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Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Prevalence and dynamics of certain morphological variants of neuroglial brain malignancies (ICD-10 C71) are unknown in the Russian Federation., Objective: To assess the incidence of neuroglial brain malignancies in 2000-2020 considering individual records of morphologically verified cases in the cancer registry of the Arkhangelsk region., Material and Methods: We analyzed overall and age-adjusted incidence of neuroglial brain malignancies in 2000-2020 considering morphological subtypes of tumor. Incidence of morphologically verified glioblastoma was assessed in detail taking into account gender, age and place of residence. Segmented regression analysis was used to assess the dynamics and significance of linear trends., Results: In total, there were 1699 brain malignancies for the period from 2000 to 2020. Morphological verification was obtained in 1289 (76%) patients including 467 (27%), 92 (5%) and 307 (18%) ones with glioblastoma, anaplastic G3 glioma and G2 glioma, respectively. Percentage of glioblastoma and anaplastic gliomas increased from 23.4% and 3.9% in 2000 to 55.3% and 9.2% in 2020, respectively. Age-adjusted incidence for the entire C71 group decreased from 5.2 to 3.2 cases per 100,000 after 2015 (annual decline 7.1%). However, incidence of glioblastoma monotonously increased from 1.0 to 2.1 per 100,000 (annual increment 6.2%). Incidence was similar in men and women. Age-adjusted incidence was 50-70% higher among rural population., Conclusion: Significant increase (>2 times) in the incidence of glioblastoma was found over the past twenty years. Probably, it is associated with improved diagnosis and registration of this disease. In-depth analysis of morbidity and survival of patients with rare neuroglial tumors is required.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schmitz, Potekhina, Belousov and Lavrentieva.)

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% O 2 ). 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 LLC.)

One of the prevalent genetic causes of idiopathic male sterility is related to micro-deletions in AZF locus located in Y-chromosome. In total population, rate of such micro-deletions makes up to 1:4000. however, in infertile males their rate varies from 2% to 10%. In AZF locus three subregions are distinguished: AZFa, AZFb and AZFc. The loss of one or several subregions can result in disorder of spermatogenesis of various degree - from decreasing of its activity to Sertoli-cell syndrome manifested by azoospermia or oligospermia of severe degree. Therefore, implementation of genetic testing for presence of micro-deletions in AZF locus is a necessary test in case of prognosis of male sterility and its treatment. The purpose of study is to develop and test a diagnostic system of detection of micro-deletions in subregions of AZF locus using multiplex polymerase chain reaction in real-time. As a reference method a technique was implemented described in guidelines of the European Academy of Andrology conjointly with European Molecular Genetics Quality Network. The technique testing specified analysis of 33 samples of DNA separated from blood of males with azoospermia and oligospermia of severe degree. No discordant results were received as compared with reference method. In 27 DNA samples the deletions were detected in AZF locus: 4 AZFa deletions (15%), 2 AZFb deletions (7%), 17 AZFc deletions (63%) and 6 combined deletions of AZFb+candи AZFa+b+с (22%). The proposed technique permits detect micro-deletions of subregions of AZF locus., Competing Interests: The authors declare no conflict of interest.

We studied the distribution of mRNAs involved in dorsoventral differentiation of Xenopus laevis embryos (Xbra, chordin, Xnot, Xvent1, Xvent2, and Xwnt11) among polyribosomes (the translated form of mRNA) and informosomes (the nontranslated form of mRNA). It was shown using molecular hybridization that all of the studied templates are in the informosomes until the midgastrula stage, thus suggesting regulation at the level of translation. At the midgastrula stage (stage 11), translation begins on mRNAs of chordin, Xnot2, Xvent1, and Xbra, although mRNAs of Xvent1 and Xbra are partially located in the informosomes. The matrices of Xwnt11 are localized predominantly in the informosomes at the midgastrula stage, while those of Xvent2 are not seen in the polyribosomes until the end of gastrulation. We propose that the fate of different mRNAs is determined by different mechanisms of r-translational regulation in different cell lineages. It cannot be excluded that the Xvent2 transcript is not involved in translation and fulfills its morphogenetic functions in the form of RNA or RNP.