Fluorescence Microplate Reader Research Articles

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Wheat Germ Agglutinin-Conjugated Disulfide Cross-Linked Alginate Nanoparticles as a Docetaxel Carrier for Colon Cancer Therapy.

PurposeIn chemotherapy, oral administration of drug is limited due to lack of drug specificity for localized colon cancer cells. The inability of drugs to differentiate cancer cells from normal cells induces side effects. Colonic targeting with polymeric nanoparticulate drug delivery offers high potential strategies for delivering hydrophobic drugs and fewer side effects to the target site. Disulfide cross-linked polymers have recently acquired high significance due to their potential to degrade in reducing colon conditions while resisting the upper gastrointestinal tract’s hostile environment. The goal of this project is, therefore, to develop pH-sensitive and redox-responsive fluorescein-labeled wheat germ agglutinin (fWGA)-mounted disulfide cross-linked alginate nanoparticles (fDTP2) directly targeting docetaxel (DTX) in colon cancer cells.MethodsfDTP2 was prepared by mounting fWGA on DTX-loaded nanoparticles (DTP2) using the two-step carbodiimide method. Morphology of fDTP2 was examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Dynamic light scattering (DLS) study was carried out to determine the mean diameter, polydispersity index (PDI) and zeta potential of fDTP2. Cellular uptake efficiency was examined using fluorescence microplate reader. Biocompatibility and active internalization of fDTP2 were conducted on HT-29.ResultsfDTP2 was found to exhibit a DTX loading efficiency of 19.3%. SEM and TEM tests revealed spherical nanoparticles. The in vitro DTX release test showed a cumulative release of 54.7%. From the DLS study, fDTP2 reported a 277.7 nm mean diameter with PDI below 0.35 and −1.0 mV zeta potential. HT-29 which was fDTP2-treated demonstrated lower viability than L929 with a half maximal inhibitory concentration (IC50) of 34.7 µg/mL. HT-29 (33.4%) internalized fDTP2 efficiently at 2 h incubation. The study on HT-29 active internalization of nanoparticles through fluorescence and confocal imaging indicated such.ConclusionIn short, fDTP2 demonstrated promise as a colonic drug delivery DTX transporter.

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

SCARF1 promotes M2 polarization of Kupffer cells via calcium-dependent PI3K-AKT-STAT3 signalling to improve liver transplantation.

ObjectivesThis study aimed to investigate the protective effect of SCARF1 on acute rejection (AR), phagocytic clearance of Kupffer cells (KCs), M2 polarization and the exact mechanism underlying these processes.MethodsAAV was transfected into the portal vein of rats, and AR and immune tolerance (IT) models of liver transplantation were established. Liver tissue and blood samples were collected. The level of SCARF1 was detected via WB and immunohistochemical staining. Pathological changes in liver tissue were detected using HE staining. Apoptotic cells were detected using TUNEL staining. KC polarization was assessed via immunohistochemical staining. Primary KCs were isolated and co‐cultured with apoptotic T lymphocytes. Phagocytosis of apoptotic cells and polarization of KCs were both detected using immunofluorescence. Calcium concentration was determined using immunofluorescence and a fluorescence microplate reader. The levels of PI3K, p‐AKT and P‐STAT3 were assessed via WB and immunofluorescence.ResultsCompared to the IT group, the level of SCARF1 was significantly decreased in the AR group. Overexpression of SCARF1 in KCs improved AR and liver function markers. Enhanced phagocytosis mediated by SCARF1 is beneficial for improving the apoptotic clearance of AR and promoting M2 polarization of KCs. SCARF1‐mediated enhancement of phagocytosis promotes increased calcium concentration in KCs, thus further activating the PI3K‐AKT‐STAT3 signalling pathway.ConclusionsSCARF1 promotes the M2 polarization of KCs by promoting phagocytosis through the calcium‐dependent PI3K‐AKT‐STAT3 signalling pathway.

Development of 96-microwell Plate Assay with Fluorescence Reader and HPLC Method with Fluorescence Detection for High-throughput Analysis of Linifanib in its Bulk and Dosage Forms

Background: Linifanib (LFB) is a tyrosine kinase inhibitor with antineoplastic activity. The existing methods for the analysis of LFB in bulk and dosage forms do not meet the requirements of quality control (QC) analysis. Objective: The present study was devoted to the development of two methods with high throughputs for determination of LFB. These methods are 96-microwell plate assay with microplate fluorescence reader (MWP-FR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Methods: The MWP-FR assay was carried out in white opaque 96-well assay plates and the native fluorescence signals of LFB were measured at 360 nm for excitation and 500 nm for emission. In the HPLC-FD, the chromatographic separation of LFB and quinine sulphate (QS) as internal standard (IS) was performed on µ-Bondapack CN HPLC column using a mobile phase consisting of acetonitrile:water (60:40, v/v) pumped at a flow rate of 1 ml/min in an isocratic mode. The fluorescence detector was set at 350 nm for excitation and 454 nm for emission. Results: The linear ranges of the MWP-FR and HPLC-FD were 1-12 µg/well and 10-500 ng/ml, respectively. The limits of detection were 0.85 µg/well and 8.24 ng/ml for MWP-FR and HPLC-FD, respectively. Both MWP-FR and HPLC-FL methods were successfully applied for the determination of LFB in both bulk and tablets. Conclusion: Both methods have high analytical throughputs, they are suitable for use in QC laboratories for analysis of large numbers of LFB samples, and are environmentally friendly as they consume low volumes of chemicals and solvents.

RNA interference targeting NOX4 protects visual function in an experimental model of retinal detachment by alleviating blood-retinal barrier damage.

To observe the effects of the inhibition of NADPH oxidase 4 (NOX4) expression on the retinal vascular barriers and visual function after retinal detachment (RD). RD model was established 3wk after adeno-associaned virus vector injection. The retinal tissue was harvested 3d after RD, and the death of retinal vascular endothelial cells and photoreceptors was observed using electron microscopy. The NOX4 expression was detected by Western blot. Confocal microscopy was used to observe a retinal patch that had been perfused with Evans blue. A modified water maze test was used to detect the time required to find the platform on the water surface. The visual function of the rats was evaluated and reactive oxygen species (ROS) expression was detected by a fluorescence microplate reader. The retinal patch showed that NOX4 interference significantly reduced the destruction of the tight junctions between the retinal endothelium of RD rats and reduced leakage. Western blotting showed decreased expression of the NOX4 protein and decreased expression of ROS in retinal tissue; the Morris water maze test results showed that NOX4 interference significantly decreased the escape latency of the rats. NOX4 interference reduces the production of ROS in retinal vascular endothelial cells after experimental RD, thereby protecting the blood-retinal barrier and protecting visual function.

Preservation of mitochondrial homeostasis is responsible for the ameliorative effects of Suhuang antitussive capsule on non-resolving inflammation via inhibition of NF-κB signaling and NLRP3 inflammasome activation

Ethnopharmacological relevanceSuhuang antitussive capsule (Suhuang), one of traditional antitussive Chinese patent medicines, has been used for the treatment of post-infectious cough and cough variant asthma in clinical practice. It has been demonstrated to show numerous biological actions including antitussive and anti-inflammatory effects. Aim of the studyThis study aims to investigate the effects of Suhuang on non-resolving inflammation and its underlying molecular mechanism. Material and methodsIn vitro, mitochondrial membrane potential and ROS were detected by flow cytometry analysis. mtDNA release and mPTP fluorescence were determined by Q-PCR and fluorescence microplate reader analysis. Cytochrome C release and 8-OHdG levels were evaluated by ELISA. Additionally, the effects of Suhuang on Drp1, MMP9, IκBα/NF-κB and NLRP3/ASC/Caspase-1 expression were determined by Q-PCR, gelatin zymography or immunoblot analysis. In vivo, C57/BL6 mice were orally administrated for 2 weeks with Suhuang, then lung injury was induced by LPS. Inflammatory mediators mRNA, histological assessment and NF-κB/Caspase-1/IL-1β levels were evaluated by Q-PCR, H&E staining and immunoblot analysis. Two sepsis models of mice were further used to evaluate its anti-inflammatory effects. ResultsSuhuang restored mitochondrial homeostasis by inhibiting Drp1 activation and mitochondrial fission. Besides, Suhuang reduced mPTP opening, mitochondrial membrane potential collapse, ROS overproduction and mtDNA release. Moreover, Suhuang down-regulated MMP9 expression. As a consequence of preserved mitochondrial homeostasis, Suhuang inhibited NF-κB pathway activation by prevention of NF-κB-p65 phosphorylation and IκBα degradation. Suhuang also limited NLRP3 inflammasome activation by blocking NLRP3-ASC interaction and promoting NLRP3 ubiquitination degradation. Drp1 knockdown in vitro diminished the inhibitory effects of Suhuang on inflammatory responses, indicating the essential role of Drp1 in the Suhuang's activity. Consistently, the therapeutic effects of Suhuang were confirmed in LPS-inhaled mice, which recapitulated the protective actions of Suhuang in mitochondrial homeostasis in vitro. Additionally, two sepsis models of mice confirmed the inhibitory effects of Suhuang on uncontrolled inflammation. ConclusionsAltogether, our work reveals that Suhuang inhibits non-resolving inflammation through inhibition of NF-κB signaling and NLRP3 inflammasome activation by preserving mitochondrial homeostasis, providing new pharmacological data for the clinical use of Suhuang. Our study also suggests mitochondrial homeostasis as a potential intrinsic regulatory strategy for treating inflammatory diseases.

A Streamlined Study on Chitosan-Zinc Oxide Nanomicelle Properties to Mitigate a Drug-Resistant Biofilm Protection Mechanism

The nosocomial multidrug resistant bacteria (MDR), are rapidly circulating from water surfaces to humans away from the clinical setting, forming a cyclical breeding ground of resistance, causing worldwide infections, and thus requiring urgent responses. The combination of chitosan and zinc oxide (CZNPs), with proven bactericidal effects on some MDRs, was further studied to set the stage for a broad-spectrum in vivo utilization of CZNPs. Toward ensuring CZNPs' uniformity and potency, when it faces not only biofilms but also their extracellular polymeric substances (EPS) defense mechanism, the size, zeta potential, and polydispersity index (PDI) were determined through dynamic light scattering (DLS). Furthermore, the efficacy of CZNPs was tested on the inhibition of MDR Gram-negative Escherichia coli BAA-2471 and Gram-positive Enterococcus faecium 1449 models, co-cultured in an Alvatex 3D fiber platform as a biofilm-like structure. The Biotek Synergy Neo2 fluorescent microplate reader was used to detect biofilm shrinkage. The biofilm protection mechanism was elucidated through detection of EPS using 3D confocal and transmission electronic microscopy. Results indicated that 200 μl/mL of CZNPs, made with 50 nm ZnO and 10,000 Da chitosan (N = 369.1 nm; PDI = 0.371; zeta potential = 22.8 mV), was the most promising nanocomposite for MDR biofilm reduction, when compared to CZNPs enclosing ZnO, 18 or 100 nm. This study depicts that CZNPs possess enough potency and versatility to face biofilms' defense mechanism in vivo.

Characterization of Chemically Activated Pyrolytic Carbon Black Derived from Waste Tires as a Candidate for Nanomaterial Precursor.

Pyrolysis is a feasible solution for environmental problems related to the inadequate disposal of waste tires, as it leads to the recovery of pyrolytic products such as carbon black, liquid fuels and gases. The characteristics of pyrolytic carbon black can be enhanced through chemical activation in order to produce the required properties for its application. In the search to make the waste tire pyrolysis process profitable, new applications of the pyrolytic solid products have been explored, such as for the fabrication of energy-storage devices and precursor in the synthesis of nanomaterials. In this study, waste tires powder was chemically activated using acid (H2SO4) and/or alkali (KOH) to recover pyrolytic carbon black with different characteristics. H2SO4 removed surface impurities more thoroughly, improving the carbon black’s surface area, while KOH increased its oxygen content, which improved the carbon black’s stability in water suspension. Pyrolytic carbon black was fully characterized by elemental analysis, inductively coupled plasma–optical emission spectrometry (ICP-OES), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray diffraction (XRD), N2 adsorption/desorption, scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM-EDS), dynamic light scattering (DLS), and ζ potential measurement. In addition, the pyrolytic carbon black was used to explore its feasibility as a precursor for the synthesis of carbon dots; synthesized carbon dots were analyzed preliminarily by SEM and with a fluorescence microplate reader, revealing differences in their morphology and fluorescence intensity. The results presented in this study demonstrate the effect of the activating agent on pyrolytic carbon black from waste tires and provide evidence of the feasibility of using waste tires for the synthesis of nanomaterials such as carbon dots.

Fluorescence Determination of Acrylamide in Snack, Seasoning, and Refreshment Food Samples with an iOS Gadget–Based Digital Imaging Colorimeter

The novelty of this research work lies in a simple, fast, and plausible analysis of acrylamide in food samples accomplished using an iOS gadgets–based digital imaging colorimeter (iOS gadgets–based DIC). This method is based on fluorescence color measurements obtained from digital images of derivatized acrylamide solution extracted from snack, seasoning, and refreshment food samples (fried banana chip, fried potato chip, fried taro chip, fried durian chip, cayenne pepper, paprika seasoning, tea, and instant coffee). This device represents a convenient and low-cost detection for immediate and simultaneous determination. In addition, there is no reported research using an iOS gadgets–based DIC for the analysis of acrylamide in food samples. In this research, acrylamide is degraded through Hofmann reaction, and a fluorescent product is produced by the vinyl amine reacting with fluorescein, resulting in strong fluorescence emission at 590 nm. Fluorescein was chosen instead of fluorescamine because fluorescein is about 10 times cheaper than fluorescamine. The linear range for the relationship between color value and acrylamide concentration is in the range 1.00–10.0 mg L−1 with the correlation coefficient (R2) of 0.9985. The limits of detection and quantitation were 0.53 and 1.78 mg L−1, respectively. The color values of acrylamide solution were tested for accuracy using food samples spiked with 1.00, 3.00, and 5.00 mg L−1 of standard acrylamide solutions. The recoveries for food samples are in the range 82.74–113.3%. There was no significant difference observed when comparing the results obtained with the DIC instrument with those obtained with fluorescence microplate reader and high-performance liquid chromatography.

Abstract P225: Rupp Rat Model Cd4+ T Cells Activate Nk Cells And Cause Mitochondrial Oxidative Stress And Hypertension In Normal Pregnant Rats

Preeclampsia (PE) is new onset hypertension during pregnancy and is associated with elevated inflammatory response such as CD4+ T cells, NK cells, and cytokines. We have previously shown women with PE exhibit increases in circulating and placental CD4+T cells and placental mitochondrial (mt) dysfunction/ROS compared to normal pregnant (NP) women. The Reduced Uterine Perfusion Pressure (RUPP) rat model produces many characteristics of PE such as hypertension, increases in CD4+ cells, increases in renal and placental NK cells, and mt dysfunction/ROS. We have previously demonstrated that RUPP CD4+T cells cause hypertension in NP rats, however the role of RUPP CD4+ T cells in stimulating NK cells to cause mt dysfunction/ROS are not elucidated. Therefore, we examined the effect of adoptive transfer of RUPP CD4+ T cells to activate NK cells in NP rats. Splenic CD4+ T cells were isolated from RUPP rats, cultured, and injected into NP rats on GD 13. On GD19, MAP values and blood/tissue samples were collected from both RUPP CD4+ T cell recipients and NP controls. Mitochondrial respiration and mtROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader, respectively. A student’s t-test was used for statistical analysis. On GD19, MAP increased to 110±2 mmHg (n=13) in RUPP CD4+ T cell recipients compared to control NP rats 102±2 mmHg (n=7, p<0.05). Circulating cytolytic NK cells increased to 3±0.6% in RUPP CD4+ T cell recipients (n=8) compared to NP controls 0.3±0.2% (n=7, p<0.05). Placental state 3 (209.3±31.3 vs 422.7 ±83.3 pmol/sec/mg, p<0.05) and maximal (152.1±46.2 vs 229.7±58.9 pmol/sec/mg) and renal state 3 (133.4 ±21.4 vs 289.8±43.4 pmol/sec/mg, p<0.05) and maximal (61.8±18 vs 242.4±27.7 pmol/sec/mg, p<0.05) respiration rates, indicative of ATP production and electron transport chain efficacy respectively, were reduced with RUPP CD4+ T cells (n=6; n=9) compared to NP (n=5; n=5). Collectively, the data indicate that the adoptive transfer of RUPP CD4+ T cells stimulates cytolytic NK cells and placental and renal mitochondrial dysfunction/ROS during pregnancy as important mechanisms of hypertension in the pathophysiology of preeclampsia. Keywords: Preeclamspia, Hypertension, Oxidative stress

Abstract P207: Mitochondrial Dysfunction And Natural Killer Cell Activation Stimulated By Il-17 Signaling From Th17 Cells In Response To Placental Ischemia During Pregnancy; Sarah Fitzgerald, Evangeline Deer, Owen Herrock, Tarek Ibrahim, Lorena Amaral, Denise Cornelius And Babbette Lamarca; 1 Department Of Pharmacology, University Of Mississippi Medical Center.

Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension associated with placental ischemia (PI), mitochondrial (mt) dysfunction, and an imbalance in T helper (TH) and Natural Killer (NK) cells during pregnancy. The reduced uterine perfusion pressure (RUPP) model is an established model for PE. We have previously shown an important role for IL-17 in hypertension and activation of NK cells in the RUPP rat. We don’t know the role for IL-17 or TH17 cells in contributing to NK cell mediated mt dysfunction and ROS associated with PI. Therefore, we hypothesize that hypertension in response to PI stimulated TH17 cells causes mt ROS mediated through IL-17 signaling to NK cells. On gestation day 12 (GD12) RUPP-induced TH17s (splenic CD4+/CD25- cells) were adoptively transferred (Ad-T) into normal pregnant (NP) rats. Recombinant mouse IL-17 receptor C (IL-17RC) (100 pg/day) was administered from GD14-19 via osmotic mini-pump. On GD19, samples were collected and mean arterial pressure (MAP) was measured. Mt respiration and mt ROS data was measured in isolated mt from renal and placental tissues using the Oxygraph 2K and fluorescent microplate reader, respectively. A one-way ANOVA with Bonferroni post hoc test was used for statistical analysis. MAP was increased in Ad-T rats (112 ± 0.72mmHg, n=14) compared to NP (92 ± 3.03mmHg, n=14) (p<0.05), and was lowered with IL-17RC (97.2± 2.14 mmHg, n=13). Circulating activated NK cells were significantly increased with Ad-T (3.326± 0.76 %gated, n=9) (p<0.0001) compared to NP (0.05± 0.05 %, n=11), and were attenuated with IL-17RC (0.42± 0.4 %, n=5) (p<0.01). Placental activated NK cells were significantly increased with Ad-T (2.5± 1.01 %, n=4) (p<0.05) compared to NP (0.03± 0.03 %, n=8) and were attenuated with IL-17RC (0.21± 0.12 %, n=4) (p<0.05). Renal mtROS increased in Ad-T (312.4 ± 44.7%, n=9) compared to NP (191.9 ± 20.5%, n=5). Placental mtROS significantly increased in Ad-T (312.5 ± 23.6%, n=9) (P<0.0005) compared to NP controls (134 ± 9.6%, n=5) (p<0.0001), and was decreased by administration of IL-17RC (174.5± 42.5 %, n=5) (p<0.0001). These data demonstrate that IL-17 signaling plays an important role in NK cell activation and tissue mt function in response to TH17 cells stimulated during PE.

Use of a Fluorescence-Based Assay To Measure Escherichia coli Membrane Potential Changes in High Throughput.

Bacterial membrane potential is difficult to measure using classical electrophysiology techniques due to the small cell size and the presence of the peptidoglycan cell wall. Instead, chemical probes are often used to study membrane potential changes under conditions of interest. Many of these probes are fluorescent molecules that accumulate in a charge-dependent manner, and the resulting fluorescence change can be analyzed via flow cytometry or using a fluorescence microplate reader. Although this technique works well in many Gram-positive bacteria, it generates fairly low signal-to-noise ratios in Gram-negative bacteria due to dye exclusion by the outer membrane. We detail an optimized workflow that uses the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC2(3)], to measure Escherichia coli membrane potential changes in high throughput and describe the assay conditions that generate significant signal-to-noise ratios to detect membrane potential changes using a fluorescence microplate reader. A valinomycin calibration curve demonstrates this approach can robustly report membrane potentials over at least an ∼144-mV range with an accuracy of ∼12 mV. As a proof of concept, we used this approach to characterize the effects of some commercially available small molecules known to elicit membrane potential changes in other systems, increasing the repertoire of compounds known to perturb E. coli membrane energetics. One compound, the eukaryotic Ca2+ channel blocker amlodipine, was found to alter E. coli membrane potential and decrease the MIC of kanamycin, further supporting the value of this screening approach. This detailed methodology permits studying E. coli membrane potential changes quickly and reliably at the population level.

Abstract 1151: Inclusion of cholesterol and A549 lipid extract in CLENs improved cellular uptake in adenocarcinoma non-small cell lung cancer target cells

Abstract Purpose:The purpose of this investigation was to determine the optimal amount of cholesterol and A549-derived lipid extract (LE) included in the initial design and development of A-549-cell membrane lipid extracted nanoliposomes (A549-CLENs) for cell targeting studies. Methods and Materials:A549 (ATCC® CCL-185™), used as a positive control and NCI-H1703 (ATCC® CRL-5889™), used as a negative control, and respective growth media were purchased from ATCC (Manassas, VA). A549-GFP (green fluorescent protein) cell line was purchased from AntiCancer, Inc. DOPC, DOTAP, cholesterol, and DSPE-PEG5000 were obtained from Avanti Polar Lipids (Alabastar, AL). Cellular membrane lipids of A549 cells were extracted by modified Bligh & Dyer method. CLENs were fluorescently labeled with rhodamine-B. Particle sizes and zeta potentials were performed using ZetaPALS (Brookhaven Instruments Corporation, Holtsville, NY, USA). For cellular uptake studies cells were seeded at 2.0 x 104 cells in a 48-well plate followed by incubation for 24 hours. A fluorescence microplate reader and/or fluorescence microscopy were used to evaluate cellular uptake as a function of relative amount of cholesterol and A549-lipid extract inclusion in CLENs. Results:In general, an increase in the inclusion of cholesterol reduced the particle size of CLENs. Amount of 10 mol% of cholesterol content in CLENs demonstrated the smallest mean size diameters and a zeta potential value recorded at 243 ± 4.4 nm and 1 ± 2.61 mV, respectively. The finding correlated also with a relatively high degree of cellular uptake of the preparation type for part I of the physicochemical characterization studies. We next maintained 10 mol% cholesterol content consistent in all future preparations and increased the total A-549 lipid extract material at the expense of DOPC. In general, the mean diameter and zeta potential for A549-CLENs were 246 ± 3.8 nm and 20.4 ± 0.84 mV, respectively. Approximately 10-15 mol% of A-549 lipid extract inclusion resulted in a consistently high degree of overall cellular uptake by GFP-A549 and A549 target cells compared to control and the non-target cell population, NCI-H1703. Conclusion: Preparation of A549-CLENs consisting of DOPC/ DOTAP/ Chol/ LE/PEG (65/10/10/10/5) established the greatest increase in cellular uptake and overall selectivity. Future studies will consider antibody conjugation and development of tumor-specific biomarkers for serodiagnostic detection and lung cancer treatment. Citation Format: Maxime Langlois, Guo-Yun Kao, Diana Lasker. Inclusion of cholesterol and A549 lipid extract in CLENs improved cellular uptake in adenocarcinoma non-small cell lung cancer target cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1151.

MiR-219a-2 relieves myocardial ischemia-reperfusion injury by reducing calcium overload and cell apoptosis through HIF1α/ NMDAR pathway

ObjectiveDuring the process of myocardial ischemia-reperfusion injury (MIRI), the intracellular Ca2+ concentration ([Ca2+]i) continues to increase, leads to the cardiomyocyte apoptosis and eventually causes myocardial damage, while the upstream regulation mechanism of calcium overload is still unknown. This study focuses on the role of miR-219a-2 in MIRI and aims to elaborate its regulatory mechanism on calcium overload that occurs during MIRI. MethodsThe expression of miR-219a-2 was determined in the heart tissues of MIRI mice by qRT-PCR. The [Ca2+]i was measured by fluo-3 using a fluorescence microplate reader. The expression of hypoxiainducible factor 1α (HIF1α) and NR1, the obligatory subunit of N-methyl-d-aspartate receptor 1 (NMDAR), were measured by qRT-PCR and western blot. The luciferase reporter assay was used to confirm the interplay between miR-219a-2 and HIF1α and the interplay between HIF1α and NR1. The cell apoptosis was measured by the expression level of B-cell lymphoma 2 interacting mediator of cell death (Bim) and the number of TUNEL-positive cells. The myocardial infarct size of mice was measured by TTC/Evans Blue staining. ResultsMiR-219a-2 was down-regulated in the heart tissues of MIRI mice. miR-219a-2 overexpression decreased [Ca2+]i and the expression of HIF1α and NR1 in hypoxia/reoxygenation (H/R)-treated HL-1 cells. Then, the luciferase reporter assay showed that miR-219a-2 inhibited the transcription of HIF1α and HIF1α promoted the transcription of NR1. Both HIF1α overexpression and NMDAR function enhancement removed the inhibitory effect of miR-219a-2 on calcium overload and cell apoptosis in H/R-treated HL-1 cells. Finally, the overexpression of miR-219a-2 decreased Ca2+ concentration, cell apoptosis, and myocardial infarction size in MIRI mice, while the NMDAR function enhancer reversed the therapeutic effect of miR-219a-2. ConclusionMiR-219a-2 reducing NMDAR-mediated calcium overload via HIF1α/NR1 axis, thus alleviating cell apoptosis in MIRI.

Nerve growth factor from Indian Russell’s viper venom (RVV-NGFa) shows high affinity binding to TrkA receptor expressed in breast cancer cells: Application of fluorescence labeled RVV-NGFa in the clinical diagnosis of breast cancer

Nerve growth factor (NGF) is a minor and neglected component of snake venom. Present study describes the purification and characterization of a NGF isoform (RVV-NGFa) from Indian Russell’s viper venom (RVV). RVV-NGFa showed a protonated molecular ion [MH+] at m/z 17388.725 Da. The RVV-NGFa induced neuritogenesis in PC-12 cells but did not show cytotoxicity in mammalian cells, hemolytic activity, platelet modulation, and interference in blood coagulation system which are the characteristic pharmacological properties of RVV. By ELISA and immunofluorescence microplate reader assay, the RVV-NGFa showed appreciable binding to TrkA receptor expressed in breast cancer MDA-MB-231 and MCF-7 cells; nevertheless, pre-incubation of cells with anti-TrkA (and not TrkB or TrkC) or anti-p75NTR antibody significantly decreased (p < 0.05) this binding. The RVV-NGFa demonstrated insignificant binding with non-cancerous cells (HEK-293, L6) lacking TrkA receptor. The binding of RVV-NGFa to TrkA receptor of breast cancer cells resulted in internalization of ligand (RVV-NGFa)-receptor (TrkA) complex to cell cytoplasm in a time-dependent manner. The spectrofluorometric study demonstrated an interaction between RVV-NGFa and cytosolic domain of the purified TrkA receptor. The fluorescence (FITC)-tagged RVV-NGFa depicted a strong fluorescence signal that was observed under a fluorescence microscope and determined by fluorescence microplate reader assay post binding to breast cancer cells; but no fluorescence signal was detected after incubating FITC-RVV-NGFa with non-cancerous L6 and HEK-293 cells. The clinical application of FITC/fluorescence nanoparticle tagged RVV-NGFa for the ex vivo and in vivo diagnosis of breast cancer is highly promising.

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

IL‐17 Signaling Stimulates Natural Killer Cell Activation and Causes Mitochondrial Dysfunction in Response to TH17 Cells Stimulated in Response to Placental Ischemia During Pregnancy

Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension that is associated with placental ischemia, mitochondrial (mt) dysfunction, and an imbalance in T helper and Natural Killer (NK) cells during pregnancy. The reduced uterine perfusion pressure (RUPP) model is an established model for PE exhibiting hypertension in response to placental ischemia associated with mt dysfunction and mt reactive oxygen species (ROS), activation of T helper and NK cells. We have previously shown an important role for IL‐17 in the hypertension and activation of NK cells in the RUPP rat. However we don’t know the role for IL‐17 or TH17 cells in contributing to NK cell mediated mt dysfunction and ROS associated with placental ischemia. Therefore, we hypothesize that hypertension in response to placental ischemia stimulated TH17 cells causes mt ROS mediated through IL‐17 signaling to NK cells. On gestation day 12 (GD12) RUPP‐induced TH17s (splenic CD4+/CD25− cells) were injected into normal pregnant (NP) rats. Recombinant mouse IL‐17 receptor C (IL‐17RC) (100 pg/day) was administered from GD14‐19 via osmotic mini‐pump. On GD19, samples were collected and mean arterial pressure (MAP) was measured. Mt respiration and mt ROS data was measured in isolated mitochondria from renal and placental tissues using the Oxygraph 2K and fluorescent microplate reader, respectively. A one‐way ANOVA with Bonferroni post hoc test was used for statistical analysis. MAP was increased in NP+RUPP TH17 recipient rats (110 ± 0.94mmHg, n=10) compared to NP controls (91 ± 3.6mmHg, n=11) (p&lt;0.05), and was lowered with IL‐17RC (100± 2.4 mmHg, n=4). Circulating activated NK cells were significantly increased with RUPP TH17 (4.74± 0.9 %, n=5) (p&lt;0.0001) compared to NP controls (0.08± 0.06 %, n=8), and were attenuated with IL‐17RC (0.00± 0.00 %, n=3) (p&lt;0.001). Placental activated NK cells were significantly increased with RUPP TH17 (2.5± 1.01 %, n=4) (p&lt;0.05) compared to NP controls (0.03± 0.03 %, n=8) and were attenuated with IL‐17RC (0.21± 0.12 %, n=4) (p&lt;0.05). Renal mtROS increased in NP RUPP TH17 recipients (593 ± 110%, n=9) compared to NP controls (289 ± 30%, n=6), and was attenuated by IL‐17RC (380± 103%, n=3). Placental mtROS significantly increased in NP RUPP TH17 recipients (3081 ± 720%, n=7) (P&lt;0.0005) compared to NP controls (1287 ± 231.3%, n=6) (p&lt;0.0001), and was decreased by administration of IL‐17RC (561.7± 105.5 %, n=3) (p&lt;0.0001). These data demonstrate that IL‐17 signaling plays an important role in NK cell activation and tissue mt function in response to TH17 cells stimulated during placental ischemia of pregnancy.Support or Funding InformationR01HD067541‐06, P20GM121334, AHA 19CDA34670055, T32HL105324

CD4+ T Cells from RUPP rat model activate NK cells and cause mitochondrial oxidative stress and hypertension in normal pregnant rats

Preeclampsia (PE) is new onset hypertension during pregnancy and is associated with elevated inflammatory response such as CD4+ T cells, NK cells, and cytokines. We have previously shown that women with PE exhibit increases in placental mitochondrial (mt) dysfunction and ROS compared to normal pregnant (NP) women. The Reduced Uterine Perfusion Pressure (RUPP) rat model produces many characteristics of PE such as hypertension, increases in CD4+ cells, renal and placental NK cells, and mt dysfunction/ROS. Moreover, we have shown an important role for mt ROS in hypertension in RUPPs. We have previously demonstrated that RUPP CD4+T cells cause hypertension in NP rats, however the role of RUPP CD4+ T cells in stimulating NK cells to cause mitochondrial dysfunction/ROS are unknown. Therefore, we examined the effect of adoptive transfer of RUPP CD4+ T cells to activate NK cells in NP rats and cause mt dysfunction and ROS as mechanisms of hypertension. Splenic CD4+ T cells were isolated from RUPP rats, cultured, and injected into NP rats on gestation day (GD) 13. On GD19, MAP blood and tissue samples were collected. Mt respiration and mtROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader, respectively. A student’s t‐test was used for statistical analysis. On GD19, MAP increased to 110±2 mmHg (n=12) in RUPP CD4+ T cell recipients compared to control NP rats 101±2 mmHg (n=7, p&lt;0.05). Circulating cytolytic NK cells increased to 3±0.6% in RUPP CD4+ T cell recipients (n=8) compared to NP controls 0.3±0.2% (n=7, p&lt;0.05). Renal state 3 (194.3±62.33 vs 1004±251.9 pmol/sec/mg, p&lt;0.05) and maximal (151.2±75.19 vs 531.5±99.97 pmol/sec/mg, p&lt;0.05) respiration rates, indicative of ATP production and electron transport chain efficacy respectively, were reduced with RUPP CD4+ T cells (n=6) compared to NP (n=4). Renal mtROS increased from 251.5±81.75% in NP controls (n=4) to 416.9±47.68% in RUPP CD4+ T cell recipients (n=9, p&lt;0.05). Collectively, the data indicate that the adoptive transfer of RUPP CD4+ T cells stimulates cytolytic NK cells and renal mitochondrial dysfunction/ROS during pregnancy as important mechanisms of hypertension in the pathophysiology of preeclampsia.Support or Funding InformationNIH Grants: RO1HD067541‐06 and P20GM121334; T32HL105324

Folate-Targeted and Oxygen/Indocyanine Green Loaded Lipid Nanoparticles for Dual-Mode Imaging and Photo-Sonodynamic/Photothermal Therapy of Ovarian Cancer in Vitro and in Vivo

We have successfully fabricated versatile folate-targeted and oxygen/indocyanine green-loaded lipid nanoparticles (FA-OINPs) for dual-mode imaging-guided therapy in ovarian cancer cells and subcutaneous xenograft models. FA-OINPs were demonstrated to have great potential as superb contrast agents to enhance ultrasound and photoacoustic (US/PA) imaging We have successfully fabricated versatile folate-targeted and oxygen/indocyanine green-loaded lipid nanoparticles (FA-OINPs) for dual-mode imaging-guided therapy in ovarian cancer cells and subcutaneous xenograft models. FA-OINPs were demonstrated to have great potential as superb contrast agents to enhance ultrasound and photoacoustic (US/PA) imaging in vitro and in vivo. Confocal laser scanning microscopy and flow cytometry analysis verified that FA-OINPs could specifically target SKOV3 ovarian cancer cells and be endocytosed with a remarkable efficiency. Compared with other therapeutic options, FA-OINPs exhibited an excellent therapeutic outcome after exposure to laser and ultrasound. The MTT assay and flow cytometry analysis confirmed that cytotoxicity effects and apoptosis/necrosis rates were significantly increased. The fluorescence microscopy and fluorescence microplate reader detection validated that the generation of intracellular reactive oxygen species (ROS) was dramatically improved. Immunohistochemical analyses of tumor tissues demonstrated the enhanced tumor apoptosis, the decreased vascular endothelial growth factor (VEGF) and microvascular density (MVD) expression, and the decreased expression of CD68 after treatment. The presented results suggest that photo-sonodynamic/photothermal mediated FA-OINPs could provide a promising strategy for synergistic therapy in ovarian cancer with the guidance of US/PA dual-mode imaging.