Stromal and Tumor Glioma-Derived Cells with Similar Characteristics have Differences in α-Smooth Muscle Actin Expression and Localization

Gliomas are solid brain tumors composed of tumor cells and recruited heterogenic stromal components. The study of the interactions between the perivascular niche and its surrounding cells is of great value in unraveling mechanisms of drug resistance in malignant gliomas. In this study, we isolated the stromal diploid cell population from oligodendroglioma and a mixed population of tumor aneuploid and stromal diploid cells from astrocytoma specimens. The stromal cells expressed neural stem/progenitor and mesenchymal markers showing the same discordant phenotype that is typical for glioma cells. Moreover, some of the stromal cells expressed CD133. For the first time, we demonstrated that this type of stromal cells had the typical myofibroblastic phenotype as the α-SMA+ cells formed α-SMA fibers and exhibited the specific function to deposit extracellular matrix (ECM) proteins at least in vitro. Immunofluorescent analysis showed diffuse or focal α-SMA staining in the cytoplasm of the astrocytoma-derived, A172, T98G, and U251MG glioma cells. We could suggest that α-SMA may be one of the main molecules, bearing protective functions. Possible mechanisms and consequences of α-SMA disruptions in gliomas are discussed

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

BACKGROUND: Cancer stem cells\u27 (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs\u27 subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. METHODS: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). RESULTS: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. CONCLUSION: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment

Appointment of lipid-lowering therapy in the Russian population: comparison of SCORE and SCORE2 (according to the ESSE-RF study)

Aim. In 2021, the European Society of Cardiology (ESC) guidelines for the prevention of cardiovascular diseases (CVDs) were published, where a new SCORE2 CVD risk assessment model was introduced. In our work, we compared approaches to determine the indications for initiating lipid-lowering therapy in the Russian population aged 25-64 years according to the guidelines for the diagnosis and treatment of lipid metabolism disorders of the Russian National Atherosclerosis Society (2020) and ESC guidelines for CVD prevention (2021).Material and methods. The ESSE-RF epidemiological study was conducted in 12 Russian regions. All participants signed informed consent and completed approved questionnaires. We performed anthropometric and blood pressure (BP) measurements, as well as fasting blood sampling. In total, 20665 people aged 25-64 years were examined. The analysis included data from 19546 respondents (women, 12325 (63,1%)).Results. Of the 19546 participants, 3828 (19,6%) were classified as high or very high CV risk based on the 9 criteria: BP ≥180/110 mm Hg, total cholesterol >8,0 mmol/l, low-density lipoprotein (LDL) >4,9 mmol/l, lipid-lowering therapy, chronic kidney disease (CKD) with glomerular filtration rate <60 ml/min/1,73 m2, type 2 diabetes, previous stroke and/or myocardial infarction. Of 3828 people, lipidlowering therapy was indicated in 3758 (98%) (criteria for LDL ≥1,8 mmol/l and LDL ≥1,4 mmol/l, respectively, high and very high risk). In addition, 5519 individuals aged <40 years were excluded from further analysis due to the lower age threshold of models. For 10199 participants aged >40 years without established CVD, diabetes, CKD, cardiovascular risk stratification was performed according to the SCORE and SCORE2. Of them, according to the Russian National Atherosclerosis Society (2020) and ESC 2021 guidelines, lipid-lowering therapy was indicated for 701 and 9487 participants, respectively.Conclusion. Using the new approach proposed by the ESC in 2021, the number of patients aged 40-64 years without CVD, diabetes and CKD with indications for lipidlowering therapy for primary prevention in Russia increases by 14 times compared with the 2020 Russian National Atherosclerosis Society guidelines

Mitochondrial Lysyl-tRNA Synthetase Independent Import of tRNA Lysine into Yeast Mitochondria

Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the in vitro import of tRNA into mitochondria. Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present for the import of lysyl tRNA in yeast

Generation of Trophoblast Stem Cells from Rabbit Embryonic Stem Cells with BMP4

Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal

FGF4 and Retinoic Acid Direct Differentiation of hESCs into PDX1-Expressing Foregut Endoderm in a Time- and Concentration-Dependent Manner

BACKGROUND: Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing beta cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm--the origin of pancreatic endoderm. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1(+) cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1(+) cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm. CONCLUSION/SIGNIFICANCE: In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling

BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin

Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast

Hypertension control during the COVID-19 pandemic: results of the MMM2021 in Russia

Repetitive quarantines and social restrictions during the coronavirus disease 2019 (COVID-19) pandemic have negatively affected the population health in general, and the control of hypertension (HTN) in particular.Aim. To evaluate the control of HTN in the Russian population during the COVID-19 period based on the results of screening for HTN May Measurement Month 2021 (MMM2021).Material and methods. During May-August 2021, 2491 participants from 11 Russian regions took part in the screening. Participation was voluntary without restrictions on sex. All participants were over 18 years of age. During the screening, blood pressure (BP) was measured three times using automatic and mechanical BP monitors. In addition, a questionnaire was filled out on behavioral risk factors, comorbidities and therapy. HTN was diagnosed with systolic BP ≥140 mmHg and/ or diastolic blood pressure ≥90 mmHg and/or taking antihypertensive therapy. The questionnaire included questions about prior COVID-19, vaccinations and their impact on the intake of antihypertensive drugs.Results. The analysis included data from 2461 respondents aged 18 to 92, of which 963 were men (39,1%). The proportion of hypertensive patients was 41,0%, while among them 59,0% took antihypertensives and 30,9% were effectively treated. In comparison with pre-pandemic period according to MMM2018-2019, the higher proportion of HTN patients in the Russian sample was revealed during MMM2021 (41,0% vs 31,3%, p<0,001) with a comparable proportion of patients receiving antihypertensive therapy (60,7% vs 59,0%, p=0,05) and treatment efficacy (28,7% vs 30,9%, p=0,36). Monotherapy was received in 44,7% of cases, while dual and triple combination therapy — in 30,9% and 14,1%, respectively. The majority of respondents (~90%) did not adjust their antihypertensive therapy during the COVID-19 pandemic.Conclusion. According to HTN screening in Russia, there is persistent ineffective control of HTN, which may be due to both the worsening pattern of behavioral risk factors, limited access to healthcare during COVID-19, and the inertia of physicians and low adherence of patients due to the asymptomatic HTN course in the majority

Lentivirus as a Tool for Lineage-Specific Gene Manipulations

The trophectoderm (TE) of blastocysts, the first epithelium established in mammalian development, (1) plays signaling, supportive, and patterning functions during preimplantation development, (2) ensures embryo implantation into the uterine wall, and (3) gives rise to extraembryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust nonpermeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics

PIAS Proteins as Repressors of Oct4 Function

The POU domain transcription factor Oct4 plays essential functions in the maintenance of pluripotent embryonic and germ cells of mammals. Molecular mechanisms of Oct4 action remain poorly understood. To isolate modulators of Oct4 activity, we performed a yeast two-hybrid screen with the Oct4 POU domain as a bait and isolated PIASy as an Oct4-interacting protein. Oct4 and PIASy interact in vivo via their POU domain and SAP-domain-containing N terminus, respectively. PIASy does not enhance Oct4 sumoylation but acts as a potent inhibitor of Oct4-mediated transcriptional activation, sequestering Oct4 protein from the vicinity of Cajal bodies and splicing speckles to the nuclear periphery. These modes of PIASy action are uncoupled from its sumoylation activity. Other PIAS family members, PIAS1 and PIAS3, can also interact with Oct4 in vivo and target Oct4 to the nuclear periphery, depending on cellular context. We propose that Oct4 inhibition, mediated by this new class of transcriptional partners, might be instrumental during mammalian development