Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel\'s protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.

A growing body of evidence suggests that anesthetics impact the outcome of patients with cancer after surgical intervention. However, the optimal dose and underlying mechanisms of co-administered anesthetics in lung tumor therapy have been poorly studied. Here, we aimed to investigate the role of combined anesthetics propofol, sufentanil, and rocuronium in treating lung cancer using an orthogonal experimental design and to explore the optimal combination of anesthetics. First, we evaluated the effects of the three anesthetics on the proliferation and invasion of A-549 cells using Cell Counting Kit 8 and Transwell migration and invasion assays. Subsequently, we applied the orthogonal experimental design (OED) method to screen the appropriate concentrations of the combined anesthetics with the most effective antitumor activity. We found that all three agents inhibited the proliferation of A-549 cells in a dose- and time-dependent manner when applied individually or in combination, with the highest differences in the magnitude of inhibition occurring 24 h after combined drug exposure. The optimal combination of the three anesthetics that achieved the strongest reduction in cell viability was 1.4 µmol/L propofol, 2 nmol/L sufentanil, and 7.83 µmol/L rocuronium. This optimal 3-drug combination produced a more beneficial result at 24 h than either single drug. Our results provide a theoretical basis for improving the efficacy of lung tumor treatment and optimizing anesthetic strategies.

The blood-brain barrier (BBB) limits the uptake of central nervous system (CNS)-targeting drugs into the brain. Engineering molecular shuttles for active transportation across the barrier has thus potential for improving the efficacy of such drugs. In vitro assessment of potential transcytosis capability for engineered shuttle proteins facilitates ranking and the selection of promising candidates during development. Herein, the development of an assay based on brain endothelial cells cultured on permeable recombinant silk nanomembranes for screening of transcytosis capability of biomolecules is described. The silk nanomembranes supported growth of brain endothelial cells to form confluent monolayers with relevant cell morphology, and induced expression of tight-junction proteins. Evaluation of the assay using an established BBB shuttle antibody showed transcytosis over the membranes with an apparent permeability that significantly differed from the isotype control antibody.

Cell migration and invasion have essential roles in both normal physiology and disease. As such, methodologies to assess cell migratory and invasive capacities are necessary to elucidate normal cell processes and underlying mechanisms of disease. Here, we describe commonly used transwell in vitro methods for the study of cell migration and invasion. The transwell migration assay involves the chemotaxis of cells through a porous membrane after the establishment of a chemoattractant gradient using two medium-filled compartments. The transwell invasion assay involves the addition of an extracellular matrix on top of the porous membrane which only permits chemotaxis of cells which possess invasive properties such as tumor cells.

Cell migration is a fundamental procedure involved in many physiological processes such as embryological development, tissue formation, immune defense or inflammation, and cancer progression. Here, we provide four in vitro assays that describe step-by-step cell adhesion, migration and invasion strategies, and their corresponding image data quantification. These methods include the following: two-dimensional wound healing assays, two-dimensional individual cell-tracking experiments by live cell imaging, and three-dimensional spreading and transwell assays. These optimized assays will facilitate physiological and cellular characterization of cell adhesion and motility, which may be used for fast screening of specific therapeutic drugs for adhesion function, novel strategies in pathophysiological diagnosis, and assaying new molecules involved in migration and invasion metastatic properties of cancer cells.

Cannabichromene (CBC) and cannabichromenic acid (CBCA) are cannabis constituents currently under evaluation for their therapeutic potential, but their pharmacological properties have not been thoroughly investigated. The most studied ATP-binding cassette (ABC) transporters, ABC subfamily G member 2 (ABCG2) and ABC subfamily B member 1 (ABCB1) limit absorption of substrate drugs in the gut and brain. Moreover, inhibitors of these proteins can lead to clinically significant drug-drug interactions (DDIs). The current study sought to examine whether CBC and CBCA affect ABCB1 and ABCG2 to advance their basic pharmacological characterisation. The plant cannabinoids CBC and CBCA were screened in vitro in a bidirectional transport assay to determine whether they were substrates and/or inhibitors of ABCB1 and ABCG2. Transwell assays with polarized epithelial Madin-Darby Canine Kidney II (MDCK) cells expressing ABCB1 or ABCG2 were used. Samples were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). CBCA was found to be an ABCB1 substrate, but not an ABCG2 substrate. CBC was not a substrate of either transporter. Neither CBCA nor CBC inhibited ABCB1 transport of prazosin or ABCG2 transport of digoxin. In silico molecular docking suggested CBCA binds ABCB1 in the access tunnel and the central binding pocket. CBC, an agent with anticonvulsant, anti-inflammatory and anti-depressant properties, is not a substrate or inhibitor of ABCB1 or ABCG2, which is favourable to its therapeutic development. CBCA is an ABCB1 substrate in vitro which might contribute to its poor absorption. These findings provide important basic pharmacological data to assist the therapeutic development of these cannabis constituents.

BACKGROUND: Adenosine monophosphate deaminase 3 (AMPD3) is an isoenzyme involved in the regulation of the energetic metabolism of mammalian cells. Cancer cells have a high demand for their energy supply. This experimental study aimed to illustrate the role of AMPD3 in human head and neck squamous cell carcinoma (HNSCC).
METHODS: Real-time quantitative reverse transcription-polymerase chain reaction was used to investigate the expression of the AMPD3 gene in human HNSCC tissues to assess the changes in cancerous and noncancerous parts and the correlation with different tumor behavior. The functions of AMPD3 were investigated using wound-healing and migration assays.
RESULTS: AMPD3 was significantly down-regulated in cancerous tissues of HNSCC (p=0.001) and this was correlated with more advanced tumor and clinical stages. Patients with high expression had better 5-year survival. AMPD3 knock-down in SCC-4 and SCC-25 cells demonstrated reduction of proliferation but increased migration and invasion.
CONCLUSIONS: To our knowledge, this is the first report evidencing the expression pattern of AMPD3 in HNSCC and demonstrated that high AMPD3 expression might represent a good prognostic biomarker. AMPD3 may have an antiproliferative potential but its down-regulation may not contribute to reducing the migration and invasion of HNSCC cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are especially useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology, and developmental biology. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. The aim of this article is to discuss the existing methods for transwell migration/invasion studies, the problems associated with this assay, and proposed modifications to this methodological approach that makes it simple to perform and improve the assay accuracy. Results of our studies demonstrated that the count of cells that had grown on top of the membrane is important to accurately evaluate the percentage of migrated/invaded cells. The results also showed that the transparent transwell insert with 4\',6-diamidino-2-phenylindole (DAPI) stained cells is the best approach to ease the analysis of cell numbers on top of the membranes. In addition, the overlay of bright light (representing membrane pores) and DAPI images can further improve the accuracy of cell count. All these modifications in combination simplify the assay performance and improve the accuracy of the transwell migration assay method.

The present study delineates the fabrication of paper-based devices for culturing liver cells and developing related bioassays. The devices were prepared by conventional lab-based LaserJet printing technology and employed for 3D cell culture. Our results demonstrated that the devices efficiently supported the growth of multiple cell types incuding HepG2, HUVEC, fibroblasts, and MSCs. We further showed that the device specifications (grade of paper or design parameters) greatly impacted the functional phenotype of the HepG2 cells. We also explored the application of the developed devices for routine cell culture, drug screening, coculture, and transwell migration assays. The cellular responses observed on the paper under different culture configurations were similar to those obtained in the case of tissue culture plate (TCP). Moreover, we showed that the paper-based devices were compatible with the immunocytochemistry and ELISA procedures (no indication of nonspecific matrix-antibody interaction). Considering the simplicity, experimental flexibility, cost-effectiveness, and multiplexibility of the paper-based liver models, it is deemed to be ideal for developing cell-based bioassays, especially in resource-limited settings.

Retinoblastoma is an intraocular malignant tumor and generally occurred in childhood. Here, we intended to appraise the functional influence of microRNA-142-5p (miR-142-5p) in retinoblastoma. MiR-142-5p was declined, and MYCN was upregulated in retinoblastoma tissues and cells. Moreover, miR-142-5p restricted cell proliferation, migration, invasion, and enhanced cell apoptosis in retinoblastoma cells. MYCN was adversely controlled by miR-142-5p. Besides, the inhibition of miR-142-5p-mediated effects on retinoblastoma progression were blocked by MYCN overexpression in retinoblastoma cells. This research illustrated that miR-142-5p restricted retinoblastoma progression via interacting with MYCN.