Live reporting for hypoxia : Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O2). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs. © 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LL

Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow

Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays. We provide a brief overview of successful, perspective, and hopeful attempts at using genetically encoded fluorescent sensors in HTS of modulators of ion channels, Ca2+ homeostasis, GPCR activity, and for screening cytotoxic, anticancer, and anti-parasitic compounds. We discuss the advantages of sensors in whole organism drug screening models and the perspectives of the combination of human disease modeling by CRISPR techniques with genetically encoded fluorescent sensors for drug screening

A Quest for Global Leadership: A Case Study of Tokyo Electron Group

In the regimes of turbulent business environment sustainability of the competitive advantage highly depends on the ability of a company to respond to macro-environmental changes, thus it very vivid reminds of the quest with the main prize called the global leadership. This paper is a case study of Japanese Company Tokyo Electron, which has being at the market of semiconductor production equipment for nearly half of a century and made its way from a trading company to one of the world leading manufacturers. In a response to the global change worldwide, TEL’s top-management developed a new strategy Global Position One, with the main objective to achieve position number one globally. By addressing today’s business environment issues, paying a fair attention to corporate social responsibility and reducing the negative environment of its operations, TEL has created new business opportunities, enlarged its product line and successfully entered new areas. Being an industry innovator and possessing an outstanding R&D abilities, Tokyo Electron keeps the right pace of new technologies delivery. This paper evaluates how the macro-environmental changes has shaped Tokyo Electron’s strategy, examines it’s dynamic capabilities, estimates strengths and weaknesses of TEL and opportunities and threats which might be harmful to achieve its major objectives. Based on the results of the research we make a forecast whether a quest for global leadership is successful for TEL and identify the issues to be addressed

Hypoxia Onset in Mesenchymal Stem Cell Spheroids: Monitoring With Hypoxia Reporter Cells

The therapeutic and differentiation potential of human mesenchymal stems cells (hMSCs) makes these cells a promising candidate for cellular therapies and tissue engineering. On the path of a successful medical application of hMSC, the cultivation of cells in a three-dimensional (3D) environment was a landmark for the transition from simple two-dimensional (2D) testing platforms to complex systems that mimic physiological in vivo conditions and can improve hMSC curative potential as well as survival after implantation. A 3D arrangement of cells can be mediated by scaffold materials where cells get entrapped in pores, or by the fabrication of spheroids, scaffold-free self-organized cell aggregates that express their own extracellular matrix. Independently from the cultivation method, cells expanded in 3D experience an inhomogeneous microenvironment. Many gradients in nutrient supply, oxygen supply, and waste disposal from one hand mimic in vivo microenvironment, but also put every cell in the 3D construct in a different context. Since oxygen concentration in spheroids is compromised in a size-dependent manner, it is crucial to have a closer insight on the thresholds of hypoxic response in such systems. In this work, we want to improve our understanding of oxygen availability and consequensing hypoxia onset in hMSC spheroids. Therefore, we utilized human adipose tissue-derived MSCs (hAD-MSCs) modified with a genetical sensor construct to reveal (I) the influence of spheroid production methods and (II) hMSCs cell number per spheroid to detect the onset of hypoxia in aggregates. We could demonstrate that not only higher cell numbers of MSCs, but also spheroid formation method plays a critical role in onset of hypoxia

Opposite changes of regulatory T cell blood content may differentially contribute to atherosclerosis or lymphoproliferative disorders

Background. Chronic autoimmune inflammation in arterial wall may lead to atherosclerosis progression. Objective. The aim of this study was to investigate the association between Treg, Th17 and B1a cell blood frequencies as well as IgM autoantibodies to oxLDL and the abundance of carotid atherosclerosis. Material and methods. 18 patients with increased IMT (intima-media thickness) and 65 patients with different severity of carotid atherosclerotic plaques were included. Treg, Th17 and B1a cell blood frequencies were assessed via direct immunofluorescence staining and flow cytometry, oxLDL as well as IgM autoantibodies to oxLDL were measured with commercial kits. Results. We observed higher values of Treg in patients without carotid atherosclerosis. Patients with intact carotid arteries as compared to patients with mild atherosclerotic plaques had decreased Th1 levels. OxLDL IgM levels were higher in patients with intact carotid arteries. Patients who received statin treatment had higher levels of Treg. Immunophenotyping of B cells revealed two cases of monoclonal B-cell lymphocytosis and 1 case of B-CLL (B-cell chronic lymphocytic leukemia) in elderly patients with intact carotid arteries. Conclusion. We hypothesize that certain parameters of cell immunity may hamper atherosclerosis while protecting from lymphoproliferative disorders

Physiological and Genomic Characterization of Actinotalea subterranea sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family Actinotaleaceae

The goal of the present work was to determine the diversity of prokaryotes involved in anaerobic oil degradation in oil fields. The composition of the anaerobic oil-degrading methanogenic enrichment obtained from an oil reservoir was determined by 16S rRNA-based survey, and the facultatively anaerobic chemoorganotrophic bacterial strain HO-Ch2T was isolated and studied using polyphasic taxonomy approach and genome sequencing. The strain HO-Ch2T grew optimally at 28 °C, pH 8.0, and 1–2% (w/v) NaCl. The 16S rRNA gene sequence of the strain HO-Ch2T had 98.8% similarity with the sequence of Actinotalea ferrariae CF5-4T. The genomic DNA G + C content of strain HO-Ch2T was 73.4%. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the genome of strain HO-Ch2T and Actinotalea genomes were 79.8–82.0% and 20.5–22.2%, respectively, i.e., below the thresholds for species delineation. Based on the phylogenomic, phenotypic, and chemotaxonomic characterization, we propose strain HO-Ch2T (= VKM Ac-2850T = KCTC 49656T) as the type strain of a new species within the genus Actinotalea, with the name Actinotalea subterranea sp. nov. Based on the phylogenomic analysis of 187 genomes of Actinobacteria we propose the taxonomic revision of the genera Actinotalea and Pseudactinotalea and of the family Actinotaleaceae. We also propose the reclassification of Cellulomonas carbonis as Actinotalea carbonis comb. nov., Cellulomonas bogoriensis as Actinotalea bogoriensis comb. nov., Actinotalea caeni as Pseudactinotalea caeni comb. nov., and the transfer of the genus Pseudactinotalea to the family Ruaniaceae of the order Ruaniales

Correction: Semenova et al. Physiological and Genomic Characterization of <i>Actinotalea subterranea</i> sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family <i>Actinotaleaceae. Microorganisms</i> 2022, <i>10,</i> 378

The authors wish to make the following correction to this paper [...

Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types