Stability of RNA sequences derived from the coronavirus genome in human cells.

Most viruses inhibit the innate immune system and/or the RNA degradation processes of host cells to construct an advantageous intracellular environment for their survival. Characteristic RNA sequences within RNA virus genomes or RNAs transcribed from DNA virus genomes contribute toward this inhibition. In this study, we developed a method called "Fate-seq" to comprehensively identify the RNA sequences derived from RNA and DNA viruses, contributing RNA stability in the cells. We examined the stabilization activity of 5,924 RNA fragments derived from 26 different viruses (16 RNA viruses and 10 DNA viruses) using next-generation sequencing of these RNAs fused 3' downstream of GFP reporter RNA. With the Fate-seq approach, we detected multiple virus-derived RNA sequences that stabilized GFP reporter RNA, including sequences derived from severe acute respiratory syndrome-related coronavirus (SARS-CoV). Comparative genomic analysis revealed that these RNA sequences and their predicted secondary structures are highly conserved between SARS-CoV and the novel coronavirus, SARS-CoV-2, which is responsible for the global outbreak of the coronavirus-associated disease that emerged in December 2019 (COVID-19). These sequences have the potential to enhance the stability of viral RNA genomes, thereby augmenting viral replication efficiency and virulence.

Establishment and characterization of patient-derived cancer models of malignant peripheral nerve sheath tumors.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are a rare subtype of soft-tissue sarcoma, derived from a peripheral branch or the sheath of the sciatic nerve, brachial plexus, or sacral plexus. The clinical outcomes for MPNST patients with unresectable or metastatic tumors are dismal, and novel therapeutic strategies are required. Although patient-derived cancer cell lines are vital for basic research and preclinical studies, few MPNST cell lines are available from public cell banks. Therefore, the aim of this study was to establish cancer cell lines derived from MPNST patients. METHODS: We used tumor tissues from five patients with MPNSTs, including one derived from a rare bone tissue MPNST. The tumor tissues were obtained at the time of surgery and were immediately processed to establish cell lines. A patient-derived xenograft was also established when a sufficient amount of tumor tissue was available. The characterization of established cells was performed with respect to cell proliferation, spheroid formation, and invasion. The mutation status of actionable genes was monitored by NCC Oncopanel, by which the mutation of 114 genes was assessed by next-generation sequencing. The response to anti-cancer agents, including anti-cancer drugs approved for the treatment of other malignancies was investigated in the established cell lines. RESULTS: We established five cell lines (NCC-MPNST1-C1, NCC-MPNST2-C1, NCC-MPNST3-C1, NCC-MPNST4-C1, and NCC-MPNST5-C1) from the original tumors, and also established patient-derived xenografts (PDXs) from which one cell line (NCC-MPNST3-X2-C1) was produced. The established MPNST cell lines proliferated continuously and formed spheroids while exhibiting distinct invasion abilities. The cell lines had typical mutations in the actionable genes, and the mutation profiles differed among the cell lines. The responsiveness to examined anti-cancer agents differed among cell lines; while the presence of an actionable gene mutation did not correspond with the response to the anticipated anti-cancer agents. CONCLUSION: The established cell lines exhibit various characteristics, including proliferation and invasion potential. In addition, they had different mutation profiles and response to the anti-cancer agents. These observations suggest that the established cell lines will be useful for future research on MPNSTs.

The Functional RNA Database 3.0: databases to support mining and annotation of functional RNAs.

We developed a pair of databases that support two important tasks: annotation of anonymous RNA transcripts and discovery of novel non-coding RNAs. The database combo is called the Functional RNA Database and consists of two databases: a rewrite of the original version of the Functional RNA Database (fRNAdb) and the latest version of the UCSC GenomeBrowser for Functional RNA. The former is a sequence database equipped with a powerful search function and hosts a large collection of known/predicted non-coding RNA sequences acquired from existing databases as well as novel/predicted sequences reported by researchers of the Functional RNA Project. The latter is a UCSC Genome Browser mirror with large additional custom tracks specifically associated with non-coding elements. It also includes several functional enhancements such as a presentation of a common secondary structure prediction at any given genomic window < or =500 bp. Our GenomeBrowser supports user authentication and user-specific tracks. The current version of the fRNAdb is a complete rewrite of the former version, hosting a larger number of sequences and with a much friendlier interface. The current version of UCSC GenomeBrowser for Functional RNA features a larger number of tracks and richer features than the former version. The databases are available at http://www.ncrna.org/.

Oxidized Perilla and Linseed Oils Induce Neuronal Apoptosis by Caspase-Dependent and -Independent Pathways.

Alpha-linolenic acid (ALA), a polyunsaturated fatty acid, is involved in bioregulatory functions. In recent years, the health-promoting effects of vegetable-derived edible oils rich in ALA have attracted attention. ALA has a variety of physiological effects such as anti-arteriosclerotic and antiallergic properties, but is prone to oxidation. Therefore, safety concerns exist with regard to adverse effects on humans induced by its oxides. However, the effects on neuronal cells induced by oxidized ALA-rich oils, such as perilla and linseed oils, have not been fully investigated. This information is very important from the viewpoint of food safety. In this study, we investigated the effects of oxidized perilla and linseed oils, which are rich in ALA, on the toxicity of neuronal SH-SY5Y cells. Perilla and linseed oils were significantly oxidized compared with other edible vegetable oils. These oxidized oils induce neuronal cell death and apoptosis via caspase-dependent and -independent pathways through reactive oxygen species (ROS) generation. Furthermore, they suppressed neurite outgrowth. These results suggest that oxidized perilla and linseed oils have the potential to cause neuronal loss and ROS-mediated apoptosis, and thus may affect the onset and progression of neurodegenerative disorders and other diseases.

Establishment and characterization of novel patient-derived extraskeletal osteosarcoma cell line NCC-ESOS1-C1.

Extraskeletal osteosarcoma (ESOS) is a rare mesenchymal malignancy producing osteoid and bone in soft tissue without skeletal attachment. ESOS exhibits chemoresistance and poor prognosis, and is distinct from osseous osteosarcoma. The biological characteristics of ESOS are not fully understood, and patient-derived cell lines of ESOS are not available from public cell banks. Here, we established a novel cell line of ESOS and characterized its genetic and biological characteristics as well as examined its response to anti-cancer reagents. The cell line was established using tumor tissue from a 58-year-old female patient with ESOS, and named as NCC-ESOS1-C1. Phenotypes relevant to malignancy such as proliferation and invasion were examined in vitro, and genetic features were evaluated using the NCC Oncopanel assay. The response to inhibitors was monitored by screening of an anti-cancer reagent library. The cells constantly proliferated, showing spheroid formation and invasion capabilities. The NCC Oncopanel revealed the presence of actionable mutations in PIK3CA. Library screening revealed the presence of anti-cancer reagents with significant anti-proliferative effects on NCC-ESOS1-C1 at a low concentration. In conclusion, we established and characterized a novel ESOS cell line, NCC-ESOS1-C1. This cell line will be a useful resource for basic research and preclinical studies.

Systematic Review of the Current Status of Human Sarcoma Cell Lines.

Sarcomas are rare mesenchymal malignant tumors with unique biological and clinical features. Given their diversity, heterogeneity, complexity, and rarity, the clinical management of sarcomas is quite challenging. Cell lines have been used as indispensable tools for both basic research and pre-clinical studies. However, empirically, sarcoma cell lines are not readily available. To understand the present status of sarcoma cell lines and identify their current challenges, we systematically reviewed reports on sarcoma cell lines. We searched the cell line database, Cellosaurus, and categorized the sarcoma cell lines according to the WHO classification. We identified the number and availability of sarcoma cell lines with a specific histology. We found 844 sarcoma cell lines in the Cellosaurus database, and 819 of them were named according to the WHO classification. Among the 819 cell lines, 36 multiple and nine single cell lines are available for histology. No cell lines were reported for 133 of the histological subtypes. Among the 844 cell lines, 148 are currently available in public cell banks, with 692 already published. We conclude that there needs to be a larger number of cell lines, with various histological subtypes, to better benefit sarcoma research.

Validity of simplified, calibration-less exercise intensity measurement using resting heart rate during sleep: a method-comparison study with respiratory gas analysis.

BACKGROUND: The recent development of wearable devices has enabled easy and continuous measurement of heart rate (HR). Exercise intensity can be calculated from HR with indices such as percent HR reserve (%HRR); however, this requires an accurate measurement of resting HR, which can be time-consuming. The use of HR during sleep may be a substitute that considers the calibration-less measurement of %HRR. This study examined the validity of %HRR on resting HR during sleep in comparison to percent oxygen consumption reserve (%VO2R) as a gold standard. Additionally, a 24/7%HRR measurement using this method is demonstrated. METHODS: Twelve healthy adults aged 29 ± 5 years underwent treadmill testing using the Bruce protocol and a 6-min walk test (6MWT). The %VO2R during each test was calculated according to a standard protocol. The %HRR during each exercise test was calculated either from resting HR in a sitting position (%HRRsitting), when lying awake (%HRRlying), or during sleep (%HRRsleeping). Differences between %VO2R and %HRR values were examined using Bland-Altman plots. A 180-day, 24/7%HRR measurement with three healthy adults was also conducted. The %HRR values during working days and holidays were compared. RESULTS: In the treadmill testing, the mean difference between %VO2R and %HRRsleeping was 1.7% (95% confidence interval [CI], - 0.2 to 3.6%). The %HRRsitting and %HRRlying values were 10.8% (95% CI, 8.8 to 12.7%) and 7.7% (95% CI, 5.4 to 9.9%), respectively. In the 6MWT, mean differences between %VO2R and %HRRsitting, %HRRlying and %HRRsleeping were 12.7% (95% CI, 10.0 to 15.5%), 7.0% (95% CI, 4.0 to 10.0%) and - 2.9% (95% CI, - 5.0% to - 0.7%), respectively. The 180-day, 24/7%HRR measurement presented significant differences in %HRR patterns between working days and holidays in all three participants. CONCLUSIONS: The results suggest %HRRsleeping is valid in comparison to %VO2R. The results may encourage a calibration-less, 24/7 measurement model of exercise intensity using wearable devices. TRIAL REGISTRATION: UMIN000034967.Registered 21 November 2018 (retrospectively registered).

Species-Specific Quantitative Proteomics Profiles of Sarcoma Patient-Derived Models Closely Reflect Their Primary Tumors.

PURPOSE: The purpose is to examine whether patient-derived sarcoma models recapitulate the spectrum of sarcoma heterogeneity seen in patients. EXPERIMENTAL DESIGN: To characterize patient-derived models for functional studies, proteomic comparisons with originating sarcomas representative of three intrinsic subtypes by MS are performed. RESULTS: Human protein profiling is found to be retained with high fidelity in patient-derived models. The protein profiles of patient sarcoma tumors and mouse stroma are characterized by species-specific quantitative proteomics. Protein-expression levels in mouse stroma are affected by the primary human tumor. The levels of stromal proteins derived from tumors are lower in PDXs and cell lines, and some human stromal proteins are replaced by the corresponding mouse proteins in PDXs. CONCLUSIONS AND CLINICAL RELEVANCE: These findings suggest that the effects of the microenvironment on drug responses may not reflect those in the primary tumor. This cross-species proteomic analysis in PDXs can potentially improve preclinical evaluation of treatment modalities and enhance the ability to predict clinical trial responses.

Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system.

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.

Fucoidan inhibits parainfluenza virus type 2 infection to LLCMK2 cells.

The effects of fucoidan and L-fucose, a fundamental major component of fucoidan, on the growth of human parainfluenza virus type 2 (hPIV-2) in LLCMK(2) cells were investigated. Fucoidan inhibited cell fusion and hemadsorption, but L-fucose only partly inhibited both. Virus RNA was not detected in the hPIV-2 infected cells cultured with fucoidan. However, L-fucose did not inhibit virus RNA synthesis. Indirect immunofluorescence study showed that virus protein synthesis was inhibited by fucoidan, but not by L-fucose. Furthermore, using a recombinant, green fluorescence protein-expressing hPIV-2, it was found that virus entry was inhibited by fucoidan, but not by L-fucose. These results suggested that fucoidan inhibited virus adsorption to the surface of the cells by binding to the cell surface and prevented infection, indicating that the sulfated polysaccharide form was important for the inhibition by fucoidan.

Simultaneous determination of isoflavones and bisphenol A in rat serum by high-performance liquid chromatography coupled with coulometric array detection.

A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU beta-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.