Corticosteroids reduce pathologic interferon responses by downregulating STAT1 in patients with high-risk COVID-19.

We do not yet understand exactly how corticosteroids attenuate hyperinflammatory responses and alleviate high-risk coronavirus disease 2019 (COVID-19). We aimed to reveal the molecular mechanisms of hyperinflammation in COVID-19 and the anti-inflammatory effects of corticosteroids in patients with high-risk COVID-19. We performed single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from three independent COVID-19 cohorts: cohort 1 was used for comparative analysis of high-risk and low-risk COVID-19 (47 PBMC samples from 28 patients), cohort 2 for longitudinal analysis during COVID-19 (57 PBMC samples from 15 patients), and cohort 3 for investigating the effects of corticosteroid treatment in patients with high-risk COVID-19 (55 PBMC samples from 13 patients). PBMC samples from healthy donors (12 PBMC samples from 12 donors) were also included. Cohort 1 revealed a significant increase in the proportion of monocytes expressing the long noncoding RNAs NEAT1 and MALAT1 in high-risk patients. Cohort 2 showed that genes encoding inflammatory chemokines and their receptors were upregulated during aggravation, whereas genes related to angiogenesis were upregulated during improvement. Cohort 3 demonstrated downregulation of interferon-stimulated genes (ISGs), including STAT1, in monocytes after corticosteroid treatment. In particular, unphosphorylated STAT-dependent ISGs enriched in monocytes from lupus patients were selectively downregulated by corticosteroid treatment in patients with high-risk COVID-19. Corticosteroid treatment suppresses pathologic interferon responses in monocytes by downregulating STAT1 in patients with high-risk COVID-19. Our study provides insights into the mechanisms underlying COVID-19 aggravation and improvement and the effects of corticosteroid treatment.

In vivo-mimicking microfluidic perfusion culture of pancreatic islet spheroids.

Native pancreatic islets interact with neighboring cells by establishing three-dimensional (3D) structures, and are surrounded by perfusion at an interstitial flow level. However, flow effects are generally ignored in islet culture models, although cell perfusion is known to improve the cell microenvironment and to mimic in vivo physiology better than static culture systems. Here, we have developed functional islet spheroids using a microfluidic chip that mimics interstitial flow conditions with reduced shear cell damage. Dynamic culture, compared to static culture, enhanced islet health and maintenance of islet endothelial cells, reconstituting the main component of islet extracellular matrix within spheroids. Optimized flow condition allowed localization of secreted soluble factors near spheroids, facilitating diffusion-mediated paracrine interactions within islets, and enabled long-term maintenance of islet morphology and function for a month. The proposed model can aid islet preconditioning before transplantation and has potential applications as an in vitro model for diabetic drug testing.

Repurposing Penfluridol in Combination with Temozolomide for the Treatment of Glioblastoma.

Despite the presence of aggressive treatment strategies, glioblastoma remains intractable, warranting a novel therapeutic modality. An oral antipsychotic agent, penflurido (PFD), used for schizophrenia treatment, has shown an antitumor effect on various types of cancer cells. As glioma sphere-forming cells (GSCs) are known to mediate drug resistance in glioblastoma, and considering that antipsychotics can easily penetrate the blood-brain barrier, we investigated the antitumor effect of PFD on patient-derived GSCs. Using five GSCs, we found that PFD exerts an antiproliferative effect in a time- and dose-dependent manner. At IC50, spheroid size and second-generation spheroid formation were significantly suppressed. Stemness factors, SOX2 and OCT4, were decreased. PFD treatment reduced cancer cell migration and invasion by reducing the Integrin α6 and uPAR levels and suppression of the expression of epithelial-to-mesenchymal transition (EMT) factors, vimentin and Zeb1. GLI1 was found to be involved in PFD-induced EMT inhibition. Furthermore, combinatorial treatment of PFD with temozolomide (TMZ) significantly suppressed tumor growth and prolonged survival in vivo. Immunostaining revealed decreased expression of GLI1, SOX2, and vimentin in the PFD treatment group but not in the TMZ-only treatment group. Therefore, PFD can be effectively repurposed for the treatment of glioblastoma by combining it with TMZ.

Spheroid Culture of Mammalian Olfactory Receptor Neurons: Potential Applications for a Bioelectronic Nose.

The olfactory system can detect many odorants with high sensitivity and selectivity based on the expression of nearly a thousand types of olfactory receptors (ORs) in olfactory receptor neurons (ORNs). These ORs have a dynamic odorant detection range and contribute to signal encoding processes in the olfactory bulb (OB). To harness the capabilities of the olfactory system and develop a biomimetic sensor, stable culture and maintenance of ORNs are required. However, in vitro monolayer culture models have several key limitations: i) short available period of cultured neurons, ii) low cultural efficiency, and iii) long-term storage challenges. This study aims to develop a technique: i) to support the spheroid culture of primary ORN precursors facilitating stable maintenance and long-term storage, and ii) to demonstrate the viability of ORN spheroid culture in developing an olfactory system mimetic bioelectronic nose. Recombinant protein (REP; TGPG[VGRGD(VGVPG)6]20WPC) was used to form the ORN spheroids. Spheroid formation enabled preservation of primary cultured ORNs without a significant decrease in viability or the expression of stemness markers for ten days. Physiological characteristics of the ORNs were verified by monitoring intracellular calcium concentration upon odorant mixture stimulation; response upon odorant stimulation were observed at least for ten days in these cultivated ORNs differentiated from spheroids. Coupling ORNs with multi electrode array (MEA) enabled the detection and discrimination of odorants by analyzing the electrical signal patterns generated following odorant stimulation. Taken together, the ORN spheroid culture process is a promising technique for the development of a bioelectronic nose and high-throughput odorant screening device.

Surface-Modified Mesh Filter for Direct Nucleic Acid Extraction and its Application to Gene Expression Analysis.

Rapid and convenient isolation of nucleic acids (NAs) from cell lysate plays a key role for onsite gene expression analysis. Here, this study achieves one-step and efficient capture of NA directly from cell lysate by developing a cationic surface-modified mesh filter (SMF). By depositing cationic polymer via vapor-phase deposition process, strong charge interaction is introduced on the surface of the SMF to capture the negatively charged NAs. The NA capturing capability of SMF is confirmed by X-ray photoelectron spectroscopy, fluorescent microscopy, and zeta potential measurement. In addition, the genomic DNAs of Escherichia Coli O157:H7 can be extracted by the SMF from artificially infected food, and fluorescent signal is observed on the surface of SMF after amplification of target gene. The proposed SMF is able to provide a more simplified, convenient, and fast extraction method and can be applied to the fields of food safety testing, clinical diagnosis, or environmental pollutant monitoring.

Effects of various extracellular matrix proteins on the growth of HL-1 cardiomyocytes.

We present the physical and biochemical effects of extracellular matrixes (ECMs) on HL-1 cardiomyocytes. ECMs play major roles in cell growth, adhesion and the maintenance of native cell functions. We investigated the effects of 6 different cell culture systems: 5 different ECM-treated surfaces (fibronectin, laminin, collagen I, gelatin and a gelatin/fibronectin mixture) and 1 nontreated surface. Surface morphology was scanned and analyzed using atomic force microscopy in order to investigate the physical effects of ECMs. The attachment, growth, viability, proliferation and phenotype of the cells were analyzed using phase-contrast microscopy and immunocytochemistry to elucidate the biochemical effects of ECMs. Our study provides basic information for understanding cell-ECM interactions and should be utilized in future cardiac cell research and tissue engineering.