Multimode Microplate Reader Research Articles

Targeted lipid nanoparticles to prevent trans-placental passage in the ex vivo human placental cotyledon perfusion model.

Medication use during pregnancy poses risks to both the mother and the fetus. These risks include an elevated potential for fetotoxicity due to placental drug transport. Nanomedicines offer a promising solution by potentially preventing trans-placental passage. Targeted nanomedicines could enhance safety and efficacy in treating maternal or placental pathophysiology. Our study investigates placental transfer kinetics of targeted lipid nanoparticles (LNPs) in an ex vivo human placenta cotyledon perfusion model. We collected human placentas for dual-side ex vivo placental perfusions. Targeted LNPs with a fluorescence tag were introduced into the maternal circuit of each placenta. To establish if there was trans-placental passage of LNPs to the fetal circuit, we collected samples from maternal and fetal circuits throughout the six hours of the perfusion. We determined the fluorescence signal using a multi-mode microplate reader and Multiphoton microscopy to localize the LNPs in the placenta tissue. Data from perfused placenta tissue showed no significant transfer of the fluorescently labeled LNPs across the placental barrier to the fetal circuit. Localization of targeted LNPs in tissue samples is mainly observed in the maternal blood space of the placenta. Our results suggest that targeted LNPs present a promising strategic approach to prevent trans-placental passage to the fetus. Our future perspectives involve investigating the efficacy of targeted LNPs as well as loading targeted LNPs with nucleic acid-based therapeutics to investigate their therapeutic potential.

Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

ГЕНЫ НЕЙРОМЕДИАТОРНЫХ СИСТЕМ МОЗГА, ДЕТЕРМИНИРУЮЩИЕ ПСИХОЭМОЦИОНАЛЬНЫЙ СТАТУС ЧЕЛОВЕКА

Background. In recent decades, there has been a significant interest worldwide in the clarification of the genetic mechanisms of the human psychoemotional status. The identification of genetic markers associated with psychoemotional status expands the possibilities of considering individual characteristics in career guidance, as well as in choosing methods of individual psychotherapy. Purpose. To identify the most informative polymorphic variants of genes associated with the human psychoemotional status. Material and methods. Psychological and molecular genetic testing was conducted among representatives of two groups of the Belarusian population: a control group (518 people) and a group of patients with personality disorders and deviant behavior (534 people). The following psychological testing methods were used: PSS-10 and PSS-14 – Russian-language versions of the Perceived Stress Scale-10, HADS-T – a test that measures symptoms of anxiety, and HADS-D – a test that measures symptoms of depression. DNA isolated from buccal epithelial cells was used as biological material for molecular genetic research. Genomic DNA was isolated using an extraction kit from Primetech LLC (Belarus). DNA concentration was assessed using a Qubit fluorimeter (Invitrogen, USA), as well as a Promega GloMax® Explorer Multimode Microplate Reader (USA). Results. A total of 33 polymorphic variants of genes of the brain neurotransmitter systems have been studied. 8 polymorphic variants were found to have statistically significant different frequencies in the studied groups, and 3 ones to have frequencies that differ at the level of tendency. Conclusions. The results obtained made it possible to form a panel of the most informative polymorphic variants of genes of the brain neurotransmitter systems, which can be used to determine individual psycho-emotional characteristics.

Elevated plasma pyruvate kinase M2 concentrations are associated with the clinical severity and prognosis of coronary artery disease.

Pyruvate kinase M2 (PKM2) was involved in the pathophysiology of atherosclerosis and coronary artery disease (CAD). We tested whether plasma PKM2 concentrations were correlated with clinical severity and major adverse cardiovascular events (MACEs) in CAD patients. A total of 2443 CAD patients and 238 controls were enrolled. The follow-up time was two years. Plasma PKM2 concentrations were detected by enzyme-linked immunosorbent assay (ELISA) kits (Cloud-Clone, Wuhan, China) using SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, USA). The predictors of acute coronary syndrome (ACS) were assessed by logistic regression analysis. The association between PKM2 concentration in different quartiles and MACEs was evaluated by Kaplan-Meier (KM) curves with log-rank test and Cox proportional hazard models. The predictive value of PKM2 and a cluster of conventional risk factors was determined by Receiver operating characteristic (ROC) curves. The net reclassification improvement (NRI) and the integrated discrimination improvement (IDI) were utilized to evaluate the enhancement in risk prediction when PKM2 was added to a predictive model containing a cluster of conventional risk factors. In CAD patients, PKM2 concentration was the independent predictor of ACS (P < 0.001). Kaplan-Meier cumulative survival curves and Cox proportional hazards analyses revealed that patients with a higher PKM2 concentration had higher incidence of MACEs compared to those with a lower PKM2 concentration (P < 0.001). The addition of PKM2 to a cluster of conventional risk factors significantly increased its prognostic value of MACEs. Baseline plasma PKM2 concentrations predict the clinical severity and prognosis of CAD.

Advancing Quantification of Water-Extractable Arabinoxylan in Beer: A High-Throughput Approach.

Water-extractable arabinoxylan (WEAX) may cause major problems during clarification processes in a brewery owing to its ability to form gel networks. However, high WEAX contents can also enhance the nutritional quality of the final product as they play an important role in the human diet. Therefore, precise quantification of WEAX is required. Current methods are very time- and resource-consuming as well as limited in the number of samples and in some cases provide low accuracy. Thus, a reproducible high-throughput method for the quantification of WEAX optimized for beer was developed, reaching recovery rates (RRs) of almost 100%. The assay is based on Douglas's colorimetric method. Hydrolysis was conducted using glacial acetic acid to induce the formation of red color complexes resulting from the interaction between pentose degradation products and phloroglucinol. The method was successfully transferred to a multi-mode microplate reader to minimize the loss of color intensity over time and to obtain a high throughput. By using 96-well plates, up to 40% of the previous analysis time could be saved, and a larger number of samples could be analyzed in one batch. The collected data determined xylose as an optimal calibration standard due to high accuracy and reproducibility. The respective AX control standards showed RR within the range of 95-105% without exception. To validate and show the ruggedness of the modified method, WEAX concentration in seven commercial German beers (e.g., lager, pilsner, wheat beer, non-alcoholic beer) was quantified. Interfering hexose sugars that lead to measurement errors when analyzing samples with high amounts of fermentable sugars (e.g., non-alcoholic beer produced by limited fermentation) were eliminated by Saccharomyces diastaticus fermentation. Further investigations were carried out by means of LC-MS in order to obtain additional information about the reddish product in the hydrolyzed samples. In this context, C16H12O6 could be identified as one of numerous condensation products, contributing to the coloring. The collected data showed the impact of diverse factors on the measured AX concentration and helped optimize the experimental procedure for a high sample throughput with precise and highly reproducible results. The proposed quantification method should be primarily used in completely fermented finished beer to emphasize the time aspect. Wort samples and non-alcoholic beer produced by limited fermentation can be also analyzed, but only after fermentation with S. diastaticus.

Advanced glycation end products and their soluble receptor (sRAGE) in patients with Hashimoto's thyroiditis on levothyroxine substitution.

Advanced glycation end products (AGEs) are heterogenous group of irreversible chemical moieties originated from non-enzymatic glycation and oxidation of proteins, nucleic acids, and lipids. The engagement of AGEs with their chief cellular receptor (RAGE) activates a myriad of signaling pathways contributing to the progression of chronic diseases like autoimmune thyroiditis, type 2 diabetes mellitus and its complications. Soluble RAGE (sRAGE) prevents AGE-RAGE interaction in a competitive manner. We investigated the association between serum AGE, sRAGE and thyroid function in 73 Hashimoto thyroiditis patients (HT) on levothyroxine substitution, and in 83 age, BMI and gender-matched healthy controls. The serum AGEs levels were determined by autofluorescence on a multi-mode microplate reader, and the serum sRAGE levels by ELISA method. Mean AGE level was lower (10.71 vs 11.45 AU/µg protein; p=0.046), while mean sRAGE level was higher (923 vs 755 pg/mL; p<0.0005) in the serum of HT patients than the controls. AGE correlated with age, while sRAGE correlated negatively with BMI in both groups. We found negative correlation between AGE and fT3 levels (r=-0.32; p=0.006) and sRAGE and TSH levels (r=-0.27; p=0.022) in HT patients, while we failed to find association between AGE, sRAGE and parameters of thyroid function in the control group. Median AGE/sRAGE ratio was lower in HT patients than in controls (2.4, IQR 1.9 - 3.1 vs 3.3, IQR 2.3 - 4.1 AU/pg; p < 0.001). In HT patients, the AGE/sRAGE ratio correlated positively with BMI and correlated negatively with fT3. According to our results in HT patients lower TSH and higher fT3 levels within the reference range is accompanied by a favorable AGE/RAGE balance. Further investigations are needed to confirm these results.

A Fluorescent Reporter-Based Evaluation Assay for Antibacterial Components Against Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) is the agent of citrus bacterial canker (CBC) disease, which has significantly reduced citrus quantity and quality in many producing areas worldwide. Copper-based bactericides are the primary products for CBC control and management, but the problems derived from copper-resistant and environmental contamination have become issues of anxiety. Thus, there is a need to find alternative antibacterial products instead of relying on a single type of agent. This study developed a method to evaluate the inhibition of antibacterial agents using the fluorescence-labeled recombinant Xcc strain (Xcc-eYFP). The optimization of timelines and parameters for the evaluation of antibacterial agents involved the use of a Spark™ multimode microplate reader. This evaluation and screening method can be applied to bactericides, cocktail-mixture formulations, antagonistic bacteria, and derived metabolites. The results showed that the minimum inhibitory concentration (MIC) of commercial bactericides determined by fluorescence agrees with the MIC values determined by the conventional method. A screened cocktail-mixture bactericide presents more activity than the individual agents during the protective effects. Notably, this method has been further developed in the screening of Xcc-antagonistic bacterial strains. In summary, we provide a validated strategy for screening and evaluation of different antibacterial components for inhibition against Xcc for CBC control and management.

Repurposing and Designing Anti‐Giardial Drugs Targeting Lipids and Membranes

Giardia lamblia is an intestinal parasite responsible for transmitting giardiasis, worldwide. This parasite exists in two morphologic forms: an infective cyst and a replicating trophozoite. The symptoms include diarrhea, vomiting, dehydration and stunted growth in children. Metronidazole (Mz) is a commonly prescribed medicine to treat giardiasis, however, the emergence of Mz resistant (Mzr) parasite is increasing rapidly. Therefore, it is imperative to identify new targets for developing non‐metronidazole classes of anti‐giardial agents. Our laboratory is interested in elucidating two novel targets for drug development against Giardia: (1) glucosylceramide transferase enzyme (gGlcT1), and (2) the lipid rafts/microvesicles assembly. Both targets are important for the attachment, growth, and encystation of Giardia and maintaining overall pathogenesis. For targeting gGlcT1, we took the ligand‐based approach to identify compounds against this sphingolipid synthesis enzyme. Our search identified several compounds (including FDA‐approved drugs) such as sugammadex, temocapril, delapril, and miglustat, which were active in blocking attachment of trophozoites and cyst formation in vitro. Another drug PPMP ((D‐threo‐1‐phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol), a well‐known inhibitor of gGlcT1 with potential anti‐cancer activity, also inhibited trophozoite attachment (IC50,~6.5 mM) and Giardia cyst production (IC50,~3.5 mM). Previously, we have demonstrated that oseltamivir (Tamiflu®) was effective in disrupting giardial raft assembly and cyst production in vitro. In the current study, we synthesized eleven oseltamivir analogues and five of them were tested on the attachment of trophozoites. Oseltamivir and derivatives were synthesized from shikimic acid using standard methods. Briefly, 1x105 trophozoites were exposed to these analogues for 48 hours at concentrations of 0, 5, 10, and 20 µM on clear‐bottom 96‐well plates. Treated cells were washed with PBS (supplemented with 5 mM glucose and L‐cysteine) to remove non‐adhered cells. The attached parasites were stained with DAPI for 20 minutes at 37°C followed by quantification with the help of Biotek’s Cytation 5 Multimode Microplate Reader (GloMax). One analogue, called HVK‐ii‐43, caused 80% inhibition of attachment of trophozoites at 10 mM concentration. At the same concentration (10 mM), a second analogue, JK‐i‐45 also inhibited the attachment by 50% (IC50, ~ 10 mM). These results suggest that synthetic derivatives of oseltamivir are pharmacologically active against Giardiaand should be considered as effective anti‐giardial therapies. Our results also support the hypothesis that active compounds in combination doses or in hybrid forms (e.g., PPMP‐oseltamivir) could be used as novel anti‐giardial agents that will be effective against Mzr cells. Future work will involve gGlcT1 characterization, monitoring lipid rafts disassembly, and microvesicle release from Giardia after exposure to these and additional analogues.

Ibuprofen and diclofenac differentially affect cell viability, apoptosis and morphology changes of human cholangiocarcinoma cell lines

ObjectivesCholangiocarcinoma is a malignant biliary epithelial duct neoplasm caused by chronic inflammation after liver fluke infection. It is a major public health concern in the Greater Mekong sub-region in northeast Thailand. Herein, the effects of the non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac on the cell proliferation activity of the human cholangiocarcinoma cell lines KKU-M139 and KKU-213B were studied. MethodsCell viability was assessed with MTT assays. Inverted phase-contrast light microscopy, scanning electron microscopy and transmission electron microscopy were used to investigate the cells’ morphological alterations. Caspase 3/7 and Annexin V/PI were detected with a multimode microplate reader. ResultsIbuprofen and diclofenac decreased viability in both cell lines, and ibuprofen-treated cells exhibited reversible cell injury. In both KKU-M139 and KKU-213B cell lines, the diclofenac-treated cells had the greatest injury. The cells exhibited features of irreversible cell injury. In addition, caspase 3/7 and Annexin V/PI detection revealed early cell apoptotic characteristics. ConclusionThese findings suggest that NSAIDs may potentially suppress cell viability. Ibuprofen and diclofenac both induced morphological changes and apoptosis.

Bacterial Growth Curve Measurements with a Multimode Microplate Reader.

Bacterial studies based on growth curves are common in microbiology and related fields. Compared to the standard photometer and cuvette based protocols, bacterial growth curve measurements with microplate readers provide better temporal resolution, higher efficiency, and are less laborious, while analysis and interpretation of the microplate-based measurements are less straightforward. Recently, we developed a new analysis method for evaluating bacterial growth with microplate readers based on time derivatives. Here, we describe a detailed protocol for this development and provide the homemade program for the new analysis method.

Measuring Intracellular H 2 O 2 in Intact Human Cells Using the Genetically Encoded Fluorescent Sensor HyPer7.

Depending on its local concentration, hydrogen peroxide (H2O2) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H 2O2 are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of H2O2 in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular H2O2 dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent H2O2sensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local H2O2 dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader. Graphical abstract: Overview of HyPer7 sensor function and measurement results.

Biomimetic dandelion-like magnetic nanoparticles for capture and detection of S. aureus and L. monocytogenes

Foodborne pathogenic microorganism pose a serious threat to public health security; thus, constructing quick and sensitive methods for early screening foodborne pathogenic microorganism is necessary for human health. Teicoplanin (Teic) is a broad-spectrum biometric molecule, which can recognize Gram-positive (G+) bacteria. In this study, using bovine serum albumin (BSA) and PEG2k as “carrier” and “linker”, respectively. Teic was wired to magnetic beads (MBs) to form the biomimetic dandelion-like magnetic nanoparticles (MBs-PEG-BSA-Teic) to capture Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes ). The MBs-PEG-BSA-Teic displayed excellent capture ability of S. aureus (> 93%) and L. monocytogenes (> 86%). After separation, the fluorescence probes were added, forming a “sandwich” structural compounds, namely, MBs-PEG-BSA-Teic~bacteria~fluorescence probes. The fluorescence intensity of the probes was measured by a multimode microplate reader. Consequently, this novel method showed high detection sensitivity to target model bacteria, and the detection limit of S. aureus and L. monocytogenes in PBS buffer solution and juice were achieved 101 CFU/mL, spinach and ground beef samples were achieved 102 CFU/g. The method has easy operation and high sensitivity, and it is less interfered by food matrix or negative bacteria; thus, this method is suitable for early screening G+ pathogen in suspected food samples.

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

EXTH-54. PHOTODYNAMIC ONCOLYTIC VIROTHERAPY INCORPORATING GENETICALLY ENGINEERED PHOTOSENSITIZER KILLERRED FOR THE TREATMENT OF CENTRAL NERVOUS SYSTEM MALIGNANCIES

Abstract BACKGROUND Photodynamic therapy (PDT) is a targeted cancer therapy utilizing tumor-specific accumulation of photosensitizers and generation of reactive oxygen species (ROS) upon receiving specific light. The deadly CNS malignancies, high-grade gliomas and malignant meningioma, represent excellent candidates for this therapeutic method due to accessibility to light irradiation at the time of surgery. On the other hand, oncolytic virotherapy using a genetically engineered oncolytic herpes simplex virus (oHSV), has been intensively investigated as a multi-mechanistic therapy against these tumors. One of the advantages of oHSV is its ability to incorporate therapeutic transgenes. Our study aims to address our hypothesis that incorporating KillerRed, the first fully genetically encoded photosensitizing fluorescent protein, into oHSV will establish photodynamic oncolytic virotherapy that enhances tumoricidal efficacy as a novel treatment approach to CNS neoplasms. METHOD The optical properties of the intracellular KillerRed protein expressed in cells were determined by scanning by a multi-mode microplate reader to determine the optimal irradiation wavelength. In vitro efficacy of KillerRed-mediated PDT was tested using human glioblastoma and malignant meningioma cell lines. oHSV G47delta expressing KillerRed was constructed by a bacterial artificial chromosome-based method. KillerRed-transduced cells were confirmed to express red fluorescence, followed by irradiation by an amber color LED. Cell death and viability were assessed by DAPI staining and MTS assay, respectively. ROS generation post light treatment was assessed by DCF-DA cellular ROS assay. RESULTS KillerRed had an excitation peak at 580-585nm in transduced cells. Light irradiation by an amber LED after infection with G47delta-KillerRed induced increased cell growth inhibition and death compared with virus infection without light or light alone. Increased ROS production was observed following KillerRed PDT. CONCLUSION G47delta-KillerRed enables a combination of oncolytic virus therapy and PDT to augment tumor killing. This approach is being tested in in vivo mouse models using potent focused laser irradiation.

Inhibition of Pseudomonas aeruginosa Biofilm Formation and Quorum Sensing System by Extracts of Prunus avium Stalk

Recently, misuse or overuse of antibiotics has led to the antibiotic resistance problem, a global healthcare problem. Most virulence factors and biofilm formation in Pseudomonas aeruginosa are controlled by quorum sensing (QS). The inhibition of QS system by inhibitor molecules has been suggested as a novel alternative antivirulence approach in which no need to kill the bacteria. In the present study, QS and biofilm inhibitory potentials of the methanol and acetone extracts of Prunus avium stalk against P. aeruginosa were evaluated. The extracts were tested at the concentrations of 240, 120, and 60 μg/ml. lasB-gfp, rhlA-gfp, pqsA-gfp biosensor strains and P. aeruginosa PAO1 were used to monitor QS and biofilm inhibition, respectively. Fluorescence and absorbance measurements were performed on Cytation 3 multimode microplate reader. QS inhibition ratios for las, rhl, and pqs systems and biofilm inhibition ratios of the acetone extracts were recorded as 70.43%, 47.25%, 76.31%, and 47.76% (±6,60) and of the methanol extracts as 74.96%, 40.10%, 71.89%, and 38.54% (±3,56) at a certain concentration of 240 μg/ml, respectively. As a result, anti-QS and anti-biofilm properties of acetone extracts were better than that of methanol extracts. Further investigations are needed to discover inhibitor compounds of P. avium and also their effects on human cells and then these compounds may be used in new drug discoveries.

Infusion or Decoction Extracts of Helianthus annuus Leaves: Potential Inhibitors for QS system and Biofilm Formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the drug-resistant opportunistic pathogens with the ability to form biofilm and to produce a number of virulence factors via Quorum Sensing (QS) regulation. Most researchers have focused on QS inhibition to overcome the drug resistance problem. QS inhibitor molecules are investigated from natural resources. In the present study, anti-QS activities of ethyl acetate extracts of decoction and infusion samples from Helianthus annuus leaves were tested on biosensor strains of P. aeruginosa (lasB-gfp, rhlA-gfp and pqsA-gfp), as well as anti-biofilm activities on PAO1 wild type. H. annuus leaf samples were firstly infused or decocted and then extracted with ethyl acetate. The efficacies of infusion or decoction extracts were examined at the concentrations of 240, 120, and 60 μg/ml in 96-well microplates and evaluated in Citation 3 multimode microplate reader (Biotek). The inhibition rates of decoction extracts were recorded as 70.61% for las, 44.09% for rhl and 83.77% pqs system at 240 μg/ml. The biofilm inhibition percentages of the extracts were determined to be 50.82% (±1.36). Moreover, inhibition rates for infusion extracts were detected as 62.08% for las, 45.15% for rhl and 77.79% for pqs, and 53.88% (±3.94) for biofilm formation. In conclusion, the potential efficacies of the extracts of decocted or infused H.annuus leaves were demonstrated on QS system and biofilm formation of P. aeruginosa. However, there is a need for more detailed investigations and determination of the active substances that have QSI and anti-biofilm effect.

The fluorescence of resin-based composites: An analysis after ten years of aging.

The long-term preservation of fluorescence qualities of resin-based composite (RBC) restorations is an absolute condition for the implementation of the fluorescence-aided identification technique (FIT) in dentistry and forensic medicine. Therefore, this study assessed the fluorescence of 244 color shades of 16 commercially available RBC brands with a monochromator-based multimode microplate reader. The specimens were stored in the dark at room temperature and reassessed ten years after the initial investigation. The mean intensity of the fluorescence maxima decreased from (31,030±936) RFU to (22,027±632) RFU. Linear regression resulted in r2=0.972 and a slope=0.701±0.005. The fluorescence intensity of the tested RBCs dropped to about 70% of the initial intensity independent of the brand, color shade and initial fluorescence intensity. On the basis of this in vitro 10-year data set, we assume that in vivo RBC fluorescence is also suitable for the detection and differentiation of clinically aged RBC restorations by FIT.

Rapid screening and evaluation of XOD inhibitors and O2 •- scavenger from total flavonoids of Ginkgo biloba leaves by LC-MS and multimode microplate reader.

Superoxide anion radical scavenger and xanthine oxidase inhibitor play an important role in the treatment of several relevant human diseases. In the present study, ultrafiltration liquid chromatography-mass spectrometry coupled to microplate reader was applied to screen and identify superoxide anion radical scavengers and xanthine oxidase inhibitors from total flavonoids of Ginkgo biloba leaves. As a result, four compounds (quercetin, apigenin, kaempferol and isorhamnetin) were screened as xanthine oxidase inhibitors by ultrafiltration LC-MS, and the 50% scavenging concentration values of the screened flavonoids were lower than those for allopurinol. Lineweaver-Burk plot results indicated that kaempferol was a competitive xanthine oxidase inhibitor; the other flavonoids were all anticompetitive inhibitors. Four flavonoids-rutin, quercetin, kaempferol and isorhamnetin-were screened as superoxide anion radical scavengers by LC-MS. The results demonstrate that the method for screening and evaluation of superoxide anion radical scavenger and xanthine oxidase inhibitor from a complex mixture system is feasible and efficient.

Assessment of Morphine Tolerance Using cAMP‐GloSensor&amp;[trade] Real‐time Detection Assay in SH‐SY5Y Human Neuroblastoma Cells

PurposeActivation of GPCRs by morphine triggers downstream effects including inhibition of adenylyl cyclase and decrease in 3′,5′‐cyclic adenosine monophosphate (cAMP). cAMP upregulation is observed in morphine tolerance and withdrawal. Endothelin‐A receptor (ETAR) antagonist, BQ123 potentiates morphine analgesia, reverses tolerance, and reduces withdrawal symptoms. However, signaling mechanisms involved in these phenomena are unknown. The objective of this study was to determine morphine‐ and BQ123‐induced changes in cAMP signaling in SH‐SY5Y neuroblastoma cells which endogenously express mu‐opioid receptors (MORs) and ETARs.MethodsSH‐SY5Y cells (ATCC®) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37°C/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104 cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and 30% transfection efficiency was achieved. Stably transfected SH‐SY5Y cell line was established using GloSensor™ −23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Promega’s real‐time detection system in live cells, the cAMP‐GloSensor™ assay was used to measure cAMP responses. Stably transfected cells were incubated with equilibrium buffer [cAMP GloSensor™ reagent (2% (v/v)), 88% CO2 independent medium, 10% FBS]. cAMP levels were determined by changes in luminescence using an EnSpire™ multimode microplate reader (PerkinElmer). In acute studies, cells were treated for 2 h with morphine (10–10−5 M) and BQ123 (1 μM). In chronic studies, cells were treated for 24 h with morphine (1 μM) in the presence and absence of BQ123 (1 μM). After induction of tolerance for 24 h, two challenge doses of morphine (10 μM and 20 μM) were tested. Isoproterenol was used as a positive control (1–10−8 M). Each experiment was repeated in triplicate and data was analyzed in GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA).ResultsA concentration‐dependent increase in cAMP was observed with isoproterenol in SH‐SY5Y cells stably transfected with −23F cAMP plasmid. Acute morphine treatment (10–10−5 M for 2 h) produced concentration‐dependent decrease in cAMP, while acute treatment with BQ123 (1–10−5 M) increased cAMP levels. Of interest, BQ123 (1 μM) appeared to enhance morphine‐induced decrease in cAMP response. Chronic morphine treatment (1 μM for 24 h) increased cAMP response, indicative of tolerance. Cells treated with combination of morphine (1 μM) and BQ123 (1 μM) for 24 h showed dose‐dependent inhibition of cAMP levels with challenge doses of morphine.ConclusionTo better understand the mechanism by which ETAR antagonists reverse morphine tolerance and reduce withdrawal symptoms, signaling mechanisms were explored in SH‐SY5Y cells endogenously expressing ETA receptors and MORs. We found that BQ123 appears to enhance morphine‐induced cAMP signaling (measured by Glo‐Sensor™ cAMP assay) through Gi‐coupled MORs in both acute and chronic studies, which support our previous work. We will further explore the involvement of beta‐arrestin and GPCR kinases (GRKs) in these phenomena.Support or Funding InformationChicago College of Pharmacy Faculty Research Grant; College of Graduate Studies, Biomedical Sciences, Midwestern University, Downers Grove IL.

Abstract C085: A new method to determine drug-target residence time of kinase inhibitors in living cells

Abstract Background: Recently, it has become clear that drug-target residence time, in addition to affinity, often drives pharmacodynamic activity and disease efficacy in vivo. In the aim of determining the dissociation properties of test compounds, the kinase field has traditionally utilized purified proteins in various competitive probe displacement formats. Using such detection formats, kinetic parameters of drug-target binding can be quantified using analytical approaches described by Motulsky and Mahan. While quantitative, such approaches may fail to translate with cellular efficacy due to reliance on pure proteins or protein fragments. Furthermore, these methods are generally not amenable to high-throughput analysis for exploring intracellular drug-target residence time. Thus, a simple and quantitative method to evaluate the residence time of compounds inside living cells is highly desirable. Method: The NanoBRET™ Target Engagement Intracellular Kinase Assay was employed to investigate the kinetics of target engagement for various test compounds in living cells. This method utilizes BRET (Bioluminescence Resonance Energy Transfer) in cells by molecular proximity of the NanoBRET™ Tracer to the NanoLuc® luciferase-fused kinase. Quantitative determination of both of apparent affinity as well as binding kinetics for unodified test compounds can be achieved by competitive displacement of the Tracer. Full-length BTK fused with the NanoLuc® luciferase was transiently transfected in HEK293 cells and the cells were incubated at 37°C, 5% CO2 overnight. Transfected cells were harvested and suspended in 1 ml of Assay Medium in a conical tube. After incubation with test compound for 2 hours, the cells were washed with Assay Medium to remove unbound compound. Dosing of test compounds was an approximately IC80-90 concentration unless otherwise specified. The cell suspension was dispensed into a 96-well plate and the NanoBRET™ Tracer K-4 or K-5 was added to the wells following the addition of the Nano-Glo® Substrate Solution. BRET was measured repeatedly with the Glomax® Discover Multimode Microplate Reader. Data were fitted to the one-phase association equation or the kinetic equation developed by Malany to obtain the dissociation rate constatns of test compounds. Results: The kinetic constants using tracer K-4 or K-5 were successfully quantitated in live cells using NanoBRET™. Though the kinetic constants as determined by simple kobs fitting using tracer K-4 or K-5 were different, the constants as determined using the Malany approach for each tracer were in gratifying agreement. The results determined using the Malany equation reveal suitable differentiation of residence time for the irreversible and reversible compounds. As expected, longer residence time was observed for the irreversible/covalent inhibitors. Some reversible inhibitors also showed protracted residence time, offering opportunities for durable target inhibition in cells. Conclusion: We propose the method by which the data in the compound wash-out experiments are fitted to the kinetic equation of Malany as a simple and quantitative method to determine intracellular residence time of kinase inhibitors. Citation Format: Takeomi Inoue, Aki Emi, James D Vasta, Matthew B Robers, Yusuke Kawase. A new method to determine drug-target residence time of kinase inhibitors in living cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C085. doi:10.1158/1535-7163.TARG-19-C085