Melatonin activates cell death programs for the suppression of uterine leiomyoma cell proliferation.

The circadian nature of melatonin has a protective effect on the progression of female reproductive cancers, including breast and ovarian cancers. However, the effect of melatonin on the growth of uterine leiomyoma is still unclear. In this study, we found that the growth of uterine leiomyoma ELT3 cells was reduced by treatment with melatonin. Treatment with melatonin increased the distribution of sub-G1 phase and increased DNA condensation in ELT3 cells. Melatonin-induced apoptosis and autophagy cell death progression were observed in ELT3 cells. Melatonin exerts a highly selective effect on primary normal human uterine smooth muscle (UtSMC) cells. The UtSMC cell cycle was arrested by melatonin treatment through up-regulation of p21, p27, and PTEN protein expression, but melatonin did not further promote apoptosis program activation. Melatonin reduced cell proliferation in ELT3 cells underlying the activation of melatonin MT1 and MT2 receptors, which in turn down-regulated the Akt-ERK1/2-NFκB signaling pathway. Melatonin reduced ELT3 tumor growth in both xenograft and orthotopic uterine tumor mice models. The extracellular matrix of the tumor was also reduced by melatonin treatment. Taken together, these results suggest that melatonin potentially plays a role in suppression of uterine leiomyoma growth.

Fabrication of a reticular poly(lactide-co-glycolide) cylindrical scaffold for the in vitro development of microvascular networks.

The microvascular network is a simple but critical system that is responsible for a range of important biological mechanisms in the bodies of all animals. The ability to generate a functional microvessel not only makes it possible to engineer vital tissue of considerable size but also serves as a platform for biomedical studies. However, most of the current methods for generating microvessel networks in vitro use rectangular channels which cannot represent real vessels in vivo and have dead zones at their corners, hence hindering the circulation of culture medium. We propose a scaffold-wrapping method which enables fabrication of a customized microvascular network in vitro in a more biomimetic way. By integrating microelectromechanical techniques with thermal reflow, we designed and fabricated a microscale hemi-cylindrical photoresist template. A replica mold of polydimethylsiloxane, produced by casting, was then used to generate cylindrical scaffolds with biodegradable poly(lactide-co-glycolide) (PLGA). Human umbilical vein endothelial cells were seeded on both sides of the PLGA scaffold and cultured using a traditional approach. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin on the cultured cell demonstrated the potential of generating a microvascular network with a degradable cylindrical scaffold. Our method allows cells to be cultured on a scaffold using a conventional culture approach and monitors cell conditions continuously. We hope our cell-covered scaffold can serve as a framework for building large tissues or can be used as the core of a vascular chip for in vitro circulation studies.

Nanostructured electrochemical biosensor for th0065 detection of the weak binding between the dengue virus and the CLEC5A receptor.

In this paper, we develop an effective method for detecting weak molecular bonding between the dengue virus (DV) and its receptor C-type lectin domain family 5, member A (CLEC5A). The CLEC5A-DV interaction is critical for DV-induced hemorrhagic fever and shock syndrome, so the sensing of CLEC5A-DV binding is crucial to realize a thorough study of the pathogenesis of dengue fever. Through a highly sensitive nanostructured sensing electrode of gold nanoparticles (GNPs) uniformly deposited on a nanohemisphere array, a label-free detection of the ultra weak binding between CLEC5A and the DV can be performed with electrochemical impedance spectroscopy (EIS). Experimental results demonstrate that the proposed approach is a highly promising method for investigating weak molecular interactions such as the ligand-receptor interaction of dengue fever, enterovirus (EV), or the interaction between cancer surface glycoproteins and their receptors. FROM THE CLINICAL EDITOR: Authors of this study investigated the ultra-weak binding between dengue virus and its CLEC5A receptor via electrochemical impedance spectroscopy and gold NP sensing electrode. Similar methods may be applicable in other infections and in cancer models as well.

A 3D Bioprinted Human Neurovascular Unit Model of Glioblastoma Tumor Growth.

A 3D bioprinted neurovascular unit (NVU) model is developed to study glioblastoma (GBM) tumor growth in a brain-like microenvironment. The NVU model includes human primary astrocytes, pericytes and brain microvascular endothelial cells, and patient-derived glioblastoma cells (JHH-520) are used for this study. Fluorescence reporters are used with confocal high content imaging to quantitate real-time microvascular network formation and tumor growth. Extensive validation of the NVU-GBM model includes immunostaining for brain relevant cellular markers and extracellular matrix components; single cell RNA sequencing (scRNAseq) to establish physiologically relevant transcriptomics changes; and secretion of NVU and GBM-relevant cytokines. The scRNAseq reveals changes in gene expression and cytokines secretion associated with wound healing/angiogenesis, including the appearance of an endothelial mesenchymal transition cell population. The NVU-GBM model is used to test 18 chemotherapeutics and anti-cancer drugs to assess the pharmacological relevance of the model and robustness for high throughput screening.

Development of human-derived, three-dimensional respiratory epithelial tissue constructs with perfusable microvasculature on a high-throughput microfluidics screening platform.

The COVID-19 pandemic has highlighted the need for human respiratory tract-based assay platforms for efficient discovery and development of antivirals and disease-modulating therapeutics. Physiologically relevant tissue models of the lower respiratory tract (LRT), including the respiratory bronchioles and the alveolar sacs, are of high interest because they are the primary site of severe SARS-CoV-2 infection and are most affected during the terminal stage of COVID-19. Current epithelial lung models used to study respiratory viral infections include lung epithelial cells at the air-liquid interface (ALI) with fibroblasts and endothelial cells, but such models do not have a perfusable microvascular network to investigate both viral infectivity and viral infection-induced thrombotic events. Using a high throughput, 64-chip microfluidic plate-based platform, we have developed two novel vascularized, LRT multi-chip models for the alveoli and the small airway. Both models include a perfusable microvascular network consisting of human primary microvascular endothelial cells, fibroblasts and pericytes. The established biofabrication protocols also enable the formation of differentiated lung epithelial layers at the ALI on top of the vascularized tissue bed. We validated the physiologically relevant cellular composition, architecture and perfusion of the vascularized lung tissue models using fluorescence microscopy, flow cytometry, and electrical resistance measurements. These vascularized, perfusable microfluidic lung tissue on high throughput assay platforms will enable the development of respiratory viral infection and disease models for research investigation and drug discovery.

Modeling SARS-CoV-2 and influenza infections and antiviral treatments in human lung epithelial tissue equivalents.

There is a critical need for physiologically relevant, robust, and ready-to-use in vitro cellular assay platforms to rapidly model the infectivity of emerging viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens.

Modeling SARS-CoV-2 and Influenza Infections and Antiviral Treatments in Human Lung Epithelial Tissue Equivalents.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.

Micropatterned topographies reveal measurable differences between cancer and benign cells.

During metastasis, cancer cells migrate away from the primary tumor-site, encountering different microenvironment topographies that may facilitate or inhibit cancer cell adherence and growth; those relate to sites of invasion and seeding. To evaluate topography effects, poly-lactic-poly-glycolic (PLGA) gels are generated as flat substrates, porous, or with rectangular microchannels of varying widths (5-100 µm) and depths (10/20 µm). The topography effect on time-dependent adherence, proliferation, morphology, alignment and long-term structural development of metastatic breast-cancer and benign cells are evaluated; adherence at short time-scales (3 h) is compared to developed morphologies and multicellular structures (>2 days) indicating function. At short time-scales, both cell types exhibit rounded morphologies, however, while the benign cells tend to cluster the cancer cells preferentially adhered as single cells at high-curvature substrate-sites (e.g. convex pore-edges or channel-edges). At long times, the benign cells develop extensive, tissue-like multicellular sheets spanning across several 10 µm deep channels or filling in single-file 20 µm-deep narrow channels (5-15 µm). Contrastingly, cancer cells mainly attach as single cells to high-curvature channel bottoms, in alignment with narrow channels. Thus, cell responses to topography, specifically their localization and growth in narrow microchannels, may provide a way to distinguish cancer from benign cells, by demonstrating their inherent function.

Development of double-generation gold nanoparticle chip-based dengue virus detection system combining fluorescence turn-on probes.

A sensing platform, combined amino acid labeling kit and a double-generation gold nanoparticle (DG-AuNP) chip, was designed to prove the existence of weak but crucial binding between the DV (dengue virus) and its CLEC5A receptor. At first, we have designed a kit combining the novel fluorescence turn-on sensors for lysine, arginine and cysteine amino acids. Advantages of this kit are that emission on-off switching can increase the signal-to-noise ratio and the virus must be fluorescently labelled with sufficient numbers of fluorophores because the lysine, arginine and cysteine residues of viral proteins are labelled simultaneously. Consequently, this kit can be used to light-on single DV spot both in solution and in cell under fluorescence microscopy. Second, the labeling kit was used to examine the DV binding to the CLEC5A-coated DG-AuNP chip. Based on our study, the double-generation gold nanoparticle construction of chip can support more coating areas for receptor CLEC5A and then, support more binding opportunities for DV. Meanwhile, the grooves between nanohemispheres will provide the extra driving force for DV stacking. We try to give a proof that this sensing platform is very useful for detection of weak binding mechanism.