A Miniaturized Pump Out Method for Characterizing Molecule Interaction with ABC Transporters

Characterizing interaction of newly synthetized molecules with efflux pumps remains essential to improve their efficacy and safety. Caco-2 cell line cultivated on inserts is widely used for measuring apparent permeability of drugs across biological barriers, and for estimating their interaction with efflux pumps such as P-gp, BCRP and MRPs. However, this method remains time consuming and expensive. In addition, detection method is required for measuring molecule passage across cell monolayer and false results can be generated if drugs concentrations used are too high as demonstrated with quinidine. For this reason, we developed a new protocol based on the use of Caco-2 cell directly seeded on 96- or 384-well plates and the use of fluorescent substrates for efflux pumps. We clearly observed that the new method reduces costs for molecule screening and leads to higher throughput compared to traditional use of Caco-2 cell model. This accelerated model could provide quick feedback regarding the molecule design during the early stage of drug discovery and therefore reduce the number of compounds to be further evaluated using the traditional Caco-2 insert method.

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Absorption and Transport Characteristics and Mechanisms of Carnosic Acid

Biology ◽

10.3390/biology10121278 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1278

Author(s):

Xuexiang Chen ◽

Meigui Huang ◽

Dongmei Liu ◽

Yongze Li ◽

Qiu Luo ◽

...

Keyword(s):

Cell Model ◽

Cell Monolayer ◽

Scientific Basis ◽

Carnosic Acid ◽

Intestinal Epithelial ◽

Apparent Permeability ◽

Beneficial Effects ◽

Single Cell Model ◽

Relevant Dose ◽

Apparent Permeability Coefficient

Carnosic acid (CA) is a phenolic diterpenoid mainly found in rosemary and sage. CA has been reported to possess health-beneficial effects in various experimental settings. Herein, a mouse experiment and Caco-2 single-cell model were used to understand the absorption and transport characteristics of CA. First, we determined the tissue distribution of CA in mice, following an oral gavage at a physiologically relevant dose. We found that CA was bioavailable systemically and present locally in the digestive tract, especially in the cecum and colon. Next, we thought to characterize the absorption and transport of CA in the Caco-2 cell monolayer model of the intestinal epithelial barrier. In the Caco-2 cell model, CA exhibited a moderate permeability and was subjected to a mild efflux. Moreover, the apparent permeability coefficient (Papp) of CA transported across Caco-2 cell monolayers was significantly changed when the inhibitors of specific active transporter and passive diffusion were added to cells, suggesting that the absorption and transport of CA involved both passive and active transportation. The present study is an important first step towards understanding the absorption, transport, and metabolic mechanisms of CA. This could provide the scientific basis for developing CA-containing functional foods or dietary supplements with improved bioavailability.

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Intestinal Transportations of Main Chemical Compositions of Polygoni Multiflori Radix in Caco-2 Cell Model

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2014/483641 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Jie Yu ◽

Na Li ◽

Pei Lin ◽

Yunfei Li ◽

Xiaojian Mao ◽

...

Keyword(s):

Gibbs Energy ◽

Cell Model ◽

Cell Monolayer ◽

Intestinal Transport ◽

Transepithelial Transport ◽

Chemical Compositions ◽

Polygonum Multiflorum ◽

Close Attention ◽

Apparent Permeability ◽

Cell Monolayers

Context. Polygoni Multiflori Radix (PMR) is originated from the root ofPolygonum multiflorumThunb. and used in oriental countries for centuries. However, little researches pay close attention to the absorption of its major constituents.Objective. Transepithelial transport of TSG, RL, PL, and four anthraquinones is carried out.Materials and Methods. Caco-2 cell monolayer, which represented a well-established model for the study of intestinal transport of nutrients and xenobiotics, was used in this paper.Results. The apparent permeability coefficients (Papp) in the Caco-2 cell monolayers were TSG (2.372 × 10−9) < EG (2.391 × 10−9) < EN (2.483 × 10−9) < PL (4.917 × 10−9) < RN (1.707 × 10−8) < RL (1.778 × 10−8) < AE (1.952 × 10−8). Thus, RN, RL, and AE were considered partly absorbed, while other constituents were hardly absorbed.Discussion and Conclusion. Glycosides showed poor permeabilities than aglycones. In the meantime, TSG and EN gave out poor recovery rates in this assay, which indicated that TSG and EN may accumulate or metabolise in the Caco-2 cells.In silicoprediction indicated that Gibbs energy (r=0.751,p<0.05) and heat of form (r=0.701,p<0.05) were strongly positively correlated withPapp.

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Bioavailability Studies of BioTurmin-WD (Water Dispersible Curcuminoids) Using Caco-2 Cell Model

Journal of Food Research ◽

10.5539/jfr.v3n3p158 ◽

2014 ◽

Vol 3 (3) ◽

pp. 158 ◽

Cited By ~ 1

Author(s):

H. N. Shivaprasad ◽

M. Bhanumathy ◽

Subrata Pandit ◽

D. Manohar ◽

B. Pavan Kumar ◽

...

Keyword(s):

Cell Model ◽

Curcuma Longa ◽

Cell Monolayer ◽

Absorption Capacity ◽

Apparent Permeability ◽

Water Dispersible ◽

Therapeutic Value ◽

Clinical Benefits ◽

Apparent Permeability Coefficient

Curcuminoids is the mixture of curcumin, demethoxycurcumin and bisdemethoxycurcumin obtained from dried rhizomes of Curcuma longa, commonly used for its wide therapeutic value. However, the absorption efficacy of curcuminoids is too low to exhibit its proper therapeutic value. Thus, a new preparation named as BioTurmin-WD (water dispersible curcuminoids) was developed for improved bioavailability. By using human Caco-2 cell monolayer, the permeability efficacy of BioTurmin-WD was evaluated and compared with that of 95% pure curcuminoids. Caco-2 model predicts the in vivo absorption of drugs across the gut wall by measuring the rate of transport of a compound across the Caco-2 cell line. BioTurmin-WD was added to the apical layer and basolateral samples were collected over 120 min to examine the concentration diffusing across the cell monolayer. Permeable curcuminoids across the cell monolayer was analysed through reverse phase high pressure liquid chromatography (RP-HPLC). Apparent permeabilities (Papp) of BioTurmin-WD and 95% curcuminoids were found to be 5.89 × 10-6 and 2.65 × 10-6 cm/s respectively. The apparent permeability coefficient of BioTurmin-WD was 7.03-fold higher than 95% pure curcuminoids. Percentage permeability of BioTurmin-WD (0.2945) was much higher than 95% curcuminoids (0.0859). Results indicated that BioTurmin-WD have a much higher absorption capacity (bioavailability) compared to 95% pure curcuminoids. Thus, BioTurmin-WD may be useful as a dietary supplement with greater bioavailability to exert clinical benefits in humans at a lower dosage.

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Improved micro-impedance spectroscopy to determine cell barrier properties

The EuroBiotech Journal ◽

10.2478/ebtj-2020-0017 ◽

2020 ◽

Vol 4 (3) ◽

pp. 150-155 ◽

Cited By ~ 1

Author(s):

Md. Mehadi Hasan Sohag ◽

Olivier Nicoud ◽

Racha Amine ◽

Abir Khalil-Mgharbel ◽

Jean-Pierre Alcaraz ◽

...

Keyword(s):

Impedance Spectroscopy ◽

Epithelial Cells ◽

Lipid Bilayers ◽

Cultured Cells ◽

Cell Model ◽

Measurement Techniques ◽

Cell Monolayer ◽

Microfluidic Channel ◽

Small Surface ◽

Resistance Pathway

AbstractThe goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm2) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.

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Foregut endoderm is required at head process stages for anteriormost neural patterning in chick

Development ◽

10.1242/dev.128.3.309 ◽

2001 ◽

Vol 128 (3) ◽

pp. 309-320 ◽

Cited By ~ 5

Author(s):

S. Withington ◽

R. Beddington ◽

J. Cooke

Keyword(s):

Heart Development ◽

Lower Layer ◽

Early Stage ◽

Cell Monolayer ◽

Homeobox Gene ◽

Primitive Streak ◽

Definitive Endoderm ◽

Gene Expressions ◽

Specific System ◽

Foregut Endoderm

Anterior definitive endoderm, the future pharynx and foregut lining, emerges from the anterior primitive streak and Hensen's node as a cell monolayer that replaces hypoblast during chick gastrulation. At early head process stages (4+ to 6; Hamburger and Hamilton) it lies beneath, lateral to and ahead of the ingressed axial mesoderm. Removal of the monolayer beneath and ahead of the node at stage 4 is followed by normal development, the removed cells being replaced by further ingressing cells from the node. However, similar removal during stages 4+ and 5 results in a permanent window denuded of definitive endoderm, beneath prechordal mesoderm and a variable sector of anterior notochord. The foregut tunnel then fails to form, heart development is confined to separated lateral regions, and the neural tube undergoes no ventral flexures at the normal positions in brain structure. Reduction in forebrain pattern is evident by the 12-somite stage, with most neuraxes lacking telencephalon and eyes, while forebrain expressions of the transcription factor genes GANF and BF1, and of FGF8, are absent or severely reduced. When the foregut endoderm removal is delayed until stage 6, later forebrain pattern appears once again complete, despite lack of foregut formation, of ventral flexure and of heart migration. Important gene expressions within axial mesoderm (chordin, Shh and BMP7) appear unaffected in all embryos, including those due to be pattern-deleted, during the hours following the operation when anterior brain pattern is believed to be determined. A specific system of neural anterior patterning signals, rather than an anterior sector of the initially neurally induced area, is lost following operation. Heterotopic lower layer replacement operations strongly suggest that these patterning signals are positionally specific to anteriormost presumptive foregut. The homeobox gene Hex and the chick Frizbee homologue Crescent are both expressed prominently within anterior definitive endoderm at the time when removal of this tissue results in forebrain defects, and the possible implications of this are discussed. The experiments also demonstrate how stomodeal ectoderm, the tissue that will, much later, form Rathke's pouch and the anterior pituitary, is independently specified by anteriormost lower layer signals at an early stage.

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Intracellular amyloid toxicity induces oxytosis/ferroptosis regulated cell death

Cell Death and Disease ◽

10.1038/s41419-020-03020-9 ◽

2020 ◽

Vol 11 (10) ◽

Cited By ~ 2

Author(s):

Ling Huang ◽

Daniel B. McClatchy ◽

Pamela Maher ◽

Zhibin Liang ◽

Jolene K. Diedrich ◽

...

Keyword(s):

Cell Death ◽

Cell Model ◽

Early Stage ◽

Metabolic Reprogramming ◽

Therapeutic Interventions ◽

Amyloid Toxicity ◽

Cell Death Pathway ◽

Regulated Cell Death ◽

Metabolomic Data ◽

Basic Biology

Abstract Amyloid beta (Aβ) accumulates within neurons in the brains of early stage Alzheimer’s disease (AD) patients. However, the mechanism underlying its toxicity remains unclear. Here, a triple omics approach was used to integrate transcriptomic, proteomic, and metabolomic data collected from a nerve cell model of the toxic intracellular aggregation of Aβ. It was found that intracellular Aβ induces profound changes in the omics landscape of nerve cells that are associated with a pro-inflammatory, metabolic reprogramming that predisposes cells to die via the oxytosis/ferroptosis regulated cell death pathway. Notably, the degenerative process included substantial alterations in glucose metabolism and mitochondrial bioenergetics. Our findings have implications for the understanding of the basic biology of proteotoxicity, aging, and AD as well as for the development of future therapeutic interventions designed to target the oxytosis/ferroptosis regulated cell death pathway in the AD brain.

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QuantitativeIn VitroandIn VivoEvaluation of Intestinal and Blood-Brain Barrier Transport Kinetics of the PlantN-Alkylamide Pellitorine

BioMed Research International ◽

10.1155/2016/5497402 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Lieselotte Veryser ◽

Nathalie Bracke ◽

Evelien Wynendaele ◽

Tanmayee Joshi ◽

Pratima Tatke ◽

...

Keyword(s):

Blood Brain Barrier ◽

Rate Constant ◽

Cell Monolayer ◽

Elimination Rate ◽

Brain Barrier ◽

Apparent Permeability ◽

Influx Rate ◽

Blood Brain ◽

Apical Side ◽

The Brain

Objective.To evaluate the gut mucosa and blood-brain barrier (BBB) pharmacokinetic permeability properties of the plantN-alkylamide pellitorine.Methods.Pure pellitorine and anAnacyclus pyrethrumextract were used to investigate the permeation of pellitorine through (1) a Caco-2 cell monolayer, (2) the rat gut after oral administration, and (3) the BBB in mice after intravenous and intracerebroventricular administration. A validated bioanalytical UPLC-MS2method was used to quantify pellitorine.Results.Pellitorine was able to cross the Caco-2 cell monolayer from the apical-to-basolateral and from the basolateral-to-apical side with apparent permeability coefficients between0.6·10-5and4.8·10-5 cm/h and between0.3·10-5and5.8·10-5 cm/h, respectively. In rats, a serum elimination rate constant of 0.3 h−1was obtained. Intravenous injection of pellitorine in mice resulted in a rapid and high permeation of pellitorine through the BBB with a unidirectional influx rate constant of 153 μL/(g·min). In particular, 97% of pellitorine reached the brain tissue, while only 3% remained in the brain capillaries. An efflux transfer constant of 0.05 min−1was obtained.Conclusion.Pellitorine shows a good gut permeation and rapidly permeates the BBB once in the blood, indicating a possible role in the treatment of central nervous system diseases.

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123 GENISTEIN TRANSPORT ACROSS THE BOVINE OVIDUCT EPITHELIUM

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab123 ◽

2013 ◽

Vol 25 (1) ◽

pp. 208 ◽

Cited By ~ 2

Author(s):

C. Simintiras ◽

F. L. Courts ◽

R. G. Sturmey

Keyword(s):

Epithelial Cells ◽

Room Temperature ◽

Cell Monolayer ◽

Barrier Properties ◽

Apparent Permeability ◽

Mammalian Embryo ◽

Significant Difference ◽

Apical Compartment ◽

Oviduct Epithelium

The oviduct plays a vital role in regulating the environment surrounding the gametes and early mammalian embryo. However, the permeability of the oviduct to circulating dietary-derived compounds remains relatively unknown. The present study has investigated the barrier properties of the oviduct epithelium in vitro to the movement of genistein, a soya isoflavone and analogue of 17β-estradiol that naturally occurs in the diet and has been reported to exert teratogenic effects. Bovine oviduct epithelial cells (BOECs) were isolated from abattoir-derived reproductive tracts at the mid-luetal phase. The purity of the cell isolate was confirmed using flow cytometry to determine the proportion of cells that expressed CK18 (epithelia), and Vimentin (fibroblasts). Cells were seeded at a density of 1 × 106 mL–1 on polyethylene terephthalate transwell porous supports (Corning) and maintained between two media-filled chambers for 10 to 12 days, until they formed a polarised confluent monolayer, as confirmed by transepithelial resistance (TEER) greater than 700 Ωcm2. To assess the rate of transport, genistein was added to the basal compartment at physiologically relevant levels (100 µM) and the apical compartment was sampled at regular time points for 120 mins. The concentration of genistein in the apical and basal media was measured by HPLC. Furthermore, we compared the rate of genistein transport at physiological (39°C) and room temperature to indicate whether transport was temperature dependent. Rates of transport are expressed as mean apparent permeability coefficient and were compared between groups by a Student’s t-test. Genistein crossed the bovine oviduct epithelium at a linear rate that was higher than spontaneous diffusion across a blank membrane support (12.7 × 10–3 cm–2 µM–1 v. 7.32 × 10–3 cm–2 µM–1, n = 4; P = 0.0075). The rate of genistein transport by epithelial cells was unchanged when cells were assayed at room temperature (12.7 × 10–3 cm–2 µM–1 v. 13.11 × 10–3 cm–2 µM–1, n = 3; P = 0.76), respectively. No significant difference in the directionality of transport was found. Furthermore, TEER was maintained at approximately 700 Ωcm2, indicating that the cells remained confluent for the duration of the experiment. These data suggest that the bovine oviduct epithelial cell monolayer facilitates genistein movement from the basal to the apical compartment in vitro. Furthermore, the observation that the rate of transport is unchanged by temperature suggests a passive, trans-cellular, physicochemical mechanism, rather than an active biological process. Regardless of the mechanism, the oviduct epithelium is permeable to genistein, and may even facilitate its transport into the lumen, suggesting that gametes and early embryos could be exposed to this compound in vivo. This is relevant given the previously reported finding indicating that this naturally occurring dietary isoflavone has detrimental effects on early development (Newbold et al. 2001 Cancer Res. 61, 4325–4328). Furthermore, the results demonstrate the potential use of this epithelial model in characterising the transport or barrier properties of the oviduct epithelium towards a range of circulating xenobiotics.

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Detecting Loosening of Bolted Connections in a Pipeline Using Changes in Natural Frequencies

Volume 1: 23rd Biennial Conference on Mechanical Vibration and Noise, Parts A and B ◽

10.1115/detc2011-47912 ◽

2011 ◽

Cited By ~ 1

Author(s):

K. He ◽

W. D. Zhu

Keyword(s):

Damage Detection ◽

Natural Frequencies ◽

Detection Method ◽

Complex Geometry ◽

Early Stage ◽

Measurement Noise ◽

Modeling Error ◽

Bolted Connections ◽

Bolted Connection ◽

Stiffness Reduction

Loosening of bolted connections in a structure can significantly reduce the load-bearing capacities of the structure. Detecting loosening of bolted connections at an early stage can avoid failure of the structure. Due to the complex geometry of a bolted connection and the material discontinuity between the clamped components, it is difficult to detect loosening of a bolted connection using conventional non-destructive test methods. A vibration-based method that uses changes in natural frequencies of a structure to detect the locations and extent of damage can be used to detect loosening of bolted connections, since the method focuses on detecting a stiffness reduction, which can result from loosening of the bolted connections. Experimental and numerical damage detection using the vibration-based method was conducted to detect the loosening of the bolted connections in a fullsize steel pipeline with bolted flanges. With the recent development of a predictive modeling technique for bolted connections in thin-walled structures, an accurate physics-based finite element model of the pipeline that is required by the vibration-based damage detection method is developed. A trust-region search strategy is employed to improve the damage detection method so that convergence of the damage detection algorithm can be ensured for under-determined systems, and the robustness of the algorithm can be enhanced when relatively large modeling error and measurement noise are present. The location and extent of the loosened bolted connections were successfully detected in experimental damage detection using changes in the natural frequencies of the first several modes; the exact location and extent of the loosened bolted connections can be detected in the numerical simulation where there are no modeling error and measurement noise.

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Early Visualization Detection of Gray Mold (Botrytis) on Eggplant Leaves Based on Multi-Spectral Image

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.741.323 ◽

2015 ◽

Vol 741 ◽

pp. 323-327

Author(s):

Yan Yang

Keyword(s):

Detection Method ◽

Early Stage ◽

Gray Mold ◽

Usual Method ◽

Excessive Amount ◽

Disease Spread ◽

Spectral Image ◽

Fungal Strains ◽

Entire Field ◽

Fungus Disease

Gray mold (Botrytis) is a common fungus disease on eggplants,it can reduce the production by 20-30% at worse. Most disease infestations are not evenly distributed across the cultivation area but in patches [1]. The usual method of prevention is spraying fungicide on entire field, which would requires an excessive amount of fungicide and resulting, increases cost of production, pollutes the environment, and improves of resistance fungal strains [2]. In fact, the fungicide can be spray only the area infected which is rather small on the early stage and it is fully capable of controlling disease spread. Therefore, rapid disease detection is the key. The accurate and effective detection method would be helpful for reducing the dosage of fungicide and preventing disease spread.

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