Oct4 differentially regulates chromatin opening and enhancer transcription in pluripotent stem cells

The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2

Hybrid e-rehabilitation services: SMART-system for remote support of rehabilitation activities and services

One of the most effective solutions in medical rehabilitation assistance is remote patient / person-centered rehabilitation. Rehabilitation also needs effective methods for the “Physical therapist – Patient – Multidisciplinary team” system, including the statistical processing of large volumes of data. Therefore, along with the traditional means of rehabilitation, as part of the “Transdisciplinary intelligent information and analytical system for the rehabilitation processes support in a pandemic (TISP)” in this paper, we introduce and define: the basic concepts of the new hybrid e-rehabilitation notion and its fundamental foundations; the formalization concept of the new Smart-system for remote support of rehabilitation activities and services; and the methodological foundations for the use of services (UkrVectores and vHealth) of the remote Patient / Person-centered Smart-system. The software implementation of the services of the Smart-system has been developed

Morphological peculiarites and functional activity of adipose-derived mesenchimal stem cells during in vitro cultivation conditions

The studies were conducted on 2-3-months-old males of C57BL/6 mice weighing 20–24 g. Obtaining and cultivating of adipose-derived mesenchimal stem cells (AD MSCs) were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. AD MSCs of the 2, 4, 7 and 12 passages were analyzed. Morphometric analysis was performed using a light microscopy. Morphometric parameters such as cell and nucleus area or nuclear-cytoplasmic ratio (NCR) were calculated using the Axiovision light microscope (Carl Zeiss, Germany) and ImageJ 1.45 software. Trypan blue dye used for investigation of the viability of MSC. The morphological characteristics of mesenchymal stem cells from adipose tissue during the process of cultivation changes: at the first passages of cultivation, the cells are spindle-shaped with two, at least three, long long cytoplasmic processes, located bipolar. Near the nucleus the Golgi complex is clearly visible – a sign of active cells. At later passages cells have a small cytoplasmic processes and the bipolar arrangement of processes changes by stellar arrangement. Golgi complex is also clearly visualized. The indicator of the nuclear-cytoplasmic ratio in MSC from adipose tissue is significantly reduced at 7 passage to 0.2189 ± 0.0122 (P < 0.01), and at 12 passage to 0.1111 ± 0.0086 (P < 0.001) compared to the 2 passage. The coefficient of proliferation of MSC from adipose tissue is significantly reduced at 12th passage. The viability of mesenchymal stem cells from adipose tissue with an increasing of a number of passages significantly reduces and at the 12th passage of cultivation reaches 84,67 ± 1,36* (P < 0.05). The content of apoptotic cells that exhibited sensitivity to serum-free significantly increased at 7 and 12 passages and was respectively 21.33 ± 1.36 (P < 0.05) and 23.67 ± 0.97% (P < 0.05)

Вміст жирних кислот в ліпідах мезенхімних стовбурових клітин культури жирової тканини

The content of fatty acids in the lipids of mesenchymal stem cells of dog adipose tissue culture was studied. Mesenchymal stem cells of dog adipose tissue culture were obtained by culturing the primary material in a CO2 incubator with a content of 5 % CO2, at a temperature of 37 °C in DMEM medium with the addition of 10–15 % fetal bovine serum and 1 % antibiotic-antimycotic. When the confluency of the monolayer reached 70–80 %, the cells were transferred to a suspension and subcultivated in order to reduce the heterogeneity of the culture and obtain a sufficient amount of biological material. The lipids of the obtained stem cells were analyzed for the content of fatty acids by the method of thin-layer gas-liquid chromatography. Determination of the content of lipids of fatty acids in FSK of a cat was carried out by the Folch method. A mixture of fatty acid methyl esters was analyzed on a Trace GC Ultra gas chromatograph with a flame ionization detector on a capillary column SPTM –2560, 100 m x 0.25 mm ID, 0.20 μm film (Supelco). Identification of fatty acids was carried out using a standard sample of Supelco 37 Сomponent FAME Mix. Quantitative assessment of the LC spectrum was carried out by the method of normalization of the peak planes of methylated LC derivatives and their content was determined as a percentage of the total content of all LC. The conducted study of the content of fatty acids in lipids made it possible to reveal certain features of the lipid metabolism of mesenchymal stem cells cultured in dog adipose tissue. A high content of oleic acid, characteristic of cells resistant to apoptosis and with high proliferative potential, was determined; a high ratio of unsaturated linoleic to saturated stearic acid (С18:1/С18.0), which reflects the high activity of the stearoyl-coenzyme-desaturase enzyme and, indirectly, the active state of the Wnt/β-catenin signaling pathway; inability to lengthen the chain of saturated fatty acids; lack or low activity of de novo synthesis of omega-6 polyunsaturated fatty acids. 18 fatty acids were found in the composition of lipids of fetal stem cells of a cat, of the saturated ones - the most palmitic acid (33.70 ± 0.02 %), of the monounsaturated ones – oleic acid (21.63 ± 0.03 %), of the polyunsaturated ones – linoleic acid (6.45 ± 0.07 %). The least amount of cis-,11,14-eicosadienoic acid (0.04 ± 0.01 %) was found in the composition of cell lipids. The total amount of saturated fatty acids in dog mesenchymal stem cell lipids was 65.65 ± 0.02 %), unsaturated fatty acids – 34.35 ± 0.02 %. Monoene fatty acids were determined in the amount of 24.46 ± 0.02 %, and polyene – 9.89 ± 0.02 %. The ratio index of polyunsaturated fatty acids ω 3 to ω 6 is 0.40. Lipids of mesenchymal stem cells of adipose tissue culture were characterized by a lower content of monoene unsaturated fatty acids 24.46 ± 0.02; (P < 0.05), with a higher content of ω3 fatty acids 3.04 ± 0.02 %; (P < 0.05), with a lower content of ω6 fatty acids 6.86 ± 0.02 %; (P < 0.05) in contrast to lipids of red bone marrow stem cells.Досліджено вміст жирних кислот в ліпідах мезенхімних стовбурових клітин культури жирової тканини собаки. Мезенхімні стовбурові клітини культури жирової тканини собаки отримували шляхом культивування первинного матеріалу в СО2 інкубаторі з вмістом 5 % СО2, за температури 37 °С у середовищі DMEM з додаванням 10–15 % фетальної сироватки великої рогатої худоби та 1 % антибіотика-антимікотика. Коли конфлюетність моношару сягала 70–80 %, клітини переводили в суспензію та проводили субкультивування з метою зниження гетерогенності культури та отримання достатньої кількості біологічного матеріалу. Ліпіди отриманих стовбурових клітин  досліджували на вміст жирних кислот методом тонкошарової газорідинної хроматографії. Визначення вмісту ліпідів жирних кислот ФСК кота проводили методом Фолча. Суміш метилових ефірів жирних кислот аналізували на газовому хроматографі Trace GC Ultra з полум’яно-іонізаційним детектором на капілярній колонці SPTM–2560, 100 m × 0,25 mm ID, 0,20 μm film (Supelco). Ідентифікацію жирних кислот проводили за допомогою стандартного зразка Supelco 37 Сomponent FAME Mix. Кількісну оцінку спектру ЖК проводили методом нормування площин піків метильованих похідних ЖК і визначали їхній вміст у відсотках від сумарного вмісту усіх ЖК. Проведене дослідження вмісту жирних кислот в ліпідах дало можливість виявити певні особливості ліпідного обміну мезенхімних стовбурових клітин культури жирової тканини собаки. Визначено високий вміст олеїнової кислоти, характерний для клітин, резистентних до апоптозу та з високим проліферативним потенціалом; високе співвідношення ненасиченої лінолевої до насиченої стеаринової кислоти (С18:1/С18.0), яке відображає високу активність фермента стеарол-коензим-десатурази та опосередковано – активний стан Wnt/β-катенін сигнального шляху; нездатність до подовження ланцюга насичених жирних кислот; відсутність або низьку активність синтезу de novo омега-6 поліненасичених жирних кислот. У складі ліпідів фетальних стовбурових клітин кота виявлено 18  жирних кислот, з насичених – найбільше пальмітинової кислоти (33,70 ± 0,02 %), з мононенасичених – олеїнової кислоти (21,63 ± 0,03 %), з поліненасичених – лінолевої кислоти (6,45 ± 0,07 %). Найменше у складі ліпідів клітин виявлено ціс-,11,14-ейкозадієнової кислоти (0,04 ± 0,01 %). Сумарна кількість насичених жирних кислот у ліпідах мезенхімниї стовбурових клітин собаки становила 65,65± 0,02%), ненасичених жирних кислот – 34,35 ± 0,02 %. Моноєнові жирні кислоти визначено у кількості 24,46± 0,02%, а полієнові – 9,89± 0,02%. Індекс співвідношення поліненасичених жирних кислот ω 3 до ω 6 становить 0,40. Ліпіди мезенхімних стовбурових клітин  культури жирової тканини  характеризувалися нижчим умістом моноєнових ненасичених жирних кислот 24,46 ± 0,02; (P < 0,05), більшим вмістом ω3 жирних кислот 3,04 ± 0,02 %; (P < 0,05), меншим вмістом ω6 жирних кислот 6,86 ± 0,02 %; (P < 0,05) на противагу ліпідам стовбурових клітин культури червоного кісткового мозку

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD