype and vector controls. As a result, root length and plant growth were more suppressed by Cd2+ in the transgenic lines. Our data suggest that transcriptional regulation of AtANN1 by PeRAX2 can be utilized to improve Cd2+ enrichment and phytoremediation, although the enriched Cd2+ affected antioxidant defense system and plant growth in the model species.

Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of β-arrestin in HSY human parotid cells

ObjectivesLocal anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and β-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor–β-arrestin complexes in the HSY human parotid cell line. MethodsCa2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and β-arrestin were examined using a β-arrestin GPCR assay kit. ResultsLidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 μM, respectively, for 50 μM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of β-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. ConclusionLocal anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of β-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.

Molecular mechanisms underlying Tao-Hong-Si-Wu decoction treating hyperpigmentation based on network pharmacology, Mendelian randomization analysis, and experimental verification

Context Hyperpigmentation, a common skin condition marked by excessive melanin production, currently has limited effective treatment options. Objective This study explores the effects of Tao-Hong-Si-Wu decoction (THSWD) on hyperpigmentation and to elucidate the underlying mechanisms. Materials and methods We employed network pharmacology, Mendelian randomization, and molecular docking to identify THSWD’s hub targets and mechanisms against hyperpigmentation. The Cell Counting Kit-8 (CCK-8) assay determined suitable THSWD treatment concentrations for PIG1 cells. These cells were exposed to graded concentrations of THSWD-containing serum (2.5%, 5%, 10%, 15%, 20%, 30%, 40%, and 50%) and treated with α-MSH (100 nM) to induce an in vitro hyperpigmentation model. Assessments included melanin content, tyrosinase activity, and Western blotting. Results ALB, IL6, and MAPK3 emerged as primary targets, while quercetin, apigenin, and luteolin were the core active ingredients. The CCK-8 assay indicated that concentrations between 2.5% and 20% were suitable for PIG1 cells, with a 50% cytotoxicity concentration (CC50) of 32.14%. THSWD treatment significantly reduced melanin content and tyrosinase activity in α-MSH-induced PIG1 cells, along with downregulating MC1R and MITF expression. THSWD increased ALB and p-MAPK3/MAPK3 levels and decreased IL6 expression in the model cells. Discussion and conclusion THSWD mitigates hyperpigmentation by targeting ALB, IL6, and MAPK3. This study paves the way for clinical applications of THSWD as a novel treatment for hyperpigmentation and offers new targeted therapeutic strategies.

The Xanthine Derivative KMUP-1 Inhibits Hypoxia-Induced TRPC1 Expression and Store-Operated Ca2+ Entry in Pulmonary Arterial Smooth Muscle Cells.

Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca2+]i through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O2/5% CO2 for 24 h to elucidate TRPC1 overexpression and observe the Ca2+ release and entry. KMUP-1 (1 μM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 μM) and the protein kinase A (PKA) inhibitor KT5720 (1 μM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 μM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 μM) and nifedipine (5 μM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca2+ entry upon SR Ca2+ depletion in hypoxic PASMCs.

Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells.

Phospholipids are vital constituents of the cell membrane and aid in signal transduction. Phospholipid profiles vary distinctively with the cell type. Notably, specific phospholipid molecules are present in significantly higher or lower concentrations in cancer cells versus normal cells. In this study, live single-cell mass spectrometry (MS) was developed for analyzing phospholipids at the single-cell level. This method facilitates rapid molecular analysis of cells under microscopic observation. For nanoelectrospray ionization, phospholipids were extracted from single cells isolated in a glass capillary through a high-efficiency process. Cell-derived phosphatidylcholines were detected with high sensitivity when trehalose C14 was added as a solubilizing reagent. Trehalose C14 can solubilize cells at low concentrations owing to its low critical micelle concentration, and exerts minimal matrix effects (such as suppressing ionization and causing peak overlap) in the MS analysis of cellular molecules. Analyses of phospholipids in Raji and HEV0070 cells using the developed method revealed specific peaks of phosphatidylcholine and sphingomyelin in the respective cells. The developed technique not only affords phospholipid profiles at the single-cell level, but also holds promise for identifying biomarkers associated with various diseases, particularly cancer.

Adsorbate coverage effects on the electroreduction of CO to acetate

CO electroreduction (COR), powered by renewable electricity, shows promise for producing green chemicals at the industrial scale. Here, we aim to understand why amongst C2 products, acetate is formed with relatively greater selectivity on Cu catalysts mounted in flow cells, as compared to in H-cells. We attribute this to improved CO transport in flow cells, which increases local pH and *CO coverage (θ*CO). The effect of local pH was verified through COR in different electrolytes, which shows a significant increase in the Faradaic efficiency (FE) of acetate as local pH rose. Higher CO concentrations in flow cells under similar local pH also substantially increased the FEacetate, as compared to FEethylene and FEethanol. This suggests that acetate formation is most sensitive to θ*CO. DFT calculations emphasize the importance of θ*CO in favoring acetate formation from the *CHCO intermediate, and reveals the role of lateral interactions in the production of C2 molecules.

Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, aphenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Biological effects and phytochemical study of the underground part of Iris scariosa Willd. ex Link extract: A new source of bioactive constituents

The expected toxicity and resistance of chemotherapeutic agents necessitate and encourage for the use of natural chemotherapeutic sources of plant origin in the clinical stage of cancer therapy. Plants of the genus Iris (Iridaceae) used by local populations for the treatment of cancer, bacterial and viral infections. In this study, an ethanol extract of rhizomes of I. scariosa was prepared and tested for the cytotoxicity using the MTT assay. The extract exhibited the most potent cytotoxicity against the breast cancer cell line MCF7 (IC50 = 9.28 ± 0.49 μg/ml, selectively index ˃5), and induced apoptosis in MCF7 lines. Notably, the extract significantly inhibited the colony formation of MCF7 and HepG2 cancer cells at a concentration range from 10.6 to 85.0 μg/ml, including non-toxic concentrations for HepG2 cells. The ethanol extract was analyzed by HPLC, revealed the identification of 5 secondary metabolites (quercetin, rutin, myricetin, apigenin, artemisetin), the content of which was shown to reach around 15% of the extract. The petroleum ether (PE) part of the extract (yield 2.62%) was analyzed by GC–MS. The composition of tert-butyl methyl ether (TBME) part of the extract (yield 23.72%) was studied. Total of 15 individual compounds: two benzophenones, eight isoflavones, four flavones and a (2R)-flavanone were isolated. The pentamethoxyflavone artemisetin and flavanone pinocembrin were isolated for the first from Iris sp. The readily available isoflavones from the TBME part of extract (irilone, iriflogenin, irigenin and tectorigenin) may serve as new leads for the discovery of anticancer drugs.

A Novel CaCu-Metal-Organic-Framework Based Multimodal Treatment Platform for Enhanced Synergistic Therapy of Hepatocellular Carcinoma.

Metal ions have attracted a lot of interest in antitumor therapy due to their unique mechanism of action. However, multiple death mechanisms associate with metal ions to synergistic antitumors have few studies mainly due to the serious challenges in designing and building metal-associated multimodal treatment platforms. Hence, a series of glutathione-activatable CaCu-based metal-organic-frameworks loaded with doxorubicin and ovalbumin are successfully designed and synthesized with an "all in one" strategy, which is modified by galactosamine-linked hyaluronic acid to prepare multimodal treatment platform (SCC/DOX@OVA-HG) for targeted delivery and synergistic antitumor therapy. SCC/DOX@OVA-HG can be rapidly degraded by the overexpressed glutathione and then releases the "cargoes" in the tumor microenvironment. The released Cu+ efficiently catalyzes H2O2 to produce highly toxic ROS for CDT, and the up-regulation of calcium ion concentration in tumor cells induced by the released Ca2+ enables calcium overload therapy, which synergically enhances the metal-related death pattern. Meanwhile, OVA combined with Ca2+/Cu2+ further activates macrophages into an M1-like phenotype to accelerate tumor cell death through immunotherapy. Besides, the released DOX can also insert into the DNA double helix for chemotherapy. Consequently, the developed SCC/DOX@OVA-HG reveals significantly improved antitumor efficacy through a multimodal synergistic therapy of chemotherapy, chemodynamic therapy, calcium overload, and immunotherapy.

Abstract 1171: Estimating sensitivity of exploratory cellular biomarkers in oncology clinical trials

Abstract Background: In the prolonged and complex process of assessing immunotherapeutic oncology drug candidates, flow cytometry plays an essential role. In oncology studies, biomarker measurement requires robust and reliable flow cytometric assays. A crucial element of these assays is to understand the lower limits of measurable surface biomarkers. Methods: We incorporated into the development and validation of flow cytometry assays the measurement of the lower limit of quantitation (LLOQ). The LLOQ is the lowest number of a reportable parameter in a blood or bone marrow study sample that can be quantitatively determined with acceptable precision and accuracy. Two steps are necessary to determine the LLOQ. First in these assessments a measurable range of lower limits of a particular cell type or receptor is estimated. That is, in validating an assay LLOQ, there are three options to select cells expressing target surface biomarkers of interest: using cultured or bioengineered cells, using samples of known high expression of a biomarker, or using pre-titered positive samples. Then decreasing concentrations of the target cell or receptor may be used to precisely determine the LLOQ of a specific assay reportable parameter. Results: By acquiring at least ten negative control sample results, the limit of the blank (LOB) and limit of detection (LOD) of a biomarker is determined. LOB is calculated as mean +1.645 SD, and LOD as mean +3 SD. The negative control samples either lack the fluorescent monoclonal antibodies against the markers of interest or lack the target cell population. Different reportable parameters, detected by one or more biomarkers have varying LLOQ. The LLOQ for a reportable parameter is a precise measurement of a parameter above or equal to the LOD. We found that typical LLOQ values are from 4 to 145 positive cells, with a frequency of 0.01 to 0.1% among a parent population. For example, in the case of tetramer assays, flow cytometry can reliably detect as few as 25 CD8+ antigen-specific cells among three million lymphocytes. Conclusions: LLOQ determinations for exploratory flow cytometry biomarkers are required for clinical research in cancer immunotherapy and cancer vaccine development. This valuable information allows for interpretation of data at the low end of a measurable range. Citation Format: Chengsen Xue, Christina D. Swenson, Thomas W. Mc Closkey. Estimating sensitivity of exploratory cellular biomarkers in oncology clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1171.

O247: In vitro exposure of gastric cancer cell line AGS to the cannabinoid cannabidiol (CBD) results in cytotoxicity in a dose-dependent manner

Abstract Introduction Current curative therapeutic regimens for gastric cancer combine chemotherapy with surgery, though this approach is associated with morbidity and mortality and relatively low long-term survival when compared to other solid organ and gastro-intestinal tumours. Cannabinoids are known to have anti-cancer properties in some tumours, though their role in gastric cancer is relatively unknown. This study aimed to determine if the non-psychoactive cannabidiol (CBD) induces cytotoxicity in the gastric cancer cell line AGS. Method The gastric cancer cell line AGS was maintained as per recommended guidelines. When cells reached 70% confluence, they were seeded in 96 well plates and treated with CBD or cisplatin at concentrations ranging from 0.2 μM to 100 μM or vehicle control. After 96 hours contact time, cell viability was assessed using a colorimetric cell viability assay (MTT assay). Experiments were repeated independently four times. Cell viability at each concentration was recorded and half maximal inhibitory concentration (IC50) was calculated. Results were expressed as the mean ± SE mean. Results Treatment with cisplatin induced significant cytotoxicity in a dose dependent manner with an IC50 concentration of 1.8 ± 0.3μM. Treatment with CBD also induced cytotoxicity in a dose dependent manner. Cytotoxic effects were observed with concentrations as low as 3.1μM (78% viability). The IC50 concentration for CBD was 10.2 ± 2.5 μM. Conclusions CBD induced significant cytotoxicity at micromolar concentration in stomach carcinoma cells. Further studies should be carried out to investigate the mechanisms of action that underpin the observed in vitro cytotoxic effect.

Nonlinear observer for online concentration estimation in vanadium flow batteries based on half-cell voltage measurements

This paper presents a nonlinear observer to estimate the active species concentrations in vanadium flow batteries. To conduct the estimation, the observer relies only on current, flow rate and two half-cell voltage measurements. In contrast to previous works in the field, the proposed observer is capable to deal simultaneously with two significant and challenging conditions: (1) a not necessarily high flow rate, which results in different concentrations for tanks and cells, and (2) presence of crossover and oxidation side reactions, that result in imbalance between the electrolytes on the positive and negative sides of the system. The stability and convergence of the observer are formally demonstrated using a Lyapunov analysis and subsequently validated through comprehensive computer simulations. Finally, utilising the information provided by the observer, a strategy to independently regulate the flow rate of each electrolyte based on their individual state of charge is developed.

Preventive effect of propolis on cognitive decline in Alzheimer’s disease model mice

Brazilian green propolis (propolis) is a chemically complex resinous substance that is a potentially viable therapeutic agent for Alzheimer’s disease. Herein, propolis induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in Neuro-2A cells; moreover, propolis-induced [Ca2+]i elevations were suppressed prior to 24-h pretreatment with amyloid-β. To reveal the effect of [Ca2+]i elevation on impaired cognition, we performed memory-related behavioral tasks in APP-KI mice relative to WT mice at 4 and 12 months of age. Propolis, at 300–1000 mg/kg/d for 8 wk, significantly ameliorated cognitive deficits in APP-KI mice at 4 months, but not at 12 months of age. Consistent with behavioral observations, injured hippocampal long-term potentiation was markedly ameliorated in APP-KI mice at 4 months of age following repeated propolis administration. In addition, repeated administration of propolis significantly activated intracellular calcium signaling pathway in the CA1 region of APP-KI mice. These results suggest a preventive effect of propolis on cognitive decline through the activation of intracellular calcium signaling pathways in CA1 region of AD mice model.