Investigation the effects of vitreous humor on proliferation and dedifferentiation of differentiated NTERA2 cells

Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

Effect of miR-384-targeting LINC00491 on proliferation, migration and invasion of tongue squamous cell carcinoma cells

Purpose: To investigate the effect of long-chain non-coding RNA LINC00491 (LncRNA LINC00491) on the proliferation, migration and invasion of tongue squamous cell carcinoma (TSCC) cells, and the underlying mechanism. Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) was applied to determine the expressions of LINC00491 and micro-RNA-384 (miR-384). Furthermore, LINC00491 and miR-384 were transfected into CAL-27 cells, while cell cycle was analyzed using flow cytometry. Cell proliferation was determined by methyl thiazolyl diphenyl-tetrazolium (MTT) assay. Cell migration and invasion were evaluated using Transwell experiments. The relationship between LINC00491 and miR-384 was confirmed using double luciferase reporting assay, while protein expression levels of P21, Ki67, E- cadherin, N-cadherin, and vimentin were assayed with Western blotting. Results: The expression of LINC00491 increased in TSCC tissues (p < 0.05). The proportion of cells in G1-phase increased, while the proportion of cells in S-phase decreased (p < 0.05). There was decrease in cell survival, cell migration and cell invasion (p < 0.05). The protein expression levels of Ki67, N- cadherin, and vimentin were lowered, while those of P21, E-cadherin protein were increased (p < 0.05). Transfection of LINC00491 and miR- 384 reduced the proportion of cells in G1 phase, but increased the proportion of cells in S-phase (p < 0.05). Moreover, cell survival, migration and invasion were increased. The protein expressions of Ki67, N-cadherin, and vimentin rose, while those of P21 and E-cadherin decreased (p < 0.05). Conclusion: LINC00491 promotes the proliferation, migration and invasion of TSCC cells by inhibiting miR-384. This finding provides a potential target for the treatment of TSCC.

FBXW7-mediated ERK3 degradation regulates the proliferation of lung cancer cells

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family, members of which play essential roles in diverse cellular processes during carcinogenesis, including cell proliferation, differentiation, migration, and invasion. Unlike other MAPKs, ERK3 is an unstable protein with a short half-life. Although deubiquitination of ERK3 has been suggested to regulate the activity, its ubiquitination has not been described in the literature. Here, we report that FBXW7 (F-box and WD repeat domain-containing 7) acts as a ubiquitination E3 ligase for ERK3. Mammalian two-hybrid assay and immunoprecipitation results demonstrated that ERK3 is a novel binding partner of FBXW7. Furthermore, complex formation between ERK3 and the S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) E3 ligase resulted in the destabilization of ERK3 via a ubiquitination-mediated proteasomal degradation pathway, and FBXW7 depletion restored ERK3 protein levels by inhibiting this ubiquitination. The interaction between ERK3 and FBXW7 was driven by binding between the C34D of ERK3, especially at Thr417 and Thr421, and the WD40 domain of FBXW7. A double mutant of ERK3 (Thr417 and Thr421 to alanine) abrogated FBXW7-mediated ubiquitination. Importantly, ERK3 knockdown inhibited the proliferation of lung cancer cells by regulating the G1/S-phase transition of the cell cycle. These results show that FBXW7-mediated ERK3 destabilization suppresses lung cancer cell proliferation in vitro.

Dynamic Activity of Histone H3-Specific Chaperone Complexes in Oncogenesis

Canonical histone H3.1 and variant H3.3 deposit at different sites of the chromatin via distinct histone chaperones. Histone H3.1 relies on chaperone CAF-1 to mediate replication-dependent nucleosome assembly during S-phase, while H3.3 variant is regulated and incorporated into the chromatin in a replication-independent manner through HIRA and DAXX/ATRX. Current literature suggests that dysregulated expression of histone chaperones may be implicated in tumor progression. Notably, ectopic expression of CAF-1 can promote a switch between canonical H3.1 and H3 variants in the chromatin, impair the chromatic state, lead to chromosome instability, and impact gene transcription, potentially contributing to carcinogenesis. This review focuses on the chaperone proteins of H3.1 and H3.3, including structure, regulation, as well as their oncogenic and tumor suppressive functions in tumorigenesis.

Komagataella phaffii Cue5 Piggybacks on Lipid Droplets for Its Vacuolar Degradation during Stationary Phase Lipophagy

Recently, we developed Komagataella phaffii (formerly Pichia pastoris) as a model for lipophagy, the selective autophagy of lipid droplets (LDs). We found that lipophagy pathways induced by acute nitrogen (N) starvation and in stationary (S) phase have different molecular mechanisms. Moreover, both types of lipophagy are independent of Atg11, the scaffold protein that interacts with most autophagic receptors and, therefore, is essential for most types of selective autophagy in yeast. Since yeast aggrephagy, the selective autophagy of ubiquitinated protein aggregates, is also independent of Atg11 and utilizes the ubiquitin-binding receptor, Cue5, we studied the relationship of K. phaffii Cue5 with differentially induced LDs and lipophagy. While there was no relationship of Cue5 with LDs and lipophagy under N-starvation conditions, Cue5 accumulated on LDs in S-phase and degraded together with LDs via S-phase lipophagy. The accumulation of Cue5 on LDs and its degradation by S-phase lipophagy strongly depended on the ubiquitin-binding CUE domain and Prl1, the positive regulator of lipophagy 1. However, unlike Prl1, which is required for S-phase lipophagy, Cue5 was dispensable for it suggesting that Cue5 is rather a new substrate of this pathway. We propose that a similar mechanism (Prl1-dependent accumulation on LDs) might be employed by Prl1 to recruit another ubiquitin-binding protein that is essential for S-phase lipophagy.

Stabilization of Dicalcium Silicate-Zn Composite Approaching Layered Double Hydroxide Structure for Bioactive Cement Applications

Layered Double Hydroxide (LDH) is ionic clay that is characterized by the union of metal cations and OH- hydroxides. LDH composites exhibit considerably high releasing and recharging capacity and have applications as bioactive cements. They can be prepared by direct co-precipitation of metal salts at controlled pH. The preparation is carried out from an acid solution of Zn (NO3)2.6H2O, Al (NO3)3.9H2O and a basic solution of Na2CO3 and NaOH, with a Zn/Al ratio = 3, the pH is stabilized between 9 and 9.5 at a constant temperature of 45°C. The objective of this study is to incorporate Zinc and Aluminum elements at different percentages in dicalcium silicate phase to produce C2S phase incorporating LDH composite. The characterizations of the developed phases by XRD and SEM indicate the formation of stoichiometric LDH phases Zn6Al2(OH)16CO3.4H2O and non-stoichiometric Zn0.61Al0.39 (OH)2(CO3)0.195.xH2O, the incorporation of Zn in the belitic C2S phase and not Al. The obtained micrographs by SEM(EDAX) analysis show new morphology of the stabilized composite.

DNMT1 regulates the timing of DNA methylation by DNMT3 in an enzymatic activity-dependent manner in mouse embryonic stem cells

DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.

Corrosion Properties of S-Phase/Cr2N Composite Coatings Deposited on Austenitic Stainless Steel

In order to study the suitability of the S-phase layers as the interlayer for Cr2N chromium nitride coatings, a number of composite coatings were deposited by the reactive magnetron sputtering (RMS) method on austenitic steel substrates with various initial surface conditions (as delivered and polished) and their corrosion resistance was assessed. Coatings with S-phase interlayer were deposited at three different nitrogen contents in the working atmosphere (15%, 30%, and 50%), which influenced the nitrogen concentration in the S-phase. Coatings with chromium, as a traditional interlayer to improve adhesion, and uncoated austenitic stainless steel were used as reference materials. Detailed microstructural and phase composition studies of the coatings were carried out by means of scanning electron microscopy (SEM), optical microscopy (LM), and X-ray diffraction (XRD) and were discussed in the context of results of corrosion tests carried out with the use of the potentiodynamic polarization method conducted in a 3% aqueous solution of sodium chloride (NaCl). The performed tests showed that the electrochemical potential of the S-phase/Cr2N composite coatings is similar to that of Cr/Cr2N coatings. It was also observed that the increase in the nitrogen content in the S-phase interlayer causes an increase in the polarization resistance of the S-phase/Cr2N composite coating. Moreover, with a higher nitrogen content in the S-phase interlayer, the polarization resistance of the S-phase/Cr2N coating is higher than for the Cr/Cr2N reference coating. All the produced composite coatings showed better corrosion properties in relation to the uncoated austenitic stainless steel.

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.