Rapid detection of FadA in Fusobacterium nucleatum using the quantitative LAMP colorimetric phenol red method in stool samples from colorectal cancer patients.

The study aimed to develop a quantitative colorimetric loop-mediated isothermal amplification technique using the phenol red indicator (QLAMP-PhR) for detecting Fusobacterium nucleatum (Fn) levels in colorectal cancer (CRC) patients and healthy individuals. QLAMP-PhR assays were conducted on 251 stool samples specific for the Fn FadA gene. Six primers were synthesized and utilized with master mix reagents, and a phenol red indicator was employed to enhance the QLAMP-PhR technique. A standard quantitative analysis curve was generated using a logarithmic function (absorbance vs. concentration) by serially diluting the copy number of genomic DNA templates (Fn ATCC25586). The CRC group exhibited a significantly higher abundance of Fn compared to the healthy control group (P < 0.001). These findings suggest that the QLAMP-PhR technique effectively identifies Fn specifically by its gene for the key virulence factor FadA. Additionally, ideas for developing a real-time QLAMP-PhR test were presented. Compared to the traditional polymerase chain reaction (PCR) technique, QLAMP-PhR offers several advantages including rapidity, simplicity, specificity, sensitivity, and cost-effectiveness method that can quantitatively screen for Fn presence in normal populations. The QLAMP-PhR method represents a sensitive and specific amplification assay for the rapid detection of the Fn pathogen. To the best of our knowledge, this study is the first to report the application of QLAMP-PhR for detecting FadA in Fn.

The phenol red compound: A potential artifact in pharmacological induction of ferroptosis.

Phenol red (PR) is a commonly used compound in culture media as a pH indicator. However, it is unknown whether this compound can interfere with the pharmacological induction of ferroptosis. Here, using high-content live-cell imaging death analysis, we determined that the presence of PR in the culture medium preconditioned normal and tumor cells to ferroptosis induced by system xc- inhibition mediated by imidazole ketone erastin (IKE) or GPX4 blockade in response to RSL-3, but had no significant effects against treatment with the endoperoxide FINO2. Mechanistically, we revealed that PR decreases the levels of the antiferroptotic genes Slc7a11, Slc3a2, and Gpx4, while promoting the overexpression de Acls4, a key inducer of ferroptosis. Additionally, through superresolution analysis, we determined that the presence of PR mislocalizes the system xc- from the plasma membrane. Thus, our results show that the presence of PR in the culture medium can be a problematic artifact for the accurate interpretation of cell sensitivity to IKE or RSL-3-mediated ferroptosis induction.

Direct Detection of SARS-CoV-2 RNA in Saliva with Colorimetric RT-LAMP.

RT-LAMP is an effective alternative to RT-PCR-based diagnostics, offering high specificity, sensitivity, and rapid results. One notable advantage is the robustness of its enzymes, allowing for direct amplification from crude samples without the need for prior isolation of RNA. Colorimetric LAMP is particularly attractive as it eliminates the need for complex instrumentation, making it suitable for point-of-care applications. Here, we present a comprehensive step-by-step protocol for establishing an RT-LAMP-based test for direct detection of SARS-CoV-2 genomic RNA in saliva samples using different colorimetric detection methods. Importantly, this versatile test can be easily adapted to detect emerging pathogens.

Development of a RP-HPLC method for simultaneous determination of atenolol, metoprolol tartrate and phenol red for in-situ rat intestinal perfusion studies.

Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76-50 µg/mL), MT (1.14-50 µg/mL), and PR (0.47-20 µg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.

Fabrication of freshness indicators based on methylcellulose-containing color indicator solutions for monitoring the quality of coconut water.

This is the first study to apply intelligent packaging to coconut water. The purpose of this study was to determine the best color indicator solution for making freshness indicator labels based on methylcellulose along with the color change profile of coconut water during storage at room temperature. Three color indicator solutions were used, namely phenol red, bromothymol blue, and methyl red, which were then continued with the fabrication of freshness indicator labels based on methylcellulose from each of these color indicator solutions and applied to coconut water at 25 °C room temperature storage for 24 h with observations every 4 h in the form of pH, total dissolved solids, total acid, turbidity, total microbes, CO2 gas, O2 gas, and freshness indicator label color changes. The values of pH, total soluble solids, and O2 gas decreased with storage time, whereas the values of total acid, turbidity, total microbes, and CO2 gas continued to increase. The methylcellulose-based phenol red freshness indicator label provides the best color change profile that matches the freshness condition of coconut water, namely purplish red (fresh), orange (immediately consumed), and yellow (damaged) so that it can be used as intelligent packaging to monitor the quality of coconut water.

Preparation and properties of polyvinyl alcohol/chitosan-based hydrogel with dual pH/NH3 sensor for naked-eye monitoring of seafood freshness.

To address the issue of food spoilage causing health and economic loss, we developed a pH/NH3 dual sensitive hydrogel based on polyvinyl alcohol/chitosan (PVA/CS) containing chitosan-phenol red (CP). The CP was synthesized via Mannich reaction and immobilized it in PVA/CS hydrogel through freezing/thawing method to prepare the final PVA/CS/CP hydrogel. The synthesis of CP was confirmed by 1H NMR, FT-IR, XRD, UV-vis, and XPS. The characteristics of hydrogel were evaluated by FT-IR, XRD, SEM, mechanical properties, thermal stability, leaching, and color stability tests. The PVA/CS/CP hydrogel showed distinctly different color at various pH and NH3 vapor levels (yellow to purple). The hydrogel exhibited obvious color changes (ΔE = 46.95) in response to shrimp spoilage, stored at 4 °C. It showed positive and strong correlation between the ΔE values of the indicator hydrogel and total volatile basic nitrogen (TVB-N) as (R2 = 0.9573) and with pH as (R2 = 0.8686), respectively. These results clearly show that the PVA/CS/CP hydrogel could be applied for naked-eye real-time monitoring of seafood freshness in intelligent packaging.

Electropolymerization of poly(phenol red) on laser-induced graphene electrode enhanced adsorption of zinc for electrochemical detection.

We present a highly sensitive and selective electrode of laser-induced graphene modified with poly(phenol red) (P(PhR)@LIG) for measuring zinc nutrition in rice grains using square wave anodic stripping voltammetry (SWASV). The physicochemical properties of P(PhR)@LIG were investigated with scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), Fourier infrared spectroscopy (FT-IR) and Raman spectroscopy. The modified electrode demonstrated an amplified anodic stripping response of Zn2+ due to the electropolymerization of P(PhR), which enhanced analyte adsorption during the accumulation step of SWASV. Under optimized parameters, the developed sensor provided a linear range from 30 to 3000 µg L-1 with a detection limit of 14.5 µg L-1. The proposed electrode demonstrated good reproducibility and good anti-interference properties. The sensor detected zinc nutrition in rice grain samples with good accuracy and the results were consistent with the standard ICP-OES method.

Evaluation of semi-quantitative colorimetric assays based on loop-mediated isothermal amplification indicators by using image analysis.

Colorimetric assays are some of the most convenient detection methods, creating discoloration in solutions that is visible to the naked eye. However, colorimetric reactions have some limitations regarding the variability in the color perception of individuals caused by factors such as color blindness, experience, and gender. Semi-quantitative chromatic analysis has been used as an alternative method to differentiate between two colors and accurately interpret the results from a numerical value, with high confidence. Therefore, we developed and determined the optimal model between Red-Green-Blue (RGB) and Commission Internationale de l'Eclairage (CIE) Lab color spaces to establish a semi-quantitative colorimetric assay via image analysis by the ImageJ program for loop-mediated isothermal amplification (LAMP), using the dyes malachite green and phenol red. The semi-quantitative colorimetric assays using the color distance values of the CIELab color space (ΔEab) were more suitable than those using the RGB color space (ΔERGB) for chromatic differentiation between positive and negative reactions in both indicator dyes, demonstrating the feasibility of this assay to be applied in the detection of a wide range of pathogens and infectious diseases.

Validation of the phenol red thread test in a Chinese population.

BACKGROUND: To investigate the validation of phenol red thread (PRT) test in a Chinese population by evaluating the intraobserver repeatability and interobserver reproducibility, determining correlations between the PRT test and other dry eye disease (DED) parameters including tear meniscus height (TMH) and Schirmer I test, and testing the accuracy of diagnosing DED when using the PRT test alone. METHODS: A total of 108 eyes were involved in this prospective and diagnostic study, and were divided into two groups (with and without DED). Each subject underwent a series of ocular surface examinations, including Ocular Surface Disease Index (OSDI) questionnaire, non-invasive tear breakup time (NIBUT), tear meniscus height (TMH) assessment, PRT test, fluorescein tear breakup time (FBUT), corneal fluorescein staining and Schirmer I test. RESULTS: In the experimental group and the control group, the intra-class correlation coefficients (ICCs) of the repeatability were 0.747 and 0.723, respectively (all P < 0.05). The ICCs of the reproducibility in both groups were 0.588 and 0.610, respectively (all P < 0.05). The PRT test correlated weakly with the Schirmer I test and the tear meniscus height, with Spearman coefficients of 0.385 and 0.306, respectively (all P < 0.05). The PRT test is available to diagnose DED, with an area under the curve of 0.806 and a Youden index of 0.556 at the cutoff point of 8.83 mm. CONCLUSIONS: The PRT test can provide patients a comfortable, timesaving and less irritating approach to screening and diagnosing DED compared to Schirmer I test.

Dye-Modified, Sonochemically Obtained Nano-SnS2 as an Efficient Photocatalyst for Metanil Yellow Removal.

We investigate the possibility of modification of SnS2 powder through sonochemical synthesis with the addition of an organic ligand. For that purpose, two organic dyes are used, Phenol Red and Anthraquinone Violet. All obtained powders are characterized using XRD, SEM, EDX, FT-IR, and UV-Vis investigations. Synthesized samples showed composition and structural properties typical for sonochemically synthesized SnS2. However, investigation with the Tauc method revealed that SnS2 powder modified with Phenol Red exhibits a significant shift in value of optical bandgap to 2.56 eV, while unmodified SnS2 shows an optical bandgap value of 2.42 eV. The modification of SnS2 powder with Anthraquinone Violet was unsuccessful. The obtained nanopowders were utilized as photocatalysts in the process of Metanil Yellow degradation, revealing that SnS2 modified with Phenol Red shows about 23% better performance than the unmodified one. The mean sonochemical efficiency of the performed synthesis is also estimated as 9.35 µg/W.

Specific identification of Mycobacterium bovis by Loop-Mediated Isothermal Amplification (LAMP) targeting the Region of Difference 12 (RD12) of the M. tuberculosis complex.

Bovine tuberculosis is a prevalent zoonotic disease that causes high risks for production animals, dairy producers and consumers, together with significant economic losses. Thus, methods for easy, fast and specific detection of Mycobacterium bovis in small and medium-sized livestock under field conditions are very required. In this work, a Loop-Mediated Isothermal Amplification LAMP-PCR targeting the Region of Difference 12 (RD12) of M. bovis genome was designed for the purpose of identification. A set of six primers designed for the isothermal amplification of five different genomic fragments led to the specific identification of M. bovis from other mycobacterial species. A basic colorimetric reaction was clearly observed at first sight under natural light, indicating positive identification of M. bovis in a maximum of 30 min of isothermal amplification at 65 °C. The limit of detection was near 50 fg of M. bovis genomic DNA, corresponding approximately to 10 copies of the genome. •The proposed LAMP-PCR amplification of M. bovis genomic DNA might be performed by untrained laboratory personnel.•Specific identification of M. bovis LAMP is possible in 30 min at 65.. C using a simple water bath.•The basic colorimetric reaction for M. bovis identification could be observed with the naked eye under natural light.

New insights on the adsorption of phenol red dyes from synthetic wastewater using activated carbon/Fe2(MoO4)3.

Water shortage is considered as one of the main challenges of human life. A practical solution to this problem is the wastewater treatment. The removal of dyes from wastewaters has received considerable critical attention by researchers due to their high volume and toxicity. In the current research, the adsorption of phenol red dyes from synthetic wastewater using the activated carbon produced from Mespilus germanica modified with Fe2(MoO4)3 was studied. The proposed adsorbent was characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), energy dispersive X-ray analysis (EDX)/Map, Brunauer-Emmett-Teller (BET), and Raman techniques. The optimal adsorption operating parameters including pH, stirring rate, temperature, dosage of adsorbent, dye initial concentration, and contact time were 3, 500 rpm, 25 °C, 1 g/L, 10 mg/L, and 60 min, respectively. Furthermore, the successful regeneration of the adsorbent for 3 times, using methanol solution as a regeneration medium, denoted its capability in performing adsorption and desorption processes. Equilibrium studies showed that the adsorption of phenol red dyes by activated carbon (AC)/Fe2(MoO4)3 was desirable and physical and the experimental data were fitted well by the Freundlich model. In addition, the kinetic behavior of the current adsorption process was well described by the pseudo-second-order kinetic model, while thermodynamic calculations showed that the process was exothermic and spontaneous.

Quantitative detection of phenol red by surface enhanced Raman spectroscopy based on improved GA-BP.

Phenol red (PR) is generally used as an acid-base indicator and a printing and dyeing colorant. When its content exceeds a certain concentration in water, it will cause great damage to the human body. Therefore, it is very important to detect the content of PR in water. The advantage of surface enhanced Raman scattering (SERS) is detecting samples quickly, non-destructive and high sensitivity without sample pre-treatment. SERS has attracted great attention in all fields of detection and analysis. In this paper, the method of attaching silver nanoparticles to metallic single-walled carbon nanotubes form carbon nanotubes/silver nanoparticles (CNTs/AgNPs) structure and then combining it with silica sheet is proposed. SERS substrate with silica/carbon nanotubes/silver nanoparticles (SiO2/CNTs/AgNPs) composite structure has extremely high reinforcement effect. In the quantitative analysis of the detected substance, mathematical fitting or machine learning is used to find the relationship between the intensity of Raman signal and the concentration of the detected substance. The BP neural network optimized by genetic algorithm (GA-BP) is designed in this study. The weights of GA-BP to enhance the robustness of BP neural network, the method of adaptive learning rate and the number of hidden nodes is set to solve the problem that GA-BP is easy to fall into local optimum, thus establishing a quantitative analysis model of PR solution concentration. The model can detect different concentrations of PR solutions with high accuracy quickly, simply and sensitively. Finally, compared with other published quantitative models, GA-BP correlation coefficient R2 determined by the training results of the model is 0.99996, and the root mean square error of the prediction is RMSEP = 0.002510, which is 0.0005 higher than the mathematical fitting method, it shows better performance. A reliable idea for the preparation of SERS substrate and online detection of PR concentration in water proposed in this study.

Phenol red dye removal from wastewater using TiO2-FSM-16 and Ni-FSM-16 photocatalysts.

In this study, the performance of Ni-FSM-16 and TiO2-FSM-16 photocatalysts in phenol red removal was explored. The XRD, FE-SEM, and BET tests were used to characterize the catalysts. All experiments were performed at ambient temperature and under UV (20 W). The parameters including dye concentration (20-80 mg/L), photocatalyst concentration (0-8 g/L), UV exposure duration, and contact time (0-160 min) were optimized using RSM software. BET values of Ni-FSM-16 and TiO2-FSM-16 were 718.63 m2/g and 844.93 m2/g, respectively. TiO2-FSM-16 showed better performance in dye removal than Ni-FSM-16. At pH 3, the maximum dye removal by TiO2-FSM-16/UV and Ni-FSM-16/UV was obtained 87% and 64%, respectively. The positive hole species had the main role in photocatalytic phenol red removal. The reusability study was done for up to 7 cycles, but the catalysts can be reused effectively for up to 3 cycles. The synergistic factor for the TiO2-FSM-16 and TiO2-FSM-16/UV processes were calculated to be 1.55 and 2.12, respectively. The dye removal efficiency by TiO2-carbon and Ni-carbon was slightly lower than those obtained by the FSM-16 ones. The TiO2-FSM-16 and Ni-FSM-16 catalysts had a suitable surface and acceptable efficiency in phenol red removal.

Bilberry-containing supplements on severe dry eye disease in young and middle-aged adults: A 3-month pilot analysis.

Purpose: To explore the effect of bilberry and fish oil combination supplement on a small clinical sample patient-base with severe dry eyes. Methods: Twenty-four subjects were recruited with twelve randomly assigned to the intervention and control groups, respectively. Inclusion criteria included severe dry eye symptoms determined by scores >33 from the Ocular Surface Disease Index (OSDI) questionnaire. The intervention group was instructed to take an oral supplement with key ingredients of 600 mg bilberry extract and 240 mg docosahexaenoic acid-refined fish oil once daily for 3 months. The control group did not take any supplements. Mean changes in OSDI score, non-invasive tear break-up time (NITBUT), phenol red thread test (PRT), and percentage of meibomian gland openings were used as outcome measures. Testing was done at baseline, 1-month, and 3-month follow-up. Comparison between the treatment and control groups, and the younger adult and middle-age groups were performed. Results: The mean baseline values for the treatment and control groups were not clinically different. The OSDI score, NITBUT, PRT, and percentage of meibomian gland openings improved after taking the supplements for 3 months. The OSDI score, NITBUT, and PRT showed clinical improvements between the intervention and control groups. These improvements were consistent between the two age groups. Conclusion: This study suggested preliminary improvements in signs and symptoms of severe dry eyes that were independent of age after taking dietary supplementation of bilberry extract and fish oil for 3 months. Further studies using more device-based measures and a placebo supplement are warranted.

The evaluation of normal ocular parameters in two breeds of hedgehogs.

OBJECTIVES: The purpose of this study was to evaluate conjunctival microflora and measure normal tear production and intraocular pressure (IOP) in two breeds of hedgehogs (long-eared hedgehogs and Brandt's hedgehogs). METHODS: Forty-eight hedgehogs from two different breeds were chosen for the study. Tear production was measured using the Schirmer tear test (STT) and phenol red thread test (PRTT) in both eyes. IOP was measured using a rebound tonometer. To perform microbiological sampling one drop of tetracaine was instilled in the eyes. Two sterile microswabs were used to collect samples for the microbial and fungal culture. All the microswab samples were transferred in phosphate-buffered saline (PBS) to the laboratory for culture. Two MacConkey and two blood agar media plates were employed for each eye. Oneplate of sabouraud dextrose agar (SDA) was used for the fungal culture for each eye. Standard biochemical tests were performed to identify the isolated organisms. RESULTS: The mean STT and PRTT values were 1.6 ± 0.1 mm/min and 2.4 ± 0.3 mm/15 s in long-eared hedgehogs and 2.2 ± 0.1 mm/min and 2.5 ± 0.3 mm/15 s in Brandt's hedgehogs, respectively. Mean (SD) Intraocular pressure of right eyes in long-eared hedgehog and Brandt hedgehog were 19.7 ± 1.4 mmHg and 19.2 ± 2.4 mmHg, respectively. In the left eyes of long-eared hedgehog and Brandt hedgehog mean (SD) IOP were 19.8 ± 1.5 mmHg and 19.5 ± 2.1 mmHg, respectively. In long-eared hedgehogs, the most common bacteria were Staphylococcus epidermidis and Bacillus spp. In Brandt's hedgehogs, 24 out of 48 eyes had Staphylococcus epidermidis, which was the most commonly isolated bacterial species. CONCLUSIONS: This study established reference intervals for IOP, STT and PRTT in hedgehogs and recognised and compared ocular conjunctival microflora in two breeds of hedgehogs.

Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

BACKGROUND: High cost of commercial RNA extraction kits limits the testing efficiency of SARS-CoV-2. Here, we developed a simple nucleic acid extraction method for the detection of SARS-CoV-2 directly from nasopharyngeal swab samples. METHODS: A pH sensitive dye was used as the end point detection method. The obvious colour changes between positive and negative reactions eliminates the need of other equipment. RESULTS: Clinical testing using 260 samples showed 92.7% sensitivity (95% CI 87.3-96.3%) and 93.6% specificity (95% CI 87.3-97.4%) of RT-LAMP. CONCLUSIONS: The simple RNA extraction method minimizes the need for any extensive laboratory set-up. We suggest combining this simple nucleic acid extraction method and RT-LAMP technology as the point-of care diagnostic tool.

Cryopreservation of undifferentiated and differentiated human neuronal cells.

The effective use of human-derived cells that are difficult to freeze, such as parenchymal cells and differentiated cells from stem cells, is crucial. A stable supply of damage-sensitive cells, such as differentiated neuronal cells, neurons, and glial cells can contribute considerably to cell therapy. We developed a serum-free freezing solution that is effective for the cryopreservation of differentiated neuronal cells. The quality of the differentiated and undifferentiated SK-N-SH cells was determined based on cell viability, live-cell recovery rate, and morphology of cultured cells, to assess the efficacy of the freezing solutions. The viability and recovery rate of the differentiated SK-N-SH neuronal cells were reduced by approximately 1.5-folds compared to that of the undifferentiated SK-N-SH cells. The viability and recovery rate of the differentiated SK-N-SH cells were remarkably different between the freezing solutions containing 10% DMSO and that containing 10% glycerol. Cryoprotectants such as fetal bovine serum (FBS), antifreeze proteins (sericin), and sugars (maltose), are essential for protecting against freeze damage in differentiated neuronal cells and parenchymal cells. Serum-free alternatives (sericin and maltose) could increase safety during cell transplantation and regenerative medicine. Considering these, we propose an effective freezing solution for the cryopreservation of neuronal cells.

Differential Impacts of Phenol Red on Benzo[a]pyrene and Dexamethasone-Modified Cytochrome P450s in Human Cancer Cells.

<b>Background and Objective:</b> Phenol red, the pH indicator in cell culture media, influences the expression of cytochrome P450s (CYPs) in cell lines. This study aimed to examine how phenol red modified CYP induction by benzo[<i>a</i>]pyrene and dexamethasone in human hepatocarcinoma (HepG2), colorectal adenocarcinoma (Caco-2) and choriocarcinoma (BeWo) cells. <b>Materials and Methods:</b> The cells (1×10⁵ cells/well in a 24-well plate) were incubated with benzo[<i>a</i>]pyrene (0.1, 1 and 10 µM) or dexamethasone (1, 5 and 10 µM) in either phenol red or phenol red-free media for 24 hrs. The mRNA expression of CYPs was determined by Real-Time Polymerase Chain Reaction (RT/qPCR). <b>Results:</b> Phenol red enhanced expression of benzo[<i>a</i>]pyrene-induced CYP1A2 inHepG2 and BeWo cells and suppressed benzo[<i>a</i>]pyrene-induced CYP2A6 expression in HepG2 and Caco-2 cells, benzo[<i>a</i>]pyrene induced CYP2B6 expression in HepG2 cells and benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP3A4 expression in HepG2 and Caco-2 cells. The expression of CYP3A5 was affected differently in HepG2 and Caco-2 cell lines. Phenol red enhanced benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP3A5 expression in Caco-2 cells but suppressed benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP3A5 expression in HepG2 cells. <b>Conclusion:</b> Phenol red differentially influenced expression of benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP1A2, CYP2A6, CYP2B6, CYP3A4 and CYP3A5 mRNAs in HepG2, Caco-2 and BeWo cells. Therefore, the inclusion of phenol red in cell culture media is of concern in studies of drug and xenobiotic metabolism via CYPs in human cell line models.

Development and characterization of a selective chromatographic approach to the rapid discovery of ligands binding to muscarinic-3 acetylcholine receptor.

The pursuit of new ligands binding to muscarinic-3 acetylcholine receptor (M3R) is viewed as challenging due to the lack of screening methods with high efficiency. To address such challenges, this work developed and characterized an approach to the rapid discovery of M3R ligands using the immobilized receptor as the chromatographic stationary phase. We fused haloalkane dehalogenase (Halo) as a tag at the C-terminus of M3R. The fusion M3R was immobilized on 6-chlorocaproic acid-activated ammino-microspheres by the specific covalent reaction between the Halo-tag and the linker. Comprehensive characterizations of the immobilized M3R were performed by scanning electron microscope, X-ray photoelectron spectroscopy, and the investigation on the binding of three specific ligands to the receptor. The feasibility of the immobilized M3R in complex matrices was tested by screening the bioactive compounds in Zhisou oral liquid, assessing the interaction between the screened compounds and the receptor using zonal elution, and evaluating the in vivo activity of the targeted compounds. The results evidenced that the immobilized M3R has high specificity, good stability, and the capacity to separate M3R ligands from complex matrices. These allowed us to identify naringin, hesperidin, liquiritigenin, platycodin D, and glycyrrhizic acid as the potential ligands of M3R. The association constants of the five compounds to M3R were 4.44 × 104, 1.11 × 104, 7.20 × 104, 4.15 × 104, and 3.36 × 104 M-1. The synergistic application of the five compounds exhibited an equivalent expectorant activity to the original formula. We reasoned that the current method is possible to provide a highly efficient strategy for the discovery of receptor ligands.