Transcriptomic and western blot characterisation of the human CLEFF4 clone, a new rapid cell line replacement for the Caco2 model.

The CLEFF4 sub clone from stock late passage Caco2 cells has a unique property of being able to develop polarised cell monolayers with high P-gp expression and tight junctions much quicker than the original cell line. Instead of being useful for transport studies 21-24 days after initiating culture, the CLEFF4 cell line matures in 5-6 days with tight junctions surpassing that of 3 week old Caco2 cells in that time frame [1]. This has enabled the CLEFF4 cell line to provide measures of apparent permeability for potential drug candidates, so important for pre-clinical drug development, 4 times faster than the original cell line. RNA samples were collected and analysed at days 4 and 7 of culture over a 3 year period and had full RNA transcriptome analysed by the ranaseq.eu open bioinformatics platform. Protein was also collected from day 4 to day 22 of culture. Differential expression data from the FASTQ files have shown significant differences in expression in multiple genes involved with drug efflux, tight junctions, phase 2 metabolism and growth factors, which have been confirmed from protein determination that may hold the key to understanding accelerated human cell maturation. These gene expression results may be significant for other tissues beyond the gastrointestinal tract, and potentially for accelerated cell growth for the new field of laboratory grown tissues for organ replacement. The data also confirms the different genetic expression in CLEFF4 cells compared to Caco2 and the stable nature of the different expression over many years.

Natural products as a source of Coronavirus entry inhibitors.

The COVID-19 pandemic has had a significant and lasting impact on the world. Four years on, despite the existence of effective vaccines, the continuous emergence of new SARS-CoV-2 variants remains a challenge for long-term immunity. Additionally, there remain few purpose-built antivirals to protect individuals at risk of severe disease in the event of future coronavirus outbreaks. A promising mechanism of action for novel coronavirus antivirals is the inhibition of viral entry. To facilitate entry, the coronavirus spike glycoprotein interacts with angiotensin converting enzyme 2 (ACE2) on respiratory epithelial cells. Blocking this interaction and consequently viral replication may be an effective strategy for treating infection, however further research is needed to better characterize candidate molecules with antiviral activity before progressing to animal studies and clinical trials. In general, antiviral drugs are developed from purely synthetic compounds or synthetic derivatives of natural products such as plant secondary metabolites. While the former is often favored due to the higher specificity afforded by rational drug design, natural products offer several unique advantages that make them worthy of further study including diverse bioactivity and the ability to work synergistically with other drugs. Accordingly, there has recently been a renewed interest in natural product-derived antivirals in the wake of the COVID-19 pandemic. This review provides a summary of recent research into coronavirus entry inhibitors, with a focus on natural compounds derived from plants, honey, and marine sponges.

Deep learning pose estimation for multi-cattle lameness detection.

The objective of this study was to develop a fully automated multiple-cow real-time lameness detection system using a deep learning approach for cattle detection and pose estimation that could be deployed across dairy farms. Utilising computer vision and deep learning, the system can analyse simultaneously both the posture and gait of each cow within a camera field of view to a very high degree of accuracy (94-100%). Twenty-five video sequences containing 250 cows in varying degrees of lameness were recorded and independently scored by three accredited Agriculture and Horticulture Development Board (AHDB) mobility scorers using the AHDB dairy mobility scoring system to provide ground truth lameness data. These observers showed significant inter-observer reliability. Video sequences were broken down into their constituent frames and with a further 500 images downloaded from google, annotated with 15 anatomical points for each animal. A modified Mask-RCNN estimated the pose of each cow to output 5 key-points to determine back arching and 2 key-points to determine head position. Using the SORT (simple, online, and real-time tracking) algorithm, cows were tracked as they move through frames of the video sequence (i.e., in moving animals). All the features were combined using the CatBoost gradient boosting algorithm with accuracy being determined using threefold cross-validation including recursive feature elimination. Precision was assessed using Cohen's kappa coefficient and assessments of precision and recall. This methodology was applied to cows with varying degrees of lameness (according to accredited scoring, n = 3) and demonstrated that some characteristics directly associated with lameness could be monitored simultaneously. By combining the algorithm results over time, more robust evaluation of individual cow lameness was obtained. The model showed high performance for predicting and matching the ground truth lameness data with the outputs of the algorithm. Overall, threefold lameness detection accuracy of 100% and a lameness severity classification accuracy of 94% respectively was achieved with a high degree of precision (Cohen's kappa = 0.8782, precision = 0.8650 and recall = 0.9209).

Adverse Drug Reactions in Breastfed Infants: A Cross-Sectional Study of Lactating Mothers.

Background: Breastfeeding-related adverse drug reactions (ADRs) are thought to be uncommon as reported cases are globally low. The nonspecific nature of these reactions and a lack of awareness and difficulty in identification of ADRs by mothers and clinicians may result in these ADRs being underreported. Aims: This study hypothesized that breastfeeding-related infant ADRs are more frequent than reported. As a first-hand account of breastfeeding mothers, this study aimed to evaluate the impact of the perceived ADRs on the continuation of breastfeeding and maternal treatment. Methods: Women currently breastfeeding or having breastfed in the last 12 months were invited to complete an online survey. The survey comprised 42 questions in 5 sections to obtain data from breastfeeding mothers, including their use of medicines during lactation, perceptions of infant adverse reactions attributable to maternal medication use and its potential impact on breastfeeding. Results: This online survey was completed by 339 women, 42% of whom reported taking at least one medication during breastfeeding. ADRs were reported in 23 infants where a possible or probable causal relationship indicated by a Naranjo score of 1-8 was established in 16 (11.3%). Antibiotics (n = 12) and opioids (n = 2), including tramadol and oxycodone were identified as the most common adverse reaction-causing drugs. The average age of infants at the time of the perceived ADR was 25.6 days (95% confidence interval; 4-85 days; median age 17.5 days). Conclusion: Suspected ADR reporting in this study was significantly greater than those reported to the regulatory body, the Australian Therapeutics Goods Administration, which shows that common breastfeeding-related infant ADRs are underreported.

Are active efflux transporters contributing to infant drug exposure via breastmilk? A longitudinal study.

Although most drugs are considered safe and compatible with breastfeeding, cases of toxic drug exposure have been reported. Active efflux transporters have been implicated as a mechanism in the transfer of drugs from mother to baby via breastmilk. Using breastmilk as a source of human mammary epithelial cells, this novel longitudinal study investigated the expression of four active transporters, namely, MDR1, MRP1, MRP2 and BCRP in the lactating human breast. BCRP gene was found to be strongly overexpressed with levels peaking at 5 months postpartum, potentially indicating a time where a breastfed infant may be at risk of inadvertent exposure to BCRP substrates. Serum albumin, a major component of human breastmilk was increasingly downregulated as lactation progresses. Xanthine oxidase/dehydrogenase, an enzyme in breastmilk attributed to a reduced risk of gastroenteritis caused by Escherichia coli and Salmonella enteritides, was downregulated. Lysozyme and fatty acid synthase are progressively upregulated. This study also shows that breastmilk-derived epithelial cells, when propagated in culture, exhibit characteristics significantly different to those derived directly from breastmilk. This serves to warn that in vitro studies are not a true representation of in vivo processes in the lactating breast; hence, application of in vitro data should be conducted with caution.

Equal but different! Improving care for older LGBT+ adults.

International human rights movements have improved the visibility and equality of lesbian, gay, bisexual and transgender+ (LGBT+) communities and their members. Health outcomes for LGBT+ people remain, however, worse than for their non-LGBT+ peers. Older LGBT+ people have experienced fewer positive changes, in part due to their lived experience of discrimination and their ongoing, unintentional invisibility in medical and social care. This article highlights the impacts of societal structure, health and social care on the lives of older LGBT+ people including physical and mental health, End of Life, Dementia, Housing and Care Settings, and a focus on the experiences of trans-people. We look at the existing improvements developed by LGBT+ communities (and their allies) and propose refreshing Person-Centred Care to improve inclusivity. Finally, we provide a framework for looking at the areas in which service challenges arise and suggest ways to address these to make health and social care services more ready to meet the needs of older LGBT+ people.

Landscape-scale drivers of pollinator communities may depend on land-use configuration.

Research into pollinators in managed landscapes has recently combined approaches of pollination ecology and landscape ecology, because key stressors are likely to interact across wide areas. While laboratory and field experiments are valuable for furthering understanding, studies are required to investigate the interacting drivers of pollinator health and diversity across a broader range of landscapes and a wider array of taxa. Here, we use a network of 96 study landscapes in six topographically diverse regions of Britain, to test the combined importance of honeybee density, insecticide loadings, floral resource availability and habitat diversity to pollinator communities. We also explore the interactions between these drivers and the cover and proximity of semi-natural habitat. We found that among our four drivers, only honeybee density was positively related to wild pollinator abundance and diversity, and the positive association between abundance and floral resources depended on insecticide loadings and habitat diversity. By contrast, our exploratory models including habitat composition metrics revealed a complex suite of interactive effects. These results demonstrate that improving pollinator community composition and health is unlikely to be achieved with general resource enhancements only. Rather, local land-use context should be considered in fine-tuning pollinator management and conservation. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Teucrium polium: Potential Drug Source for Type 2 Diabetes Mellitus.

The prevalence of type 2 diabetes mellitus is rising globally and this disease is proposed to be the next pandemic after COVID-19. Although the cause of type 2 diabetes mellitus is unknown, it is believed to involve a complex array of genetic defects that affect metabolic pathways which eventually lead to hyperglycaemia. This hyperglycaemia arises from an inability of the insulin-sensitive cells to sufficiently respond to the secreted insulin, which eventually results in the inadequate secretion of insulin from pancreatic ß-cells. Several treatments, utilising a variety of mechanisms, are available for type 2 diabetes mellitus. However, more medications are needed to assist with the optimal management of the different stages of the disease in patients of varying ages with the diverse combinations of other medications co-administered. Throughout modern history, some lead constituents from ancient medicinal plants have been investigated extensively and helped in developing synthetic antidiabetic drugs, such as metformin. Teucrium polium L. (Tp) is a herb that has a folk reputation for its antidiabetic potential. Previous studies indicate that Tp extracts significantly decrease blood glucose levels r and induce insulin secretion from pancreatic ß-cells in vitro. Nonetheless, the constituent/s responsible for this action have not yet been elucidated. The effects appear to be, at least in part, attributable to the presence of selected flavonoids (apigenin, quercetin, and rutin). This review aims to examine the reported glucose-lowering effect of the herb, with a keen focus on insulin secretion, specifically related to type 2 diabetes mellitus. An analysis of the contribution of the key constituent flavonoids of Tp extracts will also be discussed.

P-Glycoprotein-Mediated Efflux Using a Rapidly Maturing Caco2 Clone (CLEFF4) in Only 5 Days without Requiring Modified Growth Medium.

In drug discovery it is essential that one of the parameters tested for any new chemical entity is its affinity for human efflux systems, most notably P-glycoprotein (P-gp). These efflux systems affect not only rates of oral absorption but also rates of excretion through the liver, blood-brain barrier, and accumulation in potential target cells that upregulate efflux systems. Current methods to determine drugs' P-gp transport potential include in vitro bidirectional transport studies, and the two most common cell lines used are Caco2 and MDR1-transfected MDCK models. Caco2 cells are human but slow growing and require more than 3 weeks to mature, while MDCK cells are canine, but when transfected with human P-gp become a rapid model of P-gp affinity. Our laboratory has generated a Caco2 subclone called CLEFF4 that is fully human, yet now approaches the rapid nature of the MDCK model. No special medium is required. We have shown, in as little as 5 days postseeding, high transepithelial electrical resistance values of more than 1000 Ω·cm2 plus P-gp expression more than threefold higher than that of 21-day-old cells. Currently tested drugs included rhodamine 123 (Rh123), vinblastine, and doxorubicin, and all drugs exhibited P-gp-mediated efflux that was inhibited by PSC833. By day 6, bidirectional transport of Rh123 was as potent as that of mature Caco2 cells, for use in comparative P-gp affinity studies. We now have a human P-gp model that is rapid and works without any need for special accelerating medium. We believe this could be a welcome addition to the testing regime of new chemical entities.

Early warning systems in biosecurity; translating risk into action in predictive systems for invasive alien species.

Invasive alien species (IAS) are one of the most severe threats to biodiversity and are the subject of varying degrees of surveillance activity. Predictive early warning systems (EWS), incorporating automated surveillance of relevant dataflows, warning generation and dissemination to decision makers are a key target for developing effective management around IAS, alongside more conventional early detection and horizon scanning technologies. Sophisticated modelling frameworks including the definition of the 'risky' species pool, and pathway analysis at the macro and micro-scale are increasingly available to support decision making and to help prioritise risks from different regions and/or taxa. The main challenges in constructing such frameworks, to be applied to border inspections, are (i) the lack of standardisation and integration of the associated complex digital data environments and (ii) effective integration into the decision making process, ensuring that risk information is disseminated in an actionable way to frontline surveillance staff and other decision makers. To truly achieve early warning in biosecurity requires close collaboration between developers and end-users to ensure that generated warnings are duly considered by decision makers, reflect best practice, scientific understanding and the working environment facing frontline actors. Progress towards this goal will rely on openness and mutual understanding of the role of EWS in IAS risk management, as much as on developments in the underlying technologies for surveillance and modelling procedures.

Efflux transporters and tight junction expression changes in human gastrointestinal cell lines cultured in defined medium vs serum supplemented medium.

AIMS: Many gastrointestinal cell lines including Caco-2, LS174T and RKO require foetal calf serum (FCS) in culture medium. However, when isolating secreted product from conditioned medium (CM), after cell exposure to a trigger, it is better to remove FCS in the culture medium for identification of secreted products of interest. However, it is unknown whether defined medium adversely affects active efflux protein expression and tight junction formation. MATERIALS AND METHODS: Using different gastrointestinal cell lines chosen with different levels of efflux transporter expression, fully defined components, such as using transferrin, insulin, selenium and ethanolamine without FCS or with a reduced percentage of FCS (2%) were tested as an optimal choice for cell growth. In addition to morphological characteristics, the expression of the ABC efflux transporters, ABCB1 (P-glycoprotein [P-gp]), ABCC2 (multidrug resistance associated protein 2), ABCG2 (breast cancer resistance protein) and occludin was determined. KEY FINDINGS: The cells required a minimum of 2% FCS for expression of transporters. Fully defined medium with no serum adversely affected the expression of transporters, especially P-gp. An important characteristic of Caco-2 cells is its ability to form tight junctions. Caco-2 did not form adequate tight junctions without 10% FCS added in the medium, as evidenced by low TEER values and reduced occluding immunohistochemistry. SIGNIFICANCE: FCS is required for efflux protein expression and tight junction generation. Nevertheless, it is possible to use 5 fold less FCS which assists with low molecular weight secretion isolation. Passage number also contributes significantly to the presence of these transporters.

Impacts of neonicotinoid use on long-term population changes in wild bees in England.

Wild bee declines have been ascribed in part to neonicotinoid insecticides. While short-term laboratory studies on commercially bred species (principally honeybees and bumblebees) have identified sub-lethal effects, there is no strong evidence linking these insecticides to losses of the majority of wild bee species. We relate 18 years of UK national wild bee distribution data for 62 species to amounts of neonicotinoid use in oilseed rape. Using a multi-species dynamic Bayesian occupancy analysis, we find evidence of increased population extinction rates in response to neonicotinoid seed treatment use on oilseed rape. Species foraging on oilseed rape benefit from the cover of this crop, but were on average three times more negatively affected by exposure to neonicotinoids than non-crop foragers. Our results suggest that sub-lethal effects of neonicotinoids could scale up to cause losses of bee biodiversity. Restrictions on neonicotinoid use may reduce population declines.

The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation.

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10(-6) cm/sec, followed by amodiaquine around 20 x 10(-6) cm/sec; both mefloquine and artesunate were around 10 x 10(-6) cm/sec. Methylene blue was between 2 and 6 x 10(-6) cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.

Climate-driven spatial mismatches between British orchards and their pollinators: increased risks of pollination deficits.

Understanding how climate change can affect crop-pollinator systems helps predict potential geographical mismatches between a crop and its pollinators, and therefore identify areas vulnerable to loss of pollination services. We examined the distribution of orchard species (apples, pears, plums and other top fruits) and their pollinators in Great Britain, for present and future climatic conditions projected for 2050 under the SRES A1B Emissions Scenario. We used a relative index of pollinator availability as a proxy for pollination service. At present, there is a large spatial overlap between orchards and their pollinators, but predictions for 2050 revealed that the most suitable areas for orchards corresponded to low pollinator availability. However, we found that pollinator availability may persist in areas currently used for fruit production, which are predicted to provide suboptimal environmental suitability for orchard species in the future. Our results may be used to identify mitigation options to safeguard orchard production against the risk of pollination failure in Great Britain over the next 50 years; for instance, choosing fruit tree varieties that are adapted to future climatic conditions, or boosting wild pollinators through improving landscape resources. Our approach can be readily applied to other regions and crop systems, and expanded to include different climatic scenarios.

Species distribution models for crop pollination: a modelling framework applied to Great Britain.

Insect pollination benefits over three quarters of the world's major crops. There is growing concern that observed declines in pollinators may impact on production and revenues from animal pollinated crops. Knowing the distribution of pollinators is therefore crucial for estimating their availability to pollinate crops; however, in general, we have an incomplete knowledge of where these pollinators occur. We propose a method to predict geographical patterns of pollination service to crops, novel in two elements: the use of pollinator records rather than expert knowledge to predict pollinator occurrence, and the inclusion of the managed pollinator supply. We integrated a maximum entropy species distribution model (SDM) with an existing pollination service model (PSM) to derive the availability of pollinators for crop pollination. We used nation-wide records of wild and managed pollinators (honey bees) as well as agricultural data from Great Britain. We first calibrated the SDM on a representative sample of bee and hoverfly crop pollinator species, evaluating the effects of different settings on model performance and on its capacity to identify the most important predictors. The importance of the different predictors was better resolved by SDM derived from simpler functions, with consistent results for bees and hoverflies. We then used the species distributions from the calibrated model to predict pollination service of wild and managed pollinators, using field beans as a test case. The PSM allowed us to spatially characterize the contribution of wild and managed pollinators and also identify areas potentially vulnerable to low pollination service provision, which can help direct local scale interventions. This approach can be extended to investigate geographical mismatches between crop pollination demand and the availability of pollinators, resulting from environmental change or policy scenarios.

Rapid copper acquisition by developing murine mesothelioma: decreasing bioavailable copper slows tumor growth, normalizes vessels and promotes T cell infiltration.

Copper, an essential trace element acquired through nutrition, is an important co-factor for pro-angiogenic factors including vascular endothelial growth factor (VEGF). Decreasing bioavailable copper has been used as an anti-angiogenic and anti-cancer strategy with promising results. However, the role of copper and its potential as a therapy in mesothelioma is not yet well understood. Therefore, we monitored copper levels in progressing murine mesothelioma tumors and analyzed the effects of lowering bioavailable copper. Copper levels in tumors and organs were assayed using atomic absorption spectrophotometry. Mesothelioma tumors rapidly sequestered copper at early stages of development, the copper was then dispersed throughout growing tumor tissues. These data imply that copper uptake may play an important role in early tumor development. Lowering bioavailable copper using the copper chelators, penicillamine, trientine or tetrathiomolybdate, slowed in vivo mesothelioma growth but did not provide any cures similar to using cisplatin chemotherapy or anti-VEGF receptor antibody therapy. The impact of copper lowering on tumor blood vessels and tumor infiltrating T cells was measured using flow cytometry and confocal microscopy. Copper lowering was associated with reduced tumor vessel diameter, reduced endothelial cell proliferation (reduced Ki67 expression) and lower surface ICAM/CD54 expression implying reduced endothelial cell activation, in a process similar to endothelial normalization. Copper lowering was also associated with a CD4(+) T cell infiltrate. In conclusion, these data suggest copper lowering is a potentially useful anti-mesothelioma treatment strategy that slows tumor growth to provide a window of opportunity for inclusion of other treatment modalities to improve patient outcomes.

Bringing ecosystem services into economic decision-making: land use in the United Kingdom.

Landscapes generate a wide range of valuable ecosystem services, yet land-use decisions often ignore the value of these services. Using the example of the United Kingdom, we show the significance of land-use change not only for agricultural production but also for emissions and sequestration of greenhouse gases, open-access recreational visits, urban green space, and wild-species diversity. We use spatially explicit models in conjunction with valuation methods to estimate comparable economic values for these services, taking account of climate change impacts. We show that, although decisions that focus solely on agriculture reduce overall ecosystem service values, highly significant value increases can be obtained from targeted planning by incorporating all potential services and their values and that this approach also conserves wild-species diversity.

P-glycoprotein expression in Helicobacter pylori-positive patients: the influence of MDR1 C3435T polymorphism.

OBJECTIVE: The aim of this study was to determine whether the presence of Helicobacter pylori (H. pylori) infection and multidrug resistance protein 1 (MDR1) C3435T polymorphism had an influence on P-glycoprotein (P-gp) expression in the upper gastrointestinal tract. METHODS: A total of 76 patients who underwent upper gastroendoscopy at Sir Charles Gairdner Hospital in Western Australia from October 2010 to July 2011 were enrolled in the study. Antral and duodenal biopsies were collected for P-gp examination. Blood samples were taken and analyzed for MDR1 C3435T polymorphism. H. pylori infection status was confirmed by culture and polymerase chain reaction. RESULTS: A significant difference was found in P-gp expression between H. pylori-positive and H. pylori-negative patients (P = 0.028). For the MDR1 C3435T polymorphism, the TT genotype had a significantly lower P-gp expression compared with the CC genotype in antral specimens (P = 0.041). The homozygous TT genotype with H. pylori infection was also significantly different in P-gp expression compared with H. pylori-negative patients (P = 0.029). CONCLUSIONS: P-gp expression in the upper gastrointestinal tract is associated with H. pylori infection, and the TT genotype appeared to be associated with lower P-gp expression than the CC genotype in the stomach.

Development of a model for functional studies of ABCG2 (breast cancer resistance protein) efflux employing a standard BeWo clone (B24).

Human choriocarcinoma-derived BeWo cells express high levels of breast cancer resistance protein (BCRP/ABCG2) with no functional P-glycoprotein (P-gp) (ABCB1) activity, making them a potential model to study bidirectional ABCG2-mediated drug transport. However, the original BeWo clone (B24) available to researchers does not form confluent monolayers with tight junctions required by the model. Our aim was to adapt culture conditions to attempt to generate confluent BeWo monolayers for drug transport studies using the standard B24 clone. BeWo cells (B24; American Type Culture collection [ATCC]) were cultured in six-well plates or polycarbonate millicell inserts in a number of media formulations, growth supplements, and basement membrane substitutes. Cells were examined for confluence by microscopy, and transepithelial electrical resistance (TEER) was measured daily; monolayer permeability was assessed when TEER had stabilized. Optimal growth rates were achieved in culture conditions consisting of Medium 199 (M199) supplemented with epidermal growth factor (EGF; 20 ng/mL), vitamin supplements, and 10% fetal calf serum (FCS) with collagen coating. A TEER of 170 Ω in 0.6 cm(2) inserts was achieved 2 weeks after seeding under optimal conditions. The cell-impermeable diffusion marker 5(6) carboxy-2,7dichlorodihydrofluorescein (C-DCDHF) had a permeability coefficient of 3.5×10(-6) cm/s, indicative of minimal paracellular permeability. ABCG2 expression, as determined by immunoblotting, remained unaffected by confluency. In conclusion, we describe culture conditions for the B24 BeWo clone that facilitate the formation of monolayers with tighter junctions and reduced paracellular transport compared to previously published models. These growth conditions provide a good model of ABCG2-mediated drug transport in a human placental cell line.